Possible role of atypical protein kinase C activated by arachidonic acid in Ca²⁺ sensitization of rabbit smooth muscle

1. Diacylglycerol (DAG; 10 μM), an activator of conventional and novel protein kinases C (cPKCs and nPKCs), induced Ca²⁺ sensitization of force in isolated intact and α-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 (PKCα_22-30 peptide; 50 μM). Similarly, GF 109203X (600 nM), an inhibitor of cPKCs and nPKCs, completely abolished Ca²⁺ sensitization by phorbol 12,13-dibutyrate (PDBu; 1 μM). In contrast, Ca²⁺ sensitization induced by the a₁-adrenergic agonist phenylephrine (100 μM) was not inhibited by these inhibitors of cPKCs and nPKCs. 3. A pseudosubstrate peptide inhibitor of the atypical PKCs (aPKCs) PKCζ_116-124 (50 μM) significantly (about 50%) inhibited the Ca²⁺ sensitization of force and MLC₂₀ phosphorylation induced by 100 μM phenylephrine and by 300 μM arachidonic acid, but not that by DAG (10 μM) or PDBu (1 μM). 4. A phospholipase A₂ (PLA₂) inhibitor, ONO-RS-082 (1O μM), 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 ζ, λ (or ι) and an unidentified 64 kDa protein. The distribution (cytosolic and particulate) of these proteins was not affected by PDBu (1 μM). 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.