Eddlestone G T, Komatsu M, Shen L, Sharp G W
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA.
Mol Pharmacol. 1995 Apr;47(4):787-97.
The mechanisms underlying mastoparan-induced elevation of the intracellular free calcium concentration ([Ca2+]i) were investigated in the insulin-secreting cell lines RINm5F and HIT. In both cell types, micromolar 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 organic calcium channel blockers; the increase of [Ca2+]i caused by high glucose concentrations or tolbutamide was not enhanced by mastoparan. These data indicate the involvement of voltage-dependent calcium channels and suggest that depolarization, rather than a direct effect on the channels, mediates the response to mastoparan. This proposition was supported 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 potentiometric indicator bis-oxonol, and it was shown not to be additive with the depolarization induced by high glucose concentrations or tolbutamide. 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 channels [K(ATP) channels] in cell-attached and excised membrane patches. Responsiveness to mastoparan in excised patches demonstrated the membrane-delimited character of K(ATP) channel inhibition. The observation that the response persisted in the absence of exogenous GTP and in the presence of 250 microM GDP or guanosine-5'-O-(2-thio)diphosphate suggested that this effect is not mediated via enhancement of G protein activity. Partial suppression of channel activity by mastoparan did not prevent the action of tolbutamide, which fully suppressed the remaining activity in excised patches. In summary, the increase of [Ca2+]i in the insulin-secreting tumor cell lines RINm5F and HIT in response to mastoparan is mediated via G protein-independent suppression of K(ATP) channel activity, cell depolarization, and activation of voltage-dependent calcium channels.
在胰岛素分泌细胞系RINm5F和HIT中研究了mastoparan诱导细胞内游离钙浓度（[Ca2+]i）升高的机制。在这两种细胞类型中，微摩尔浓度的mastoparan均能迅速诱导[Ca2+]i升高，以fura-2荧光增加来衡量。该反应依赖于细胞外钙内流，并被有机钙通道阻滞剂所抑制；高葡萄糖浓度或甲苯磺丁脲引起的[Ca2+]i升高不会因mastoparan而增强。这些数据表明电压依赖性钙通道参与其中，并提示去极化而非对通道的直接作用介导了对mastoparan的反应。使用制霉菌素通透膜片技术测量的全细胞钙电流不受mastoparan影响这一观察结果支持了这一观点。使用电位指示剂双羟萘酚观察到mastoparan诱导的去极化，并且发现它与高葡萄糖浓度或甲苯磺丁脲诱导的去极化没有叠加作用。在单通道膜片钳实验中确定了mastoparan诱导去极化的机制，结果表明mastoparan导致细胞贴附膜片和切除膜片中ATP敏感性钾通道（K(ATP)通道）关闭。在切除膜片中对mastoparan的反应证明了K(ATP)通道抑制的膜限定特性。在没有外源性GTP以及存在250μM GDP或鸟苷-5'-O-(2-硫代)二磷酸的情况下反应仍然存在这一观察结果表明该效应不是通过增强G蛋白活性介导的。mastoparan对通道活性的部分抑制并未阻止甲苯磺丁脲的作用，甲苯磺丁脲能完全抑制切除膜片中剩余的活性。总之，胰岛素分泌肿瘤细胞系RINm5F和HIT中对mastoparan反应时[Ca2+]i的升高是通过G蛋白非依赖性抑制K(ATP)通道活性、细胞去极化以及电压依赖性钙通道的激活介导的。
