Mariot P, Gilon P, Nenquin M, Henquin J C
Unité d'Endocrinologie et Métabolisme, University of Louvain, Brussels, Belgium.
Diabetes. 1998 Mar;47(3):365-73. doi: 10.2337/diabetes.47.3.365.
Sulfonylureas stimulate insulin secretion by blocking ATP-sensitive K+ channels (K+-ATP channels) of the beta-cell membrane, thereby causing depolarization, Ca2+ influx, and rise in cytoplasmic Ca2+ concentration ([Ca2+]i), whereas diazoxide inhibits insulin secretion by opening K+-ATP channels. It has been suggested recently that these drugs also respectively increase and decrease the efficacy of Ca2+ on exocytosis. This hypothesis was tested here with intact islets or single beta-cells from normal mice. Depolarizing islet cells by raising extracellular K+ from 4.8 to 15, 30, and 60 mmol/l progressively raised [Ca2+]i and stimulated insulin secretion. The magnitude of the [Ca2+]i rise produced by a subsequent addition of 100 micromol/l tolbutamide decreased as the concentration of K+ was increased. The effect on insulin secretion paralleled that on [Ca2+]i. Similarly, the magnitudes of the [Ca2+]i drop and of the inhibition of insulin secretion produced by 250 micromol/l diazoxide were inversely related to the concentration of K+. Either drug was effective on secretion only when it increased or decreased [Ca2+]i. Exocytosis of insulin granules from single, voltage-clamped beta-cells was also studied by measuring cell capacitance changes. In the perforated patch configuration, exocytosis was evoked by depolarizing pulses. Addition of tolbutamide to the extracellular medium did not affect the Ca2+ current and the resulting change in cell capacitance. In the whole-cell configuration, cell capacitance increased with the concentration of free Ca2+ in the solution diffusing from the pipette into the cell. It was markedly potentiated by cAMP, was inhibited by activation of alpha2-adrenoceptors with clonidine, and was strongly augmented by acetylcholine. In contrast, tolbutamide was ineffective whether applied intra- or extracellularly, at low or high free Ca2+, and with or without cAMP. Diazoxide also failed to interfere directly with exocytosis. These results indicate that tolbutamide and diazoxide affect insulin secretion by changing the concentration, not the action, of Ca2+ in beta-cells.
磺脲类药物通过阻断β细胞膜上的ATP敏感性钾通道（K⁺-ATP通道）来刺激胰岛素分泌，从而引起去极化、Ca²⁺内流以及细胞质Ca²⁺浓度（[Ca²⁺]i）升高，而二氮嗪则通过开放K⁺-ATP通道来抑制胰岛素分泌。最近有人提出，这些药物还分别增加和降低Ca²⁺对胞吐作用的效能。本研究用来自正常小鼠的完整胰岛或单个β细胞对这一假说进行了验证。通过将细胞外K⁺浓度从4.8 mmol/L提高到15、30和60 mmol/L使胰岛细胞去极化，可逐渐升高[Ca²⁺]i并刺激胰岛素分泌。随后加入100 μmol/L甲苯磺丁脲所引起的[Ca²⁺]i升高幅度随着K⁺浓度的增加而减小。对胰岛素分泌的影响与对[Ca²⁺]i的影响平行。同样，250 μmol/L二氮嗪所引起的[Ca²⁺]i下降幅度和胰岛素分泌抑制程度与K⁺浓度呈负相关。只有当这两种药物增加或降低[Ca²⁺]i时，它们才对分泌有效。还通过测量细胞电容变化研究了单个电压钳制β细胞中胰岛素颗粒的胞吐作用。在穿孔膜片钳模式下，去极化脉冲可诱发胞吐作用。向细胞外培养基中加入甲苯磺丁脲不影响Ca²⁺电流及由此引起的细胞电容变化。在全细胞膜片钳模式下，细胞电容随着从微电极扩散到细胞内的溶液中游离Ca²⁺浓度的增加而增大。它被cAMP显著增强，被可乐定激活α₂肾上腺素能受体所抑制，并被乙酰胆碱强烈增强。相反，无论在细胞内还是细胞外应用甲苯磺丁脲，在低游离Ca²⁺或高游离Ca²⁺条件下，以及有无cAMP时，甲苯磺丁脲均无效。二氮嗪也未能直接干扰胞吐作用。这些结果表明，甲苯磺丁脲和二氮嗪通过改变β细胞中Ca²⁺的浓度而非其作用来影响胰岛素分泌。
