The Na+,K+,2Cl-cotransport system in HeLa cells: aspects of its physiological regulation.

We have previously reported on the biochemical properties of a Na+,K+,2Cl-cotransport system in HeLa cells and here we deal with aspects of its physiological regulation. Na+,K+,2Cl-cotransport in HeLa cells was studied by 86Rb+ influx and 86Rb+/22Na+ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on 86Rb+ flux, mediated by the bumetanide-sensitive Na+,K+,2Cl-cotransport system and the ouabain-sensitive Na+/K(+)-pump, were investigated. ANP reduced bumetanide-sensitive 86Rb+ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive 86Rb+ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a beta-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive 86Rb+ influx. Furthermore, efflux of 86Rb+ and 22Na+ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168: 148-154, 1990), this study indicates that ANP participates in the regulation of the Na+,K+,2Cl-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative alpha-methyl-aminoisobutyric acid (metAIB, 1 and 5 mM, respectively) also reduced Na+,K+,2Cl-cotransport-mediated 86Rb+ uptake and diminished the stimulatory effect of hypertonicity on the contransporter. In addition, the Na+/K(+)-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-GMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na+/K(+)-pump.