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 (PO2) over the physiological range modulated the transporter, with a PO2 for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 microgr;M). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or beta-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 microM) 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 PO2. 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.
Muzyamba
M C
MC
Department of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 3BX, UK.
Cossins
A R
AR
Gibson
J S
JS
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000097:blood*; D004229:Dithiothreitol / Q000494:pharmacology; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010100:Oxygen / Q000097:blood*; D010313:Partial Pressure; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine / Q000494:pharmacology; D014422:Turkeys
D000318:Adrenergic beta-Agonists; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine; D007545:Isoproterenol; D005033:Ethylmaleimide; D010100:Oxygen; D004229:Dithiothreitol