NAD +-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD + hydrolysis

Abstract

We previously demonstrated inhibition of Na +-dependent 32P i transport in canine renal brush-border membranes in association with NAD +-induced ADP ribosylation of membrane protein(s) and postulated that NAD + inhibits P i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD + resulted in release of P i which was prevented by EDTA. It was proposed that NAD +-mediated inhibition of 32P i transport might occur through this mechanism. To determine whether NAD + inhibited 32P i transport by a mechanism other than or in addition to release of P i, we compared Na +-dependent 32P i counterflow in brush-border membrane equilibrated with P i or with P i generated from NAD +. Release of P i from NAD + incubated with brush-border membrane was confirmed. The increased uptake of 32P i which was demonstrated in brush-border membrane equilibrated with P i was not measured when intravesicular P i was generated from a concentration of NAD + which effected ADP-ribosylation of brush border membranes (100 μM NAD +). In contrast, increased uptake of 32P i was demonstrated when intravesicular P i was generated from 1 μM NAD + which did not effect ADP ribosylation. Mg 2+-dependent ADP ribosylation of brush-border membrane incubated with NAD + was demonstrated which persisted during the time interval of 32P i uptake measurements. Our findings are compatible with the hypothesis that NAD +-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg 2+ and decreased ADP ribosylation.