RAPID NON-GENOMIC STIMULATION OF VACUOLAR H+-ATPase ACTIVITY IN OUTER MEDULLARY COLLECTING DUCT INTERCALATED CELLS BY ALDOSTERONE.

C. Winter (1,2), N. Schulz (1), G. Giebisch (2), J. P. Geibel (2,3), C. A. Wagner (1).
(1) Institute of Physiology, University of Zurich, Zurich, Switzerland, Departments of (2) Cellular and Molecular Physiology and (3) Surgery, School of Medicine, Yale University, New Haven, CT, USA.

Systemic sodium and acid-base homeostasis are tightly linked as evident from inborn and acquired syndromes of disturbed Na+ homeostasis leading to metabolic acidosis or alkalosis, respectively. One major mechanism connecting Na+ and acid-base homeostasis is the renin-angiotensin-aldosterone system controlling Na+-(re)absorption in the colon and kidney and thus blood pressure. Besides its role in blood pressure regulation, angiotensin II has also been shown to exert stimulatory effects on renal transporters involved in acid-base homeostasis such as Na+/H+-exchange, Na+/HCO3-cotransport and vacuolar H+-ATPase activity. In addition, chronic elevation of aldosterone levels stimulates renal bicarbonate reabsorption and proton secretion. This effect has been mainly attributed to indirect coupling of Na+-reabsorption and H+-secretion. Here we investigated the direct, short-time effect of aldosterone on vacuolar H+-ATPase activity in intercalated cells of freshly isolated mouse outer medullary collecting ducts (OMCD). H+-ATPase activity was measured using the intracellular pH-sensitive dye BCECF to monitor intracellular pH recovery after an acid-load (NH4Cl-pulse) which was sensitive to the vacuolar H+-ATPase specific inhibitor concanamycin (200 nM). Exposure to aldosterone (10 nM) for 20 min increased vacuolar H+-ATPase activity approximately 2-3 fold. The mineralocorticoid receptor agonist spironolactone (10 mM) did not prevent the aldosterone induced stimulation. Moreover, inhibition of transcription or translation with actinomycin D or cycloheximide also failed to prevent aldosterone induced vacuolar H+-ATPase stimulation. Spironolactone, actinomycin D or cycloheximide alone had no effect on vacuolar H+-ATPase activity. Western blotting of medullary membrane protein fractions from mice injected with aldosterone (30 min) showed no change in ATP6V0A4 (a4 subunit isoform) protein abundance. Incubation of OMCDs with colchicine (100 mM), however, abolished the stimulatory effect of aldosterone suggesting that the integrity of the microtubular network was necessary for vacuolar H+-ATPase stimulation. Immunohistochemistry in kidneys from aldosterone injected mice was suggestive of increased apical H+-ATPase staining in OMCD intercalated cells. In summary, we demonstrate rapid non-genomic stimulation of vacuolar H+-ATPase activity in OMCD intercalated cells, an additional mechanism by which aldosterone could link Na+ and acid-base homeostasis.