CONTROL OF CYTOSOLIC-FREE CALCIUM IN CULTURED HUMAN PANCREATIC BETA-CELLS OCCURS BY EXTERNAL CALCIUM-DEPENDENT AND INDEPENDENT MECHANISMS

Changes in cytosolic intracellular free Ca2+ ([Ca2+]i) in response to glucose, glyburide, cholinergic agonists, and elevated [K+]o (external potassium concentration) were measured in cultured human islet beta-c ells. In the absence of glucose, the mean resting [Ca2+]i in single be ta-cells was 84.5 +/- 4.7 nM (n = 86) and remained unchanged in low ex ternal [Ca2+]o (Ca2+ concentration) (<0.2 muM) at 23-25 C. Glucose (5. 6-33 mM) induced a slow dose-related [Ca2+]i rise up to 300.0 +/- 50.6 nm (n = 19). This [Ca2+]i rise always occurred with a delay that vari ed from cell to cell (approximately 10-120 sec), and the steady state [Ca2+]i exhibited a sigmoidal dependence on glucose concentration (mid point at 14.9 mM). The glucose-induced rise in [Ca2+]i was attenuated by about 62% in low external [Ca2+]o and was not affected by dantrolen e, a drug that inhibits Ca2+ release from the endoplasmic reticulum. I n the absence or presence of glucose, cholinergic receptor agonists ev oked a biphasic increase in [Ca2+]i up to 350 nM; the delayed componen t of the [Ca2+]i rise was blocked by dantrolene. A rapid elevation of [K+]o to 40 mM also elicited a biphasic rise in [Ca2+]i, which peaked at about 250 nm and was inhibited by the Ca2+ channel antagonist nifed ipine. Glyburide (4 muM) in the absence of glucose also induced a [Ca2 +]o-dependent rise in [Ca2+]i. Increasing the concentration of glucose from 4 to 16.7 mm evoked a biphasic pattern of insulin secretion from perifused isolated islets at 37 C. Finally, in the presence of 4 mm g lucose, a cholinergic muscarinic receptor agonist stimulated insulin s ecretion. A glucose-stimulated [Ca2+]i rise was also studied at 24 and 37 C in cultured rat islet cells. Our results suggest that the Ca2+ r equired for glucose-induced and muscarinic agonist-potentiated insulin release enters the cytosol from both extracellular and intracellular Ca2+ stores.