Aldosterone stimulates vacuolar H⁺-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

Abstract

Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(αq) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(αq) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.

Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis

“…Both our in vivo and in vitro data demonstrate that effectors of the PKA (cpt-cAMP) and PKC (DOG) pathways mimic the effect of aldosterone on caput clear cell activity. These results are in agreement with our previous studies showing activation of V-ATPase-dependent proton secretion in renal intercalated cells by these agonists (63). Previous results from our group have also shown that luminal delivery of drugs that stimulate either the PKA or PKC pathways increase clear cell activity (6,40).…”

“…These results indicate an amplification of the amount of V-ATPase present at the cell surface via either activation of the exocytosis of V-ATPase-rich vesicles or via inhibition of endocytosis. As a positive control we also analyzed sections from the kidney of mice treated with aldosterone, cpt-cAMP, or DOG and observed increased V-ATPase-rich microvilli projections in intercalated cells (data not shown), similar to our recently published observations (43,63). In addition to being regulated via recycling mechanisms, previous studies conducted mainly in yeast and insects have shown that V-ATPase activity can also be regulated via local assembly/disassembly of V-ATPase subunits (12,30,57).…”

“…Previous results from our group have also shown that luminal delivery of drugs that stimulate either the PKA or PKC pathways increase clear cell activity (6,40). In the kidney it was suggested that the PKC pathway contributes more than the PKA pathway to the nongenomic effect of aldosterone on intercalated cell V-ATPase activity (63). In the epididymis we show here that the effect of aldosterone is PKC but not PKA dependent because chelerythrine, but not mPKI inhibited the stimulatory effect of aldosterone on V-ATPase activity.…”

“…A 200-l bolus of either PBS, aldosterone (200 nM, Sigma-Aldrich), 8-(4-chlorophenylthio)adenosine 3=,5=-cylic monophosphate (cpt-cAMP; 20 mM, Sigma-Aldrich), or 1,2-dioctanoyl-sn-glycerol (DOG; 20 M, Sigma-Aldrich) was first injected and was then followed by an infusion of 600 l of either PBS or drug over 15 min using an infusion pump (KdScientific, Holliston, MA) at a rate of 2.4 ml/h. The doses used here are based on previously published studies showing activation of V-ATPase-proton secretion in renal intercalated cells by these agonists in vivo (43,63,64). The final aldosterone concentration injected into the animals was estimated to be ϳ1.55 nM, based on an average extracellular fluid volume of 103 ml for an adult rat (21,33,36,60).…”

“…Traditionally the cellular responses to aldosterone have been associated with genomic effects. However, recent studies have also shown a direct nongenomic regulation of intercalated cell function in the kidney by aldosterone (63,64). Intercalated cells are similar to epididymal clear cells; they can also express high levels of V-ATPase in their plasma membrane and are involved in luminal acidification in the kidney.…”