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 ( I-p) generated by the Na,K-ATPase was measured in A6 epithelia apicall y permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate ( PMA) produced a decrease in I-p carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunit s (similar to 30% reduction within 25 min, maximum after 40 min) indep endent of the PKC phosphorylation site (T15A/S16A). In addition to thi s major effect of PMA, which was independent of the intracellular sodi um concentration and was prevented by the PKC inhibitor bisindolylmale imide GF 109203X (BIM), another BIM-resistant, PKC site-independent de crease was observed when the I-p was measured at low sodium concentrat ions (total reduction similar to 50% at 5 mM sodium). Using ouabain bi nding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatm ent stimulated fluid phase endocytosis sevenfold and decreased by 16.5 % the basolateral cell surface area measured by transepithelial capaci tance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, t o a withdrawal of sodium pumps from the basolateral cell surface indep endent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.