Nutrient modulation of polarized and sustained submembrane Ca2+ microgradients in mouse pancreatic islet cells.
The intracellular calcium concentration ([Ca2+]i) near the plasma membrane was measured in mouse pancreatic islet cells using confocal spot detection methods. Whereas small cytosolic Ca2+ gradients were observed with 3 mM glucose, a steeper sustained gradient restricted to domains beneath the plasma membrane (space constant, 0.67 micrometer) appeared with 16.7 mM glucose. When the membrane potential was clamped with increasing K+ concentrations (5, 20 and 40 mM), no [Ca2+]i gradients were observed in any case. Increasing glucose concentration (0, 5 and 16.7 mM) in the presence of 100 microM diazoxide, a K+ channel opener, plus 40 mM K+ induced steeper [Ca2+]i gradients, confirming the role of membrane potential-independent effects of glucose. Prevention of Ca2+ store refilling with 30 microM cyclopiazonic acid (CPA) or blockade of uniporter-mediated Ca2+ influx into the mitochondria with 1 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or 1 microM Ru-360 significantly reduced the steepness of the 16.7 mM glucose-induced [Ca2+]i gradients. Measured values of [Ca2+]i reached 6.74 +/- 0.67 microM at a distance of 0.5 micrometer from the plasma membrane and decayed to 0.27 +/- 0.03 microM at a distance of 2 micrometer. Mathematically processed values at 0.25 and 0 micrometer gave a higher [Ca2+]i, reaching 8.18 +/- 0.86 and 10.05 +/- 0.98 microM, respectively. The results presented indicate that glucose metabolism generates [Ca2+]i microgradients, which reach values of around 10 microM, and whose regulation requires the involvement of both mitochondrial Ca2+ uptake and endoplasmic reticulum Ca2+ stores.
Quesada
I
I
Department of Physiology, Institute of Bioengineering, Campus de San Juan, Aptdo. 18, University Miguel Hernandez, 03550 San Juan, Alicante, Spain.
Martín
F
F
Soria
B
B
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003981:Diazoxide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D007211:Indoles; D014966:Xanthenes; C059715:Fluo-3; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000252:Calcium-Transporting ATPases; D005947:Glucose; D003981:Diazoxide; D011188:Potassium; D002118:Calcium; C000543:cyclopiazonic acid