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Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non‐specific attenuation of receptor‐dependent cell activation?
Author(s) -
Hecker Markus,
Lückhoff Andreas,
Busse Rudi
Publication year - 1993
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041560317
Subject(s) - protein kinase c , bradykinin , phorbol , bisindolylmaleimide , pkc alpha , cytosol , microbiology and biotechnology , biology , intracellular , receptor , chemistry , phosphorylation , biochemistry , enzyme
Abstract Receptor‐mediated elevations of intracellular Ca 2+ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI 2 ) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol‐12‐myristate‐13‐acetate (PMA) (3‐300 nM) or 1‐oleyl‐2‐acetyl‐glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI 2 from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol‐12, 13‐didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN‐01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin‐stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN‐01) elevation of intracellular Ca 2+ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca 2+ or basal endothelial autacoid release. Several isoforms of PKC (namely PKC α , PKC δ , PKC ϵ , and PKC ζ ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype‐specific anti‐PKC antibodies, which, except PKC ϵ , were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane‐bound PKC α immunoreactivity, whereas there was no translocation of PKC α from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor‐dependent agonist. © 1993 Wiley‐Liss, Inc.