Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle.
1. Diacylglycerol (DAG; 10 microM), an activator of conventional and novel protein kinases C (cPKCs and nPKCs), induced Ca2+ sensitization of force in isolated intact and alpha-toxin-permeabilized femoral artery (FA) and portal vein (PV), and increased the phosphorylation of myosin light chain (MLC20) at the same peptides phosphorylated by myosin light chain kinase. 2. Ca2+ sensitization by DAG was specifically inhibited by a pseudosubstrate peptide inhibitor of cPKCs (PKC alpha(22-30) peptide; 50 microM). Similarly, GF 109203X (600 nM), an inhibitor of cPKCs and nPKCs, completely abolished Ca2+ sensitization by phorbol 12,13-dibutyrate (PDBu; 1 microM). In contrast, Ca2+ sensitization induced by the alpha1-adrenergic agonist phenylephrine (100 microM) was not inhibited by these inhibitors of cPKCs and nPKCs. 3. A pseudosubstrate peptide inhibitor of the atypical PKCs (aPKCs) PKC zeta(116-124) (50 microM) significantly (about 50%) inhibited the Ca2+ sensitization of force and MLC20 phosphorylation induced by 100 microM phenylephrine and by 300 microM arachidonic acid, but not that by DAG (10 microM) or PDBu (1 microM). 4. A phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (10 microM), abolished the release of arachidonic acid and partially (by 40%) inhibited the Ca2+ sensitization induced by phenylephrine in FA smooth muscle. This effect was not additive to the inhibition observed with the aPKC inhibitor peptide, suggesting that arachidonic acid and aPKCs exert their effects via the same pathway, probably through activation of aPKC(s) by arachidonic acid. 5. Western blot analysis with antibodies to aPKCs revealed aPKCs zeta, lambda (or iota) and an unidentified 64 kDa protein. The distribution (cytosolic and particulate) of these proteins was not affected by PDBu (1 microM). 6. Our results are consistent with a significant role for atypical (or related) PKCs through a PLA2-arachidonic acid-aPKC pathway in agonist-induced Ca2+ sensitization, in parallel with a similar, but minor role of the DAG-cPKC cascade. The inability of the combination of the two (aPKC and cPKC) inhibitors to completely eliminate Ca2+ sensitization also suggests the presence of a third, still unidentified, pathway of this mechanism.
Gailly
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Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
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D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry; D007537:Isometric Contraction / Q000187:drug effects*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011169:Portal Vein; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D010738:Type C Phospholipases
D000316:Adrenergic alpha-Agonists; D004075:Diglycerides; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D018994:Myosin Light Chains; D010446:Peptide Fragments; C044295:1,2-dioctanoylglycerol; D010656:Phenylephrine; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010738:Type C Phospholipases; C070515:bisindolylmaleimide I; D002118:Calcium