Endothelin (ETA) receptor number and calcium signalling are up-regulated by protein kinase C-beta 1 overexpression.
To evaluate the role of protein kinase C (PKC) in regulation of cellular responsiveness to mitogens, we used rat 6 (R6) fibroblasts that stably overexpress the beta 1 isoenzyme of protein kinase C (PKC-beta 1). The potent vasoconstrictor and mitogen endothelin-1 (ET-1; 100 nM) was substantially more effective in stimulating InsP3 accumulation in PKC-beta 1-overexpressing fibroblasts (PKC3 cells) than in control fibroblasts lacking the PKC-beta 1 cDNA insert. PKC3 cells were found to express a 7-fold greater number of endothelin receptors than did control cells, whereas both cell lines showed equivalent Kd values. These receptors were of the ETA subtype, as defined by a 1000-fold greater affinity for ET-1 than for ET-3. Changes in intracellular free Ca2+ levels ([Ca2+]i) in response to ET-1 measured with the fluorescent Ca2+ indicator fura-2 showed that ET-1 was more potent and efficacious in stimulating [Ca2+]i in PKC3 cells than in control fibroblasts. The ET-1-induced Ca2+ rise was completely blocked by the selective ETA antagonist BQ123, but only slightly diminished by extracellular application of 2 mM EGTA. In contrast with the effects of PKC-beta 1 overexpression on responsiveness to ET-1, alpha-thrombin, which was previously found to have a weaker effect on InsP3 accumulation in PKC-beta 1-overexpressing cells, was also a less effective stimulator of [Ca2+]i in PKC3 cells than in control cells. These results demonstrate that, although the Ca2+ response to alpha-thrombin is diminished by PKC-beta 1 overexpression, ETA receptor number and cellular responsiveness to ET-1 are increased in PKC-beta 1-overexpressing cells.
Pachter
J A
JA
Schering-Plough Research Institute, Kenilworth, NJ 07033-0539.
Mayer-Ezell
R
R
Cleven
R M
RM
Fawzi
A B
AB
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D007295:Inositol Phosphates / Q000096:biosynthesis; D007527:Isoenzymes / Q000378:metabolism*; D011493:Protein Kinase C / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017466:Receptors, Endothelin / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D015854:Up-Regulation
D007295:Inositol Phosphates; D007527:Isoenzymes; D017466:Receptors, Endothelin; D004247:DNA; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium