Inhibition of mitochondrial function affects cellular Ca²⁺ handling in pancreatic B-cells

Abstract.

The mitochondrial inhibitors NaN₃ and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were used to study the role of mitochondria in pancreatic B-cell Ca²⁺ homeostasis. In glucose-stimulated B-cells NaN₃ and FCCP both increased the K_ATP current and thus hyperpolarized the cell membrane potential, as expected for agents depleting cellular ATP. NaN₃ and FCCP stopped the glucose-induced oscillations in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) and elicited a biphasic response. After a first rapid and transient increase, [Ca²⁺]_c rose in a second slow phase to a sustained level. In cells pretreated with thapsigargin the first inhibitor-induced rise in [Ca²⁺]_c was absent, suggesting that it may be due to Ca²⁺ mobilization from intracellular stores. The glucose-induced oscillations were terminated again by NaN₃ and FCCP, respectively, but the slow increase in [Ca²⁺]_c of the second phase was still present. A minute increase in [Ca²⁺]_c elicited by NaN₃ or FCCP was even visible after the removal of extracellular Ca²⁺, suggesting that the inhibitors also mobilize Ca²⁺ from mitochondria. NaN₃ and FCCP induced Ca²⁺ influx into B-cells treated with low glucose concentrations whose voltage-dependent Ca²⁺ channels are closed. Experiments with thapsigargin-preincubated cells indicate that disturbance of mitochondrial function stimulates Ca²⁺ influx through voltage-independent Ca²⁺ pathways. During the NaN₃-induced increase in [Ca²⁺]_c, K⁺-elicited depolarizations of the cells did not further augment [Ca²⁺]_c. Evidently, this is due to a direct inhibitory effect of azide on L-type Ca²⁺ channels. The data demonstrate that disturbing the mitochondrial function affects cellular Ca²⁺ homeostasis in B-cells at several sites. Thus, it is concluded that intact mitochondrial function is a prerequisite for regular Ca²⁺ handling in B-cells.