Tsuura Y, Ishida H, Hayashi S, Sakamoto K, Horie M, Seino Y
Department of Metabolism and Clinical Nutrition, Kyoto University Faculty of Medicine, Japan.
J Gen Physiol. 1994 Dec;104(6):1079-98. doi: 10.1085/jgp.104.6.1079.
Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde-induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL-penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.
一氧化氮（NO）是许多组织细胞内信号转导系统中的一种强效信使。在胰腺β细胞中，据报道NO由L-精氨酸通过一氧化氮合酶生成。为了阐明NO对胰岛素分泌的影响并研究其作用的细胞内机制，我们使用硝普钠（SNP）作为NO供体。SNP以剂量依赖的方式抑制葡萄糖诱导的胰岛素分泌，其作用可被已知的NO清除剂血红蛋白逆转。然而，甘油醛诱导的胰岛素分泌不受SNP影响。由于ATP敏感性钾通道（KATP通道）的关闭已被确认为葡萄糖诱导胰岛素分泌的关键步骤，我们使用膜片钳技术直接评估了SNP对KATP通道活性的影响。发现葡萄糖降低的KATP通道活性在添加SNP后可被可逆性激活，并且通过应用另一种NO生成剂S-亚硝基-N-乙酰-DL-青霉胺（SNAP）能够类似地重现这种激活。此外，根据对葡萄糖诱导的胰岛素释放抑制的可逆性，这些激活作用被血红蛋白完全消除。然而，SNP不能影响施加到质膜内侧的ATP对KATP通道的抑制作用。因此，NO对KATP通道的激活似乎是由于β细胞中葡萄糖代谢受损导致ATP生成减少。由于SNP对甘油醛诱导的KATP通道抑制没有影响，NO可能干扰了磷酸丙糖之前的糖酵解步骤。然而，即使在存在SNP的情况下，2-脱氧葡萄糖通过可能因葡萄糖激酶磷酸化而消耗ATP对KATP通道的激活也不受影响。但是，在暴露于低浓度（0.02 U/ml）链球菌溶血素O制成的通透β细胞（开放细胞贴附构型）中，SNP重新打开已被6-磷酸果糖消除的KATP通道，而在被1,6-二磷酸果糖抑制的KATP通道中未观察到这种作用。另一方面，在大鼠心室肌细胞中，即使在低浓度葡萄糖下，KATP通道也不会被SNP激活。从这些观察结果来看，磷酸果糖激酶活性的抑制可能是胰腺β细胞中NO诱导葡萄糖代谢受损的作用位点。因此，NO似乎是通过独特的细胞内机制导致胰腺β细胞葡萄糖诱导的胰岛素分泌恶化的一个因素。
