Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane

Although in LLC-PK cells ATP depletion has been shown to result in alterati ons of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, t here is little information concerning the regulation of this exchanger in t he distal luminal membrane by ATP and actin filaments. The present study ex amined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 muM ATP in the luminal membrane vesicles from rabbit proximal tubule s did not influence the Ethyl Isopropyl Amiloride sensitive Nat uptake by t hese membranes. in contrast, the same treatment of luminal membranes from d istal tubules significantly enhanced the exchanger activity from 0.22 +/- 0 .04 to 0.39 +/- 0.08 pM/mug/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATP<gamma>s, the effect on the distal luminal membra ne was strongly diminished suggesting that the action of the nucleotide imp licates a phosphorylation step. Confirming this hypothesis, addition of 300 -muM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described b etween ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 muM cyto chalasin B in the distal luminal membrane vesicles induced, as observed wit h ATP, a significant increase in the Na+ uptake. However, the actions of AT P and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal lumi nal isoform through an intramembranous mechanism and secondly, a phosphoryl ation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mec hanisms. (C) 2001. Wiley-Liss. Inc.