Jean-Pierre Raufman

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.

Use of 125I-[Y39]exendin-4 to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig

We synthesized and iodinated an exendin-4 analogue, [Y39]exendin-4 (700 Ci/mmol), for use as a radioligand to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig. Binding of this bioactive radioligand was rapid, temperature-dependent and specific (not inhibited by other pancreatic or gastric secretagogues). Measurement of the ability of exendin-4 to inhibit the binding of 125I-[Y39]exendin-4 indicated the presence of two classes of receptors. Pancreatic acini had 12.5.10(10) binding sites/mg acinar protein of which 6% were high affinity (Kd = 0.5 nM) and 94% were low affinity (Kd = 0.1 microM). Chief cells had 3370 binding sites/cell of which 9% were high affinity (Kd = 0.3 nM) and 91% were low affinity (Kd = 0.2 microM). Washing with 0.2 M acetic acid (pH 2.5), 0.2 M glycine (pH 10.5), or trypsin (100 micrograms/ml) after 30 min incubation at 37 degrees C, indicated that 63 and 49% of radioligand was internalized in acini and chief cells, respectively. Truncated glucagon-like peptide-1 (tGLP-1), a mammalian peptide sharing 53% homology with exendin-4, inhibited radioligand binding at the same concentrations that altered secretion from acini and chief cells. Glucagon, GLP-1 and GLP-2 inhibited 125I-[Y39]exendin-4 binding only at concentrations > or = 100 nM. Exendin(9-39)NH2, a specific exendin-receptor antagonist, potently inhibited 125I-[Y39] exendin-4 binding (IC50 = 6.1 and 3.5 nM in acini and chief cells, respectively). In pancreatic acini and gastric chief cells from guinea pig, exendin-3, exendin-4 and tGLP-1 increase cellular cAMP and modulate enzyme secretion by interacting with high-affinity exendin receptors. 125I-[Y39] exendin-4 is a useful radioligand for studying exendin receptors.

Gastric chief cells: Receptors and signal-transduction mechanisms

Elucidation of receptors and mediators regulating gastric pepsinogen secretion has lagged behind understanding of the factors that control acid secretion. During the past decade, as a consequence of the development of in vitro models for studying the control of pepsinogen secretion at the cellular level, much information about chief cell receptors and signal-transduction mechanisms has been obtained, including the identification and characterization of receptors for secretin, vasoactive intestinal polypeptide, cholinergic agonists, gastrin, cholecystokinin, peptide YY, and cholera toxin. Moreover, these cell preparations have permitted secretagogue-induced changes in chief-cell calcium concentration, protein kinase C distribution, and phosphoinositide and cyclic nucleotide content to be measured and related to changes in pepsinogen secretion. This article reviews these advances, discusses areas of uncertainty and controversy, and indicates areas for future investigation.

PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas

Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N-terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38-induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion.

Increases in cellular calcium concentration stimulate pepsinogen secretion from dispersed chief cells

Intracellular calcium concentration ([Ca]i) and pepsinogen secretion from dispersed chief cells from guinea pig stomach were determined before and after stimulation with calcium ionophores. [Ca]i was measured using the fluorescent probe quin2. Basal [Ca]i was 105 +/- 4 nM. Pepsinogen secretion was measured with a new assay using 125I-albumin substrate. This assay is 1000-fold more sensitive than the widely-used spectrophotometric assay, technically easy to perform, rapid, and relatively inexpensive. The kinetics and stoichiometry of ionophore-induced changes in [Ca]i and pepsinogen secretion were similar. These data support a role for calcium as a cellular mediator of pepsinogen secretion.

Expression of Rab3D in dispersed chief cells from guinea pig stomach

Rab3 proteins are low molecular weight GTP-binding proteins that are expressed in neurons and other secretory cells. These proteins are localized to secretory vesicles and may play a role in regulated exocytosis. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have been identified. We examined the expression of Rab3 isoforms in dispersed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cytosolic and membrane fractions, but staining was more intense in membrane fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To determine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obtained by reverse transcription and subjected to PCR using degenerate primers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced amino acid sequence was identical to that of mouse Rab3D (amino acids 16-83). Moreover, Rab3D was the only isoform detected in chief cells by these methods. To identify rab3 transcripts, the guinea pig rab3D fragment obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in chief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These results indicate that Rab3D is expressed in gastric chief cells.

Calcineurin Mediates Calcium-induced Potentiation of Adenylyl Cyclase Activity in Dispersed Chief Cells from Guinea Pig Stomach FURTHER EVIDENCE FOR CROSS-TALK BETWEEN SIGNAL TRANSDUCTION PATHWAYS THAT REGULATE PEPSINOGEN SECRETION

In cholera toxin-treated gastric chief cells, incubation with a cholinergic agonist (carbamylcholine), a regulatory peptide (cholecystokinin), or a calcium ionophore (A23187) causes a dose- and time-dependent potentiation of cAMP levels. Because this augmented response is calcium/calmodulin-dependent, we hypothesized that it was mediated by calcineurin (protein phosphatase 2B). To test this hypothesis, we examined the actions of calcineurin inhibitors on secretagogue-induced potentiation of cAMP levels in guinea pig chief cells. Preincubation of cells with 0.1 μM FK-506 completely prevented carbachol-induced augmentation of cAMP levels and pepsinogen secretion from cholera toxin-treated cells. Cyclosporin-A, another calcineurin inhibitor, also prevented the augmented cAMP response. FK-506 and cyclosporin inhibited augmentation of cAMP levels following treatment with cholecystokinin(26-33) and A23187, but not the smaller increase in cAMP following treatment with a phorbol ester that activates protein kinase C. Hence, the actions of calcineurin inhibitors were limited to secretagogues that increase cellular calcium. Rapamycin, an agent that competes with FK-506 for the immunophilin, FK binding protein 12, does not inhibit calcineurin. In the present study, preincubation with rapamycin did not prevent carbachol-induced augmentation of cAMP levels in cholera toxin-treated chief cells. However, a molar excess of rapamycin reversed the inhibitory actions of FK-506. These experiments provide further evidence that the actions of FK-506 on cholera toxin-treated gastric chief cells are caused by its inhibitory actions on calcineurin. FK-506 also inhibited potentiation of cAMP levels when carbachol was added to cells that were preincubated with forskolin, an agent that directly activates adenylyl cyclase. We conclude that, in gastric chief cells, calcineurin mediates cross-talk between the calcium/calmodulin and adenylyl cyclase signaling pathways.

Gastrointestinal endoscopy in patients with acquired immune deficiency syndrome: an evaluation of current practices

To determine attitudes and practices associated with gastrointestinal endoscopy in patients with acquired immune deficiency syndrome (AIDS), we surveyed the chiefs of gastroenterology at 196 medical centers in the United States. The response rate was 61% overall and 100% from New York and San Francisco. Our results revealed a broad spectrum of perceived risk and precautions. Seventy-three percent of respondents believed that endoscopy places personnel at risk for contracting AIDS. At 35% of institutions endoscopic procedures are exclusively or frequently performed at the bedside of the patient with AIDS. At 46% of institutions procedures on patients with AIDS are performed with instruments used only in these patients. Endoscopic equipment used in patients with AIDS is gas sterilized at 85% of institutions. One third of respondents noted reluctance of personnel to participate in endoscopy for patients with AIDS. Reluctance was related to the respondents perception of risk and did not differ among nurses, fellows, attendings, and gastrointestinal assistants. These data indicate that concern regarding transmission of the AIDS virus during endoscopy has resulted in costly and inefficient endoscopic practices.

Cat gastrinoma and the sequence of cat gastrins

Following the curative resection of a pancreatic gastrinoma in a cat, gastrin peptides were purified from the tissue and sequenced. The sequence of cat gastrinoma G17 (18-34) confirms the previously published sequence. The sequence of cat G34 (1-34) is reported for the first time. The NH2-terminal portion of cat G34 differs from that of dog by having a Q (Gln) for L (Leu) at position 10 from the NH2-terminus.

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxins actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.