Raat N J, Hartog A, van Os C H, Bindels R J
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Am J Physiol. 1994 Jul;267(1 Pt 2):F63-9. doi: 10.1152/ajprenal.1994.267.1.F63.
The presence of Na(+)-K(+)-2Cl- cotransport in rabbit kidney proximal tubule cells in primary culture was demonstrated by bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake studies. After addition of 10 microM bumetanide, 86RB+ uptake was inhibited from 11.1 +/- 0.8 to 1.1 +/- 0.1 nmol.mg protein-1.min-1 under isotonic (300 mosM) conditions. Na(+)-K(+)-2Cl- cotransport activities ranged from 2.2 +/- 0.3 to 13.2 +/- 1.0 nmol.mg protein-1.min-1 depending on the osmolarity of the medium (150-500 mosM). Decreasing extracellular pH to 6.5 inhibited, whereas increasing to 8.5 stimulated, transport at all osmolarities. Decreasing intracellular pH (pHi) by the NH4Cl pulse method showed similar results, suggesting a possible regulatory role of pHi on cotransport activity. Ca(2+)-free medium increased cotransport activity 35 and 20% at iso- and hypertonicity, respectively. At 300 mosM, ionomycin (5 microM) inhibited cotransport by 25%. The combination of forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM) resulted in inhibition of cotransport activity by 38% at hypertonic conditions. Calyculin (1 microM) increased cotransport activity 134 and 128% at 150 and 300 mosM, respectively. In hypertonic medium calyculin did not influence cotransport activity. Okadaic acid (1 microM) had no effect on cotransport activity at all three osmolarities. NaF (10 mM) increased cotransport at all osmolarities tested.
通过布美他尼敏感、哇巴因不敏感的⁸⁶Rb⁺摄取研究，证实了原代培养的兔肾近端小管细胞中存在Na⁺-K⁺-2Cl⁻共转运。在等渗（300 mosM）条件下，加入10 μM布美他尼后，⁸⁶RB⁺摄取从11.1±0.8降至1.1±0.1 nmol·mg蛋白⁻¹·min⁻¹。Na⁺-K⁺-2Cl⁻共转运活性范围为2.2±0.3至13.2±1.0 nmol·mg蛋白⁻¹·min⁻¹，具体取决于培养基的渗透压（150 - 500 mosM）。将细胞外pH降至6.5会抑制转运，而升至8.5则会刺激所有渗透压下的转运。通过氯化铵脉冲法降低细胞内pH（pHi）显示出类似结果，表明pHi可能对共转运活性具有调节作用。无钙培养基在等渗和高渗条件下分别使共转运活性增加35%和20%。在300 mosM时，离子霉素（5 μM）使共转运受到25%的抑制。福斯可林（10 μM）和3 - 异丁基 - 1 - 甲基黄嘌呤（1 mM）的组合在高渗条件下使共转运活性受到38%的抑制。在150和300 mosM时，花萼海绵诱癌素（1 μM）分别使共转运活性增加134%和128%。在高渗培养基中，花萼海绵诱癌素不影响共转运活性。冈田酸（1 μM）在所有三种渗透压下对共转运活性均无影响。氟化钠（10 mM）在所有测试的渗透压下均增加共转运。
