Vol. 286, Issue 1, 36-43, July 1998

Protein Kinase C Does Not Mediate Phenylephrine-Induced Down-regulation of Madin-Darby Canine Kidney Cell Alpha-1B Adrenoceptors¹

Ming Yang, Rainer Büscher, Katsunari Taguchi, Burghard Grübbel, Paul A. Insel and Martin C. Michel

Department of Medicine, University of Essen, 45122 Essen, Germany (M.Y., K.T., B.G., M.C.M.), and Department of Pharmacology, University of California San Diego, La Jolla, California (R.B., P.A.I.)

We examined the down-regulation of alpha-1B adrenoceptors in Madin-Darby canine kidney D₁ (MDCK) cells with an emphasis on a possible role of protein kinase C. The alpha-1 adrenoceptor agonist phenylephrine (1-100 µM) concentration-dependently down-regulated alpha-1B adrenoceptors in MDCK cells. Down-regulation by 100 µM phenylephrine was detectable after 2 hr and maximal after 8 to 24 hr. The receptor down-regulation was accompanied by a decrease in phenylephrine-stimulated inositol phosphate formation but not by an altered expression of immunodetectable G_q/11 alpha subunits. Even though alpha-1B adrenoceptor and P₂ purinergic receptor stimulation promote prostaglandin E₂ formation, receptor down-regulation was not prevented by indomethacin (10 µM) treatment but was partly mimicked by treatment with the purinergic receptor agonists adenosine-5'-O-(3-thio)triphosphate and 2-methylthio-ATP (300 µM each). Phorbol-12-myristate-13-acetate (1-100 nM) concentration-dependently down-regulated MDCK alpha-1B adrenoceptors to a greater extent than did phenylephrine. Three protein kinase C inhibitors, H7 (100 µM), staurosporine (100 nM) and KT5926 (1 µM), markedly attenuated receptor down-regulation promoted by phorbol ester but did not affect that by phenylephrine. Two inhibitors of Ca⁺⁺/calmodulin protein kinase pathways, KT5926 (1 µM) and W-7 (30 µM), also failed to prevent phenylephrine-induced down-regulation of alpha-1B adrenoceptors. We conclude that agonist-induced down-regulation of MDCK cell alpha-1B adrenoceptors is mimicked by a protein kinase C-activating phorbol ester but that the second messenger kinases protein kinase C and Ca⁺⁺/calmodulin protein kinase do not mediate agonist-induced down-regulation of the alpha-1B adrenoceptor.