The Mechanism Of Calcium Regulating Insulin Secretion In Pancreatic β Cells

The activity of secretion is the basis for transmitters release from neurons, hormones secretion from endocrine cells and setting or endocytosis of functional membrane proteins, which are absolutely necessary in maintaining normal physiological function of organisms. For example, the absolute or relative deficiency of insulin secretion from pancreatic ? cells will result in diabetes. Intracellular Ca²⁺ acts as a second messenger that palys important role in regulating vesicles secretion. So, it is very meaningful for study the molecular mechanism on which intracellular Ca²⁺ signal regulates vesicles secretion. In this study, using membrane capacitance, microfluorometric measurement of intracellular free Ca²⁺ concentration, flash photolysis of caged Ca²⁺, FM1-43 staining and carbon fiber technique, we studied the exact mechanism of insulin secretion and the source of intracellular Ca²⁺ induced by extracellular ATP in rat pancreatic ? cells. Our results suggest extracellular ATP promptly triggers vigorous exocytosis under resting condition via localized Ca²⁺ release from secretory granules rather than ER. In addition, GIP1-39, a novel chain length form of GIP (gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide), has been purified from porcine intestine and demonstrated that is capable of stimulating insulin secretion and elevating of intracellular Ca²⁺ concentration from rat pancreatic βcells. Main results of the study are as follows: (1) Extracelluar ATP could trigger vigorous exocytosis under resting condition via intracellular Ca²⁺ store release in rat pancreatic ? cells. We performed intracellular dialysis experiments with pipette solutions containing EGTA or BAPTA to chelate intracellular Ca²⁺ and concluded that the majority of ATP-induced exocytosis was due to localized Ca²⁺ release near the vesicle fusion sites. To address the question of whether endoplasmic reticulum (ER) Ca²⁺ pool plays a role in the ATP stimulated exocytosis, single ? cells were pre-incubated with thapsigargin and found SERCA inhibition hardly reduced ATP-induced capacitance increase in Ca2+-free bath solution. Then we further studied whether the granule Ca2+ stores contribute to the TG-insensitive exocytosis and [Ca2+]i elevation induced by ATP using ionomycin and monensin which have been shown to dissipate Ca2+ gradient from all acidic organelles and found pretreatment with ionomycin and monensin significantly reduced ATP-induced Cm increment and [Ca2+]i elevation. Furthermore, we pre-treated cells with IP3 receptor blockers, 2-APB and heparin, as well as ryanodine receptor blocker, dantrolene, respectively, and found IP3 coupled extracellular ATP stimulation to exocytosis. In addition, using flash photolysis of caged Ca2+, and found ATP application didn't act directly on the secretory machinery. All these results suggest that ATP-induced exocytosis is mainly stimulated by localized Ca2+ release from secretory granules in rat pancreatic ? cells. (2) GIP1-39, a novel chain length form of GIP, has been purified recently from porcine intestine. We have characterized that GIP1-39 is an insulinotropic peptide, and demonstrated that GIP1-39 is more potent in stimulating insulin secretion from rat pancreatic islets than GIP1-42. Therefore, we have further investigated some aspects on the mechanism behind the insulinotropic effect of GIP1-39 in single rat pancreatic ? cells. GIP1-39 was able to significantly increase intracellular Ca2+ concentration ([Ca2+]i ), and capable of enhancing exocytosis assessed by membrane capacitance measurement. The novel GIP1-39 might be a more optimal molecular pattern in stimulating insulin secretion and deserves to be further investigated biologically and clinically.