HCO 3_ secretion was investigated in interlobular duct segments isolated from guinea-pig pancreas using a semi-quantitative fluorometric method. Secretagogue-induced decreases in intracellular pH, following blockade of basolateral HCO 3 _ uptake with a combination of amiloride and DIDS, were measured using the pH-sensitive fluoroprobe BCECF. Apparent secretory HCO 3 _ fluxes were calculated from the initial rate of intracellular acidification.2. In the presence of HCO 3 _ , stimulation with secretin (10 nM) or forskolin (5 µM) more than doubled the rate of intracellular acidification. This effect was abolished in the absence of HCO 3 _ . It was also abolished in the presence of HCO 3 _ when DIDS and NPPB were applied to the luminal membrane by microperfusion. We therefore conclude that the increase in acidification rate is a useful index of secretagogue-induced HCO 3 _ secretion across the luminal membrane.3. Secretin, cholecystokinin (CCK) and bombesin each stimulated HCO 3 _ secretion in a dosedependent fashion. They evoked comparable maximal responses at about 10 nM and the EC 50 values were 0.5 nM for secretin, 0.2 nM for CCK and 30 pM for bombesin. Acetylcholine (ACh) was also effective, with a maximum effect at 10 µM.4. The stimulatory effect of CCK was blocked completely by the CCK 1 receptor antagonist devazepide but not by the CCK 2 receptor antagonist L365,260. The CCK analogue JMV-180 (Boc-Tyr(SO 3 H)-Nle-Gly-Trp-Nle-Asp-phenylethyl ester), which is an agonist of the highaffinity CCK 1 receptor but an antagonist of the low-affinity receptor, also stimulated HCO 3 _ secretion but with a smaller maximal effect than CCK. JMV-180 partially inhibited the response to a high concentration of CCK but not to a lower concentration, suggesting that both high-and low-affinity states of the CCK 1 receptor evoke HCO 3 _ secretion.5. The stimulatory effect of bombesin was blocked completely by the gastrin-releasing peptide (GRP) receptor antagonist D-Phe 6 -bombesin(6-13)-methyl ester (BME) but not by the neuromedin B (NMB) receptor antagonist D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH 2 (BIM-23127).6. Secretagogue-evoked fluid secretion was also examined using video microscopy to measure the rate of swelling of ducts whose ends had sealed during overnight culture. Secretin, CCK, bombesin and ACh all evoked fluid secretion with maximal rates of approximately 0.6 nl min _1 mm _2, and with concentration dependences similar to those obtained for HCO 3 _ secretion.7. We conclude that CCK, bombesin and ACh stimulate the secretion of a HCO 3 _ -rich fluid by direct actions on the interlobular ducts of the guinea-pig pancreas and that these responses are mediated by CCK 1 receptors, GRP receptors and muscarinic cholinoceptors, respectively.
We have raised four region-specific antisera in rabbits against rat prosecretin or secretin using synthetic peptides such as prosecretin(]--41), prosecretin(Z-]]), prosecretin(98~]13) and secretin and developed three region-specific radioimmunoassays (RIAs) for prosecretin(]-41), prosecrtin(1-11) and secretin. The results showed clearly the presence of prosecretin-like peptides in rat gastrointestine, whereas only a trace of prosecretin-like IR were recognized, if any, in the brain. In the Wistar male rat gastrointestine, the highest concentration of both prosecretin(1~41)-like and secretin-like immunoreactivity (IR) were detected in the ileum rather than in the duodenum. It is noteworthy that serial section of rat deodenum revealed the positive staining of prosecretin(98--113)-like IR in the cells identical to those stained with both anti-prosecretin(l-4]) serum and anti-secretin serum. These result, clearly indicate the presence of the C-terminal fragments of prosecretin as the processing products. In contrast, anti-prosecretin(]-1]) serum did not recognize the immunoreactivity in any ofthe cells in the brain and gastrointestine.
In the present study, we examined the effects of the C-terminal fragments and analogues of rat and porcine galanin on 13.9 mM glucose-induced insulin release from the isolated perfused rat pancreas. The results revealed clearly that [His23, Tyrzé]-rat galanin( I5-29) and [His23, Leu29]-rat galanin(]5-29) at lO'8 M as well as porcine galanin(]5-29) exhibits insulinotropic activity on glucose-induced insulin release from the isolated perfused pancreas, whereas rat galanin(l5-29) exhibited little effect on glucose-induced insulin release. The secondary structural analysis of the I5-29 sequence of rat and porcine galanins suggests that the conformation of the 15-29 sequences may be of importance for the insulinotropic activity of the C-terminal fragments.Galanin has been known to cause a significant suppression of glucose-induced insulin release from the isolated perfused rat pancreas (20). Subsequent structure-function studies using synthetic porcine galanin fragments indicate that galanin(2-29) exhibited a complete loss of the inhibitory action, and further shortening of the peptide chain gave peptides having insulinotropic effects, and the maximum potency occurred at the 15-29 sequence (16). This is the first demonstration of a neuropeptide and its fragments that possess opposite effect in action on a single biological system such as insulin release.In addition, the modified peptides, [D-Trpz} porcine galanin and [Pheg]-porcine galanin, did not cause a significant change in the effect on glucose-induced insulin release, while [Ala2]-porcine galanin was found to exhibit a complete loss of the suppressing effect on insulin release, similar to that seen with shorter porcine galanin fragments lacking the N-terminal amino acid sequence.Based on the secondary structures of the analogues and fragments as predicted by the ChouFasman method (3, 4), a high incidence offl-sheet structure in the a1ni11o terminal region of galanin was indicated to be essential for the inhibitory effect of galanin on glucose-induced insulin release. Both galanin(l-15)-OH and galanin(]-15)-ol showed little effect on glucose-induced i
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