Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to i nvestigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity i n microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct ce lls. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase fr om rat CCDs as well as the ouabain-sensitive component of Rb-86(+) uptake b y rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinyl ation procedure revealed that db-cAMP increased the amount of Na,K-ATPase a t the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1 .3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degreesC) prevented bo th the db-cAMP-dependent increase in cell surface expression and activity o f Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly sugge st that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD resul ts from the translocation of active pump units from an intracellular compar tment to the plasma membrane.