Inhibitory effect of a new proglumide derivative, loxiglumide, on CCK action in isolated rat pancreatic acini

The effect of a new proglumide derivative, loxiglumide (DL-4-(3,4-dichloro-benzoyl-amino)-5-(N-3-methoxy-propyl-pentylamino+ ++)-5-oxo-pentanic acid; CR 1505), on binding of 125I-CCK-8 and amylase release stimulated by CCK-8 was investigated in isolated rat pancreatic acini. Loxiglumide inhibited CCK-8-stimulated amylase release and binding of 125I-CCK-8 to rat pancreatic acini in a dose-dependent manner. Loxiglumide caused a concentration-dependent rightward shift of the dose-response curve for CCK-8-stimulated amylase release without altering the maximal response. Schild plots showed a slope of 0.82 and pA2 value of 7.05. The inhibitory effect of loxiglumide on amylase release was reversible. Loxiglumide significantly inhibited amylase release in response to CCK-8, caerulein and gastrin-I. However, loxiglumide had no effect on amylase release stimulated by other receptor secretagogues (bombesin, carbamylcholine, secretion and vasoactive intestinal polypeptide) or by agents bypassing receptors (A23187 and TPA). These results indicate that loxiglumide acts as a potent, competitive and specific CCK antagonist on the pancreatic acini.     

Characterization of a new diphenylpyrazolidinone cholecystokinin antagonist in vitro isolated rat pancreatic acini

Summary The effects of a newly developed diphenylpyrazolidinone cholecystokinin (CCK) antagonist LY219,057 were examined in the isolated rat pancreatic acini and compared with those of devazepide (previously designated L364,718 or MK-329). LY219,057 caused a concentration-dependent inhibition of 100 pM CCK octapeptide (CCK-8)-stimulated amylase release, with a half-maximal inhibition (ID₅₀) at 287.5±28.4 nM and was 200 times less potent than devazepide (ID₅₀=1.4±0.2 nM). The antagonism was competitive in nature because LY219,057 caused a parallel rightward shift of the dose-response curve for CCK-8-stimulated amylase secretion without altering the maximal increase. LY219,057 significantly inhibited amylase release in response to CCK-8 and cerulein but had no effect on amylase release stimulated by other receptor secretagogs or agent bypassing receptors. LY219,057, whether added at the beginning or 20 min after the CCK-8 stimulation, inhibited amylase release. This compound caused a residual inhibition of the action of CCK-8. Acini preincubated with 1.0 μM LY219,057 for 30 min at 37°C were threefold less sensitive to CCK-8 than the acini preincubated without LY219,057. Thee results indicate that LY219,057 acts as a potent, competitive, and specific CCK receptor antagonist of the action of CCK on the exocrine pancreas.     

Loxiglumide

D,l-4-(3,4-dichlorobenzoylamino)-5-(N-3-methoxypropyl-pentylamino)-5-oxopentanoic acid (CR 1505; loxiglumide) is a newly developed analog of proglumide. We examined the inhibitory effects of loxiglumide on pancreatic exocrine function in the isolated pancreatic acini and the isolated perfused pancreata of rats. Loxiglumide inhibited cholecystokinin octapeptide (CCK-8)-stimulated amylase release and, similarly, binding of[ ¹²⁵ I]CCK-8 to isolated rat pancreatic acini. Loxiglumide was about 3000 times more potent than the reference substance proglumide, but was about 1000 times less potent than L-364,718, another new CCK antagonist having benzodiazepine ring, in inhibiting CCK-8-stimulated amylase release. The inhibitory effect of loxiglumide displayed competitive kinetics and was specific for CCK in that the effects of other receptor secretagogues or agents bypassing receptors were not altered. The inhibitory effect of loxiglumide was fully reversible in isolated acini. However, the pancreata perfused with 10 M loxiglumide for 20 min did not respond to CCK-8 for more than 20 min even after the removal of loxiglumide infusion. In contrast, an immediate increase in pancreatic exocrine secretion was observed after proglumide removal. Loxiglumide appeared to be bound to the receptors on acinar cells in a slowly dissociating state. These results indicate that loxiglumide acts as a potent, competitive, and specific CCK antagonist on the exocrine pancreas and, because of its prolonged inhibitory action, may be useful as a therapeutic agent in pancreatic disease.This work was supported in part by a grant from the Japanese Ministry of Health and Welfare (Intractable Diseases of the Pancreas).     

CCK receptor antagonist loxiglumide alters uptake of CCK in perfused liver and pancreatic acini of the rat

The aim of the present study was to analyze the effect of the specific cholecystokinin (CCK) receptor antagonist loxiglumide on hepatic and pancreatic processing of CCK-8 and the CCK analogue cerulein. Rat liver perfusion was performed in a non-recirculating system. CCK concentrations were measured by radioimmunoassay in perfusates from the inflow cannula (portal vein) and the outflow cannula (hepatic vein). In rat pancreatic acini, the effect of loxiglumide on internalization and surface-binding of radiolabelled CCK-8 was determined. Cerulein (20 nM, 2 nM) was extracted in a single pass through the liver by 29.7 and 25.4%, respectively. The hepatic uptake of CCK-8 (50 pM, 2 nM) was more than 90 and 89.9%, respectively. Loxiglumide drastically inhibited hepatic extraction of both peptides and reduced internalization of 125I-CCK-8 in pancreatic acini dose dependently by 39-93%. These results demonstrate that the potent CCK receptor antagonist loxiglumide significantly decreased CCK uptake by the liver and pancreas.     

Effects of L-364,718 on pancreatic exocrine and endocrine secretion in the rat.

We examined the inhibitory effect of L-364,718, a nonpeptide cholecystokinin (CCK) antagonist, on CCK stimulation of pancreatic exocrine and endocrine secretion in both the isolated pancreatic acini and the isolated perfused pancreata of rats. In the isolated acini, L-364,718 inhibited CCK octapeptide (CCK-8)-stimulated amylase release and binding of 125I-CCK-8 in a dose-dependent manner without appreciable effects on the basal amylase secretion. L-364,718 also inhibited amylase release in response to caerulein and gastrin I, but had no effect on amylase release stimulated by other secretagogues or by agents bypassing receptors. Similarly, binding of N-methylscopolamine to pancreatic acini was not inhibited by L-364,718. In the isolated perfused pancreata, L-364,718 inhibited CCK-8-stimulated pancreatic exocrine secretion and insulin release. The inhibitory effects of L-364,718 were more potent for insulin release than for exocrine secretion and persisted even after the removal of L-364,718 infusion. These results clearly demonstrate that L-364,718 is a specific, potent, and prolonged antagonist of CCK's stimulatory actions on pancreatic acinar and B cells.     

Effect of L364718, a new CCK antagonist, on amylase secretion in isolated rat pancreatic acini

We examined the effect of L364718, a new cholecystokinin (CCK) receptor antagonist, on amylase release stimulated by CCK or different secretagogues in isolated rat pancreatic acini. L364718 caused a parallel rightward shift of the dose-response curve of CCK8. Schild plots showed a slope of 1.05 +/- 0.15 and a pA2 value of 10.01 +/- 0.31. L364718 inhibited maximally stimulated amylase release by CCK in a dose-dependent manner, with half maximal inhibition (ID50) at 1.7 nM and complete inhibition at 30 nM. Asperlicin, a prototype compound of L364718, also caused dose-dependent inhibition, but L364718 was approximately 400 times more potent than asperlicin (ID50 = 761 nM). L364718 significantly inhibited amylase release in response to CCK33 and CCK8 but had no effect on amylase release stimulated by other receptor secretagogues or agents by passing receptors. The results indicate that L364718 acts as an extremely potent, competitive, and specific antagonist of CCK's action on pancreatic acini.     

Bioactivity of synthetic human cholecystokinin (CCK)-33 in vitro and in vivo.

The relative potencies of synthetic human cholecystokinin (h-CCK)-33, porcine CCK-33 (p-CCK-33) and CCK-8 were examined by measuring pancreatic secretion in the conscious rat (in vivo) and amylase release from rat pancreatic acini using a perifusion study (in vitro). The increments of protein output during an 1-hr infusion of 100 pmol/kg/hr of h-CCK-33, p-CCK-33 and CCK-8 were 27.0 +/- 2.9 mg/hr (M +/- SE), 19.3 +/- 2.8 and 14.0 +/- 1.8 mg/hr, respectively. H-CCK-33 and p-CCK-33 showed significantly higher responses of protein output than CCK-8 in a same molar ratio, in vivo. In vitro, the stimulation with 10(-10) M h-CCK-33, p-CCK-33 and CCK-8 led to a similar biphasic amylase release in a perifusion study. Twenty-five microM CR-1409, an antagonist for CCK receptor, completely inhibited the 10(-10) M h-CCK-33-stimulated amylase release. Although it was found that h-CCK-33 and p-CCK-33 were more potent than CCK-8 in vivo, 10(-10) M CCK-8, h-CCK-33 and p-CCK-33 were equipotent on rat pancreatic acini in vitro. It is suggested that the discrepancy in potencies of the large molecular form and small molecular form of CCK in vivo and in vitro may be attributed to the delay of degradation of the large molecular form of CCK in vivo.     

Effects of a new proglumide analogue CR 1392 on pancreatic exocrine secretion in the rat

The effects of proglumide analogue. CR 1392, on pancreatic exocrine secretion were studied in the isolated pancreatic acini and the isolated perfused pancreata of rats. In the isolated acini, CR 1392 caused a parallel rightward shift of the dose-response curve for amylase secretion stimulated by cholecystokinin octapeptide (CCK-8). CR 1392 inhibited maximally stimulated amylase release by CCK-8 (100 pM) in a concentration-dependent manner, with a half maximal inhibition (ID50) at 8.0 +/- 0.6 microM. CR 1409, another proglumide analogue, also caused a concentration-dependent inhibition (ID50: 3.2 +/- 0.4 microM). Although CR 1409 was about 2.5-fold more potent than CR 1392 in inhibiting the stimulated amylase release, 1 mM CR 1409 caused 107.4 +/- 0.9% increase in amylase release, suggesting acinar cell damage. CR 1392 (1 mM) also caused 19.9 +/- 2.3% increase in amylase release, but was less toxic than CR 1409. The antagonism produced by CR 1392 was selective for CCK and had no effect on amylase release stimulated by other receptor secretagogues or by agents bypassing receptors. CR 1392 added 20 min after the CCK-8 stimulation rapidly abolished pancreatic exocrine secretion in both isolated acini and isolated perfused pancreas. Although the inhibitory effect of CR 1392 was fully reversible in the isolated acini, the pancreata perfused with 100 microM CR 1392 for 20 min did not respond to the subsequent stimulation with CCK-8 for more than 20 min. These results indicate that CR 1392 is a potent, competitive, specific and long acting antagonist of CCK in rat pancreas.     

Effect of intrinsic CCK and CCK antagonist on pancreatic growth and pancreatic enzyme secretion in pancreaticobiliary diversion rats

When pancreaticobiliary diversion (PBD) surgery was performed in rats, plasma CCK level increased, the pancreas grew mainly by proliferation, and pancreatic trypsinogen showed a remarkable increase, although amylase and lipase synthesis were somewhat decreased. The sensitivity of amylase release against CCK-8 in the pancreatic acini decreased when plasma CCK level was high. These changes in pancreatic growth and pancreatic enzyme secretion caused by PBD were completely inhibited by the CCK-receptor antagonist loxiglumide. From these results, intrinsic CCK was considered to play an important role in both pancreatic enzyme synthesis and proliferation     

Interaction of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus--secretion coupling in pancreatic acinar cells

BACKGROUND: The effect of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus-secretion coupling was investigated in isolated rat pancreatic acini. METHODS AND RESULTS: Ortho-vanadate (10(3)M) reduced cholecystokinin (CCK)-8 (10(10) M)-stimulated amylase release by 40% (IC50 = 5 x 10(4) M). In contrast, preincubation with 10(3) M ortho-vanadate increased secretin (5 x 10(9) M) and vasoactive intestinal peptide (VIP) (10(7) M)-induced amylase release by 65% and 80% (IC50= 3 x 10(-4) M), respectively. 8-Bromo-cyclic adenosine-5-monophosphate (cAMP) (10(-4) M) and phorbol ester (10(-5) M)-induced secretion was increased by 60% and 50%, respectively, whereas thapsigargin-induced amylase release was not affected. Ortho-vanadate did not affect CCK-8 binding or VIP-induced cAMP synthesis in isolated acini. In contrast, preincubation with 10(-4) M ortho-vanadate resulted in a significant reduction of CCK-8-induced intracellular calcium release. In streptolysin-O-permeabilized acini, ortho-vanadate reduced calcium-induced amlyase secretion by 50%. CONCLUSIONS: The present data provide indirect evidence of a differential involvement of protein tyrosine dephosphorylation in both cAMP- and IP3/Ca(2+)-mediated pancreatic secretion. The differential effects of ortho-vanadate on cAMP- versus calcium-mediated secretion correspond to the results obtained with receptor-independent intracellularly acting secretagogues. Further experiments must define the tyrosine phosphatases involved in both signal-transduction pathways.     

Studies on human pancreatic acini: action of secretagogues on amylase release and cellular cyclic AMP accumulation

A technique for preparing a suspension of dispersed functional acini from human pancreas has been developed. The changes in pancreatic enzyme secretion and accumulation of cellular cyclic AMP caused by various secretagogues have been studied. Ca2+-mobilizing agents stimulated amylase release from human pancreatic acini. The relative potencies with which secretagogues increased amylase release were as follows: gastrin-releasing peptide's potency (Ec50, 0.1 +/- 0.01 nM) was greater than bombesin 14's (Ec50, 0.2 +/- 0.01 nM), which was greater than litorin's (Ec50, 0.6 +/- 0.18 nM), which was greater than bombesin 9's (Ec50, 6 +/- 0.1 nM). For CCK-peptides, the relative potencies were as follows: CCK-39's potency (Ec50, 0.28 +/- 0.15 microM) was equal to cerulein's (Ec50, 0.3 +/- 0.07 microM). Both of these potencies were greater than CCK-8's (Ec50, 1.6 +/- 0.1 microM), which was greater than that of CCK-4. Carbamyl choline was poorly potent (Ec50 greater than 1 mM). The 12-O-tetradecanoylphorbol-13-acetate (TPA) was active from 0.1 nM to 0.1 microM. Neither secretin nor VIP increased amylase release from human pancreatic acini but they did cause an accumulation of cellular cyclic AMP, secretin (Ec50, 0.5 +/- 0.2 nM) being more potent than VIP.     

Effect of ethanol on pancreatic exocrine secretion in rats.

The effect of ethanol on pancreatic exocrine secretion was studied in isolated rat pancreatic acini. Ethanol caused a dose-dependent stimulation of amylase release, and a twofold increase of amylase release was observed with 600 mM ethanol. Ethanol inhibited cholecystokinin octapeptide (CCK-8)- and carbamylcholine-stimulated amylase release and similarly inhibited binding of [125I]CCK-8 and [N-methyl-3H]scopolamine to isolated rat pancreatic acini in a dose-dependent manner. The inhibitory effect of ethanol was fully reversible with respect to CCK-8-induced amylase release. On the other hand, ethanol potentiated secretin- and vasoactive intestinal peptide (VIP)-stimulated amylase release. Ethanol induced a small but significant increase in Ca2+ efflux, whereas CCK-8 induced an immediate and large increase, but ethanol significantly inhibited CCK-8-stimulated Ca2+ efflux. The present study clearly demonstrates the dual effects of ethanol on pancreatic exocrine function: stimulation and inhibition. We suggest that mobilization of intracellular Ca2+ may be involved in the mechanism of ethanol's action on isolated rat pancreatic acini.     

Continuous infusion of cholecystokinin leads to down-regulation of the cholecystokinin-A receptor in the rat pancreas

BACKGROUND: Infusion of sulphated cholecystokinin-8 (CCK-8S) in rats transiently increased the proliferation of pancreatic acinar cells, whereas the CCK-A receptor antagonist devazepide decreased such proliferation. This effect ceased after 3 days. CCK-8S or devazepide injected twice daily induced a persistent effect on the cell proliferation involving the major cells of the exocrine pancreas. The aim of this study was to examine the effect of continuous infusion of CCK-8S and devazepide on CCK-A receptor gene expression. METHODS: Male Sprague-Dawley rats received subcutaneous continuous infusion of 5 microg/kg/h CCK-8S, 200 microg/kg/h devazepide, or 1% bovine serum albumin (BSA) by means of osmotic minipumps. The rats were killed after 4 days; I h before being killed they received 5-bromo-2-deoxyuridine (BrdU) intraperitoneally. Plasma was collected for analysis of CCK. The pancreas was dissected, and indirect immunofluorescence for BrdU and CCK-A receptor was performed. In situ hybridization to CCK-A receptor mRNA was performed for examination and semiquantification of receptor gene expression. RESULTS: Continuous infusion of CCK-8S led to a sixfold increase in plasma CCK and a 40% increase in pancreatic weight. Devazepide did not affect the CCK level but decreased the pancreatic weight by 24% compared with BSA-infused rats. The BrdU labeling indicated that CCK-8S had no effect on cell proliferation. Immunofluorescence for the CCK-A receptor showed a decreased labeling intensity after CCK-8S infusion. The mean optical density of in situ hybridization labeling of the sections from CCK-8S-treated rats was decreased to 37% +/- 3% of that in controls. Devazepide did not affect the CCK-A receptor gene expression. CONCLUSIONS: Continuous stimulation of the CCK-A receptor led to a downregulation of the receptor gene expression in pancreatic acinar cells and decreased labeling of the receptor at immunohistochemistry. The results suggest that down-regulation of the receptor is a protective mechanism against overstimulation.     

The changes in the rat parotid glands following total parenteral nutrition and pancreatico-biliary diversion are not mediated by cholecystokinin

Summary Conclusions The results of the present study suggest that the pancreas and parotid glands both respond with hypoplasia during absence of food in the digestive tract and with hyperplasia following pancreactico-biliary diversion (PBD). Factors other than cholecystokinin (CCK) are, however, involved in the effects on the parotid glands, since infusion of CCK-8S and devazepide was without influence. Background and Aim Total parenteral nutrition (TPN) causes reduced pancreatic weight, whereas PBD evokes hyperCCKemia and enlargement of the rat pancreas. The pancreas and parotid glands have in part similar morphology and function. Therefore, we studied the possible presence of alterations also in the parotid glands during TPN, after PBD and during infusion of sulfated cholecystokinin (CCK-8S) and the CCK-A receptor antagonist devazepide, respectively. Materials and Results Rats either received TPN for 7 d, or were kept under otherwise identical conditions with free access to food and water. TPN markedly reduced both pancreatic and parotid wet weight and thereby also the protein and amylase contents, and pancreatic DNA content was decreased. There was a significant correlation between the pancreas and parotid glands when comparing these parameters. The concentration of plasma CCK was unaffected by TPN. PBD caused a sevenfold increase in plasma CCK and a threefold increase in the pancreatic weight compared to control rats 28 d after the operation. The protein and DNA contents in the pancreas were found to be increased. The parotid glands increased twofold in weight, but their protein and amylase contents were not affected. There was a significant correlation between the pancreas and parotid glands when comparing weight, and protein and amylase concentrations. Infusion of CCK-8S during 28 d caused a marked increase in pancreatic wet wt and protein and DNA contents. The CCK-A receptor antagonist devazepide induced a reduction in protein and DNA contents in the pancreas. The parotid glands were not affected by either treatment. No effect on the labeling index of serous and ductal cells of the parotid gland was seen at 36 h, 3, 7, and 28 d of infusion with CCK-8S or devazepide.     

Characterization of cholecystokinin receptors in gastric chief cells--effect of CCK analogs and CCK receptor antagonists on chief cells

In isolated guinea pig gastric chief cells, gastrin-I and cholecystokinin-hexapeptide (CCK-4) stimulated pepsinogen release. However, the efficacies of these two peptide were 51% of that observed with CCK-octapeptide (CCK8). CR1409 and L-364718, both of which are new CCK receptor antagonists in pancreatic acinar cells, also inhibited 10(-8) M CCK8-stimulated pepsinogen release with a half-maximal inhibitory concentration (IC50) observed at 3 x 10(-9) M, respectively. The dose response curve to CCK8 for pepsinogen release shifted to the right in the presence of CR1409 or L-364718. The IC50 of these two antagonists for the CCK8-stimulated increase in cytosolic Ca2+ concentration monitored by Fura-2 were equal to those for CCK8-stimulated pepsinogen release. By contrast, the IC50 of dibutyryl cyclic GMP, a well-known CCK receptor antagonist, for CCK8-stimulated pepsinogen release was less than that for CCK8-stimulated increase in cytosolic Ca2+ concentration. Results suggested that CCK receptors in gastric chief cells are unique and may be different from CCK receptors in other tissues previously reported.     

Proglumide analogues CR 1409 and CR 1392 inhibit cholecystokinin-stimulated insulin release more potently than exocrine secretion from the isolated perfused rat pancreas

The effects of proglumide-related cholecystokinin (CCK) receptor antagonists CR 1409 and CR 1392 on CCK-octapeptide (CCK-8)-stimulated immunoreactive insulin (IRI) release and exocrine secretion were examined simultaneously in the isolated perfused rat pancreas. The CR 1409, at concentrations of 10-100 nM, significantly inhibited CCK-8 (100 pM) stimulation on IRI release but failed to inhibit the stimulatory effect of CCK-8 on both pancreatic juice flow and protein secretion. Increasing concentrations of CR 1409 inhibited both CCK-8-stimulated IRI release and exocrine secretion. Half-maximal inhibition was observed with approximately 2 nM for IRI release and 1 microM for protein secretion. When a higher dose (1 nM) of CCK-8 was used, the inhibitory effect of 10 nM CR 1409 on CCK-8-stimulated IRI release was abolished, whereas 10 microM CR 1409 retained significant inhibitory effect. Furthermore, 1 microM carbachol-induced IRI release was not altered by the addition of 10 microM CR 1409. The CR 1392 also had an inhibitory effect on both CCK-8-stimulated IRI release and exocrine secretion. The concentration of CR 1392 that caused half-maximal inhibition of CCK-8-stimulated IRI release was 300 times lower than that of exocrine secretion. In addition, 1 microM carbachol-stimulated IRI release was not altered by 100 microM CR 1392. Thus, the inhibitory effects of CR 1409 and CR 1392 on IRI release were mediated through the interaction at the CCK receptor and were more potent than those on juice and protein secretion. This study suggests, therefore, that CCK receptors on B cells might be different from those on acinar cells in terms of their relative affinities for antagonists.     

Cyclic cholecystokinin analogues with high selectivity for central receptors.

Taking as a model the N-terminal folding of the cholecystokinin tyrosine-sulfated octapeptide [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] deduced from conformational studies, two cyclic cholecystokinin (CCK) analogues were synthesized by conventional peptide synthesis: Boc-D-Asp-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 [compound I (Ahx, 2-aminohexanoic acid)] and Boc-gamma-D-Glu-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 (compound II). The binding characteristics of these peptides were investigated on brain cortex membranes and pancreatic acini of guinea pig. Compounds I and II were competitive inhibitors of [3H]Boc[Ahx28,31]CCK-(27-33) binding to central CCK receptors and showed a high degree of selectivity for these binding sites (compound I: Ki for pancreas/Ki for brain, 179; compound II: Ki for pancreas/Ki for brain, 1979). This high selectivity was associated with a high affinity for central CCK receptors (compound I: Ki, 5.1 nM; compound II: Ki, 0.49 nM). Similar affinities and selectivities were found when 125I Bolton-Hunter-labeled CCK-8 was used as a ligand. Moreover, these compounds were only weakly active in the stimulation of amylase release from guinea pig pancreatic acini (EC50 greater than 10,000 nM) and were unable to induce contractions in the guinea pig ileum (to 10(-6) M). The two cyclic CCK analogues, therefore, appear to be synthetic ligands exhibiting both high affinity and high selectivity for central CCK binding sites. These compounds could help clarify the respective role of central and peripheral receptors for various CCK-8-induced pharmacological effects.     

A novel micromethod for pancreatic acinar secretion. Time course of CCK stimulation and its inhibition by L-364,718.

Dispersed acini have proven to be particularly valuable in the study of pancreatic enzyme secretion. Complex time course studies or experiments requiring large numbers of replicates have proven difficult, however, with currently available techniques. Using a custom-designed incubation chamber, a miniaturized incubation method has been devised, which allows for continuous oxygenation of acini in 96-well microtiter plates and rapid separation of medium from acini by vacuum filtration. The filtrates from individual wells are collected into the wells of a second microtiter plate for pancreatic enzyme measurement. Using the above method, the dose response and time course of cholecystokinin (CCK-8)-stimulated amylase secretion was investigated. During a 1-h incubation, unstimulated amylase secretion was 4.1 +/- 0.3% of total acini content. Response to CCK was very sensitive, being detected at 10(-13) M (p less than 0.05), half-maximal at 10(-11) M CCK (14.0 +/- 0.6%, p less than 0.001) and maximal at 10(-9) M CCK (24.8 +/- 1.0%, p less than 0.001). In the time course experiments, an increase in amylase secretion was detected by 2.5 min and continued to increase steadily to a plateau at 40 min, with both submaximal (10(-11) M) and maximal (10(-9) M) CCK concentrations. The potent and specific CCK-receptor antagonist, L-364,718, caused a dose-dependent decrease in CCK-stimulated amylase secretion, with a half-maximal effect at 10(-10) M. The receptor antagonist, L-364,718, at 10(-8) M completely abolished CCK-stimulated amylase secretion. This microtechnique provides a simple, reliable, and reproducible method for the study of dispersed pancreatic acini.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dibutyltin dichloride modifies amylase release from isolated rat pancreatic acini

INTRODUCTION: Dibutyltin dichloride (DBTC) is widely used as a stabilizer for polyvinylchloride plastics and is of particular toxicologic interest. AIM: To examine the effects of DBTC on pancreatic exocrine function in isolated rat pancreatic acini. METHODOLOGY: Isolated rat pancreatic acini were incubated with various secretagogues in the presence or absence of DBTC. We investigated the effects of DBTC on amylase release, receptor binding, and protein kinase C (PKC) enzyme activity. RESULTS: DBTC reduced cholecystokinin octapeptide (CCK-8)-stimulated and carbamylcholine-stimulated amylase release and the binding of [(125)I]CCK-8 to isolated rat pancreatic acini. Conversely, DBTC potentiated secretin-stimulated amylase release, although it slightly inhibited [(125)I]secretin binding to its receptors. In addition, DBTC potentiated amylase release stimulated by vasoactive intestinal peptide, 8-bromoadenosine 3', 5'-monophosphate (8Br-cAMP) or calcium ionophore A23187, whereas it had no influence on amylase release stimulated by 12-O-tetradecanoylphorbol 13-acetate. The protein kinase C (PKC) inhibitor calphostin C abolished the DBTC-induced potentiation of amylase release stimulated by 8Br-cAMP or A23187. Moreover, DBTC caused a significant translocation of PKC enzyme activity from cytosol to membrane fraction. CONCLUSIONS: These results indicate that DBTC reduces CCK-8- and carbamylcholine-stimulated amylase release by inhibiting their receptor bindings to pancreatic acini, whereas it potentiates cAMP-mediated amylase release by activating PKC in isolated rat pancreatic acini.     

Carbachol does not down-regulate substance P receptors in pancreatic acini.

In a previous study, we found that first incubating guinea pig pancreatic acini with carbachol caused desensitization of the enzyme secretory response to cholecystokinin-octapeptide (CCK-8), bombesin, and carbachol but not that to substance P. This carbachol-induced desensitization could be accounted for by carbachol-induced down-regulation of receptors for CCK-8, bombesin, and carbachol. Although carbachol did not desensitize the enzyme secretory response to substance P, an effect of carbachol on substance P receptors was not examined. In the present study, in dispersed acini from guinea pig pancreas, substance P caused a twofold increase in amylase secretion. Stimulation was half-maximal at 0.7 nM and was maximal at 10 nM. Analysis of the ability of substance P to inhibit binding of 125I-substance P to substance P receptors indicated that acini possess a single class of receptors for substance P (Kd = 0.8 +/- 0.1 nM; Bmax = 1,037 +/- 145 fmol/mg of DNA). There was a close correlation between the relative potency with which substance P stimulated amylase secretion (0.7 nM) and the potency for inhibiting binding of 125I-substance P (Kd = 0.8 nM). First incubating pancreatic acini with carbachol did not alter either substance P-stimulated enzyme secretion or binding of 125I-substance P to substance P receptors, whereas in the same experiments, carbachol reduced binding of 125I-CCK-8 to cholecystokinin receptors by 50% and decreased in CCK-8-stimulated enzyme secretion by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)