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Abstract

Full-grown prophase-arrested oocytes ofXenopus laevis were treated with 50nm phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50nm 4α-phorbol 12,13-didecanoate (4αPDD) that does not activate protein kinase C. The effects on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed.

(i) During application of PMA, current generated by the Na⁺/K⁺ pump decreases; in addition, Cl⁻ and K⁺ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4αPDD was ineffective.
(ii) Ouabain binding experiments demonstrate that PMA (K_1/2=7nm), but not 4αPPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The K_D value for ouabain binding is not influenced. 4αPDD was ineffective.
(iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area.

From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.