Research ArticleCNP-pGC-cGMP-PDE3-cAMP Signal Pathway Upregulated inGastric Smooth Muscle of Diabetic Rats

Ying-Lan Cai1 Mo-Han Zhang1 Xu Huang2 Jing-Zhi Jiang1 Li-Hua Piao1

Zheng Jin1 and Wen-Xie Xu2

1Department of Physiology Yanbian University School of Medicine 977 Gongyuan Road Yanji Jilin 133002 China2Department of Physiology Shanghai Jiaotong University School of Medicine 800 Dongchuan Road 328 Wenxuan Medical BuildingShanghai 200240 China

Correspondence should be addressed to Zheng Jin zhengjinybueducn and Wen-Xie Xu wenxiexusjtueducn

Received 13 August 2014 Accepted 1 October 2014

Academic Editor Jae Yeoul Jun

Copyright copy 2015 Ying-Lan Cai et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Our previous studies have shown that CNP-NPR-BpGC-cGMP is upregulated in the diabetic ratsThe present study was designedto determine whether the upregulation of CNP-NPR-BpGC-cGMP signal pathway affects cGMP-PDE3-cAMP signal pathwayin diabetic gastric smooth muscle The gastric smooth muscle motility was observed by using isometric measurement PDEsexpressions in diabetic gastric smooth muscle tissue were observed by using immunohistochemistry Western blotting and RT-PCRmethods The results demonstrated that the inhibitory effect of CNP on the spontaneous contraction of gastric antral circularsmooth muscle was potentiated in STZ-induced diabetic rat CNP-induced increase of cGMP and cAMP was much higher indiabetic gastric smoothmuscle tissue than in controlsThe expression of PDE3 is downregulated while the levels of gene expressionof PDE1 PDE2 PDE4 and PDE5were not altered in the diabetic gastric smoothmuscle tissueThe results suggest that the sensitivityof gastric smooth muscle to CNP is potentiated via activation of CNP-pGC-cGMP-PDE3-cAMP signal pathway in STZ-induceddiabetic rats which may be associated with diabetes-induced gastric motility disorder

1 Introduction

Gastrointestinal motility disorders are common complica-tions of diabetes which can happen in all regions of thegastrointestinal tract It has been reported that 75of patientswith diabetes are often accompanied by gastrointestinalsymptoms such as abdominal distention epigastric discom-fort and constipation [1] Diabetic gastrointestinal motilitydisorders and gastric emptying delay significantly affect thepharmacokinetics of hypoglycemic and other drugs result-ing in absorption delay of these drugs and poor glycemiccontrol Meanwhile the delay of absorption probably leadsto hypoglycemia which also affects the curative effect ofhypoglycemic drugs However the mechanism of diabetes-induced gastric motility disorders is still unclear [1 2]

C-type natriuretic peptide (CNP) is an important mem-ber of natriuretic peptides (NPs) family which was firstlydiscovered in the gastrointestinal tract byKomatsu et al [3] in1991Then Gower Jr et al [4] found three kinds of natriuretic

peptide receptors (NPRs) in the mucosa and smooth muscletissues of gastric antrum in 2001 which were named NPR-ANPR-B and NPR-C The NPs-NPR-A BpGC-cGMP signalpathway was found to be involved in the inhibitory effect ofNPs on spontaneous contraction in gastric smooth muscle ofguinea pig rat and human [5 6] Elevation of intracellularcAMP and cGMP has been associated with smooth musclerelaxation in several regions of the gastrointestinal tractincluding the lower oesophageal sphincter ileum proximalcolon taenia coli and internal anal sphincter [7ndash13] Theintracellular levels of cAMP and cGMP reflect a balancebetween their synthesis and catabolism the latter beingregulated by the enzymes of phosphodiesterase (PDE) fam-ily At least five families designated PDEs 1ndash5 have beenclassified according to their substrate preference cofactorrequirements and sensitivity to endogenous inhibitors andactivators [14ndash16] In the heart PDE1 PDE2 PDE3 PDE4PDE5 PDE8 and PDE9 have been described PDE1 PDE2and PDE3 can hydrolyze both cAMP and cGMP whereas

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 305258 9 pageshttpdxdoiorg1011552015305258

2 Gastroenterology Research and Practice

PDE4 and PDE8 are selective for cAMP and PDE5 and PDE9are selective for cGMP However cGMP can inhibit cAMPhydrolysis by PDE3 and possibly PDE1 whereas cGMP canactivate PDE2 [17]

We have found that both NPR-B expression in smoothmuscle of gastric antrum and the activity of membrane-bound guanylate cyclase (pGC) were significantly increasedin diabetic rats which indicate the upregulation of CNP-NPR-BpGC-cGMP signal pathway in diabetic gastricantrum [18 19] And we also found that cGMP producedthrough CNP-NPR signal pathway induced the generationof cAMP via cGMP-PDE3-cAMP signal pathway followedby the activation of PKA resulting in the inhibition ofL-type calcium current which inhibited the spontaneouscontraction of gastric smooth muscle together with cGMP[20]

According to our above studies we are wonderingwhether the upregulation of CNP-NPR-BpGC-cGMP signalpathway affects cGMP-PDE3-cAMP signal pathway in dia-betic animals In the present study we used the diabetic ratmodel induced by streptozotocin (STZ) to explore the activityof cGMP-PDE3-cAMP signal pathway in the gastric antrum

2 Materials and Methods

21 Animals and STZ-Induced Diabetic Rat Model Wistarrats (either sex weighed 220sim250 g) used in the present studywere provided by Experimental Animal Center of YanbianUniversity Animals were fasted overnight with ad libitumaccess to water before intraperitoneal administration of STZ(Sigma-Aldrich St Louis MO USA) which was freshlyprepared in citrate buffer at a dose of 65mgKg bodyweight toproduce diabetic model Control group was intraperitoneallyadministered the same volume of citrate buffer One weekafter administration blood was withdrawn from the rat tailvein to measure glucose concentration Diabetes was definedwhen the blood glucose level was above 350mgdL Thecontrol and diabetic animals were raised separately with freeaccess to food and water for four weeks

22 Preparation of Muscle Strips and Isometric Tension Mea-surement Rats were anaesthetized and the stomachs wereremoved quickly and placed in aerated (95 O

2and 5

CO2) Krebs solution which contains (mM) NaCl 118 KCl

475 CaCl2254 KH

2PO4119 MgSO

4119 NaHCO

325 and

glucose 10 The stomach was cut along the lesser curvatureand the mucosa was removed carefully The muscle strips (2times 12mm) were cut along the circular axis of the antrum andthenmounted in a vertical organ bath containing oxygenatedKrebs solution maintained at 37∘C One end of the strip wasfixed onto a platinum hook and the other end was fixed ontoan isometric force transducer connected to the RM6240Cbiological signal processing system (Chengdu EquipmentFactory Chengdu China) to record the mechanical activityA tension of 1 g was applied to the tissues and they were equi-librated for 40min before the experiments when rhythmicspontaneous contractions were recorded All experimentalprotocols performed were approved by the local Animal

Care Committee and conformed to the Guide for the Careand Use of Laboratory Animals published by the Scienceand Technology Commission of China (STCC Publicationnumber 2 revised 1988)

After recording the baseline contraction of control ordiabetic rats 01 120583M CNP (Sigma-Aldrich St Louis MOUSA) was added to the organ bath The amplitude andfrequency of contraction before and after administration ofCNP were recorded for 5min to observe the sensitivity ofgastric smooth muscle to CNP in control and diabetic rats

The bath solution after administration was collected andfrozen in liquid nitrogen and then kept at minus80∘C for lateranalysis of the content of cAMP and cGMP

23 cGMP and cAMP Content The bath solution of 300 120583Lwas treated with 300120583L 6 triethylamine (TCA) and thenincubated for 15min at room temperature The samples werewashed three times with water-saturated ethyl ether andlyophilized with lyophilizer (Savant Farmingdale NY) Theintracellular contents of cGMP and cAMP in the sampleswere determined using cGMP or cAMPDirect ImmunoassayKit (BioVision Research Products USA) after being recon-stituted in the assay buffer The standards and samples wereacetylated firstly according to themanufacturerrsquos instructionsto increase the sensitivity of the assay

24 Immunohistochemistry The stomachs of control anddiabetic rats were obtained as described above After beingwashed with physiological saline to remove the gastric con-tent the antrum was fixed with 4 paraformaldehyde for24 h at 4∘C and dehydrated in ethanol and then embeddedwith paraffinThe sections were cut mounted on glass slidesand dried overnight After being deparaffinized with xyleneand rehydrated in ethanol the sections were washed in PBSfor 5min Endogenous peroxidase activity was blocked withincubation in 3 hydrogen peroxide at room temperaturefor 30min The sections were then incubated at 100∘C in10mmoLL citrate buffer (pH 60) for 10min to retrieveantigens cooled for 20min and then washed in PBS Afterbeing incubated in PBS containing 10 normal goat serumat 37∘C for 45min the sections were incubated with rabbitanti-PDE2 antibody (1 500 Abcam Cambridge MA USA)or rabbit anti-PDE3 antibody (1 500 Abcam CambridgeMA USA) at 4∘C overnight After washing the sections wereincubated with biotinylated goat anti-rabbit IgG at 37∘C for30min followed by incubationwith streptavidin-horseradishperoxidase at 37∘C for 30min 331015840-Diaminobenzidine wasused as a chromogen and hematoxylin was used for counter-staining The sections for which the primary antibodies wereomitted in the same procedure were used as controls

25 Western Blotting Analysis Rat antral tissues obtainedfrom the protocol described above were homogenized inRIPA lysis buffer (Beyotime Jiangsu China) supplementedwith 1mM PMSF (Beyotime Jiangsu China) on ice Thehomogenate was then centrifuged at 12000timesg for 15min at

Gastroenterology Research and Practice 3

Control

Diabetes

075g

40 s

01 120583molL CNP

01 120583molL CNP

(a)

0

10

20

30

40

50

60

70

80

90

Inhi

bitio

n of

ampl

itude

()

lowast

Control Diabetic

(b)

0

10

20

30

40

50

60

Control Diabetic

Inhi

bitio

n of

freq

uenc

y (

)

lowast

(c)

0

Tens

ion

tissu

e wei

ght

lowast

Control Diabetic

minus20

minus40

minus60

minus80

(d)

0

2

4

6

8

10

12

Tim

e of i

nhib

ition

(min

)

lowast

Control Diabetic

(e)

Figure 1 Effect of CNP on the spontaneous contraction of gastric antral smooth muscle in control and diabetic rats (a) CNP significantlyinhibited the spontaneous contraction of gastric antral smooth muscle in both control and diabetic rats (b) CNP caused significantly moreinhibition on the amplitude of the contraction in diabetic rats than in controls (119899 = 8 119875 lt 001) (c) CNP caused significantly more inhibitionon the frequency of the contraction in diabetic rats than in controls (119899 = 8 119875 lt 001) (d) CNP decreased the basal tension which was moresignificant in diabetic rats than in controls (119899 = 8119875 lt 001) (e)Theduration of CNP-induced inhibition of gastric smoothmuscle contractionwas significantly longer in diabetic rats than in controls (119899 = 8 119875 lt 001)

4 Gastroenterology Research and Practice

Vehicle

Perf

usat

e cG

MP(p

mol120583

L)

CNP (01mmolL)

25

20

15

10

05

00

ControlDiabetes

lowast

lowast

(a)

000

005

010

015

020

025

030

035

040

045

Vehicle

Perf

usat

e cA

MP(p

mol120583

L)

CNP (01mmolL)

ControlDiabetes

lowast

lowast

(b)

Figure 2 Effect of CNP on intracellular cGMP and cAMP levels in gastric smooth muscle of diabetic and control rats (a) CNP increasedintracellular cGMP level in gastric smooth muscle which was more significant in the diabetic rats than in controls (119899 = 8 119875 lt 001 versusvehicle lowast119875 lt 001 versus control) (b) CNP increased intracellular cAMP level in gastric smooth muscle which was more significant in thediabetic rats than in controls (119899 = 8 119875 lt 001 versus vehicle lowast119875 lt 001 versus control)

4∘C After determining the protein concentration the super-natant was mixed with loading buffer and heat denatured at100∘C for 10min An equal amount of protein (30 120583g) of eachsample was separated by 10 SDS-PAGE gel electrophoresisand then electrotransferred onto a nitrocellulose membrane(Amersham Pharmacia Biotech Piscataway NJ USA) Themembranes were blocked with 5 non-fat dry milk in Tris-buffered saline (TBS pH 76) containing 05 Tween-20 for2 h at room temperature and then incubated with a primaryrabbit anti-PDE2 antibody (1 500 Abcam Cambridge MAUSA) rabbit anti-PDE3 antibody (1 500 Abcam Cam-bridge MA USA) or mouse anti-120573-actin antibody (1 10000Abcam Cambridge MA USA) at 4∘C overnight After beingwashed with TBST three times (15min each) the membraneswere incubated with HRP-labeled goat anti-rabbit IgG orHRP-labeled goat anti-mouse IgG at room temperature for2 h 331015840-Diaminobenzidine was used as a chromogen Theimage from each Western blot was quantitatively analyzedusing Quantity One software (Bio-Rad) and normalized bythat of 120573-actin

26 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of PDE1 PDE2 PDE3 PDE4 and PDE5 GeneExpression Tissues of the rat stomach were obtained asdescribed above Total RNA was extracted from the wholestomach according to the manufacturerrsquos instructions forthe TRIzol Reagent (TaKara Japan) RNA concentration wasdetermined by absorbance reading at 260280 nm whichwas between 18 and 20 Primer sequences for rat PDE1PDE2 PDE3 PDE4 PDE5 and GAPDH were as followsPDE1A (sense) 51015840 -AGATGACTGGAGGGATCTTCGG-31015840 PDE1A (antisense) 51015840-AGCTTCCACGTTTTGGCTGG-31015840

PDE2A (sense) 51015840 -GATCAAAAGGATGAGAAGGG-31015840PDE2A (antisense) 51015840-TTGCACATCGTCAGAGGTTAGG-31015840 PDE3A (sense) 51015840-TGAGACCAACAACAACAGTGA-31015840PDE3A (antisense) 51015840-GAGTATAGGTGCCACAAGCC-31015840PDE4A (sense) 51015840-GCGGGACCTACTGAAGAAATTCC-31015840 PDE4A (antisense) 51015840-CAGGGTGGTCCACATCGTGG-31015840 PDE5A (sense) 51015840-AACACGCACTGCATCAGAAG-31015840 PDE5A (antisense) 51015840-CGCTGTTTCCAGATCAGA-CA-31015840 GAPDH (sense) 51015840-GATTTGGCCGTATCGGAC-31015840 GAPDH (antisense) 51015840-GAAGACGCCAGTAGACTC-31015840 Reverse transcription was performed according to themanufacturerrsquos instructions The following conditions wereused for PCR amplification 95∘C for 5min followed by30 cycles at 95∘C for 30 sec 58sim60∘C for 1min 72∘C for2min followed by 72∘C for 10min The PCR products wereseparated on a 1 agarose gel Detectable fluorescent bandswere visualized by an ultraviolet transilluminator (Bio-Rad)and quantified using Quantity One image software ThemRNA expression level for PDE1 PDE2 PDE3 PDE4 andPDE5 was normalized by that of GAPDH

27 Data Analysis The average amplitude and frequency ofthe contraction recorded before and after administration ofdrugs were considered as control and effect size respectivelyThe percentage of the change was expressed as (effect sizeminus control)control times 100 The level of the baseline wasconsidered as the basal tension (g) which was 0 g beforetreatment The tension after treatment was expressed as thechange of baseline (g)weight of the muscle strip (g) Theduration from the amplitude of contraction suppressed tominimum by CNP to contraction recovery was considered asthe time of complete inhibition of CNP Data was analyzed

Gastroenterology Research and Practice 5

CO DM

(a)

PDE2

GDPH

Control Diabetes

PDE2

GD

PH

09

08

07

06

05

04

03

02

01

00

(b)

PDE2

Diabetes

Diabetes

08

06

04

02

00

PDE2

120573-a

ctin

120573-Actin

10

12

Control

Control

(c)

Figure 3 Expression of PDE2 in the gastric smooth muscle (a) PDE2 immunoreactivity in gastric smooth muscle was not different betweenthe diabetic rats and controls (bars = 20 120583m) (b) The mRNA level of PDE2 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005) (c) The protein level of PDE2 detected by Western blotting analysis in the gastric smooth muscle was notsignificantly different between diabetic rats and controls (119899 = 9 119875 gt 005)

using Origin 60 software and expressed as means plusmn SEMData recordings were evaluated using a Studentrsquos 119905-test 119875values less than 005 were considered statistically significant

3 Results

31 Changes in BloodGlucoseConcentration Fourweeks afterinjection of STZ all the animals treated with STZ exhibitedhyperglycemia The mean blood glucose concentration ofanimals defined as diabetes was 4552 plusmn 328mgdL whichwas significantly higher than controls (1064 plusmn 169mgdL119875 lt 005)

32 Effect of CNP on the Spontaneous Contraction of GastricSmooth Muscle After equilibration spontaneous contrac-tions of gastric antral circular smooth muscle from bothcontrol and diabetic rats were recorded CNP (01 120583molL)significantly inhibited the spontaneous contractions in both

control and diabetic rats however the inhibitory response toCNPwasmuch stronger in diabetic group (Figure 1(a)) CNP(01 120583molL) inhibited the amplitudes of the contraction by5792plusmn 466 in control rats and 8015 plusmn 310 in diabeticrats (Figure 1(b) 119899 = 8 119875 lt 001) CNP (01 120583molL)inhibited the frequencies of the contraction by 2585 plusmn 657in controls and 5437 plusmn 535 in diabetic rats (Figure 1(c)119899 = 8 119875 lt 001) Meanwhile the tensions were significantlydecreased to 4788 plusmn 362 and 7005 plusmn 321 after the admin-istration of CNP in normal and diabetic groups respectively(Figure 1(d) 119899 = 8 119875 lt 001)The durations of CNP-inducedinhibitionwere 152plusmn 050min in control and 898 plusmn 088minin diabetic group (Figure 1(e) 119899 = 8 119875 lt 001) The resultssuggest that the inhibitory effect of CNP on spontaneouscontraction is potentiated in diabetic rats

33 Effect of CNP on cGMP and cAMP Generations in GastricSmooth Muscle CNP binds to the NPR-A or NPR-B in

6 Gastroenterology Research and Practice

CO DM

(a)

PDE3

GD

PH

PDE3

GDPH

08

06

04

02

00

10

12

14

16

lowast

DiabetesControl

DiabetesControl

(b)

PDE3

Diabetes

08

06

04

02

00

10

12

14

lowastPDE3

120573-a

ctin

120573-Actin

Control

DiabetesControl

(c)

Figure 4 Expression of PDE3 in the gastric smooth muscle (a) PDE3 immunoreactivity in gastric smooth muscle was lower in the diabeticrats than in controls (bars = 20120583m) (b)ThemRNA level of PDE3 in the gastric smoothmuscle was downregulated in the diabetic rats (119899 = 9119875 lt 005) (c) The protein level of PDE3 detected by Western blotting analysis in the gastric smooth muscle was significantly lower in thediabetic rats than in controls (119899 = 9 119875 lt 005)

smooth muscle cell membrane and causes the productionof cGMP by activating pGC Since the inhibitory effect ofCNP on spontaneous contraction was potentiated in diabeticrats in succession the effects of CNP on cGMP and cAMPgenerations were observed Our experiments demonstratedthat CNP significantly increased intracellular cGMP andcAMP concentrations in the gastric smooth muscles of bothcontrol and diabetic rats cGMP and cAMP productions incontrol rats were increased from 071 plusmn 009 pmol120583L and015 plusmn 005 pmol120583L before to 137 plusmn 012 pmol120583L and 023plusmn 003 pmol120583L after the administration of CNP respectivelyand the increase percentage was 9378 and 5616 respec-tively (Figures 2(a) and 2(b) 119899 = 8 119875 lt 001) while inthe diabetic group the cGMP and cAMP productions wereincreased from 083 plusmn 022 pmol120583L and 017 plusmn 005 pmol120583Lbefore to 215 plusmn 016 pmol120583L and 037 plusmn 007 pmol120583L afterthe administration of CNP respectively and the increasepercentage was 159 and 118 respectively (Figures 2(a) and

2(b) 119899 = 8 119875 lt 001) The CNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats

34 Expressions of PDEs in the Gastric Smooth Muscle Thecellular levels of the cyclic nucleotides reflect a balancebetween their synthesis and catabolism and the latter wasregulated by the enzymes of phosphodiesterase (PDE) familyOur previous study has shown that the generation of cAMPinduced by CNP is via CNP-cGMP-PDE3-cAMP signal path-way in control rats [9] Since theCNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats we were wondering whether theexpression of PDEwas changed in the diabetic rats Firstly weobserved the expressions of PDE2 and PDE3 in the gastricsmooth muscle of both control and diabetic rats by usingimmunohistochemistry technique RT-PCR and Western

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

Gastroenterology Research and Practice 3

Control

Diabetes

075g

40 s

01 120583molL CNP

01 120583molL CNP

(a)

0

10

20

30

40

50

60

70

80

90

Inhi

bitio

n of

ampl

itude

()

lowast

Control Diabetic

(b)

0

10

20

30

40

50

60

Control Diabetic

Inhi

bitio

n of

freq

uenc

y (

)

lowast

(c)

0

Tens

ion

tissu

e wei

ght

lowast

Control Diabetic

minus20

minus40

minus60

minus80

(d)

0

2

4

6

8

10

12

Tim

e of i

nhib

ition

(min

)

lowast

Control Diabetic

(e)

Figure 1 Effect of CNP on the spontaneous contraction of gastric antral smooth muscle in control and diabetic rats (a) CNP significantlyinhibited the spontaneous contraction of gastric antral smooth muscle in both control and diabetic rats (b) CNP caused significantly moreinhibition on the amplitude of the contraction in diabetic rats than in controls (119899 = 8 119875 lt 001) (c) CNP caused significantly more inhibitionon the frequency of the contraction in diabetic rats than in controls (119899 = 8 119875 lt 001) (d) CNP decreased the basal tension which was moresignificant in diabetic rats than in controls (119899 = 8119875 lt 001) (e)Theduration of CNP-induced inhibition of gastric smoothmuscle contractionwas significantly longer in diabetic rats than in controls (119899 = 8 119875 lt 001)

4 Gastroenterology Research and Practice

Vehicle

Perf

usat

e cG

MP(p

mol120583

L)

CNP (01mmolL)

25

20

15

10

05

00

ControlDiabetes

lowast

lowast

(a)

000

005

010

015

020

025

030

035

040

045

Vehicle

Perf

usat

e cA

MP(p

mol120583

L)

CNP (01mmolL)

ControlDiabetes

lowast

lowast

(b)

Figure 2 Effect of CNP on intracellular cGMP and cAMP levels in gastric smooth muscle of diabetic and control rats (a) CNP increasedintracellular cGMP level in gastric smooth muscle which was more significant in the diabetic rats than in controls (119899 = 8 119875 lt 001 versusvehicle lowast119875 lt 001 versus control) (b) CNP increased intracellular cAMP level in gastric smooth muscle which was more significant in thediabetic rats than in controls (119899 = 8 119875 lt 001 versus vehicle lowast119875 lt 001 versus control)

4∘C After determining the protein concentration the super-natant was mixed with loading buffer and heat denatured at100∘C for 10min An equal amount of protein (30 120583g) of eachsample was separated by 10 SDS-PAGE gel electrophoresisand then electrotransferred onto a nitrocellulose membrane(Amersham Pharmacia Biotech Piscataway NJ USA) Themembranes were blocked with 5 non-fat dry milk in Tris-buffered saline (TBS pH 76) containing 05 Tween-20 for2 h at room temperature and then incubated with a primaryrabbit anti-PDE2 antibody (1 500 Abcam Cambridge MAUSA) rabbit anti-PDE3 antibody (1 500 Abcam Cam-bridge MA USA) or mouse anti-120573-actin antibody (1 10000Abcam Cambridge MA USA) at 4∘C overnight After beingwashed with TBST three times (15min each) the membraneswere incubated with HRP-labeled goat anti-rabbit IgG orHRP-labeled goat anti-mouse IgG at room temperature for2 h 331015840-Diaminobenzidine was used as a chromogen Theimage from each Western blot was quantitatively analyzedusing Quantity One software (Bio-Rad) and normalized bythat of 120573-actin

26 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of PDE1 PDE2 PDE3 PDE4 and PDE5 GeneExpression Tissues of the rat stomach were obtained asdescribed above Total RNA was extracted from the wholestomach according to the manufacturerrsquos instructions forthe TRIzol Reagent (TaKara Japan) RNA concentration wasdetermined by absorbance reading at 260280 nm whichwas between 18 and 20 Primer sequences for rat PDE1PDE2 PDE3 PDE4 PDE5 and GAPDH were as followsPDE1A (sense) 51015840 -AGATGACTGGAGGGATCTTCGG-31015840 PDE1A (antisense) 51015840-AGCTTCCACGTTTTGGCTGG-31015840

PDE2A (sense) 51015840 -GATCAAAAGGATGAGAAGGG-31015840PDE2A (antisense) 51015840-TTGCACATCGTCAGAGGTTAGG-31015840 PDE3A (sense) 51015840-TGAGACCAACAACAACAGTGA-31015840PDE3A (antisense) 51015840-GAGTATAGGTGCCACAAGCC-31015840PDE4A (sense) 51015840-GCGGGACCTACTGAAGAAATTCC-31015840 PDE4A (antisense) 51015840-CAGGGTGGTCCACATCGTGG-31015840 PDE5A (sense) 51015840-AACACGCACTGCATCAGAAG-31015840 PDE5A (antisense) 51015840-CGCTGTTTCCAGATCAGA-CA-31015840 GAPDH (sense) 51015840-GATTTGGCCGTATCGGAC-31015840 GAPDH (antisense) 51015840-GAAGACGCCAGTAGACTC-31015840 Reverse transcription was performed according to themanufacturerrsquos instructions The following conditions wereused for PCR amplification 95∘C for 5min followed by30 cycles at 95∘C for 30 sec 58sim60∘C for 1min 72∘C for2min followed by 72∘C for 10min The PCR products wereseparated on a 1 agarose gel Detectable fluorescent bandswere visualized by an ultraviolet transilluminator (Bio-Rad)and quantified using Quantity One image software ThemRNA expression level for PDE1 PDE2 PDE3 PDE4 andPDE5 was normalized by that of GAPDH

27 Data Analysis The average amplitude and frequency ofthe contraction recorded before and after administration ofdrugs were considered as control and effect size respectivelyThe percentage of the change was expressed as (effect sizeminus control)control times 100 The level of the baseline wasconsidered as the basal tension (g) which was 0 g beforetreatment The tension after treatment was expressed as thechange of baseline (g)weight of the muscle strip (g) Theduration from the amplitude of contraction suppressed tominimum by CNP to contraction recovery was considered asthe time of complete inhibition of CNP Data was analyzed

Gastroenterology Research and Practice 5

CO DM

(a)

PDE2

GDPH

Control Diabetes

PDE2

GD

PH

09

08

07

06

05

04

03

02

01

00

(b)

PDE2

Diabetes

Diabetes

08

06

04

02

00

PDE2

120573-a

ctin

120573-Actin

10

12

Control

Control

(c)

Figure 3 Expression of PDE2 in the gastric smooth muscle (a) PDE2 immunoreactivity in gastric smooth muscle was not different betweenthe diabetic rats and controls (bars = 20 120583m) (b) The mRNA level of PDE2 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005) (c) The protein level of PDE2 detected by Western blotting analysis in the gastric smooth muscle was notsignificantly different between diabetic rats and controls (119899 = 9 119875 gt 005)

using Origin 60 software and expressed as means plusmn SEMData recordings were evaluated using a Studentrsquos 119905-test 119875values less than 005 were considered statistically significant

3 Results

31 Changes in BloodGlucoseConcentration Fourweeks afterinjection of STZ all the animals treated with STZ exhibitedhyperglycemia The mean blood glucose concentration ofanimals defined as diabetes was 4552 plusmn 328mgdL whichwas significantly higher than controls (1064 plusmn 169mgdL119875 lt 005)

32 Effect of CNP on the Spontaneous Contraction of GastricSmooth Muscle After equilibration spontaneous contrac-tions of gastric antral circular smooth muscle from bothcontrol and diabetic rats were recorded CNP (01 120583molL)significantly inhibited the spontaneous contractions in both

control and diabetic rats however the inhibitory response toCNPwasmuch stronger in diabetic group (Figure 1(a)) CNP(01 120583molL) inhibited the amplitudes of the contraction by5792plusmn 466 in control rats and 8015 plusmn 310 in diabeticrats (Figure 1(b) 119899 = 8 119875 lt 001) CNP (01 120583molL)inhibited the frequencies of the contraction by 2585 plusmn 657in controls and 5437 plusmn 535 in diabetic rats (Figure 1(c)119899 = 8 119875 lt 001) Meanwhile the tensions were significantlydecreased to 4788 plusmn 362 and 7005 plusmn 321 after the admin-istration of CNP in normal and diabetic groups respectively(Figure 1(d) 119899 = 8 119875 lt 001)The durations of CNP-inducedinhibitionwere 152plusmn 050min in control and 898 plusmn 088minin diabetic group (Figure 1(e) 119899 = 8 119875 lt 001) The resultssuggest that the inhibitory effect of CNP on spontaneouscontraction is potentiated in diabetic rats

33 Effect of CNP on cGMP and cAMP Generations in GastricSmooth Muscle CNP binds to the NPR-A or NPR-B in

6 Gastroenterology Research and Practice

CO DM

(a)

PDE3

GD

PH

PDE3

GDPH

08

06

04

02

00

10

12

14

16

lowast

DiabetesControl

DiabetesControl

(b)

PDE3

Diabetes

08

06

04

02

00

10

12

14

lowastPDE3

120573-a

ctin

120573-Actin

Control

DiabetesControl

(c)

Figure 4 Expression of PDE3 in the gastric smooth muscle (a) PDE3 immunoreactivity in gastric smooth muscle was lower in the diabeticrats than in controls (bars = 20120583m) (b)ThemRNA level of PDE3 in the gastric smoothmuscle was downregulated in the diabetic rats (119899 = 9119875 lt 005) (c) The protein level of PDE3 detected by Western blotting analysis in the gastric smooth muscle was significantly lower in thediabetic rats than in controls (119899 = 9 119875 lt 005)

smooth muscle cell membrane and causes the productionof cGMP by activating pGC Since the inhibitory effect ofCNP on spontaneous contraction was potentiated in diabeticrats in succession the effects of CNP on cGMP and cAMPgenerations were observed Our experiments demonstratedthat CNP significantly increased intracellular cGMP andcAMP concentrations in the gastric smooth muscles of bothcontrol and diabetic rats cGMP and cAMP productions incontrol rats were increased from 071 plusmn 009 pmol120583L and015 plusmn 005 pmol120583L before to 137 plusmn 012 pmol120583L and 023plusmn 003 pmol120583L after the administration of CNP respectivelyand the increase percentage was 9378 and 5616 respec-tively (Figures 2(a) and 2(b) 119899 = 8 119875 lt 001) while inthe diabetic group the cGMP and cAMP productions wereincreased from 083 plusmn 022 pmol120583L and 017 plusmn 005 pmol120583Lbefore to 215 plusmn 016 pmol120583L and 037 plusmn 007 pmol120583L afterthe administration of CNP respectively and the increasepercentage was 159 and 118 respectively (Figures 2(a) and

2(b) 119899 = 8 119875 lt 001) The CNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats

34 Expressions of PDEs in the Gastric Smooth Muscle Thecellular levels of the cyclic nucleotides reflect a balancebetween their synthesis and catabolism and the latter wasregulated by the enzymes of phosphodiesterase (PDE) familyOur previous study has shown that the generation of cAMPinduced by CNP is via CNP-cGMP-PDE3-cAMP signal path-way in control rats [9] Since theCNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats we were wondering whether theexpression of PDEwas changed in the diabetic rats Firstly weobserved the expressions of PDE2 and PDE3 in the gastricsmooth muscle of both control and diabetic rats by usingimmunohistochemistry technique RT-PCR and Western

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

4 Gastroenterology Research and Practice

Vehicle

Perf

usat

e cG

MP(p

mol120583

L)

CNP (01mmolL)

25

20

15

10

05

00

ControlDiabetes

lowast

lowast

(a)

000

005

010

015

020

025

030

035

040

045

Vehicle

Perf

usat

e cA

MP(p

mol120583

L)

CNP (01mmolL)

ControlDiabetes

lowast

lowast

(b)

Figure 2 Effect of CNP on intracellular cGMP and cAMP levels in gastric smooth muscle of diabetic and control rats (a) CNP increasedintracellular cGMP level in gastric smooth muscle which was more significant in the diabetic rats than in controls (119899 = 8 119875 lt 001 versusvehicle lowast119875 lt 001 versus control) (b) CNP increased intracellular cAMP level in gastric smooth muscle which was more significant in thediabetic rats than in controls (119899 = 8 119875 lt 001 versus vehicle lowast119875 lt 001 versus control)

4∘C After determining the protein concentration the super-natant was mixed with loading buffer and heat denatured at100∘C for 10min An equal amount of protein (30 120583g) of eachsample was separated by 10 SDS-PAGE gel electrophoresisand then electrotransferred onto a nitrocellulose membrane(Amersham Pharmacia Biotech Piscataway NJ USA) Themembranes were blocked with 5 non-fat dry milk in Tris-buffered saline (TBS pH 76) containing 05 Tween-20 for2 h at room temperature and then incubated with a primaryrabbit anti-PDE2 antibody (1 500 Abcam Cambridge MAUSA) rabbit anti-PDE3 antibody (1 500 Abcam Cam-bridge MA USA) or mouse anti-120573-actin antibody (1 10000Abcam Cambridge MA USA) at 4∘C overnight After beingwashed with TBST three times (15min each) the membraneswere incubated with HRP-labeled goat anti-rabbit IgG orHRP-labeled goat anti-mouse IgG at room temperature for2 h 331015840-Diaminobenzidine was used as a chromogen Theimage from each Western blot was quantitatively analyzedusing Quantity One software (Bio-Rad) and normalized bythat of 120573-actin

26 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of PDE1 PDE2 PDE3 PDE4 and PDE5 GeneExpression Tissues of the rat stomach were obtained asdescribed above Total RNA was extracted from the wholestomach according to the manufacturerrsquos instructions forthe TRIzol Reagent (TaKara Japan) RNA concentration wasdetermined by absorbance reading at 260280 nm whichwas between 18 and 20 Primer sequences for rat PDE1PDE2 PDE3 PDE4 PDE5 and GAPDH were as followsPDE1A (sense) 51015840 -AGATGACTGGAGGGATCTTCGG-31015840 PDE1A (antisense) 51015840-AGCTTCCACGTTTTGGCTGG-31015840

PDE2A (sense) 51015840 -GATCAAAAGGATGAGAAGGG-31015840PDE2A (antisense) 51015840-TTGCACATCGTCAGAGGTTAGG-31015840 PDE3A (sense) 51015840-TGAGACCAACAACAACAGTGA-31015840PDE3A (antisense) 51015840-GAGTATAGGTGCCACAAGCC-31015840PDE4A (sense) 51015840-GCGGGACCTACTGAAGAAATTCC-31015840 PDE4A (antisense) 51015840-CAGGGTGGTCCACATCGTGG-31015840 PDE5A (sense) 51015840-AACACGCACTGCATCAGAAG-31015840 PDE5A (antisense) 51015840-CGCTGTTTCCAGATCAGA-CA-31015840 GAPDH (sense) 51015840-GATTTGGCCGTATCGGAC-31015840 GAPDH (antisense) 51015840-GAAGACGCCAGTAGACTC-31015840 Reverse transcription was performed according to themanufacturerrsquos instructions The following conditions wereused for PCR amplification 95∘C for 5min followed by30 cycles at 95∘C for 30 sec 58sim60∘C for 1min 72∘C for2min followed by 72∘C for 10min The PCR products wereseparated on a 1 agarose gel Detectable fluorescent bandswere visualized by an ultraviolet transilluminator (Bio-Rad)and quantified using Quantity One image software ThemRNA expression level for PDE1 PDE2 PDE3 PDE4 andPDE5 was normalized by that of GAPDH

27 Data Analysis The average amplitude and frequency ofthe contraction recorded before and after administration ofdrugs were considered as control and effect size respectivelyThe percentage of the change was expressed as (effect sizeminus control)control times 100 The level of the baseline wasconsidered as the basal tension (g) which was 0 g beforetreatment The tension after treatment was expressed as thechange of baseline (g)weight of the muscle strip (g) Theduration from the amplitude of contraction suppressed tominimum by CNP to contraction recovery was considered asthe time of complete inhibition of CNP Data was analyzed

Gastroenterology Research and Practice 5

CO DM

(a)

PDE2

GDPH

Control Diabetes

PDE2

GD

PH

09

08

07

06

05

04

03

02

01

00

(b)

PDE2

Diabetes

Diabetes

08

06

04

02

00

PDE2

120573-a

ctin

120573-Actin

10

12

Control

Control

(c)

Figure 3 Expression of PDE2 in the gastric smooth muscle (a) PDE2 immunoreactivity in gastric smooth muscle was not different betweenthe diabetic rats and controls (bars = 20 120583m) (b) The mRNA level of PDE2 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005) (c) The protein level of PDE2 detected by Western blotting analysis in the gastric smooth muscle was notsignificantly different between diabetic rats and controls (119899 = 9 119875 gt 005)

using Origin 60 software and expressed as means plusmn SEMData recordings were evaluated using a Studentrsquos 119905-test 119875values less than 005 were considered statistically significant

3 Results

31 Changes in BloodGlucoseConcentration Fourweeks afterinjection of STZ all the animals treated with STZ exhibitedhyperglycemia The mean blood glucose concentration ofanimals defined as diabetes was 4552 plusmn 328mgdL whichwas significantly higher than controls (1064 plusmn 169mgdL119875 lt 005)

32 Effect of CNP on the Spontaneous Contraction of GastricSmooth Muscle After equilibration spontaneous contrac-tions of gastric antral circular smooth muscle from bothcontrol and diabetic rats were recorded CNP (01 120583molL)significantly inhibited the spontaneous contractions in both

control and diabetic rats however the inhibitory response toCNPwasmuch stronger in diabetic group (Figure 1(a)) CNP(01 120583molL) inhibited the amplitudes of the contraction by5792plusmn 466 in control rats and 8015 plusmn 310 in diabeticrats (Figure 1(b) 119899 = 8 119875 lt 001) CNP (01 120583molL)inhibited the frequencies of the contraction by 2585 plusmn 657in controls and 5437 plusmn 535 in diabetic rats (Figure 1(c)119899 = 8 119875 lt 001) Meanwhile the tensions were significantlydecreased to 4788 plusmn 362 and 7005 plusmn 321 after the admin-istration of CNP in normal and diabetic groups respectively(Figure 1(d) 119899 = 8 119875 lt 001)The durations of CNP-inducedinhibitionwere 152plusmn 050min in control and 898 plusmn 088minin diabetic group (Figure 1(e) 119899 = 8 119875 lt 001) The resultssuggest that the inhibitory effect of CNP on spontaneouscontraction is potentiated in diabetic rats

33 Effect of CNP on cGMP and cAMP Generations in GastricSmooth Muscle CNP binds to the NPR-A or NPR-B in

6 Gastroenterology Research and Practice

CO DM

(a)

PDE3

GD

PH

PDE3

GDPH

08

06

04

02

00

10

12

14

16

lowast

DiabetesControl

DiabetesControl

(b)

PDE3

Diabetes

08

06

04

02

00

10

12

14

lowastPDE3

120573-a

ctin

120573-Actin

Control

DiabetesControl

(c)

Figure 4 Expression of PDE3 in the gastric smooth muscle (a) PDE3 immunoreactivity in gastric smooth muscle was lower in the diabeticrats than in controls (bars = 20120583m) (b)ThemRNA level of PDE3 in the gastric smoothmuscle was downregulated in the diabetic rats (119899 = 9119875 lt 005) (c) The protein level of PDE3 detected by Western blotting analysis in the gastric smooth muscle was significantly lower in thediabetic rats than in controls (119899 = 9 119875 lt 005)

smooth muscle cell membrane and causes the productionof cGMP by activating pGC Since the inhibitory effect ofCNP on spontaneous contraction was potentiated in diabeticrats in succession the effects of CNP on cGMP and cAMPgenerations were observed Our experiments demonstratedthat CNP significantly increased intracellular cGMP andcAMP concentrations in the gastric smooth muscles of bothcontrol and diabetic rats cGMP and cAMP productions incontrol rats were increased from 071 plusmn 009 pmol120583L and015 plusmn 005 pmol120583L before to 137 plusmn 012 pmol120583L and 023plusmn 003 pmol120583L after the administration of CNP respectivelyand the increase percentage was 9378 and 5616 respec-tively (Figures 2(a) and 2(b) 119899 = 8 119875 lt 001) while inthe diabetic group the cGMP and cAMP productions wereincreased from 083 plusmn 022 pmol120583L and 017 plusmn 005 pmol120583Lbefore to 215 plusmn 016 pmol120583L and 037 plusmn 007 pmol120583L afterthe administration of CNP respectively and the increasepercentage was 159 and 118 respectively (Figures 2(a) and

2(b) 119899 = 8 119875 lt 001) The CNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats

34 Expressions of PDEs in the Gastric Smooth Muscle Thecellular levels of the cyclic nucleotides reflect a balancebetween their synthesis and catabolism and the latter wasregulated by the enzymes of phosphodiesterase (PDE) familyOur previous study has shown that the generation of cAMPinduced by CNP is via CNP-cGMP-PDE3-cAMP signal path-way in control rats [9] Since theCNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats we were wondering whether theexpression of PDEwas changed in the diabetic rats Firstly weobserved the expressions of PDE2 and PDE3 in the gastricsmooth muscle of both control and diabetic rats by usingimmunohistochemistry technique RT-PCR and Western

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

Gastroenterology Research and Practice 5

CO DM

(a)

PDE2

GDPH

Control Diabetes

PDE2

GD

PH

09

08

07

06

05

04

03

02

01

00

(b)

PDE2

Diabetes

Diabetes

08

06

04

02

00

PDE2

120573-a

ctin

120573-Actin

10

12

Control

Control

(c)

Figure 3 Expression of PDE2 in the gastric smooth muscle (a) PDE2 immunoreactivity in gastric smooth muscle was not different betweenthe diabetic rats and controls (bars = 20 120583m) (b) The mRNA level of PDE2 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005) (c) The protein level of PDE2 detected by Western blotting analysis in the gastric smooth muscle was notsignificantly different between diabetic rats and controls (119899 = 9 119875 gt 005)

using Origin 60 software and expressed as means plusmn SEMData recordings were evaluated using a Studentrsquos 119905-test 119875values less than 005 were considered statistically significant

3 Results

31 Changes in BloodGlucoseConcentration Fourweeks afterinjection of STZ all the animals treated with STZ exhibitedhyperglycemia The mean blood glucose concentration ofanimals defined as diabetes was 4552 plusmn 328mgdL whichwas significantly higher than controls (1064 plusmn 169mgdL119875 lt 005)

32 Effect of CNP on the Spontaneous Contraction of GastricSmooth Muscle After equilibration spontaneous contrac-tions of gastric antral circular smooth muscle from bothcontrol and diabetic rats were recorded CNP (01 120583molL)significantly inhibited the spontaneous contractions in both

control and diabetic rats however the inhibitory response toCNPwasmuch stronger in diabetic group (Figure 1(a)) CNP(01 120583molL) inhibited the amplitudes of the contraction by5792plusmn 466 in control rats and 8015 plusmn 310 in diabeticrats (Figure 1(b) 119899 = 8 119875 lt 001) CNP (01 120583molL)inhibited the frequencies of the contraction by 2585 plusmn 657in controls and 5437 plusmn 535 in diabetic rats (Figure 1(c)119899 = 8 119875 lt 001) Meanwhile the tensions were significantlydecreased to 4788 plusmn 362 and 7005 plusmn 321 after the admin-istration of CNP in normal and diabetic groups respectively(Figure 1(d) 119899 = 8 119875 lt 001)The durations of CNP-inducedinhibitionwere 152plusmn 050min in control and 898 plusmn 088minin diabetic group (Figure 1(e) 119899 = 8 119875 lt 001) The resultssuggest that the inhibitory effect of CNP on spontaneouscontraction is potentiated in diabetic rats

33 Effect of CNP on cGMP and cAMP Generations in GastricSmooth Muscle CNP binds to the NPR-A or NPR-B in

6 Gastroenterology Research and Practice

CO DM

(a)

PDE3

GD

PH

PDE3

GDPH

08

06

04

02

00

10

12

14

16

lowast

DiabetesControl

DiabetesControl

(b)

PDE3

Diabetes

08

06

04

02

00

10

12

14

lowastPDE3

120573-a

ctin

120573-Actin

Control

DiabetesControl

(c)

Figure 4 Expression of PDE3 in the gastric smooth muscle (a) PDE3 immunoreactivity in gastric smooth muscle was lower in the diabeticrats than in controls (bars = 20120583m) (b)ThemRNA level of PDE3 in the gastric smoothmuscle was downregulated in the diabetic rats (119899 = 9119875 lt 005) (c) The protein level of PDE3 detected by Western blotting analysis in the gastric smooth muscle was significantly lower in thediabetic rats than in controls (119899 = 9 119875 lt 005)

smooth muscle cell membrane and causes the productionof cGMP by activating pGC Since the inhibitory effect ofCNP on spontaneous contraction was potentiated in diabeticrats in succession the effects of CNP on cGMP and cAMPgenerations were observed Our experiments demonstratedthat CNP significantly increased intracellular cGMP andcAMP concentrations in the gastric smooth muscles of bothcontrol and diabetic rats cGMP and cAMP productions incontrol rats were increased from 071 plusmn 009 pmol120583L and015 plusmn 005 pmol120583L before to 137 plusmn 012 pmol120583L and 023plusmn 003 pmol120583L after the administration of CNP respectivelyand the increase percentage was 9378 and 5616 respec-tively (Figures 2(a) and 2(b) 119899 = 8 119875 lt 001) while inthe diabetic group the cGMP and cAMP productions wereincreased from 083 plusmn 022 pmol120583L and 017 plusmn 005 pmol120583Lbefore to 215 plusmn 016 pmol120583L and 037 plusmn 007 pmol120583L afterthe administration of CNP respectively and the increasepercentage was 159 and 118 respectively (Figures 2(a) and

2(b) 119899 = 8 119875 lt 001) The CNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats

34 Expressions of PDEs in the Gastric Smooth Muscle Thecellular levels of the cyclic nucleotides reflect a balancebetween their synthesis and catabolism and the latter wasregulated by the enzymes of phosphodiesterase (PDE) familyOur previous study has shown that the generation of cAMPinduced by CNP is via CNP-cGMP-PDE3-cAMP signal path-way in control rats [9] Since theCNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats we were wondering whether theexpression of PDEwas changed in the diabetic rats Firstly weobserved the expressions of PDE2 and PDE3 in the gastricsmooth muscle of both control and diabetic rats by usingimmunohistochemistry technique RT-PCR and Western

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

6 Gastroenterology Research and Practice

CO DM

(a)

PDE3

GD

PH

PDE3

GDPH

08

06

04

02

00

10

12

14

16

lowast

DiabetesControl

DiabetesControl

(b)

PDE3

Diabetes

08

06

04

02

00

10

12

14

lowastPDE3

120573-a

ctin

120573-Actin

Control

DiabetesControl

(c)

Figure 4 Expression of PDE3 in the gastric smooth muscle (a) PDE3 immunoreactivity in gastric smooth muscle was lower in the diabeticrats than in controls (bars = 20120583m) (b)ThemRNA level of PDE3 in the gastric smoothmuscle was downregulated in the diabetic rats (119899 = 9119875 lt 005) (c) The protein level of PDE3 detected by Western blotting analysis in the gastric smooth muscle was significantly lower in thediabetic rats than in controls (119899 = 9 119875 lt 005)

smooth muscle cell membrane and causes the productionof cGMP by activating pGC Since the inhibitory effect ofCNP on spontaneous contraction was potentiated in diabeticrats in succession the effects of CNP on cGMP and cAMPgenerations were observed Our experiments demonstratedthat CNP significantly increased intracellular cGMP andcAMP concentrations in the gastric smooth muscles of bothcontrol and diabetic rats cGMP and cAMP productions incontrol rats were increased from 071 plusmn 009 pmol120583L and015 plusmn 005 pmol120583L before to 137 plusmn 012 pmol120583L and 023plusmn 003 pmol120583L after the administration of CNP respectivelyand the increase percentage was 9378 and 5616 respec-tively (Figures 2(a) and 2(b) 119899 = 8 119875 lt 001) while inthe diabetic group the cGMP and cAMP productions wereincreased from 083 plusmn 022 pmol120583L and 017 plusmn 005 pmol120583Lbefore to 215 plusmn 016 pmol120583L and 037 plusmn 007 pmol120583L afterthe administration of CNP respectively and the increasepercentage was 159 and 118 respectively (Figures 2(a) and

2(b) 119899 = 8 119875 lt 001) The CNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats

34 Expressions of PDEs in the Gastric Smooth Muscle Thecellular levels of the cyclic nucleotides reflect a balancebetween their synthesis and catabolism and the latter wasregulated by the enzymes of phosphodiesterase (PDE) familyOur previous study has shown that the generation of cAMPinduced by CNP is via CNP-cGMP-PDE3-cAMP signal path-way in control rats [9] Since theCNP-induced productions ofcGMP and cAMP in gastric smoothmuscle were significantlypotentiated in diabetic rats we were wondering whether theexpression of PDEwas changed in the diabetic rats Firstly weobserved the expressions of PDE2 and PDE3 in the gastricsmooth muscle of both control and diabetic rats by usingimmunohistochemistry technique RT-PCR and Western

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

Gastroenterology Research and Practice 7PD

E1G

DPH

GAPDH

PDE1

Control Diabetes

Control Diabetes

08

06

04

02

00

10

12

(a)

PDE4

PDE4

GD

PH

GAPDH

Control Diabetes

08

06

04

02

00

10

12

Control Diabetes

(b)

GAPDH

PDE5

GD

PH

PDE5

Control Diabetes

09

08

07

06

05

04

03

02

01

00

10

Control Diabetes

(c)

Figure 5 Expression of other PDEs in the gastric smoothmuscle (a)ThemRNA level of PDE1 in the gastric smoothmuscle was not differentbetween the diabetic rats and controls (119899 = 9 119875 gt 005) (b)ThemRNA level of PDE4 in the gastric smoothmuscle was not different betweendiabetic rats and controls (119899 = 9 119875 gt 005) (c) The mRNA level of PDE5 in the gastric smooth muscle was not different between diabeticrats and controls (119899 = 9 119875 gt 005)

blotting methods We found that the expression of PDE2 wasnot significantly different between the two groupsThe PDE2immunoreactive staining of gastric smoothmuscle tissue wasnot different between control and diabetic rats (Figure 3(a))The relative PDE2 mRNA levels (PDE2GAPDH) were 065plusmn 012 and 063 plusmn 046 in the control and diabetic ratsrespectively (Figure 3(b) 119899 = 9 119875 gt 005) At the proteinlevel the ratios of PDE2120573-actin were 104 plusmn 007 in controland 110 plusmn 002 in diabetic rats (Figure 3(c) 119899 = 9 119875 gt 005)However PDE3 expression was lower in the diabetic groupthan in the control group (Figure 4(a)) At the gene level

the ratios of PDE3GAPDH were 125 plusmn 013 and 061 plusmn011 in control and diabetic rats respectively (Figure 4(b)119899 = 9 119875 lt 005) and at the protein level the ratiosof PDE3120573-actin were 119plusmn 011 and 043 plusmn 005 in controland diabetic rats respectively (Figure 4(c) 119899 = 9 119875 lt005) To further determine whether the expressions of otherPDEs were changed in diabetic rat stomach we also observedthe expressions of PDE1 PDE4 and PDE5 in the gastricsmooth muscle of both normal and diabetic rats The resultsdemonstrated that the mRNA levels of PDE1 PDE4 andPDE5 were not changed in diabetic rat stomach The ratios

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

8 Gastroenterology Research and Practice

of PDE2GAPDH were 091 plusmn 047 081 plusmn 041 and 072 plusmn007 in control rats respectively and 088 plusmn 045 090 plusmn 052and 077 plusmn 009 in diabetic rats respectively (Figure 5 119899 = 9119875 gt 005) These results suggest that only the expression ofPDE3 was downregulated in gastric smooth muscle tissues ofdiabetic rats

4 Discussion

NPs as important regulatory peptides in the gastrointestinaltract have been reported to regulate the spontaneous contrac-tion of gastric smooth muscle via NPs-NPR-BpGC-cGMPsignal pathway in the guinea pig rat and human [5 6] Ourprevious studies have demonstrated that CNP-NPR-BpGC-cGMP signal pathway is abnormal in diabetic rats which isassociated with the development of gastric motility disorder[18 19]

Intracellular cAMP and cGMP are maintained to aproper level through the regulation of their generation andhydrolysis processes Generation of cAMP and cGMP iscatalyzed by the activation of adenylate cyclase and guanylatecyclase while phosphodiesterase (PDE) is responsible forthe hydrolysis of cAMP and cGMP [21 22] Via hydrolysisPDE family can regulate cAMP and cGMP levels proteinphosphorylation and thus intervene in the signal trans-duction process while intracellular cAMP and cGMP canalso modulate the activity of PDE For example cGMP canincrease intracellular cAMP through the inhibition of PDE3to enhance the cardiac muscle contractility [23 24] And wealso found that cAMP generation induced by CNP throughcGMP-PDE3-cAMP signal pathway activated PKA signalpathway and subsequent inhibition of L-type calcium currentto inhibit the gastric smooth muscle motility together withcGMP while cGMP-PDE2-cAMP signal pathway was notinvolved in the process [20] Nevertheless whether cGMP-PDE-cAMP signal pathway is involved in the mechanismof diabetes-induced gastric motility disorder has not beenstudied so far

In the present study in order to determine the respon-siveness of gastric smoothmuscle to CNP we firstly observedthe effect of CNP on the spontaneous contraction of gastricantral circular smooth muscle in normal and diabetic ratsThe results demonstrated that CNP-induced inhibitory effectwas significantly potentiated in diabetic rats (Figure 1)Mean-while productions of cGMP and cAMP in gastric smoothmuscle induced by CNP were more pronounced in diabeticrats than in controls These results suggest that the gastricsmooth muscle in diabetic rats is more sensitive to CNPthan in control rats In the previous studies we found thatNPR-A NPR-B and NPR-C expressions were upregulated ingastric smooth muscle in diabetic mice and rats [18 19 25]Therefore we suppose that diabetes-induced upregulationof NPRs in gastric smooth muscle potentiates CNPNPR-ABpGCcGMP-PDE3-cAMP signal pathway

To further confirm our speculation we subsequentlyobserved PDEs expressions in gastric smooth muscle ofcontrol and diabetic rats by using immunohistochemistry

and Western blotting methods Our results showed that onlyPDE3 expression in gastric smooth muscle was downregu-lated in diabetic rats (Figure 4) however the other PDEsexpressions for example PDE1 PDE2 PDE4 and PDE5were not significantly changed in diabetic rats We havealso found that CNP induced relaxation of gastric smoothmuscle via CNPNPR-A BpGCcGMP-PDE3-cAMP signalpathway in normal rats [20] In heart cGMP can inhibitcAMPhydrolysis by PDE3 andpossibly PDE1whereas cGMPcan activate PDE2 [17] Our results suggest that in gastroin-testinal smooth muscle PDE3 may also regulate intracellularcAMP level

In conclusion CNP-induced simultaneous increase ofcGMP and cAMP productions results in the inhibition ofgastric smooth muscle spontaneous contraction in controland STZ-induced diabetic rats However diabetes inducesupregulation of NPRspGCcGMP signal pathway and thesubsequent increase of cGMP is responsible for the down-regulation of PDE3 expression which finally inhibits cAMPhydrolysis and increases intracellular cAMP in gastrointesti-nal smooth muscle Our results suggest that CNPNPR-ABpGCcGMPPDE3cAMP signal pathway may be associ-ated with the diabetes-induced gastric motility disorder

Disclosure

Xu Huang is co-first author

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This paper is supported by the National Natural ScienceFoundation of China (nos 81060036 and 81360070)

References

[1] J B Zhao J B Froslashkjaeligr A M Drewes and N EjskjaerldquoUpper gastrointestinal sensory-motor dysfunction in diabetesmellitusrdquo World Journal of Gastroenterology vol 12 no 18 pp2846ndash2857 2006

[2] N J Talley L Young P Bytzer et al ldquoImpact of chronic gas-trointestinal symptoms in diabetes mellitus on health-relatedquality of liferdquo The American Journal of Gastroenterology vol96 no 1 pp 71ndash76 2001

[3] Y Komatsu K Nakao S-I Suga et al ldquoC-type natriureticpeptide (CNP) in rats and humansrdquo Endocrinology vol 129 no2 pp 1104ndash1106 1991

[4] W R Gower Jr K F Salhab W L Foulis et al ldquoRegulation ofatrial natriuretic peptide gene expression in gastric antrum byfastingrdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 278 no 3 pp R770ndashR7802000

[5] H-S Guo X Cui Y-G Cui et al ldquoInhibitory effect of C-typenatriuretic peptide on spontaneous contraction in gastric antral

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013

Gastroenterology Research and Practice 9

circular smooth muscle of ratrdquoActa Pharmacologica Sinica vol24 no 10 pp 1021ndash1026 2003

[6] H-S Guo Z Jin Z-Y Jin et al ldquoComparative study in theeffect of C-type natriuretic peptide on gastricmotility in variousanimalsrdquo World Journal of Gastroenterology vol 9 no 3 pp547ndash552 2003

[7] M S Barnette F C Barone M Grouse T J Torphy and H SOrmsbee III ldquoSmooth muscle cyclic GMP (cGMP) is elevatedby activating enteric neurones in the lower esophageal sphincter(LES) muscle of three specierdquo Gastroenterology vol 95 article85 1988

[8] M Barnette T J Torphy M Grous C Fine and H S OrmsbeeIII ldquoCyclic GMP a potential mediator of neurally- and drug-induced relaxation of opossum lower esophageal sphincterrdquoJournal of Pharmacology and Experimental Therapeutics vol249 no 2 pp 524ndash528 1989

[9] M S Barnette C D Manning W J Price and F CBarone ldquoInitial biochemical and functional characterization ofcyclic nucleotide phosphodiesterase isozymes in canine colonicsmooth musclerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 264 no 2 pp 801ndash812 1993

[10] A F Joslyn M S Barnette M Grous et al ldquoCyclic nucleotidesincrease during neuronally induced relaxation of sphinctericand nonsphincteric gastrointestinal smooth musclerdquo Journal ofGastrointestinal Motility vol 2 no 1 pp 65ndash72 1990

[11] A F Joslyn M Fudge M S Barnette C D Manning FC Barone and H S Ormsbee III ldquoRelaxation of guinea-pigproximal colon (PC) and taenia coli (TC) smooth muscle isregulated by cyclic nucleotide (CN) contentrdquo Gastroenterologyvol 94 article A212 1988

[12] RC Small J P Boyle K R F Elliott RW Foster andA JWattldquoInhibitory effects of AH 21ndash132 in guinea-pig isolated ileumand taenia caecirdquo British Journal of Pharmacology vol 97 no4 pp 1174ndash1181 1989

[13] M Grous A F Joslyn W Thompson and M S BarnetteldquoChange in intracellular cyclic nucleotide content accompaniesrelaxation of the isolated canine internal anal sphincterrdquo Journalof Gastrointestinal Motility vol 3 no 1 pp 46ndash52 1991

[14] J A Beavo and D H Reifsnyder ldquoPrimary sequence ofcyclic nucleotide phosphodiesterase isozymes and the design ofselective inhibitorsrdquo Trends in Pharmacological Sciences vol 11no 4 pp 150ndash155 1990

[15] C D Nicholson R A J Challiss and M Shahid ldquoDifferen-tial modulation of tissue function and therapeutic potentialof selective inhibitors of cyclic nucleotide phosphodiesteraseisoenzymesrdquo Trends in Pharmacological Sciences vol 12 no 1pp 19ndash27 1991

[16] W J Thompson ldquoCyclic nucleotide phosphodiesterases phar-macology biochemistry and functionrdquo Pharmacology andTher-apeutics vol 51 no 1 pp 13ndash33 1991

[17] M Zaccolo and M A Movsesian ldquocAMP and cGMP signalingcross-talk role of phosphodiesterases and implications forcardiac pathophysiologyrdquo Circulation Research vol 100 no 11pp 1569ndash1578 2007

[18] Y-L Cai D-Y Xu X-L Li Z-X Qiu Z Jin and W-X XuldquoC-type natriuretic-peptide-potentiated relaxation response ofgastric smooth muscle in streptozotocin-induced diabetic ratsrdquoWorld Journal of Gastroenterology vol 15 no 17 pp 2125ndash21312009

[19] D Y Xu L Liu Y L Cai et al ldquoNatriuretic peptide-dependentcGMP signal pathway potentiated the relaxation of gastric

smooth muscle in streptozotocin-induced diabetic ratsrdquo Diges-tive Diseases and Sciences vol 55 no 3 pp 589ndash595 2010

[20] Y-L Cai Q Sun X Huang et al ldquocGMPndashPDE3ndashcAMP signalpathway involved in the inhibitory effect of CNP on gastricmotility in ratrdquo Regulatory Peptides vol 180 no 1 pp 43ndash492013

[21] G A Robison R W Butcher and E W Sutherland ldquoCyclicAMP and hormone actionrdquo inCyclic AMP pp 17ndash47 Academicpress New York NY USA 1971

[22] J P Gray G I Drummond D W T Luk J G Hardman andE W Sutherland ldquoEnzymes of cyclic nucleotide metabolism ininvertebrate and vertebrate spermrdquoArchives of Biochemistry andBiophysics vol 172 no 1 pp 20ndash30 1976

[23] J F Wen X Cui J Y Jin et al ldquoHigh and Low Gain Switchesfor Regulation of cAMP Efflux Concentration distinct Rolesfor Particulate GC- and Soluble GC-cGMP-PDE3 Signaling inRabbit Atriardquo Circulation Research vol 94 no 7 pp 936ndash9432004

[24] E Qvigstad L R Moltzau J M Aronsen et al ldquoNatri-uretic peptides increase 1205731-adrenoceptor signalling in failinghearts through phosphodiesterase 3 inhibitionrdquo CardiovascularResearch vol 85 no 4 pp 763ndash772 2010

[25] Y-S Wu H-L Lu X Huang et al ldquoDiabetes-induced lossof gastric ICC accompanied by up-regulation of natriureticpeptide signaling pathways in STZ-induced diabetic micerdquoPeptides vol 40 pp 104ndash111 2013