Na(+)-K+(NH4+)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PKC, and 20-HETE

Abstract

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.

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid ω-hydroxylase activity

“…An increased formation of 20-HETE in the SHR kidney relative to age-matched normotensive WKY rats has been associated with increased renal interlobular and afferent arteriolar resistance (12,15,19). On the other hand, the natriuretic and diuretic effects of 20-HETE would oppose the vascular effects of this eicosanoid and are considered antihypertensive (3,30,38). The overall effect of 20-HETE on blood pressure will be a function of the relative rates of 20-HETE production and the inhibitor concentration in the renal vasculature and tubules.…”

“…It has been reported that 20-HETE inhibits Na ϩ -K ϩ -ATPase, a Na ϩ -K ϩ -2Cl Ϫ cotransporter, and 70-pS K ϩ channels in renal tubules and results in natriuresis and diuresis (3,30,38). Urine volume and urinary Na ϩ , K ϩ , and creatinine levels were measured before and after 10-SUYS treatment to assess its effect on renal function.…”

“…Additional roles for 20-HETE include mediation of the myogenic response of small cerebral and renal arteries to elevations in transmural pressure, and the autoregulation of cerebral and renal blood flow, glomerular filtration rate, and tubular glomerular feedback (13,22,58). In renal tubules 20-HETE inhibits Na ϩ -K ϩ -ATPase, a Na ϩ -K ϩ -2Cl Ϫ cotransporter, and 70-pS K ϩ channels, leading to natriuresis and diuresis (3,30,38). EETs and DHETs are generally considered vasodilatory, although they exhibit both vasodilatory and vasoconstrictive properties in vitro, depending on the vascular bed and species that are studied (20,55,57).…”

NKCC2 Surface Expression in Mammalian Cells

“…Calibration of the 2Ј,7Ј-bis(carboxyethyl)-5,6-carboxyfluorescein excitation ratio was accomplished using the nigericin technique. Na-K2Cl co-transport activity was measured as bumetanide-sensitive NH 4 influx, as described previously (31,32). Cells were first bathed at 37°C in a CO 2 -free Hepes/Tris-buffered medium containing (in mM) 135 NaCl, 10 Hepes (pH 7.4), 5 KCl, 1 MgCl 2 , 0.8 KH 2 PO 4 , 0.2 K 2 HOP 4 , 1 CaCl 2 , and 10 BaCl 2 , to measure base-line pH i .…”

“…Na-K ϩ (NH 4 ϩ )-2Cl transport activity was assessed by estimating the rate of intracellular acidification caused by entry into the cells of NH 4 ϩ via this transport mechanism after abrupt application of 20 mM NH 4 Cl to the cells (31,32,59). Importantly, previous studies conducted in MTAL (31,32) and OK cells (60) clearly demonstrated that neither Na/H (NH 4 ϩ ) exchanger nor Na-K(NH 4 ϩ )-ATPase are involved in the NH 4 ϩ -induced intracellular acidification observed under these experimental conditions. More importantly, Chen and Kempson (60) reported that under control conditions, NH 4 ϩ entry into OK cells occurs mainly via a barium inhibitable component.…”