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 Rb-86(+) 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 mu M). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or P-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 mu M) inhibited deoxygenated-stimulated K+ 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.