Novel protein kinase C, nPKC, inhibition of murine bumetanide-sensitive Na+,K+,2Cl- cotransporter BSC1 in Xenopus oocyte.
In vitro transcribed RNAs obtained for the absorptive type of Na,K, 2Cl cotransporter BSC1, isoform F, and either of three deletion mutants were 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 deletion 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 constant (aKi) of 6 nM. A 15 nM PMA-elicited inhibition of Rb flux was blocked by the PKC inhibitor calphostin C (200 nM) to approximately 80%, while PKC inhibitors staurosporine (40 nM) and Gö6976 (50 nM) were ineffective. The phosphatase inhibitor okadaic acid (4.6 nM) did not influence PMA-induced flux inhibition. The PKC activator sn-1, 2-dioctanoyl glycerol (DOG), even at 2 microM, 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 microM forskolin, and/or 0. 5 mM theophylline had no effect on bumetanide-sensitive Rb fluxes. Activating endogenous muscarinic receptors with 20 microM acetylcholine had no effect on expressed BSC1 fluxes. The sensitivity to bumetanide of induced Rb flux had an aKi of around 70 nM. Attempts to follow the expression of BSC1 in plasma membranes with either 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 transport function.
Baekgaard
A
A
Dept. of Medical Physiology, Panum Institute, 2200 Copenhagen N, Denmark.
Bindslev
N
N
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018014:Gene Transfer Techniques; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins; D012413:Rubidium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014981:Xenopus
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011994:Recombinant Proteins; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D012413:Rubidium; D013755:Tetradecanoylphorbol Acetate