Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice

Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na(+)/H(+) exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO(2)/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH(2)O by the addition of mannitol. NHE activity was assessed by Na(+)-dependent acid extrusion rates (J(H)) after an acid load with NH(4)Cl prepulse. Under isosmotic conditions, J(H) values at a wide intracellular pH (pH(i)) range of 6.20-6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on J(H) at the wide pH(i) range. In contrast, in KO mice, hyperosmotic mannitol increased J(H) at a pH(i) range of 6.20-6.45 and shifted the J(H)-pH(i) relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na(+). Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased J(H) (at pH(i) 6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in J(H). The stimulatory effect of hyperosmotic mannitol on J(H) was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased J(H). We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.