Na(+)-K+(NH4+)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PKC, and 20-HETE.
Cell pH was monitored in suspensions of medullary thick ascending limbs (MTALs) of rat kidney to determine possible effects of various transduction pathways on apical Na(+)-K+ (NH4+)-2Cl- cotransport, the activity of which was measured as the bumetanide-sensitive component of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8-Bromoadenosine 3',5'-cyclic monophosphate stimulated cotransport activity through activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), since the cAMP effect was abolished by N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89); stimulation by cAMP (P < 0.02) was observed even when other Na+, Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2-carboxylate, and barium, which indicates that cotransport was directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated cotransport activity (P < 0.03), which was abolished by protein kinase C (PKC) blockade by staurosporine. In contrast, cotransport activity was reduced (P < 0.001) by arachidonic acid or 20-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic acid or ionomycin was abolished by econazole and SKF-525A that inhibit cytochrome P-450-dependent monoxygenase, which produces 20-HETE from arachidonic acid in the MTAL, and the ionomycin effect was prevented when phospholipase A2 (PLA2) was blocked by 4-bromophenacyl bromide or oleyloxyethyl phosphorylcholine. The results demonstrate that MTAL apical Na(+)-K+(NH4+)-2Cl- cotransport is stimulated by PKA and PKC and inhibited by 20-HETE that may be produced after a rise in [Ca2+]i through PLA2 activation.
Amlal
H
H
Institut National de la Santé et de la Recherche Médicale Unité 356, Université Pierre et Marie Curie, Paris, France.
Legoff
C
C
Vernimmen
C
C
Paillard
M
M
Bichara
M
M
D000641:Ammonia / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D007679:Kidney Medulla; D008138:Loop of Henle / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D006893:Hydroxyeicosatetraenoic Acids; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D016718:Arachidonic Acid; D000641:Ammonia; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C