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Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

The mammalian Harderian glands are lipid-secreting glands. In an unstimulated condition, the glandular cells frequently exocytose the lipid materials; however, no intracellular calcium ion ([Ca2+]c) changes are detectable. Cholinergic (muscarinic) secretagogues induce secretory activity and increase of [Ca2+]c. A G-protein activator, sodium fluoride, enhances the secretory activity and increase of [Ca2+]c. Removal of extracellular calcium ions inhibits the secretion enhanced by cholinergic stimulation. Under pharmacologic stimulation, glandular cells may show an apocrine-like secretory pattern. Cholinergic stimulation also induces contraction of the myoepithelial cells covering glandular end pieces; however, the reduction in volume of glandular end pieces is not prominent. Catecholamines have no effect on the release of lipid materials. These results indicate the involvement of G-proteins linking with muscarinic receptors and Ca2+ dynamics (increase of [Ca2+]c and Ca2+ influx) in lipid secretion by glandular cells and in contraction of myoepithelial cells of mammalian Harderian glands. However, the increase of [Ca2+]c in Harderian glands was less when compared with other cells—for instance, those which secrete protein. © 1996 Wiley-Liss, Inc.

1. Procaine (0.03-10 mM) inhibited carbachol (CCh)-induced amylase release from rat isolated pancreatic acini in a competitive manner. Kinetic analysis of the relation between CCh concentrations and the amount of amylase released in the presence of various procaine concentrations indicated that procaine caused competitive inhibition with the affinity constant (pA2) value of 5.00 +/- 0.08. 2. Receptor binding assay confirmed that procaine (0.01-10 mM) competitively inhibited [N-methyl-3H]-scopolamine chloride ([3H]-NMS) binding to its receptor with binding affinity (pKi) of 4.63 +/- 0.10. 3. Procaine transformed CCh-evoked [Ca2+]i dynamics: the initial rise in [Ca2+]i followed by a gradual decay during continuous stimulation with 3 microM CCh was transformed by 0.3 mM procaine to the oscillatory [Ca2+]i dynamics, which resembled the response to 0.3 microM CCh in the absence of procaine. The initial phase of [Ca2+]i oscillation corresponded to the initial phase of CCh-induced amylase release in isolated perfused acini. 4. Procaine (0.3-3 mM) did not inhibit the secretory response to cholecystokinin octapeptide (CCK-8) in isolated incubated acini. A higher concentration of procaine (10 mM) caused weak but significant inhibition of the response to only limited concentrations of CCK-8, 30 and 100 pM. Procaine lower than 10 mM was ineffective on [125I]-BH-CCK-8 binding, although procaine (10 mM) caused weak but significant inhibition of the binding.

1. The effects of chlorobutanol, a widely used drug preservative, on exocrine response and intracellular Ca2+ dynamics were examined in isolated pancreatic acini of the rat. 2. Chlorobutanol (1 mg ml-1) markedly inhibited the secretory response to cholecystokinin octapeptide (CCK-8), carbamylcholine chloride (carbachol), or sodium fluoride, a direct G-protein activator. However, chlorobutanol itself induced a maximal release of amylase when the dose was increased to 4 mg ml-1. 3. An oscillatory fluctuation of cytoplasmic Ca2+ concentration, [Ca2+]c, induced by 5 pM CCK-8 or 0.3 microM carbachol was totally abolished in the presence of 1 mg ml-1 chlorobutanol. 4. A biphasic change in [Ca2+]c induced by 100 pM CCK-8, a rapid rise followed by a gradual decay, was transformed to an oscillatory fluctuation by the preservative. 5. Chlorobutanol inhibited 13 pM [125I]-CCK-8 or 0.5 nM [3H]-methylscopolamine chloride binding to the acinar cells in a dose-dependent manner. 6. These results indicate that chlorobutanol produces discernible pharmacological effects on the secretory response in rat pancreatic acinar cells through changes in the Ca2+ dynamics. Possible sites of action could be at a binding process of secretagogues to their receptors, at an activation process of a G-protein located in the plasma membrane, or at the processes following G-protein activation. However, the possibility that the preservative may distort the Ca(2+)-transport function of the plasma membrane or the membrane of intracellular organella, especially Ca(2+)-sequestering pools, cannot be excluded.

The tritriacontapeptide amide, [27-Tyr]-CCK-PZ, was synthesized by successive azide condensation of 4 peptide fragments, (1-5), (6-11), (12-16), and (17-33), followed by exposure to hydrogen fluoride to remove all protecting groups. Though the CCK-PZ activity of synthetic peptide was only 1/250 of the standard from the natural source, it was smoothly labeled with radioactive iodine. Chain length activity relationship of CCK-PZ was also discussed.