Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.
The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.
Beron
J
J
Institute of Physiology, University of Zurich, Switzerland.
Forster
I
I
Beguin
P
P
Geering
K
K
Verrey
F
F
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D004357:Drug Synergism; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004848:Epithelium / Q000201:enzymology / Q000502:physiology; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology*; D014982:Xenopus laevis
D026941:Sodium Channel Blockers; D015222:Sodium Channels; D000584:Amiloride; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D013755:Tetradecanoylphorbol Acetate