| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1993: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
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| Research Abstract |
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.
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