Regulation of Na⁺-K⁺-2Cl^- cotransport in turkey red cells: The role of oxygen tension and protein phosphorylation

1. Na⁺-K⁺-2Cl^- cotransport (NKCC) was studied in turkey red cells using Na⁺ dependence or bumetanide sensitivity of ⁸⁶Rb⁺ influx to monitor activity of the transporter. 2. Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (P(O₂) over the physiological range modulated the transporter, with a P(O₂) for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 μM). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or β-adrenergic stimulation. 3. Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N-Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 μM) inhibited deoxygenated-stimulated KC influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or P(O₂). 4. The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K⁺-Cl^- cotransport.