Hormones - Cytokines - Signaling

Kidney International (1997) 52, 329–337; doi:10.1038/ki.1997.338

Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells

Andrea Huwiler, Verena A Briner, Doriano Fabbro and Josef Pfeilschifter

Department of Pharmacology, Biozentrum, University of Basel, Basel, Department of Medicine, Kantonsspital, Luzern, and Research Department, Pharmaceuticals Division, Novartis, Basel, Switzerland; and Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

Correspondence: Prof Josef Pfeilschifter, Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: Pfeilschifter@em.uni-frankfurt.de

Received 11 January 1995; Revised 3 March 1997; Accepted 3 March 1997.

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Abstract

Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells. In glomerular endothelial cells, extracellular ATP stimulates a phospholipase C with subsequent hydrolysis of polyphosphoinositides and an increase in cytosolic free Ca2+ concentration ([Ca2+]i). Short-term (30 min) pretreatment of endothelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), decreases the ATP-stimulated phosphoinositide degradation and Ca2+ mobilization. However, this inhibition was lost after incubating the cells for four hours with TPA. Longer-term pretreatment (10 to 48 hr) even potentiated ATP-induced phosphoinositide breakdown and Ca2+ mobilization. In addition, pretreating the cells for 30 minutes with the specific PKC inhibitor Ro 31–8220 dose-dependently increased ATP-stimulated phosphoinositide hydrolysis, thus clearly indicating a regulatory role for PKC in the inositol lipid signaling pathway in glomerular endothelial cells. By using specific antibodies recognizing the different PKC isoenzymes, it is observed that glomerular endothelial cells express five isoenzymes: PKC-alpha, -delta, -epsilon, -zeta and -theta. No PKC-beta, -gamma, -eta and -mu isoenzymes were detected. On exposure to TPA, a complete depletion of PKC-alpha is observed within four hours. In contrast, PKC-epsilon was more resistant to phorbol ester, and even after 48 hours of TPA treatment, only 60% of PKC-epsilon was down-regulated. PKC-zeta decreased very slowly from the cytosol (47% left after 24 hr of phorbol ester treatment) and translocated to the Triton XlOO-insoluble fraction. Moreover, PKC-delta and PKC-zeta were not significantly affected by 48 hours of phorbol ester incubation. Thus, only PKC-alpha is depleted with a kinetic that corresponds to the loss of feedback inhibition of ATP-stimulated phosphoinositide turnover. In the next step, [Ca2+]i changes were measured in single cells loaded with Fura-2 after microinjection of neutralizing PKC isoenzyme-specific antibodies. Injection of antibodies specific for PKC-alpha potently increased Ca2+ mobilization in response to ATP stimulation when compared to cells injected with buffer only or antibodies specific for PKC-epsilon. These results provide evidence that PKC-alpha mediates feedback inhibition of ATP-stimulated phosphoinositide hydrolysis in glomerular endothelial cells.

Keywords:

feedback regulation, desentization, adenosine triphosphate, protein kinase C, endothelial cells, kinetics

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