effect of insulin on acetylcholine-evoked amylase release and calcium mobilization in...

Effect of Insulin onAcetylcholine-Evoked AmylaseRelease and Calcium Mobilizationin Streptozotocin-Induced DiabeticRat Pancreatic Acinar CellsREKHA PATEL,a JOSE A. PARIENTE,b MARIA A. MARTINEZ,b

GINES M. SALIDO,b AND JAIPAUL SINGHa

aDepartment of Biological Sciences, University of Central Lancashire,Preston, Lancashire, PR1 2HE UKbDepartment of Physiology, University of Extremadura, Caceres, 10071 Spain

ABSTRACT: This article investigated the effect of acetylcholine (ACh) onamylase secretion and cellular calcium homeostasis [Ca2+]i in strepto-zotocin (STZ; 60 mg kg−1, intraperitoneally)-induced diabetic rats com-pared to age-matched controls in an attempt to understand the cellularmechanism of exocrine pancreatic insufficiency. ACh-evoked markeddose-dependent increases in amylase release from isolated pancreaticacini and acinar cells in healthy control rats. In diabetic acini and aci-nar cells, the ACh-evoked amylase release was significantly (P < 0.05)reduced compared to healthy acini and acinar cells. Insulin (10−6M)stimulated amylase release in both control and diabetic acini and acinarcells but with a much reduced effect in diabetic tissues. Combining in-sulin with ACh had no significant effect on amylase release compared tothe effect of ACh alone. In fura-2 loaded pancreatic acinar cells of normalrats, ACh (10−5M) evoked a large initial rise (peak) in [Ca2+]i followedby a decline into a plateau phase. This effect of ACh was significantly(P < 0.05) reduced in fura-2 loaded diabetic acinar cells. In control cells,insulin had no significant effect on either basal or ACh evoked [Ca2+]icompared to the effect of ACh alone. In contrast, in diabetic acinar cells,insulin significantly (P < 0.05) attenuated the effect of ACh. In a nor-mally free extracellular Ca2+ medium [Ca2+]o containing 1 mM EGTA,the ACh-evoked [Ca2+]i in normal healthy fura-2 loaded acini was sim-ilar to the response obtained with ACh in fura-2 loaded diabetic acini.Together, the results indicated that exocrine pancreatic insufficiency isassociated with decreased [Ca2+]i due to less Ca2+ released from internalstores and less Ca2+ entering the cell from the extracellular medium.

KEYWORDS: rats; acetylcholine; insulin; amylase; calcium; diabetes

Address for corresspondence: Prof. Jaipaul Singh, Department of Biological and Forensic Sciences,University of Central Lancashire, Preston, Lancashire, PR1 2HE UK. Voice: 0044-1772-893515; fax:0044-1772-892929.

e-mail: [email protected]

Ann. N.Y. Acad. Sci. 1084: 58–70 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1372.027

58

PATEL et al.: EFFECT OF ACH ON THE DIABETIC PANCREAS 59

INTRODUCTION

The exocrine pancreas secretes an isotonic fluid that is rich in digestive en-zymes and bicarbonate.1,2 Pancreatic juice secretion is controlled by the guthormones cholecystokinin and secretin and predominantly by the parasympa-thetic neurotransmitter acetylcholine (ACh).1–4 Upon stimulation of the vagalnerve, ACh is released and activates muscarinic receptors on acinar cells,resulting in the metabolism of inositol bisphosphate (PIP2) leading to theproduction of inositol trisphosphate (IP3), inositol tetraphosphate (IP4), anddiacylglycerol (DG). IP3 in turn stimulates Ca2+ release from intracellularstores, whereas IP4 with IP3 induce Ca2+ influx into pancreatic acinar cells.5,6

Ca2+ in turn activates calmodulin that phosphorylates regulatory proteins onzymogen granules resulting in the influx of fluid into the granules that swelland migrate to the luminal pole of acinar cells where they release enzymesby exocytosis. Similarly, DG stimulates protein kinase C that also activateszymogen granules to release digestive enzymes.7,8

There is now much evidence that the endocrine hormone insulin can interactwith the parasympathetic neurotransmitter ACh both in vivo and in vitro toproduce voluminous and sustained digestive enzyme secretion.9 In the in vitrostudies we had previously employed pancreatic segments to measure amylaseoutput.10 However, this interaction between insulin and ACh is impaired duringdiabetes mellitus (DM) leading to the long-term condition that is referred to asexocrine pancreatic insufficiency.11 This derangement in pancreatic secretionis believed to be associated with a decrease in the synthesis and production ofamylase.12 The precise mechanism for exocrine pancreatic insufficiencies isstill not fully understood. Some workers have suggested that gene expressionfor amylase synthesis is impaired while others have shown that diabetic acinarcells are unable to take up glucose and there is also a decrease in Na/K-ATPase activity.11 In a preliminary study employing pancreatic acinar cells,we have shown that pancreatic insufficiency is associated with a decrease inCCK-evoked Ca2+ homeostasis.12 Since ACh uses the same stimulus-secretioncoupling mechanism as CCK, we have decided to investigate the effect of AChon Ca2+ mobilization and amylase release in single acini and acinar cells ofdiabetic and age-matched control rats.

METHODS

General Procedures

All experiments were performed on adult male Wistar rats weighing about150–450 g and relevant ethical approvals were obtained from the EthicsCommittees of the University of Extremadura and University of CentralLancashire for the use of animals in experimental research. Young adult rats

60 ANNALS NEW YORK ACADEMY OF SCIENCES

(150–200 g) were rendered diabetic by using a single intraperitoneal (i.p.)injection of streptozotocin (STZ) (60 mg kg−1 body weight) dissolved in acitrate buffer.13 Age-matched control animals received an equivalent volumeof citrate buffer i.p. Both control and DM-induced rats were tested for diabetes4 days following STZ injection and 6–8 weeks later on the day of the experi-ment using a Glucometer (Accu Chek; Roche Diagnostic, East Sussex, UK).Blood glucose concentration in excess of 300 mg dL−1 confirmed DM.

Both age-matched control and STZ-induced diabetic animals were humanelykilled by a blow on the head followed by cervical dislocation and the pancreaswas quickly removed and placed in a modified Krebs–Henseleit (K–H) so-lution of the following composition (mM): NaCl, 103; KCl, 4.7; CaCl2, 2.6;MgCl2, 1.1; NaHCO3, 25; NaH2PO4, 1.1; D-glucose, 2.8; sodium pyruvate,4.9; sodium fumarate, 2.7; and sodium glutamate, 4.9. The solution was keptat pH 7.4 while being continuously gassed with a mixture of 95% O2–5% CO2

and maintained at 37◦C.

Preparation of Isolated Pancreatic Acini

Pancreatic acinar cells were isolated as described previously.14 The pan-creas was incubated in the presence of collagenase for 10 min at 37◦C. Thisenzymatic digestion of the tissue was followed by gently pipetting the cell sus-pension through tips of decreasing diameter for mechanical dissociation of theacinar cells. After centrifugation, cells were resuspended in HEPES-bufferedsaline (HBS) containing (in mM): HEPES, 10; NaCl, 140; KCl, 4.7; CaCl2,1.3; MgCl2, 1.1; and glucose, 10 (pH 7.4). With this isolation procedure, singlecells as well as small clusters consisting of up to five cells were obtained. Cellviability monitored with trypan blue was greater than 95%.

Measurement of Amylase Release

For the measurement of amylase secretion, aliquots (500 �L) of fresh acinarcells were incubated with ACh at 37◦C for 30 min followed by centrifugationat 500 g for 2 min (4◦C). Acini exposed to the incubation medium alone servedas unstimulated controls (basal release). Amylase release and activities in thesupernatant were determined using the Phadebas blue starch method15 andexpressed as a percentage of total amylase content at the beginning that wasreleased into the extracellular medium during the incubation.

Cell Loading and Ca2+ Analysis

The cell suspension was incubated with 2 �M fura-2-AM in the presenceof 0.025% pluronic acid at room temperature for 25 min using an establishedmethod.14 Following loading, the cells were centrifuged for 3 min at 700 rpm


and resuspended in fresh HBS and used within 2–3 h. For the determination offluorescence, 200–300 �L of cell suspension was placed on a poly-D-lysine(20 �g mL−1) coated thin glass cover slip attached to a Perspex perfusion cham-ber, which was continuously perfused with HBS containing 2.5 mM CaCl2

(approx. at a rate of 1.5 mL min−1) at room temperature.14 The perfusion cham-ber was placed on the stage of an inverted fluorescence microscope (NikonDiaphot 200; Yokohama, Tokyo, Japan). The cells (50 individual cells werechosen) were alternatively excited at 340 and 380 nm by computer-controlledfilter wheel (Lambda-2; Sutter Instruments, Novato, CA) and the emitted im-ages (>515 nm) were captured by a high-speed cooled digital CCD camera(C-4880-81; Hamamatsu Photonics, Hamamatsu City, Japan), and recordedusing appropriate software (Argus-HiSca; Hamamatsu Photonics). In someexperiments extracellular Ca2+ ([Ca2+]o) was removed from the superfusingmedium but the solution contained 1 mM EGTA. Cells were superfused withdifferent concentrations of ACh alone (10−8–10−4 M) or in combination withinsulin. All values were measured in ratio units (F340/F380) and subsequentlyconverted into concentrations using an established method.16

Statistical Analysis

All data provided were expressed as means ± standard error of the mean(SEM). Data were compared by analysis of variance (ANOVA) and only valueswith P < 0.05 were accepted as significant.

RESULTS

General Characteristics of Control and Diabetic Rats

TABLE 1 shows the general characteristics of age-matched and STZ-induceddiabetic rats. The results show that diabetic rats and the pancreas weighed

TABLE 1. General characteristics of age-matched control and STZ-induced diabetic rats

Experimental conditions Age-matched control STZ-induced DM

Weight of animals (g) 391.83 ± 37.91 (n = 10) 190.12 ± 5.41 (n = 10)∗Blood glucose 92.40 ± 2.42 (n = 10) >500 (n = 10)∗

level (mg dL−1)Weight of the pancreas 1.30 ± 0.07 (n = 10) 1.02 ± 0.05 (n = 10)∗Plasma insulin (ng mL−1) 20.63 ± 7.52 (n = 10) 4.80 ± 1.28 (n = 10)∗Basal (Ca2+)i (nM) 246.09 ± 7.53 (n = 98 cells) 169 ± 4.62 (n = 138 cells)∗% of Amylase release 9.01 ± 1.19 (n = 8) 3.25 ± 2.1 (n = 8)∗

with 10−6M insulin

Data are mean ± SEM with n values shown in brackets except for [Ca2+]i, which indicates cellsfrom 8–10 animals. ∗P < 0.05 comparing control with diabetic animals.


FIGURE 1. Dose–response bar charts showing the effect of 10−8–10−4 M ACh on totalamylase output from superfused healthy control and STZ-induced diabetic pancreatic acinarcells. Each point is mean ± SEM. n = 6–10 rats for each, ∗P < 0.05 (independent samplesStudent’s t-test). Note that stimulation with all ACh concentrations resulted in a significantdifference between control and diabetic groups in the % of total amylase released.

significantly (P < 0.05) less compared to the age-matched control. Moreover,the diabetic animals have significantly elevated blood glucose levels (P < 0.05)and significantly reduced (P < 0.05) plasma insulin concentration compared tothe age-matched control. Basal [Ca2+]i in single diabetic pancreatic acinar cellsdecreased significantly (P < 0.05) compared to control. Similarly, amylaserelease following incubation with 10−6 M insulin was significantly (P < 0.001)less in diabetic acini compared to age-matched control acinar cells.

Measurement of Amylase Release

FIGURE 1 shows the effect of different concentrations (10−8–10−4M) of AChon the percentage of amylase release from age-matched healthy control andSTZ-induced diabetic pancreatic acini and acinar cells. The results show thatcontrol acinar cells released significantly (P < 0.05) higher levels of amylasecompared to STZ-induced diabetic rats at all concentrations of ACh. Maximumtotal amylase release was achieved following incubation of acini with 10−6MACh.

FIGURE 2 shows the effect of different ACh concentrations (10−7–10−4M) onamylase release in diabetic and control pancreatic acinar cells in the absence


FIGURE 2. Bar charts showing the effect of combining 10−6 M insulin with differentconcentrations of ACh (10−7–10−4 M) on total amylase output from healthy age-matchedcontrol and STZ-induced diabetic pancreatic acinar cells. Each bar is mean ± SEM. n =6–10 rats for each, ∗P < 0.05 as compared to the respective control cells (independentsamples Student’s t-test).

and presence of 10−6M insulin. The results show that ACh alone can elicit amarked increase in amylase output. Combining ACh with insulin resulted inonly a slightly larger increase (but not significant) in amylase release comparedto the effect of ACh alone. In diabetic acinar cells either ACh or ACh incombination with 10−6M insulin evoked significantly (P < 0.05) less amylaserelease compared to the amylase release from age-matched control cells.

Measurement of [Ca2+]i

FIGURE 3 shows original chart recordings of time course mean changesin [Ca2+]i in STZ-induced diabetic and age-matched control pancreaticacini following stimulation with either 10−5M ACh alone (A), or ACh incombination with 10−6M insulin (B). The results show that ACh evoked a


0 100 200 300 400 500 600 7000

1000

2000

3000

4000

Ach Control

Ach Diabetic

Ach 10-5

M

Time (sec)

[Ca

2+]

(nM

)

0 200 400 600 8000

500

1000

1500

2000

2500

3000

3500

4000

Ins+Ach Control

Ins+Ach Diabetic

Ach 10-5

M

Insulin 10-6

M

Time (sec)

[Ca

2+]

(nM

)

(A)

(B)

FIGURE 3. (A) Original chart recording of the time course of mean changes in [Ca2+]i

before and after ACh (10−5 M) application in healthy control and STZ-induced diabeticsingle acini in the absence (A) and presence (B) of 10−6 M insulin (IN). Traces are typicalof 26–29 cells taken from 5–6 control and 5–6 diabetic rats.

rapid increase (initial peak) in [Ca2+]i and followed a slow time course decline(plateau phase) to almost control level after 6–7 min to ACh application.

The mean (± SEM) basal, ACh-evoked peak and plateau phases of the Ca2+transient in the absence and presence of 10−6M insulin are shown in FIGURE 4.The control [Ca2+]i in the absence and presence of 10−6M insulin is also shownin FIGURE 4 for comparison. The results show that ACh can evoke a markedand significant (P < 0.05) increase in the peak [Ca2+]i either in the absence or


FIGURE 4. Bar charts showing mean (± SEM) basal, ACh (10−5 M)-evoked peak andplateau phases (2 min and 5 min after the peak response) of [Ca2+]i in normal and diabeticsingle acini in the absence and presence of 10−6 M insulin (+IN). Each bar is mean ± SEM.n = 26–29 acinar cells taken from 5–6 rats for each. ∗P < 0.05 as compared to the respectivecontrol and #P < 0.05 as compared to the respective effect of insulin (independent samplesStudent’s t-test).

presence of 10−6M insulin in control pancreatic acinar cells. In diabetic acinarcells, the ACh-induced [Ca2+]i transient was significantly reduced (P < 0.05)either in the absence or presence of insulin compared to age-matched controlpancreatic acinar cells. Insulin (10−6 M) was shown to significantly increasethe ACh-evoked plateau response 2 min after the peak response (794.2 ± 41.8nM, n = 26 in healthy control acini compared to the effect of ACh (10−6 M)administration alone (434.3 ± 30.3 nM, n = 26).

In zero [Ca2+]o basal [Ca2+]i was 144.20 ± 44.72 nM (n = 29) and 44.72 ±9.44 nM (n = 26) in control and diabetic acinar cells, respectively. The ACh-evoked peak [Ca2+]i was 1744.20 ± 315.17 nM (n = 29) in control and 747.74± 7.41 nM (n = 26) in diabetic acinar cells, respectively. The ACh-evokedplateau phase after 2 min in zero [Ca2+]o was 125.56 ± 7.75 nM (n = 29) and8.31 ± 1.32 nM (n = 26) in control and diabetic acinar cells, respectively.

FIGURE 5 shows the integral rise (area under the traces) in [Ca2+]i abovebasal following 5 min of the ACh (10−5M) stimulation of age-matched healthyand STZ-induced diabetic acinar cells in the absence and presence of 10−6Minsulin during normal (1.2 mM) [Ca2+]o and in a nominally free [Ca2+]o +1mM EGTA. The results show that diabetic acinar cells contain significantly(P < 0.05) less amount of [Ca2+]i compared to control in normal [Ca2+]o.However, when [Ca2+]o was removed and 1 mM EGTA was added to the


FIGURE 5. Bar charts showing the integral of rise above basal in [Ca2+]i following 5min of 10−5 M ACh stimulation of age-matched healthy control and STZ-induced diabeticacinar cells in the absence and presence of 10−6 M insulin (INS) during normal [Ca2+]0

and nominally free [Ca2+]0 + 1 mM EGTA. Data are mean ± SEM. n = 26–29 in each barchart taken from 5–6 control and 5–6 diabetic rats.

superfusing medium, [Ca2+]i was also significantly (P < 0.05) reduced inage-matched control acinar cells similar to that seen in diabetic acinar cellsin normal [Ca2+]o. The results suggest that diabetes is associated with andecrease in cytosolic Ca2+ in pancreatic acinar cells and this may be due toboth its release from intercellular stores and its influx into pancreatic cells.

DISCUSSION

The results of this study have shown marked differences in the characteristicsof age-matched healthy control and STZ-induced diabetic rats and the functionsof the pancreas. Like other investigations,12,17 the present results show thatdiabetic rats and the pancreas weigh significantly less and the animals haveelevated blood glucose (hyperglycemia) and reduced plasma insulin levelscompared to the healthy control. It is well known that diabetic rats eat lessand moreover, they produce less digestive enzymes, especially amylase, forthe digestion of food stuffs.11,12,17–20 This in turn will naturally lead to weightloss and atrophy as well as other long-term complications resulting from thehyperglycemia.21

The results have shown that ACh can elicit marked dose-dependent increasesin amylase release from control healthy pancreatic acinar cells. In contrast, indiabetic acinar cells, the effect of ACh was significantly reduced at all concen-trations of ACh employed in this study. These data support previous findingsby other workers who have shown that STZ-rendered DM is associated withreduced amylase secretion.9,11,12,22–24 The reduction in ACh-evoked amylaseoutput in the diabetic pancreatic acinar cells is not due to a direct effect of STZ


since it has no effect on amylase secretion when it is applied either alone or incombination with secretagogues.25

In contrast to previous studies employing either isolated pancreatic seg-ments,9,26 intact isolated pancreas27–29 or the anaesthetized rat,17 the presentresults have shown that insulin at 10−6 M can stimulate amylase release fromsingle pancreatic acinar cells. When insulin was combined with ACh, amylaserelease increased only slightly compared to the effect of ACh alone. This isin contrast to previous studies employing isolated pancreatic segments andthe isolated intact pancreas in which insulin potentiated the effects of secre-tagogues.1,9,26,27,29 One possible explanation for this discrepancy is that pan-creatic acinar cells respond differently to insulin compared to segments or theintact pancreas. This may be due to the fact that insulin has more access to cellsurface receptors.

This study also measured [Ca2+]i in single fura-2 loaded pancreatic acinarcells in order to determine the precise role of Ca2+ in the stimulus-secretioncoupling process in normal and diabetic conditions. The results have shownthat basal [Ca2+]i was markedly reduced in diabetic acinar cells compared tocontrol. ACh alone evoked a rapid and large increase in [Ca2+]i in control anddiabetic acinar cells reaching a peak within 15–20 s followed by a rapid declineto a plateau phase that remained above basal level throughout the time courseresponse. In diabetic acinar cells, the ACh-evoked peak and plateau phases ofthe responses were significantly reduced compared to control. Insulin alonehad no significant effect on [Ca2+]i in either the control or diabetic acinarcells compared to the respective controls in the absence of insulin. Combininginsulin with ACh resulted in only a small increase in [Ca2+]i compared tothe effect of ACh alone in normal healthy acinar cells. In contrast, insulinsignificantly attenuated the ACh-evoked peak and plateau phases of the Ca2+transient in diabetic acinar cells compared with the effect of ACh and insulin incontrol cells. These results are in complete agreement employing suspensionsof pancreatic acinar cells.9,12,19

This study has also shown that in the absence of extracellular Ca2+ [Ca2+]o,ACh evoked significantly less [Ca2+]i compared to the response obtained withACh in normal [Ca2+]o. These results are similar to those obtained with AChin diabetic acinar cells. It is now well known that [Ca2+]o plays a major role infilling up the intracellular stores and in elevating [Ca2+]i during stimulation toenhance sustained enzyme secretion. It is also well known that in the absenceof [Ca2+]o ACh produces significantly less amylase output.20 These interestingfindings reveal a close relationship between amylase release and the levels of[Ca2+]i available in the cytoplasm to mediate enzyme secretion.

The question that now arises is: what is responsible for the reduced [Ca2+]i

and amylase secretion during the diabetic state? In a previous study we haveshown that both [Ca2+]i and [Mg2+]i are reduced in diabetic acinar cells follow-ing secretagogue stimulation compared to control.12 It is possible that cellularCa2+ homeostasis is directly linked to changes in [Mg2+]i.

30 Moreover, Mg2+


plays a major role in cellular regulation by controlling ion transport and Ca2+-dependent enzyme systems.30 Another possible explanation is that amylasemRNA is reduced during DM leading to less synthesis and less release.31,32

Moreover, in diabetic conditions pancreatic acinar cells are unable to take upeither glucose or amino acids that are associated with reduced Na+K+ATPaseactivity.33 It is conceivable that reduced [Ca2+]i may be linked to all theseprocesses since it is the mediator and promotor in cellular regulation.34

In conclusion, the results have demonstrated that both ACh and insulin canstimulate amylase release from age-matched control and diabetic acinar cellswith much reduced effect in diabetic acinar cells. Similarly, ACh can alsoelevate [Ca2+]i in fura-2 loaded control and diabetic acinar cells with lesseffect in diabetic cells. The responses in diabetic acinar cells resemble thoseobtained with ACh in a nominally free [Ca2+]i in combination with 1 mMEGTA. The results indicate that cellular Ca2+ homeostasis is decreased in DMand this in turn may result in reduced amylase secretion and exocrine pancreaticinsufficiency.

REFERENCES

1. WILLIAMS, J.A. & I.D. GOLDFINE. 1993. The insulin acinar relationship. In TheExocrine Pancreas: Biology, Pathobiology, and Disease, 2nd ed. V.L.M. Go, E.P.DiMagno, J.D. Gardner, et al., Eds.: 789–802. Raven Press. N.Y.

2. VANDER, A., J. SHERMAN & D. LUCIANO. The digestion and absorption of food.1998. In Human Physiology, The Mechanisms of Body Function, 7th ed.A. Vander, J. Sherman & D. Luciano, Eds.: 551–556, 576–577. WCB McGraw-Hill. Boston.

3. RICHINS, C.A. 1945. The innervation of the pancreas. J. Comp. Neurol. 82: 223–236.

4. BROWN, J.H. & P.M. MCDONOUGH. 1989. Muscarinic cholinergic receptors of in-ositol phospholipids metabolism and calcium mobilization. In The MuscarinicReceptors. J.H. Brown, Ed.: 259–307. Humana Press. NJ.

5. BERRIDGE, M.J. 1993. Inositol trisphosphate and calcium signalling. Nature 361:315–325.

6. PUTNEY, J.W., JR. 1988. The role of phosphoinositide metabolism in signal trans-duction in secretory cells. J. Exp. Biol. 139: 135–150.

7. JENSEN, R.T. & J.D. GARDNER. 1981. Identification and characterization of recep-tors for secretagogues on pancreatic acinar cells. Fed. Proc. 40: 2486–2496.

8. NISHIZUKA, Y. 1988. Studies and prospectives of protein kinase C in signal trans-duction. Nippon Ketsueki Gakkai Zasshi 51: 1321–1326.

9. SINGH, J. & E. ADEGHATE. 1998. Effects of islet hormones on nerve-mediated andacetylcholine-evoked secretory responses in the isolated pancreas of normal anddiabetic rats. Int. J. Mol. Med. 1: 627–634.

10. JUMA, L.M., J. SINGH, D.J. PALLOT, et al. 1997. Interactions of islet hormones withacetylcholine in the isolated rat pancreas. Peptides 18: 1415–1422.

11. SINGH, J., E. ADEGHATE, S. APARICO, et al. 2004. Exocrine pancreatic insufficiencyin diabetes mellitus. Int. J. Diabetes Metab. 12: 35–43.


12. PATEL, R., M.D. YAGO, M. MANAS, et al. 2004a. Mechanism of exocrine pancre-atic insufficiency in streptozotocin-induced diabetes mellitus in rat: effect ofcholecystokinin-octapeptide. Mol. Cell. Biochem. 261: 83–89.

13. SHARMA, A.K., I.G. DUGUID, D.S. BLANCHARD, et al. 1985. The effect of insulintreatment on myelinated nerve fibre maturation and integrity and on body growthin streptozotocin-diabetic rats. J. Neurol. Sci. 67: 285–297.

14. PARIENTE, J.A., P.C. REDONDO, M.P. GRANADOS, et al. 2003. Calcium signalling innon-excitable cells. E. C. Qua. L. 1: 29–43.

15. GARDNER, J.D. & M.J. JACKSON. 1977. Regulation of amylase release from dis-persed pancreatic acinar cells. J. Physiol. 270: 439–454.

16. GRYNKIEWICZ, G., M. POENIE & R.Y. TSIEN. 1985. A new generation of Ca2+indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450.

17. PATEL, R., J. SINGH, M.D. YAGO, et al. 2004b. Effect of insulin on exocrine pan-creatic secretion in healthy and diabetic anaesthetised rats. Mol. Cell. Biochem.261: 105–110.

18. AHMED, I., E. ADEGHATE, A.K. SHARMA, et al. 1998. Effects of Momordica charan-tia fruit juice on islet morphology in the pancreas of the streptozotocin-diabeticrat. Diabetes Res. Clin. Pract. 40: 145–151.

19. SINGH, J., L.M.O. JUMA, E. ADEGHATE, et al. 1999. Interactive adaptation of en-docrine and exocrine pancreas: effects of islet hormones and secretagogues. InAdaptation Biology and Medicine. K.B. Pandolf, N. Takeda & P.K. Singal, Eds.:223–235. Narosa Publishing House. New Delhi, India.

20. SINGH, J., M.D. YAGO & E. ADEGHATE. 2001. Involvement of cellular calcium in ex-ocrine pancreatic insufficiency during streptozotocin-induced diabetes mellitus.Arch. Physiol. Biochem. 109: 252–259.

21. KUMAR, P.J. & M.L. CLARK. 2002. Diabetes mellitus and other disorders ofmetabolism. In Clinical Medicine. P.J. Kumar & M.L. Clark, Eds.: 1069–1122.WB Saunders. London.

22. SOFRANKOVA, A. & G.J. DOCKRAY. 1983. Cholecystokinin- and secretin-inducedpancreatic secretion in normal and diabetic rats. Am. J. Physiol. 244: G370–G374.

23. OTSUKI, M., T. AKIYAMA, H. SHIROHARA, et al. 1995. Loss of sensitivity to chole-cystokinin stimulation of isolated pancreatic acini from genetically diabetic rats.Am. J. Physiol. 268: E531–E536.

24. YAGO, M.D., E. ADEGHATE & J. SINGH. 1999. Interactions between the endocrineand exocrine pancreas. Effects of islet hormones, secretagogues, and nerve stim-ulation. In Neural Regulation in the Vertebrate Endocrine System: Neuroen-docrine Regulation. R.A. Prasada Rao, R. Peters Eds.: 197–217. Kluwer Aca-demic/Plenum. N.Y.

25. MAHAY, S., E. ADEGHATE, M.Z. LINDLEY, et al. 2004. Streptozotocin-inducedtype 1 diabetes mellitus alters the morphology, secretory function and acyl lipidcontents in the isolated rat parotid salivary gland. Mol. Cell. Biochem. 261:175–181.

26. SINGH, J. & G.T. PEARSON. 1984. Effects of nerve stimulation on enzyme se-cretion from the in vitro rat pancreas and 3H-release after preincubation withcatecholamines. Naunyn Schmiedebergs Arch Pharmacol. 327: 228–233.

27. KANNO, T. & A. SAITO. 1976. The potentiating influences of insulin onpancreozymin-induced hyperpolarization and amylase release in the pancreaticacinar cell. J. Physiol. 261: 505–521.


28. SAITO, A., J.A. WILLIAMS & T. KANNO. 1980a. Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolatedand perfused rat pancreata. J. Clin. Invest. 65: 777–782.

29. SAITO, A., J.A. WILLIAMS & T. KANNO. 1980b. Potentiation by insulin ofacetylcholine-induced secretory response of the perfused rat pancreas. Biomed.Res. 1: 101–103.

30. YAGO, M.D., M. MANAS & J. SINGH. 2000. Intracellular magnesium: transport andregulation in epithelial secretory cells. Front. Biosci. 5: D602–D618.

31. DUAN, R.D. & C. ERLANSON-ALBERTSSON. 1990. Altered synthesis of some secre-tory proteins in pancreatic lobules isolated from streptozotocin-induced diabeticrats. Pancreas 5: 136–143.

32. DUAN, R.D. & C. ERLANSON-ALBERTSSON. 1992. The effect of pretranslationalregulation on synthesis of pancreatic colipase in streptozotocin-induced diabetesin rats. Pancreas 7: 465–471.

33. YANG, Y.K. & W.Y. ZHU. 1995. Effect of insulin on the function of pancreaticexocrine. Sheng. Li. Xue. Bao. 47: 238–244.

34. BERRIDGE, M.J. 1995. Inositol trisphosphate and calcium signalling. Ann. N. Y.Acad. Sci. 766: 31–43.

effect of insulin on acetylcholine-evoked amylase release and calcium mobilization in...

Documents