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-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.
Rojas
E
E
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.
Carroll
P B
PB
Ricordi
C
C
Boschero
A C
AC
Stojilkovic
S S
SS
Atwater
I
I
D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D003620:Dantrolene / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D009994:Osmolar Concentration; D010277:Parasympathomimetics / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D007328:Insulin; D010277:Parasympathomimetics; D011976:Receptors, Muscarinic; D003620:Dantrolene; D005947:Glucose; D005905:Glyburide; D002118:Calcium