Correlation between phospholipid breakdown, intracellular calcium mobulization and enzyme secretion in rat pancreatic acini treated with Boc-[Nle28, Nle31]-CCK-7 and JMV180, two cholecystokinin analogues (original) (raw)
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Biochimica Et Biophysica Acta-molecular Cell Research, 1989
When pancreatic acini are incubated with increasing concentrations of cholecystokinin octapeptide (CCK-8) the dose-response curve for stimulation of enzyme secretion increases, reaches a maximum and then decreases. The upstroke of the dose-response curve reflects occupation of high-affinity, stimulatory CCK receptors (Kd 69 pM), whereas the downstroke of the dose-response curve reflects occupation of low-affinity inhibitory CCK receptors (Kd 10 nM). In the present study, we used dispersed acini from rat pancreas to examine the effects of CCK-JMV-180, an analogue of the C-terminal heptapeptide of CCK (CCK-7) having the structure BOC-Tyr(SO3) Ahx-Gly-Trp-Ahx-Asp2 phenylethyl ester. CCK-JMV-180 inhibited binding of 125I-labelled CCK-8 to pancreatic acini. Computer analysis of the dose-inhibition curve indicated that CCK-JMV-180 interacted with both classes of CCK receptor and had a Kd of 2.2 nM for high-affinity CCK receptors and a Kd of 19 nM for low-affinity CCK receptors. Occupation of high-affinity CCK receptors by CCK-JMV-180 caused a 14-fold increase in amylase secretion, the same increase caused by occupation of these high-affinity receptors by CCK-7 or CCK-8. Occupation of low-affinity CCK receptors by CCK-JMV-180 did not alter amylase secretion, in contrast to occupation of these low-affinity receptors by CCK-7 or CCK-8, each of which caused inhibition of amylase secretion. Furthermore, occupation of the low-affinity CCK receptors by CCK-JMV-180 reversed the inhibition of amylase secretion caused by a supramaximal concentration of CCK-8. The present results indicate that CCK-JMV-180 interacts with high-affinity CCK receptors and with low-affinity CCK receptors, and has a functionally distinct action at each class of receptor: CCK-JMV-180 is an agonist at the high-affinity receptors and a competitive antagonist at low-affinity receptors.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2016
Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane t...
Pancreas, 1997
To determine the effect of long-term blockade of cholecystokinin (CCK) on both pancreatic storage and secretion processes, L 364,718 (a CCK receptor antagonist) was administered to rats at 0.1 mg/kg/day for 3, 7, and 15 days. Zymogen granules were analyzed by flow cytometry to determine their light scattering properties-forward scatter and side scatter-as well as their amylase content measured by a specific antiserum. The mean number of zymogen granules per cell was counted on pancreatic sections using electron microscopy. DNA content, pancreatic weight, and enzyme secretion were also studied under both basal conditions and CCK infusion at a dose of 1.25 p g k g h , which is able to displace the CCK receptor antagonist. Two subpopulations of zymogen granules (Z, and Z,) were identified on the basis of their light scattering parameters, in both control and L 364,718-treated rats. L 364,718 administered for 3, 7, and 15 days induced a significant reduction in the amylase content of individual zymogen granules, for both 2, and Z, zymogen granule subsets. In contrast, the number of zymogen granules per cell increased from It is well established that long-term administration of exogenous cholecystokinin (CCK) or its analogue cerulein increases the pancreatic content of both DNA and protein (1-3). Similar effects have been observed in experimental situations in which endogenous CCK was increased such as feeding with trypsin inhibitors (4) or bile-pancreatic juice diversion (5).
Regulatory Peptides, 2005
Background and aims: The neuroendocrine hormone amylin, cosecreted with insulin from pancreatic h-cells in response to nutrient ingestion, has several physiologic actions to limit the rate of nutrient uptake, including the slowing of gastric emptying. Methods: To investigate whether amylin might modulate digestive enzyme secretion from the exocrine pancreas, anesthetized Sprague Dawley rats were cannulated via the pancreatic duct and the secretory response (flow, amylase and lipase) to cholecystokinin (1 Ag s.c.) was measured in the absence and in the presence of 0.1, 0.3 and 1 Ag s.c. doses of amylin. Results: Amylin alone did not affect pancreatic secretion, but it dose-dependently inhibited cholecystokinin-stimulated amylase secretion by up to 58% and lipase secretion by up to 67%. The ED 50 's for these responses were 0.21 Ag F 0.18 log and 0.11 Ag F 0.05 log, respectively, doses that result in excursions of plasma amylin concentration that are within the reported physiological range. Amylin did not evoke cell signalling in the Ar42j model of pancreatic acinar cells, and responses to amylin were not observed in either Ar42j cells or isolated pancreatic acini in a microphysiometer indicating that the effect of amylin was indirect. Conclusions: Inhibition of stimulated pancreatic enzyme secretion is likely to be a physiological, extrapancreatic, action of amylin. Amylinergic mechanisms modulating both gastric emptying and pancreatic enzyme secretion may thus match, respectively, the appearance of substrate and enzymes in the gut lumen.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998
Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125 FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21 Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125 FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125 FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPAstimulated amylase release. These results show that tyrosine phosphorylation of p125 FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin. 0167-4889 / 98 / seefrontmatterß1998ElsevierScienceB.V.Allrightsreserved.GTPbindingprotein0167−4889/98/^see front matter ß 1998 Elsevier Science B.V. All rights reserved. GTP binding protein 0167-4889 / 98 / seefrontmatterß1998ElsevierScienceB.V.Allrightsreserved.GTPbindingprotein0167−4889/98/^see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 8 9 ( 9 8 ) 0 0 0 7 2 -X
2000
We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of 32 P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [ 32 P]phosphatidylethanol and [ 32 P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32 P-phosphatidylalcohol, yet they decreased [ 32 P]phosphatidylcholine content and stimulated the generation of [ 32 P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity. ß
The Journal of Physiology, 1990
1. A comparative study was made of the effect of the phorbol ester, 12-0tetradecanoylphorbol-13-acetate (TPA) on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the anaesthetized rat and isolated permeabilized pancreatic acinar cells. 2. Cholecystokinin octapeptide (CCK8; 0-10-6-40 nmol (kg body weight)-') induced dose-dependent increases in pancreatic juice flow, total protein output and amylase release in the anaesthetized rat. 3. Administration of TPA (10-8 mol (kg body weight)-') in combination with CCK8 resulted in marked attenuation of the CCK8-evoked secretory response. 4. Simultaneous injection of polymyxin B (10-8 mol (kg body weight)-'), an inhibitor of protein kinase C, with TPA and CCK8 reversed the inhibitory effect of the phorbol ester on CCK8-induced pancreatic juice flow, total protein output and amylase release. 5. In permeabilized rat pancreatic acini CCK8 (10-13-10-9 M) elicited dosedependent increases in [3H]leucine-labelled protein secretion (3H-labelled protein release). Combining TPA (10-8 M) with CCK8 resulted in an inhibition of the CCK8induced 3H-labelled protein release especially at lower concentrations of CCK8. At higher concentrations of CCK8, TPA was unable to inhibit the CCK8-evoked 3Hlabelled protein release. Again, polymyxin B reversed the TPA-induced inhibition of CCK8-evoked 3H-labelled protein output. 6. The results indicate that protein kinase C activation may play an important physiological role in modulating the CCK8-evoked secretory response in rat pancreas in vivo and in vitro.
Proceedings of the National Academy of Sciences, 1989
Rats infused with a supramaximally stimulating dose of the cholecystokinin (CCK) analog caerulein develop acute edematous pancreatitis. Using CCK-JMV-180, a recently developed CCK analog that acts as an agonist at high-affinity CCK receptors but antagonizes the effect of CCK at low-affinity receptors, we have determined that caerulein induces pancreatitis by interacting with low-affinity CCK receptors. Those low-affinity receptors mediate CCK-induced inhibition of digestive enzyme secretion from the pancreas. Our observations, therefore, suggest that this form of experimental pancreatitis results from the inhibition of pancreatic digestive enzyme secretion.