title: Complete inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase.
abstract: This study examines the involvement of hormones and neuromediators in the regulation of Na+, K+, Cl- cotransport in renal epithelial cells using Madin-Darby canine kidney cells with low transepithelial electrical resistance (194+/-47 Omega/cm2). In this cell line, Na+, K+, Cl- cotransport measured as bumetanide-sensitive 86Rb influx was inhibited up to 50-60% with agonists of P2-purinoceptors (ATP approximately ADP>UTP>AMP), 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, bradykinin, methacholine, propranolol, vasopressin, adenosine, dopamine and histamine. Thirty min of preincubation of MDCK cells with 0.1 microM PMA completely blocked the activity of Na+, K+, Cl- cotransport whereas down-regulation of this enzyme by 24 h of preincubation with 1 microM 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, an inhibitor of protein kinase activity, staurosporine, abolished the effect of 0.1 microM PMA but did not change inhibition of this carrier by ATP. Thus, these results show for the first time that P2-purinoceptors and PMA-sensitive isoforms of protein kinase C play a key role in the regulation of Na+, K+, Cl- cotransport in MDCK cells. These results also show that neither PMA- nor staurosporine-sensitive forms of protein kinase are involved in the inhibition of Na+, K+, Cl- cotransport by activators of P2-purinoceptors.
authors.affiliation: Centre de Recherche du CHUM, Pavillon Hôtel-Dieu, Laboratory of Molecular Pathophysiology, 3850 St. Urbain St., Montréal, Qué., Canada.
authors.forename: F
authors.initials: F
authors.lastname: Gagnon
authors.forename: S N
authors.initials: SN
authors.lastname: Orlov
authors.forename: J
authors.initials: J
authors.lastname: Tremblay
authors.forename: P
authors.initials: P
authors.lastname: Hamet
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002460:Cell Line; D004285:Dogs; D004789:Enzyme Activation; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D028021:Sodium-Potassium-Chloride Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D002352:Carrier Proteins; D058910:Purinergic P2 Receptor Agonists; D028021:Sodium-Potassium-Chloride Symporters; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate