Indian Journal of Experimental Biology Vol. 43, February 2005, pp. 167-171

Inhibitory effect of sildenafil on gastrointestinal smooth muscle: Role of NO-cGMP transduction pathway

Chandrashekhar S Patil , Yijay Pal Singh, Naveen K Jain" & Shrinivas K Kulkarni *

Pharmacology Divi sion, University Institute o f Pharmaceutical Sciences Panjab University, Chandigarh 160 014, India

"R&D Divi sion, Panacea Biotec Ltd. , Chandigarh Road , Lalru & 140 501 , Indi a

Received 28 January 2004, revised 4 October 2004

Nitri c ox ide (NO) is an important neurotransmitter in the gut and has been demonstrated to be a key physiolog ical mediator of non-adrenergic no n-cholinergic (NANC) rel axa tion of gastrointestinal smooth muscle. In the present study the effec t of POE 5 inhibitor sildenafil on the gastrointestinal functi o n (gastric emptying and intes tinal transit) has been demonstrated in mice. Sildenafil (0.5-2 mg/kg, po) did not a lter the perce nt gastric emptying however, in higher doses (5, 10 and 30 mg/kg, po) it inhibited the gastric emptying. On acute administration (0.5-5 mg/kg, po) it did not alter the intestinal transit but in higher doses ( I 0 and 30 mg/kg, p.o. ) delayed the intestinal transit. Further, the inhibitory effect of silde nafil was significantly blocked by L-NAME (10 mg/kg, ip), a non-se lective NOS inhibitor and methylene blue ( I mg/kg, ip), a guanylate cyclase inhibito r. These findings suggest the partic ipation of NO-cGMP transduction pathway in the inhibitory e ffect of sildenafi l (higher doses) on the gastrointestina l smooth muscles and its potenti al application in patients with nutcracker oesophagus, hyperte nsive lower oesophageal sphincter (LOS), achalsia and diabetic gastroparesis or coliti s where there is a loss of nNOS.

Keywords: Gastric e mptying, Intestinal motility, Non-adrenergic no n-cholinergic transmi ss ion, Sildenafil

Nonadrenergic noncholinergic (NANC) mediators and other transmitters like vasoactive intestinal peptide (V IP), and calcitonin gene-related peptide (CGRP) have been suggested in the relaxation of gastrointestinal smooth muscle 1

-3

• Nitric oxide (NO) is the principle inhibitory neurotransmitter released after deglutition by NANC inhibitory neurons. It is responsible for hyperpolarization4

·6 and regulation of

peristaltic contractions in gastrointestinal smooth muscles via elevation of cyclic guanosine mono phosphate (cGMP) and activation of cyclic GMP­dependent protein kinase (PKG) system7

. NANC­mediated relaxati on is significantly antagonized by nitric-oxide biosynthesis inhibitors NG-nitro-L­arginine (L-NNA) and NG-nitro-L-arg inine-methyl ester (L-NAME).

The amplitude and duration of the cGMP signal in the smooth muscle is regulated mainly by cGMP­specific cyclic nucleotide phosphodiesterase (PDES) . Sildenafil , a PDES inhibitor influences NO-cGMP

*Correspondent author: Phone: +91-1 72-534114 Fax: +91-172-541142 E-mail : skpu @yahoo.com

pathway by blocking PDES enzyme that degrades NO stimu lated cGMP. PDES a predominant cGMP­specific POE is expressed in skeletal, cardiac, pylorus

8 '1 and smooth muscle of the gut · . The NO-cGMP has been shown to cause relaxation

of lower oesophageal sphincter and regulates peristaltic contractions in animals and humans 10

-13

.

In vivo studies in human oesophagus have focused on the consequences of reducing the effect of N0 12

-14

•

The efficacy of PDES inhibitors in the treatment of esophageal motility associated with achalasia (a disease with decreased NO levels) has been reported 15

•16

. PDES inhibitors (sildenafil) activates the NO/cGMP pathway and relaxes the gut smooth muscle cell s.

The present study has been aimed to investigate the possible effect of acute administration sildenafil on NO-cGMP pathway in gastric emptying and gastrointest inal motility in albino mice.

Materials and Methods Animals-Swiss albino mice (20-30 g), of either

sex (Central Animal House, Panjab University , Chandigarh, India) were housed under standard

168 INDIAN J EXP BIOL, FEBRUARY 2005

laboratory conditions and kept under a 12:12 hr L:D cycle. Experiments were carried out between 0900 and 1800 hrs . All the experimental protocols were approved by the Institutional Animal Ethics Committee.

Gastric emptying--Gastric emptying was determined by as per by Scarpignato et al 17

• A test meal (0.05 % phenol red in a 1.5% aqueous methylcellulose) of 0.25 ml/mice was given through an intragastric tube. After 30 min the animals were sacrificed, stomach was re moved and homogenized in 100 ml of 0.1 N NaOH. Stomach tissue proteins (in 5 ml homogenate) were precipitated with 0.5 ml of tricholoroacetic acid (20%w/v) and centrifuged. The supernatant was mixed with 4 ml of 0.5 N NaOH and absorbance of the sampl e was read at 560 nm wavelength . Phenol red recovered fro m the stomach of mice sacrificed immediately after administration of the methylcellulose meal served as standard stomach.

Charcoal mealiest-The charcoal meal test 18 was used to assess the effect of sildenafil on intestinal transit. Briefly, mice were starved for 24 hr prior to the experiment but had free access to water. Thereafter, the tes t drug was administered alone. A charcoal meal (0.25 mil mouse consi st ing of I 0% charcoal in 5% gum acacia) was administered orally. The animals were sacrificed 20 min after charcoal meal administration . The abdomen was opened and the entire small intestine starting from the pyloric end was removed and placed on the blotting paper. All care was taken to prevent any damage to the gut and the distance traveled by the charcoal was measured and expressed as percent gastrointestinal (Gl) transit.

The gas tric emptying for each mouse was calculated according to the following formula :

amount of phenol red recovered from stomach

Gastric emptying (%)=1--------::--:----:---:-- X 100 average amount o f phenol red

recovered from standard stomach

Six to seven animals were used per dose to determine the dose response curve. Gastric emptying or gastrointestinal motility effect was expressed as percent gastric emptying or GI transit inhibition in comparison to that observed in the saline treated controls.

Statistical analysis--Results were expressed as the mean ± SE. Difference between the mean values in

groups of each drug treatment and control was analyzed by the nonparametric Kruskal Walli s, one­way analysis of variance with multiple range test.

Results Effect of sildenafil on gastric emptying in mice­

Acute administration (0.5, I , 2 , and 5 mg/kg, po) of sildenafil to mice did not alter the gastric emptying as compared to control group. However, high (I 0 and 30 mg/kg, po) produced a significant (?<0.05) decrease in gastric emptying (Fig. I).

Si ldenafil-induced decrease in e mptying was significantly blocked by L-NAME (10 mg/kg, ip) and methylene blue (I mg/kg, ip) (Fig 2), which on per se also delayed the gastric emptying in mice (Fig. 2).

Effect of sildenafil on gastrointestinal motility in mice-Acute administration (0.5, 1, 2 , and 5 mg/kg, po) of sildenafil did not alter the intestinal motility as compared to the control group. However, high doses (I 0 and 30 mg/kg, po) significantly (?<0.05) delayed the intestinal transit (Fig. 1).

Sildenafil-induced delay in intestinal transit was significantly blocked by L-NAME (10 rng/kg, ip) and methylene blue (I mg/kg, ip) (Fig. 2), which on per se administration also delayed the intesti nal transit time (Fig 2).

100

D Gastric emptying IIIGI trans it 90

80

70 ,. 60

c CIJ 50 ~ CIJ * c..

40

30

20

10

0 Conlrol ~iii (0.5) Sil (I) Sil (2) Sil (5) Si l (10) Sil (20)

Treatment (mglkg)

Fig. !- Effect of sildenafil (Sil; 0.5-30 mg/kg, po) on gas tric emptying and gastrointestinal transit in mice. Values are as mean ± SE from 6 to 8 mice per group. *P <0.05 as compared with control.

PATIL eta/: SILDENAFIL AND GASTROINTESTINAL MOTILITY 169

100 0 Gastric emptying • Gl transit

90 b b b

b

80

70 c a

60 a c

c Q) u so '-Q)

0.. a

b b

b c

c

b

40 a

30

20

10

0 Control Sit (10) Sit (30) Sit (10) <· Sil (10) + Sil (30) + Sit (30) + L-NAME MB (1)

L-NAME MB (1) L-NAME MB (1) (10) (10) (10)

Treatment

Fi g. 2-Effect of sildenafi l (Sil: 10 or 30 mg/kg, po) and its reversal by L-NAME (10 mg/kg, ip) and methylene blue (MB: I mg/kg, ip) on gastric emptying and gastrointestinal transit in mice. P <0.05 as compared with aandccontrol and bsildenafil (10 and 30 mg/kg, po)

Discussion Phosphodiesterases (PDEs) are a large group of

structurally related enzymes that catalyse the hydrolysis of 3', 5'-cyc lic nucleotides to the correspondi ng inacti ve nucleotide 5'-monophosphate by cleaving the phosphod ies ter bond between the phosphorus and oxygen atoms at the 3' position 19

•20

.

The levels of cGMP and cAMP in gastrointestinal smooth muscle are determined by the synthetic activities of soluble guanylyl cyclase (GC) and adenylyl cyclase (AC) respectively , and the degradative activities of specific PDEs, mainly cAMP- preferring PDE3, cAMP-specifi c PDE4 and cGMP-specific PDE5, respectivel / 1

• Eleven famili es (PDEI-11) of PDEs have been identified with each fami ly being the product of a separate gene and usually comprise several isoforms. PDE5 is a predominant cG MP-specific POE, expressed in skeletal, cardiac and smooth muscles22 and is

responsible for the hydrolysis of cGMP23. Sildenafil, a

potent, selective and reversible phosphodiesterases 5 inhibitor24 increases the effect of cyclic GMP, which displays an inhibitory effect on the smooth muscle cells. The inhibitory effect of sildenafil is due to the blockade of PDE5, which inactivates the intracellular cGMP stimulated by NO.

The NO released by the non-adrenergic non­cholinergic (NANC) inhibitory neurons of the myenteric plexus is also the principal inhibitory neurotransmitter in the gut smooth muscle through the prQduction of cGMP. Molecular biological studies hav~ indicated that the mechanisms for NO-mediated neurotransmission in intestine has inhibitory effects.

It is well documented that peri staltic phase leading to interdigestive motility of gastroi ntesti nal tract is characterized by the cyclic occutTence of the ' migrating motor complex ' consisting of three phases of motor activity; Phase I of motor silence, Phase II of

170 INDIAN J EXP BIOL, FEBRUARY 2005

increasili1g in·egular motility, and Phase III of sequence of propulsive motor waves, followed by phase 115

. The recent clinical findings suggested that sildenafil inhibited gastroduodenal motility by virtue of activation of NO-cGMP pathway which prolongs the phase I and prevents the occurrence of phase III of the normal peristaltic phase 15

•

In the present study sildenafil in lower doses (0.5-5 mg/kg, po) neither altered gastric emptying nor gastrointestinal motility which may be due to the insuffic ient production of cGMP to alter the normal peristal tic movements. However, in higher doses it significantly delayed the gastric emptying and intesti nal motility in both the models. The delayed effect of sildenafil at higher doses may be due to its inhibitory effect on gastrointestinal tract as PDE5 enzyme is present in pylorus and throughout the gas trointestinal smooth muscles. Sildenafil may be altering the peristaltic movement in higher doses by prolongi ng the phase I of motor si lence, and inhibiting the phase lii throughout the gas trointestinal tract. Further, the evidence of major participation of NO-cGMP pathway in the inhibitory effect of silclenafil may be ruled out as L-NAME and methylene blue significantly inhibited the inhibitory effect of silclenafil on gas tric emptying and intestinal motility. Based on these findings it is suggested that silclenafil in lower doses did not alter the gastric function in mice but at higher doses it showed inhibitory effect.

The inhibitory effect of si ldenafil on the oesophagus and the gastrointestinal smooth muscles may have deleterious or beneficial effects depending upon the clinical conditions and the close employed. The inhibitory effect of silclenafil lowers LOS pres sura and propulsive forces in the oesophageal body in healthy subjects and in patients with nutcracker oesophagus, hypertensive LOS, and achalsia. However, the inhibitory effect on the gastrointestinal tract may be responsible for the dyspeptic complaints . Interes ting ly, it may have beneficial effect in patients wi th dumping-type syndromes or accelerated gastric emptying or in conditions of diabetic gastroparesis or colitis where there is loss of nNOS that is altered with delayed gastric emptying and intestinal transit. Further, it is necessary to monitor the side effects associated with sildenafil clue to inhibitory effects 111 clinical conditions .

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