NOVEL PROTEIN-KINASE-C, NPKC, INHIBITION OF MURINE BUMETANIDE-SENSITIVE NA-COTRANSPORTER BSC1 IN XENOPUS OOCYTE(,K+,2CL)

In vitro transcribed RNAs obtained for the absorptive type of Na,K,2Cl cotransporter BSC1, isoform F, and either of three deletion mutants w ere injected into oocytes, while oocytes injected with water served as controls. Wild-type cRNA induced a bumetanide-sensitive Rb flux after 18 h which rose to a maximum of about 600 pmol.(h.oocyte)(-1) during the next 24 h. This level of flux lasted for at least 31/2 days. All d eletion mutants were either not expressed and/or were non-functional. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate ( PMA) eliminated the induced flux, with an apparent dissociation consta nt (aK(i)) of 6 nM. A 15 nM PMA-elicited inhibition of Rb flux was blo cked by the PKC inhibitor calphostin C (200 nM) to approximate to 80%, while PKC inhibitors staurosporine (40 nM) and Go6976 (50 nM) were in effective. The phosphatase inhibitor okadaic acid (4.6 nM) did not inf luence PMA-induced flux inhibition. The PKC activator sn-1,2-dioctanoy l glycerol (DOC), even at 2 mu M, did not inhibit bumetanide-sensitive Rb influx. The induced Rb fluxes were not affected by PKA or PKG, as stimulation by either 0.5 mM dibutyryl-cAMP, 0.5 mM dibutyryl-cGMP, 10 mu M forskolin, and/or 0.5 mM theophylline had no effect on bumetanid e-sensitive Rb fluxes. Activating endogenous muscarinic receptors with 20 mu M acetylcholine had no effect on expressed BSC1 fluxes. The sen sitivity to bumetanide of induced Rb flux had an aK(i) of around 70 nM . Attempts to follow the expression of BSC1 in plasma membranes with e ither immunoblotting or radio-methionine pulse-chase were unsuccessful . We conclude that a PKC, likely of the novel type, nPKC, seems to be involved in PMA-induced reduction of expressed BSC1 and/or its ion tra nsport function.