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Nutrient modulation of polarized and sustained submembrane Ca²⁺ microgradients in mouse pancreatic islet cells

Ivan Quesada *†, Franz Martín †‡ and Bernat Soria *†

1. The intracellular calcium concentration ([Ca²⁺]_i) near the plasma membrane was measured in mouse pancreatic islet cells using confocal spot detection methods.

2. 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 µm) appeared with 16·7 mM glucose.

3. When the membrane potential was clamped with increasing K⁺ concentrations (5, 20 and 40 mM), no [Ca²⁺]_i gradients were observed in any case.

4. Increasing glucose concentration (0, 5 and 16·7 mM) in the presence of 100 µM diazoxide, a K⁺ channel opener, plus 40 mM K⁺ induced steeper [Ca²⁺]_i gradients, confirming the role of membrane potential-independent effects of glucose.

5. Prevention of Ca²⁺ store refilling with 30 µM cyclopiazonic acid (CPA) or blockade of uniporter-mediated Ca²⁺ influx into the mitochondria with 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or 1 µM Ru-360 significantly reduced the steepness of the 16·7 mM glucose-induced [Ca²⁺]_i gradients.

6. Measured values of [Ca²⁺]_i reached 6·74 ± 0·67 µM at a distance of 0·5 µm from the plasma membrane and decayed to 0·27 ± 0·03 µM at a distance of 2 µm. Mathematically processed values at 0·25 and 0 µm gave a higher [Ca²⁺]_i, reaching 8·18 ± 0·86 and 10·05 ± 0·98 µM, respectively.

7. The results presented indicate that glucose metabolism generates [Ca²⁺]_i microgradients, which reach values of around 10 µM, and whose regulation requires the involvement of both mitochondrial Ca²⁺ uptake and endoplasmic reticulum Ca²⁺ stores.

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