Endothelin (ETA) receptor number and calcium signalling are up-regulated by protein kinase C-β 1 overexpression

Abstract

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.

Activation of Endothelin ETA Receptors Induces Phosphorylation of α1b-Adrenoreceptors in Rat-1 Fibroblasts

“…Besides, pp42 and pp44 forms of mitogen-activated protein kinase are also phosphorylated in residues of tyrosine, and this effect is reduced by pretreatment with PTX, whereas the mentioned tyrosine phosphorylation of focal adhesion kinase remained intact (24). On the other hand, rat-6 fibroblasts, overexpressing PKC-␤1, showed an increased calcium signaling in the presence of endothelin; in this case, an increase in the number of ET A receptors was detected (47). It is possible that GRKs could be activated by ET A receptors; however, GRKs are reported to phosphorylate only receptors in the agonist-occupied state; the participation of these kinases in the heterologous phosphorylation of ␣ 1b -ARs seems unlikely.…”

Endothelin 1 Activation of Endothelin A Receptor/NADPH Oxidase Pathway and Diminished Antioxidants Critically Contribute to Endothelial Progenitor Cell Reduction and Dysfunction in Salt-Sensitive Hypertension

“…More importantly, the up-regulated expression of ET A receptors, but not ET B receptors, suggests that ET A receptors may be the therapeutic target for EPC dysfunction in salt-sensitive hypertension. It has been reported that protein kinase C (PKC) induces ET A receptors at a transcriptional level in rat fibroblasts 15 or in cardiac cells from DOCA-salt hypertensive 16 . However, whether these possibilities exist in EPCs remain unknown.…”

Osmolar regulation of endothelin signaling in rat renal medullary interstitial cells.

“…One possible mechanism could be homologous down-regulation by increased local production of the ET peptide. This is unlikely since RMICs have not been shown to produce ET in vivo (28) (30)(31)(32)(33). This is also unlikely since H-7, a PKC inhibitor, was without effect on the reduction in ET receptors following incubation with 600 mOsm/kg urea.…”