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  • 1
    ISSN: 1432-1424
    Keywords: K+ channel ; intracellular pH ; Na+-K+ ATPase ; patch-clamp ; amphibian kidney ; aldosterone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K+ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K+ conductance (gK+) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK+ in ouabain-treated cells in the presence of furosemide. We tested whether changes in intracellular pH (pH i ) could be a signal for cells to regulategK+. After removal of aldosterone, the increase ingK+ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH i and doubledgK+. Using the patch-clamp technique, three types of K+-selective channels were identified, which had conductances of 24, 45 and 59 pS. Aldosterone had no effect on the conductance or open probability (P o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P o was increased by raising pH. These results indicate that prolonged aldosterone treatment raises pH i and increasesgK+ by promoting insertion of K+ channels into the cell membrane. Channel insertion is itself triggered by raising both pH i and increasing the activity of the Na+/K+ pump in early distal cells of frog kidney.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: fused MDCK cells ; K+ channel ; minoxidil-sulfate ; Ca+2 ; protein kinase C
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We studied the mechanism of K++ channel activation by minoxidil-sulfate (MxSO4) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca+2+-dependent inward-rectifying K++ channel with slope conductances of 53±3 (negative potential range) and 20±3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO4 initiated both transient channel activation and an increase of intracellular Ca+2+ (from 94.2±9.1 to 475±12.6 nmol/liter). The observation that K++ channel activity of excised inside-out patches was detected only at Ca+2+ concentrations in excess of 10 μmol/liter suggests the involvement of additional mechanisms during channel activation by MxSO4. Transient K++ channel activity was also induced in cell-attached patches by 10 μmol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 μmol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca+2 sensitivity of the K+ channel in inside-out patches significantly by lowering the K mfor Ca+2 from 100 μmol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO4 on K+ channel activation. We conclude that MxSO4-induced K+ channel activity is mediated by the synergistic effects of an increase in intracellular Ca+2 and a PKC-mediated enhancement of the K+ channel's sensitivity to Ca+2.
    Type of Medium: Electronic Resource
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