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Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P 2Y ‐ and P 2U ‐purinoceptor‐stimulated prostacyclin release
Author(s) -
Patel Viral,
Brown Colin,
Boarder Michael R.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15374.x
Subject(s) - protein kinase c , prostacyclin , phospholipase c , phorbol , purinergic receptor , gene isoform , medicine , inositol , endocrinology , calphostin c , cytosol , gq alpha subunit , biology , chemistry , microbiology and biotechnology , receptor , g protein , biochemistry , adenosine , kinase , enzyme , gene
1 . Enhanced synthesis of prostacyclin (PGI 2 ) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co‐existing P 2Y ‐ and P 2U ‐purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2 . Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of α, ∍ and ζ, while no immunoreactivity was found for β, γ, δ, η and θ isoforms. PKC‐α was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X‐100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC‐∍ was always in a Triton X‐100 insoluble membrane fraction, while PKC‐ζ was found in both soluble and membrane bound (Triton X‐100 soluble) forms in the unstimulated cells and was unaffected by PM A. 3 . Treatment with PM A for 6 h led to a 90% downregulation of PKC‐α, while the immunoreactivity to the e and ζ isoforms remained largely unchanged. 4 . After either 10 min or 6 h exposure to PM A the PGI 2 response to activation of both receptors was enhanced, while the inositol 1,4,5‐trisphosphate response to P 2Y ‐purinoceptor activation was substantially attenuated and the P 2U ‐purinoceptor response was unchanged. Thus the PGI 2 response to PM A under conditions when 90% of the PKC‐α was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI 2 response does not correlate with the phospholipase C response. 5 . Inhibition of PKC with the isoform non‐selective inhibitors, Ro 31–8220 and Go 6850 abolished the PGI 2 response to both P 2U ‐ and P 2Y ‐purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca 2+ sensitive isoforms (such as PKC‐α) and not Ca 2+ insensitive isoforms (such as PKC‐∍), had no effect on the PGI 2 response. 6 . The results show that there is a requirement for PKC in the stimulation of PGI 2 production by endothelial P 2Y ‐ and P 2U ‐purinoceptors. Both downregulation and inhibition studies show that PKC‐α is not responsible for the regulation of the response to P 2 ‐purinergic stimulation, and imply that the response is mediated by PKC‐∍ (PKC‐ζ is unresponsive to PMA), or an as yet uncharacterized PKC isoform.