PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization.
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 Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), 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.
Smooth Muscle Research Group, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr NW, T2N4N1 Calgary, Alberta, Canada; Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, P.O. Box 21521 El-Mesallah, Alexandria, Egypt. Electronic address: ahmed.fawzy.aly@alexu.edu.eg.
Ahmed F
AF
El-Yazbi
Smooth Muscle Research Group, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr NW, T2N4N1 Calgary, Alberta, Canada. Electronic address: kshabdel@ucalgary.ca.
Khaled S
KS
Abd-Elrahman
Smooth Muscle Research Group, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr NW, T2N4N1 Calgary, Alberta, Canada. Electronic address: amoreno@ucalgary.ca.
Alejandro
A
Moreno-Dominguez
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000098:blood supply* / Q000187:drug effects / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D004789:Enzyme Activation; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D008297:Male; D020768:Middle Cerebral Artery / Q000187:drug effects / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D058105:Polymerization; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D014655:Vascular Resistance; D014661:Vasoconstriction / Q000187:drug effects
D019086:Bridged Bicyclo Compounds, Heterocyclic; D006576:Heterocyclic Compounds, 4 or More Rings; D018994:Myosin Light Chains; D053778:Thiazolidines; C472645:blebbistatin; D012701:Serotonin; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; C037068:latrunculin B
Actin cytoskeleton polymerization; Ca(2+) sensitization; Cerebral arteries; PKC; Thin filament regulation