PROTEIN-KINASE-A INDUCES RECRUITMENT OF ACTIVE NA-ATPASE UNITS TO THEPLASMA-MEMBRANE OF RAT PROXIMAL CONVOLUTED TUBULE CELLS(,K+)

1. The aim of this study was to investigate the mechanism of control o f Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10(-3) M dibutyryl-cAMP (db-cAMP) or 8 -bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclas es by 10(-5) M forskolin) on Na+,K+-ATPase activity and membrane traff icking. 2. PKA activation stimulated both the cation transport and hyd rolytic activity of Na+,K+- ATPase by about 40%. Transport activity st imulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive Rb-86(+) uptake in a time- and dose- dependent fashion; (2) this effect was abolished by addition of H-89 o r Rp-cAMPS, two structurally different PKA inhibitors; and (3) this st imulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activit y of Na+,K+-ATPase was accounted for by an increased maximal ATPase ra te (V-max) without alteration of the efficiency of the pump, suggestin g that cAMP-PKA pathway was implicated in membrane redistribution cont rol. 3. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase act ivity by db-cAMP was associated with an increase (+40%) in Na+,K+-ATPa se units expressed at the cell surface which was assessed by Western b lotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30%) in pump units located in the subcellular fraction correspondin g to early endosomes. 4. In conclusion, PKA stimulates Na+,K+-ATPase a ctivity, at least in part, by increasing the number of Na+-K+ pumps in the plasma membrane in proximal convoluted tubule cells.