Endocrinology, Vol 134, 1771-1781, Copyright © 1994 by Endocrine Society

ARTICLES

Control of cytosolic free calcium in cultured human pancreatic beta- cells occurs by external calcium-dependent and independent mechanisms

E Rojas, PB Carroll, C Ricordi, AC Boschero, SS Stojilkovic and I Atwater
Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, and the Endocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, Maryland 20892.

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- cells. In the absence of glucose, the mean resting [Ca2+]i in single beta-cells was 84.5 +/- 4.7 nM (n = 86) and remained unchanged in low external [Ca2+]o (Ca2+ concentration) (< 0.2 microM) 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 varied from cell to cell (approximately 10-120 sec), and the steady state [Ca2+]i exhibited a sigmoidal dependence on glucose concentration (midpoint 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 dantrolene, a drug that inhibits Ca2+ release from the endoplasmic reticulum. In the absence or presence of glucose, cholinergic receptor agonists evoked a biphasic increase in [Ca2+]i up to 350 nM; the delayed component 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 nifedipine. Glyburide (4 microM) 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 glucose, a cholinergic muscarinic receptor agonist stimulated insulin secretion. 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+ required for glucose- induced and muscarinic agonist-potentiated insulin release enters the cytosol from both extracellular and intracellular Ca2+ stores.

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