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

Abstract

The mitochondrial inhibitors NaN(3) 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 NaN(3) and FCCP both increased the K(ATP) current and thus hyperpolarized the cell membrane potential, as expected for agents depleting cellular ATP. NaN(3) 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 NaN(3) 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 NaN(3) or FCCP was even visible after the removal of extracellular Ca2+, suggesting that the inhibitors also mobilize Ca2+ from mitochondria. NaN(3) 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 NaN(3)-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.

Oscillations of membrane potential and cytosolic Ca2+ concentration in SUR1−/− beta cells

“…Remarkably, NaN 3 influenced the electrical activity of SUR1 −/− beta cells by decreasing spike activity without concomitant membrane hyperpolarization. We recently showed that NaN 3 inhibits L-type Ca 2+ currents in SUR1 +/+ beta cells using the standard whole-cell configuration [44]. This implies the reduction of Ca 2+ action potentials in SUR1 −/− beta cells could result from a direct effect of NaN 3 on the L-type Ca 2+ channel.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Thus, while pyruvate is clearly the major physiologically relevant substrate in the intact cell, for unexplained reasons the isolated mitochondria appear to have difficulty oxidizing NAD-linked substrates, the failure being either stated explicitly (2), or inferred by the authors only reporting data obtained with substrates bypassing complex I, such as ␣-glycerophosphate and succinate (137,269,276,324,426). The basis of this failure, which has also been reported for permeabilized ␤-cells (see below) merits further investigation, since it may suggest that a critical component is lost.…”

ABCC8 and ABCC9: ABC transporters that regulate K+ channels

“…3. In contrast to wild-type β cells, the application of sodium azide did not result in hyperpolarization of Sur1KO β cells but did reduce the amplitude of Ca 2+ -dependent action potentials by directly inhibiting Ca 2+ channels [44,45]. Neither tolbutamide nor diazoxide had any effect on V m oscillations in K ATP null islets.…”