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.

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na+-K+([Formula: see text])-2Cl−cotransport. ANG II at 10−16to 10−12 M inhibited 30–50% ( P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10−12 M ANG II level cotransport activity; eventually, 10−6 M ANG II stimulated 34% cotransport activity ( P < 0.003). Inhibition by 10−12M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10−12 M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10−6 M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus ≤10−12 M ANG II inhibits via 20-HETE, whereas ≥5 × 10−11 M ANG II stimulates via PKC Na+-K+([Formula: see text])-2Cl−cotransport; all ANG II effects involve AT1 receptors and PLC activation.