PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization

• Published in: Biochemical pharmacology 2015-06, Vol.95 (4), p.263-278
• Main Authors: El-Yazbi, Ahmed F., Abd-Elrahman, Khaled S., Moreno-Dominguez, Alejandro
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - metabolism; Actin cytoskeleton polymerization; Animals; Brain - blood supply; Brain - drug effects; Brain - metabolism; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Ca2+ sensitization; Cerebral arteries; Enzyme Activation; Heterocyclic Compounds, 4 or More Rings - pharmacology; Male; Middle Cerebral Artery - drug effects; Middle Cerebral Artery - physiology; Myosin Light Chains - metabolism; Phorbol 12,13-Dibutyrate - pharmacology; Phosphorylation; PKC; Polymerization; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Rats, Sprague-Dawley; Serotonin - pharmacology; Thiazolidines - pharmacology; Thin filament regulation; Vascular Resistance; Vasoconstriction - drug effects
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2015.04.011

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca2+-dependent PKC, and in the absence of extracellular Ca2+. Biochemical evidence indicates that either + or − extracellular Ca2+, PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.