Ling B N, Seal E E, Eaton D C
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
J Clin Invest. 1993 Nov;92(5):2141-51. doi: 10.1172/JCI116815.
We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.
我们采用膜片钳技术研究肾小球系膜细胞（GMC）如何去极化，从而刺激电压依赖性Ca2+通道并引发GMC收缩。在添加100 mU/ml胰岛素培养的大鼠GMC中，12%的细胞贴附式膜片中含有一种Ca(2+)依赖性、4皮西门子的Cl-通道。在静息膜电位下，基础NPo（通道数量乘以开放概率）< 0.1。急性施加1 - 100 nM血管紧张素II（AII）或0.25 μM毒胡萝卜素（以释放细胞内Ca2+储存）可增加NPo。在无胰岛素培养的GMC中，Cl-通道很少见（4%），在细胞贴附式膜片中对AII或毒胡萝卜素无反应，在切除式膜片中对细胞内Ca2+的敏感性较低。GMC还含有受AII、毒胡萝卜素或细胞内Ca2+刺激的27皮西门子非选择性阳离子通道（NSCC），但同样仅在有胰岛素存在时出现。在无胰岛素培养的GMC中，暴露于胰岛素15分钟可增加NPo（胰岛素≥100 μU/ml），并恢复对AII和细胞内Ca2+的反应性（胰岛素≥1 μU/ml），对Cl-通道和NSCC均如此。GMC的AII受体结合研究表明，在无胰岛素时，Bmax（结合位点）为2.44±0.58 fmol/mg蛋白，Kd（结合解离常数）为3.02±2.01 nM。长期（数天）暴露于胰岛素后，Bmax增加86%，Kd不变。相比之下，急性（15分钟）暴露对Kd和Bmax均无显著影响。因此，我们得出以下结论：（a）大鼠GMC培养物中含有受AII刺激的Ca(2+)依赖性Cl-通道和NSCC。（b）Cl-外流和阳离子内流分别会促进GMC去极化，导致电压依赖性Ca2+通道激活和GMC收缩。（c）Cl-通道和NSCC对AII的反应性取决于胰岛素暴露情况；AII受体密度在长期而非急性胰岛素作用下增加，通道对细胞内Ca2+的敏感性在急性和慢性胰岛素作用下均增加。（d）GMC收缩性降低可能导致胰岛素缺乏或胰岛素抵抗的糖尿病患者出现肾小球高滤过。
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