Naturally Occurring Variants of the Dysglycemic Peptide Pancreastatin: DIFFERENTIAL POTENCIES FOR MULTIPLE CELLULAR FUNCTIONS AND STRUCTURE-FUNCTION CORRELATION*

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca²⁺ levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by ∼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in ∼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.     

Distribution and partial characterisation of immunoreactivity to the putative C-terminus of rat pancreastatin

The presumptive C-terminal nonapeptide of rat pancreastatin was synthesised based upon the sequence of rat chromogranin A (CGA) analogous to that of porcine pancreastatin as contained within porcine CGA. Antisera were produced which were used to determine the qualitative and quantitative distribution of pancreastatin-like immunoreactivity in rat tissues by immunocytochemistry and radioimmunoassay respectively. Pancreastatin-like immunoreactivity was most abundant in pituitary, adrenal, gastric corpus and thyroid with considerably lower levels detected in the remainder of the gastroentero-pancreatic system and brain. Immunoreactivity was localised exclusively in endocrine cells and the relative abundance of immunoreactive cells paralleled the levels obtained radioimmunometrically. Chromatographic characterisation of pancreastatin-like immunoreactivity revealed molecular heterogeneity. Immunoreactive peptides of similar size to synthetic rat pancreastatin were present in gastrointestinal tissues and thyroid. These data indicate a tissue specific processing of CGA in the rat.     

Distribution and coexistence of chromogranin A-, serotonin-and pancreastatin-like immunoreactivity in endocrine-like cells of the human anal canal

Summary The comparative distribution and coexistence of chromogranin A (CGA)-, serotonin (5-hydroxytryptamine; 5-HT)- and pancreastatin (PST)-like immunoreactivity in endocrine-like cells of the human anal canal was investigated by light-microscopic immunocytochemistry. The largest population of colorectal endocrine-like cells consisted of CGA-immunoreactive (ir) cells, followed by the 5-HT-ir and PST-ir cell population. In the anal transitional zone (ATZ), CGA-and 5-HT-immunoreactivity was equally distributed; ir-PST was confined to a smaller endocrine-like cell population. In the squamous zone and the perianal skin, Merkel cells in the basal layer of the epidermis and hair follicles exhibited ir-CGA and ir-PST but no ir-5-HT. Double immunofluorescence on identical sections revealed distinct coexistence patterns. In the colorectal zone, about 2/3 of the CGA-ir endocrine-like cells also stained for 5-HT, whereas in the ATZ epithelium, CGA- and 5-HT-immunoreactivity completely overlapped. No 5-HT-immunoreactivity could be detected in CGA-ir Merkel cells of the squamous zone of the anal canal and the perianal skin. PST-immunoreactivity was present in about 1/3 of the CGA-ir colorectal and anal transitional endocrine-like cells and in about 1/4 of the Merkel-cell population staining for CGA. These chemically heterogeneous phenotypes of the anal endocrine-like and Merkel cells may reflect a specific regulatory role of these cells in the various epithelial linings of the human anal canal and the perianal skin.     

Reserpine-Induced Processing of Chromogranin A in Cultured Bovine Adrenal Chromaffin Cells

The effect of reserpine on the processing of the secretory granule protein chromogranin A (CgA) in isolated bovine adrenal chromaffin cells was investigated using two radioimmunoassays employing site-specific antisera. The two antisera were directed against closely associated regions of the CgA molecule which would be exposed by specific processing: antiserum L331 was raised against the C-terminus of the regulatory peptide pancreastatin, and the second antiserum, L300, was raised against the synthetic peptide [Tyr0]CgA306-313 (YLSKEWEDA), a sequence that lies immediately C-terminal to pancreastatin and adjacent to a dibasic amino acid cleavage site. Chronic reserpine treatment of chromaffin cells produced a time- and dose-dependent increase in processing, as demonstrated by an increase in pancreastatin- and YLSKEWEDA-immunoreactivity (ir). The reserpine-induced rise in pancreastatin-ir was due predominantly to an increase in pancreastatin 1-47, whereas the rise in YLSKEWEDA-ir was due to increases in three polypeptides: a 51-kDa YLSKEWEDA-ir polypeptide, CgA297-313, and CgA248-313. The latter predominated. The action of reserpine on both pancreastatin- and YLSKEWEDA-ir was found to be largely inhibited by the protein synthesis inhibitor cycloheximide. The results show that treatment of isolated chromaffin cells with reserpine induces both the selective proteolytic processing and peptidyl-glycine amidation of CgA and its derived fragments. As reserpine has a similar effect on proenkephalin in chromaffin cells, the results suggest that reserpine induces a general increase in the activity of the processing enzymes, partially by an increase in protein synthesis.