EFFECTS OF PH ON PHOSPHATE-TRANSPORT BY FLOUNDER RENAL TUBULE PRIMARY CULTURES

Transepithelial phosphate (P(i)) fluxes were determined in primary monolayer cultures of the winter flounder proximal tubule in Ussing chambers. Net P(i) secretion, peritubular (P)-to-lumen (L) net flux (J(net) = 17.0 +/- 3.77 nmol.cm-2.h-1), was strongly stimulated by lowering peritubular pH to 6.5 (pH(P) 6.6 vs. pH(L) 7.5) compared with control tissues at pH 7.5 (pH(P) 7.5 vs. pH(L) 7.5) where net reabsorption (L-to-P J(net) = 1.10 +/- 0.36 nmol.cm-2.h-1) occurred. The stimulation of net secretion by pH(P) 6.5 was inhibited to 27% of control by 200-mu-M amiloride. The imposition of a reversed pH gradient (pH(L) 6.5 vs. pH(P) 7.5) did not stimulate P(i) secretion. Preincubation with 0.5 U/ml phospholipase C caused a nearly fivefold stimulation of P(i) secretion compared with the untreated controls. H-7 (100-mu-M), a protein kinase inhibitor, caused a 2.5-fold reduction in phorbol ester (PE)-induced stimulation of P(i) secretion. H-7 also significantly inhibited P(i) secretion (50% reduction) when peritubular pH was lowered to 6.5. PE-induced net P(i) secretion was significantly lower when luminal pH was lowered to 5.5 compared with controls were luminal pH was kept at 7.5. Amiloride (200-mu-M) significantly inhibited the PE-induced net P(i) secretion in the presence of luminal pH 7.5 but had no significant effect at luminal pH 5.5. Replacement of luminal NaCl with LiCl or isosmolar mannitol significantly reduced net phosphate secretion under the conditions of lowered peritubular pH. Net P(i) secretion was also dependent on the maintenance of a cellular Na+ gradient, since 100-mu-M ouabain inhibited PE-induced net P(i) secretion. It was concluded that low peritubular pH may stimulate P(i) secretion in the flounder proximal tubule in part through an amiloride-sensitive protein kinase C-activated process.