STIMULATED CA2-CELL LINE - POSSIBLE ROLE IN GLUCOSE AND AGONIST-INDUCED INSULIN-SECRETION( INFLUX RAISES MITOCHONDRIAL FREE CA2+ TO SUPRAMICROMOLAR LEVELS IN A PANCREATIC BETA)

The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intram itochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic b eta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected wit h non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transf ection with aequorin targeted to the mitochondria by fusion of the gen e in frame with the signal peptide of cytochrome c oxidase subunit VII I. Two physiological stimuli of native beta-cells, glucose and ATP, ra ised [Ca2+]c in INS-1 cells largely by stimulating Ca2+ influx. Thus, glucose (20 mM) induced repetitive transient increases in [Ca2+]c (0.4 2 min-1, mean amplitude 229 nM above 102 nM basal). These transients w ere largely due to periodic stimulation of Ca2+ influx through voltage -sensitive Ca2+ channels, since they could be rapidly and reversibly b locked by chelation of external Ca2+, by addition of the hyperpolarizi ng agent diazoxide, or with the Ca2+ channel blocker SR 7037. ATP, by contrast, caused single transient [Ca2+]c increases, to about 300 nM a bove basal levels, which could be inhibited by >90% upon external Ca2 chelation. Challenge of aequorin-transfected cells with ATP increased [Ca2+]m to 4 muM or above, an effect blocked by EGTA. Furthermore, pl asma membrane depolarization with high K+, used as a glucose surrogate to mimic, in a synchronized fashion, the influx-induced Ca2+ transien ts observed at the single-cell level, also increased [Ca2+]m to >4 muM . Similar increases in [Ca2+]m were also measured in other aequorin-tr ansfected insulin-secreting cells, RINm5F, during mobilization of inte rnal Ca2+ with carbachol. In contrast, glucose-induced changes in [Ca2 +]m were below the level of detection in INS-1 cell populations, consi stent with the asynchrony of the [Ca2+]c transients induced by this nu trient at the single-cell level, and the consequent small average [Ca2 +]c rise. These data are in line with the view that stimulated Ca2+ in flux into excitable cells raises [Ca2+]m as efficiently as internal Ca 2+ mobilization in nonexcitable cells. In the case of INS-1 and pancre atic beta-cells, this may be important both to enhance oxidative metab olism, hence fueling the secretory process, and also to maintain the p roduction of metabolic signaling molecules.