Control of cytosolic free calcium in cultured human pancreatic β-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 β-cells. In the absence of glucose, the mean resting [Ca²⁺](i) in single β-cells was 84.5 ± 4.7 nM (n = 86) and remained unchanged in low external [Ca²⁺](o) (Ca²⁺ concentration) (<0.2 μM) 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 (~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 μM) 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.