Dopamine-induced inhibition of Na+-K+-ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells.
The present study evaluated the importance of the association between Na+-K+-ATPase and the actin cytoskeleton on dopamine-induced inhibition of Na+-K+-ATPase activity. The approach used measures the transepithelial transport of Na+ in monolayers of opossum kidney (OK) cells, when the Na+ delivered to Na+-K+-ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 microg mL-1) induced increase in short-circuit current (Isc) was prevented by ouabain (100 microM) or removal of apical Na+. Dopamine (1 microM) applied from the apical side significantly decreased (29 +/- 5% reduction) the amphotericin B-induced increase in Isc, this being prevented by the D1-like receptor antagonist SKF 83566 (1 microM) and the protein kinase C (PKC) inhibitor chelerythrine (1 microM). Exposure of OK cells to cytochalasin B (1 microM) or cytochalasin D (1 microM), inhibitors of actin polymerization, from both cell sides reduced by 31 +/- 4% and 36 +/- 3% the amphotericin B-induced increase in Isc and abolished the inhibitory effect of apical dopamine (1 microM), but not that of the PKC activator phorbol-12,13-dibutyrate (PDBu; 100 nM). Colchicine (1 microM) failed to alter the inhibitory effects of dopamine. The relationship between Na+-K+-ATPase and the concentration of extracellular Na+ showed a Michaelis-Menten constant (Km) of 44.1 +/- 13.7 mM and a Vmax of 49.6 +/- 4.8 microA cm-2 in control monolayers. In the presence of apical dopamine (1 microM) or cytochalasin B (1 microM) Vmax values were significantly (P < 0.05) reduced without changes in Km values. These results are the first, obtained in live cells, showing that the PKC-dependent inhibition of Na+-K+-ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na+-K+-ATPase.
Gomes
P
P
Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.
Soares-da-Silva
P
P
D000199:Actins / Q000378:metabolism*; D000470:Alkaloids; D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D003572:Cytochalasins / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology*; D004553:Electric Conductivity; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D016015:Logistic Models; D009893:Opossums; D010617:Phenanthridines / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000199:Actins; D000470:Alkaloids; D053119:Benzophenanthridines; D003572:Cytochalasins; D010617:Phenanthridines; D015240:Phorbol 12,13-Dibutyrate; D000666:Amphotericin B; C016299:chelerythrine; D000254:Sodium-Potassium-Exchanging ATPase; D003078:Colchicine; D004298:Dopamine