Ouabain-induced cell swelling in rabbit connecting tubule: evidence for thiazide-sensitive Na(+)-Cl- cotransport

Mechanisms of Na+ entry across the luminal membrane of the rabbit connecting tubule (CNT) and cortical collecting duct (CCD) were investigated in vitro by analyzing factors that block the ouabain-induced tubular swelling. In the CNT and CCD, cell swelling caused by 100 microM ouabain added to the bath was completely blocked by luminal Na+ removal, suggesting that the main factor inducing cell swelling is Na+ entry through the luminal membrane. Trichlormethiazide (100 microM) and amiloride (10 microM) inhibited the swelling in CNT when applied in combination to the lumen, but not when given separately. The swelling was also inhibited by Cl- omission from the lumen in the presence of amiloride. By contrast, no effect was noted when furosemide (100 microM), 4-acetamide-4'-isothiocyanatostilben-2,2'-disulfonic acid (1 mM) or 5-(N-ethyl-N-isopropyl)amiloride (100 microM) was added to the lumen in the presence of amiloride, indicating the absence of any influence of the Na(+)-K(+)-2Cl- cotransporter and the parallel Na+/H+, Cl-/HCO3- exchanger. The cell swelling in the CCD was blocked by luminal addition of amiloride alone with no effect from trichlormethiazide. In CNT, when the ouabain-induced cell swelling was prevented by both diuretics, addition of parathyroid hormone (PTH, 3 nM) to the bath induced cell swelling, suggesting that another Na+ entry pathway is newly generated by PTH. These results demonstrate that ouabain-induced cell swelling depends on Na+ entry across the luminal membrane. In the CNT, the pathways include an amiloride-sensitive Na+ channel, thiazide-sensitive Na(+)-Cl- cotransport and a PTH-stimulated Na+ pathway, whereas the CCD has only the amiloride-sensitive Na+ channel.