Receptor-mediated inhibition of renal Na(+)-K(+)-ATPase is associated with endocytosis of its alpha- and beta-subunits.
The mechanisms involved in receptor-mediated inhibition of Na(+)-K(+)-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na(+)-K(+)-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na(+)-K(+)-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the alpha-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the beta-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na(+)-K(+)-ATPase activity in proximal tubules is associated with internalization of its alpha- and beta-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na(+)-K(+)-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.
Chibalin
A V
AV
Department of Molecular Medicine, Karolinska Institutet, Rolf Luft Center for Diabetes Research, Karolinska Hospital, Stockholm, Sweden.
Katz
A I
AI
Berggren
P O
PO
Bertorello
A M
AM
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002966:Clathrin / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D004298:Dopamine / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008954:Models, Biological; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D002966:Clathrin; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase; D004298:Dopamine