The roles of G-proteins in the functional regulation of beta-intercalated cells of the kidney cortex

为探讨G蛋白在皮质集合管β-插层细胞(β-IC)功能调节中的作用，采用体外微灌流技术和膜片钳技术对分离细胞进行了研究。首先，利用荧光染料测量细胞pH值，研究了激素因素对β-IC中pH调节系统的影响。证实了异丙肾上腺素能刺激根尖膜上的Cl~+-HCO3~-交换。此外，虽然霍乱毒素和百日咳毒素对异丙肾上腺素的作用无明显影响，但前列腺素E_2可抑制异丙肾上腺素的这种兴奋作用。并观察了酸碱平衡的变化对内纹外髓集合管细胞功能的影响。OMCDis与碱性溶液体外孵育可引起H^+-ATPase活性下降，而霍乱和百日咳毒素对此无影响。根据这些结果，体外微灌流系统可能不适合揭示G蛋白在调节β-IC中的作用。因此，尝试使用荧光激活细胞分选仪分离β-IC。花生凝集素是一种已知能与β-IC结合的凝集素。分离的细胞对异丙肾上腺素的反应显示环磷酸腺苷显著增加。这些细胞被培养在胶原涂层的培养皿上，这些培养的细胞对异丙肾上腺素和花生凝集素凝集素的结合也表现出反应，表明培养的β-IC保持了原来的特性。膜片钳研究表明，培养细胞存在氯离子通道，该通道可通过G蛋白和腺苷环化酶系统被异丙肾上腺素激活。还发现这些培养的β-IC含有被霍乱毒素核糖化的蛋白质。

To investigate the roles of G-proteins in the functional regulation of beta-intercalated cells(beta-IC) in the cortical collecting ducts, in vitro microperfusion studies and patch clamp studies with isolated cells were performed. Firstly, the effects of hormonal factors on the pH regulation systems in the beta-IC was investigated, using with cell pH measurements by fluorescent dye. It was confirmed that isoproterenol stimulated Cl^-HCO_3^- exchanger in the apical membrane. Furthermore, it was shown that prostaglandin E_2 inhibited this stimulatory effect of isoproterenol, although cholera toxin and pertussis toxins failed to show any significant effects on the action of isoproterenol. The effects of the changes in the acid-base balance on the function of the cells of the outermedullary collecting ducts in the inner stripe (OMCDis) were also observed. The in vitro incubation of OMCDis with alkali solution induced a decrease in H^+-ATPase activity, although cholera and pertussis toxins did not have any effects, either. From these results, it was possible that in vitro microperfusion systems might be inappropriate to reveal the roles of G-proteins in the regulation of beta-IC.Therefore, isolation of beta-IC was tried, using with fluorescent activated cell sorter. Peanut lectin agglutinin, which is known to bind beta-IC, was used as a marker. Isolated cells showed a significant increase in cyclic AMP in response to isoproterenol. These cells were cultured on the collagen-coated dishes, and these cultured cells also showed the response to isoproterenol and peanut lectin agglutinin binding, suggesting that cultured beta-IC retained its original characters. By patch-clamp studies, it was shown that cultured cells had Cl^-channel and that this channels was activated by isoproterenol via G-protein and adenylate cyclase systems. It was also revealed that these cultured beta-IC had the proteins, which was ribosylated by cholera toxin.