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Cholecystokinin octapeptide inhibits Ca2+-dependent amylase secretion from permeabilized pancreatic acini by blocking the MgATP-dependent priming of exocytosis.

ABSTRACT: At present little is known about how the low-affinity cholecystokinin receptor inhibits secretagogue-stimulated amylase secretion from pancreatic acinar cells. To examine this question we have determined how cholecystokinin octapeptide (CCK8) influences Ca2+-dependent amylase secretion from alpha-toxin-permeabilized pancreatic acini. CCK8 significantly inhibited Ca2+-stimulated amylase secretion. The inhibitory actions of CCK8 were completely blocked by the addition of JMV-180, a specific antagonist for the low-affinity CCK8 receptor. Previous studies have shown that Ca2+-dependent amylase secretion from alpha-toxin-permeabilized acini has two distinct phases [Padfield and Panesar (1997) Am. J. Physiol. 36, G655-660]. There is an initial rapid phase of secretion which represents release from exocytotic sites primed by MgATP prior to permeabilization. This is followed by a slower sustained phase of secretion which, in part, reflects the MgATP-dependent repriming of the exocytotic machinery. CCK8 did not influence the initial rapid phase of the Ca2+-dependent secretory response, but inhibited the second slower sustained phase. Moreover, CCK8 was shown to inhibit the MgATP-dependent priming of exocytosis in the acini. These results indicate that the low-affinity CCK receptor blocks stimulated amylase secretion by inhibiting the MgATP-dependent repriming of exocytosis.

SUBMITTER: Padfield PJ

PROVIDER: S-EPMC1219144 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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Protein kinase A modulates Ca(2+)- and protein kinase C-dependent amylase release in permeabilized rat pancreatic acini.

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Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis.

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Project description:To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.

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Complexin 2 modulates vesicle-associated membrane protein (VAMP) 2-regulated zymogen granule exocytosis in pancreatic acini.

Project description:Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca(2+)-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central ?-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central ?-helical domain did not alter SNARE binding and moreover, augmented Ca(2+)-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca(2+)-sensitive regulatory pathway for zymogen granule exocytosis.

| S-EPMC2975180 | biostudies-literature

A cytosolic splice variant of Cab45 interacts with Munc18b and impacts on amylase secretion by pancreatic acini.

Project description:We identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.

| S-EPMC1924827 | biostudies-literature

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

Project description:The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.

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