POSSIBLE ROLE OF ATYPICAL PROTEIN-KINASE-C ACTIVATED BY ARACHIDONIC-ACID IN CA2-MUSCLE( SENSITIZATION OF RABBIT SMOOTH)

1. Diacylglycerol (DAG; 10 mu M), an activator of conventional and nov el 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 lig ht chain kinase. 2. Ca2+ sensitization by DAG was specifically inhibit ed by a pseudosubstrate peptide inhibitor of cPKCs (PKC alpha 22-30 pe ptide; 50 mu M). Similarly, GF 109203X (600 nM), an inhibitor of cPKCs and nPKCs, completely abolished Ca2+ sensitization by phorbol 12,13-d ibutyrate (PDBu; 1 mu M). In contrast, Ca2+ sensitization induced by t he alpha(1)-adrenergic agonist phenylephrine (100 mu M) was not inhibi ted by these inhibitors of cPKCs and nPKCs. 3. A pseudosubstrate pepti de inhibitor of the atypical PKCs (aPKCs) PKC zeta(116-124) (50 mu M) significantly (about 50 %) inhibited the Ca2+ sensitization of force a nd MLC20 phosphorylation induced by 100 mu M phenylephrine and by 300 mu M arachidonic acid, but not that by DAG (10 mu M) or PDBu (1 mu M). 4. A phospholipase A(2) (PLA(2)) inhibitor, ONO-RS-082 (10 mu M), abo lished the release of arachidonic acid and partially (by 40%) inhibite d 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 t heir effects via the same pathway, probably through activation of aPKC (s) by arachidonic acid. 5. Western blot analysis with antibodies to a PKCs revealed aPKCs zeta, lambda (or iota) and an unidentified 64 kDa protein. The distribution (cytosolic and particulate) of these protein s was not affected by PDBu (1 mu M). 6. Our results are consistent wit h a significant role for atypical (or related) PKCs through a PLA(2)-a rachidonic 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.