COMPLETE INHIBITION OF NA- COTRANSPORT IN MADIN-DARBY CANINE KIDNEY-CELLS BY PMA-SENSITIVE PROTEIN-KINASE(, K+, CL)

This study examines the involvement of hormones and neuromediators in the regulation of Na+, K+, Cl- cotransport in renal epithelial cells u sing Madin-Darby canine kidney cells with low transepithelial electric al resistance (194 +/- 47 Omega/cm(2)). In this cell line, Na+, K+, Cl - cotransport measured as bumetanide-sensitive Rb-86 influx was inhibi ted up to 50-60% with agonists of P-2-purinoceptors (ATP approximate t o ADP > UTP > AMY), slightly (15-30%) increased by activators of cAMP signaling (forskolin, 8-Br-cAMP) and was insensitive to activators of cGMP signaling (8-Br-cGMP, nitroprusside), EGF, angiotensin II, bradyk inin, methacholine, propranolol, vasopressin, adenosine, dopamine and histamine. Thirty min of preincubation of MDCK cells with 0.1 mu M PMA completely blocked the activity of Na+, K+, Cl- cotransport whereas d own-regulation of this enzyme by 24h of preincubation with 1 mu M PMA activated Na+, K+, Cl- cotransport by 60% and abolished the effect of short-term treatment with PMA. Regulation of Na+, K+, Cl- cotransport by ATP was insensitive to down-regulation of PMA-sensitive isoforms of protein kinase C. In addition, in inhibitor of protein kinase activit y, staurosporine, abolished the effect of 0.1 mu M PMA but did not cha nge inhibition of this carrier by ATP. Thus, these results show for th e first time that P-2-purinoceptors and PMA-sensitive isoforms of prot ein kinase C play a key role in the regulation of Na+, K+, Cl- cotrans port in MDCK cells. These results also show that neither PMA- nor stau rosporine-sensitive forms of protein kinase are involved in the inhibi tion of Na+, K+, Cl- cotransport by activators of P-2-purinoceptors. ( C) 1998 Elsevier Science B.V.