MASTOPARAN INCREASES THE INTRACELLULAR FREE CALCIUM-CONCENTRATION IN 2 INSULIN-SECRETING CELL-LINES BY INHIBITION OF ATP-SENSITIVE POTASSIUM CHANNELS

The mechanisms underlying mastoparan-induced elevation of the intracel lular free calcium concentration ([Ca2+](i)) were investigated in the insulin-secreting cell lines RINm5F and HIT. In both cell types, micro molar concentrations of mastoparan induced a prompt increase of [Ca2+] (i), measured as an increase in fura-2 fluorescence. This response was dependent on extracellular calcium entry and was suppressed by organi c calcium channel blockers; the increase of [Ca2+](i) caused by high g lucose concentrations or tolbutamide was not enhanced by mastoparan, T hese data indicate the involvement of voltage-dependent calcium channe ls and suggest that depolarization, rather than a direct effect on the channels, mediates the response to mastoparan. This proposition was s upported by the observation that whole-cell calcium currents measured using the nystatin-permeabilized patch technique were not affected by mastoparan. Mastoparan-induced depolarization was observed using the p otentiometric indicator bis-oxonol, and it was shown not to be additiv e with the depolarization induced by high glucose concentrations or to lbutamide. The mechanism underlying mastoparan-induced depolarization was identified in single-channel patch-clamp experiments, where it was shown that mastoparan caused closure of ATP-sensitive potassium chann els [K(ATP) channels] in cell-attached and excised membrane patches. R esponsiveness to mastoparan in excised patches demonstrated the membra ne-delimited character of K(ATP) channel inhibition. The observation t hat the response persisted in the absence of exogenous GTP and in the presence of 250 mu M GDP Or guanosine-5'-O-(2-thio)diphosphate suggest ed that this effect is not mediated via enhancement of G protein activ ity. Partial suppression of channel activity by mastoparan did not pre vent the action of tolbutamide, which fully suppressed the remaining a ctivity in excised patches. In summary, the increase of [Ca2+](i) in t he insulin-secreting tumor cell lines RINm5F and HIT in response to ma stoparan is mediated via G protein-independent suppression of K(ATP) c hannel activity, cell depolarization, and activation of voltage-depend ent calcium channels.