The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5´-[γ-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AlF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca2+-free conditions. In ileum, PDBu translocated PKCs α, β1, β2, ϵ and θ to the membrane fraction, and GTP[S] caused a small translocation of PKC-ϵ. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-ϵ was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs α, β1, β2, ϵ and θ do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.
Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle
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Peter E. JENSEN, Ming C. GONG, Avril V. SOMLYO, Andrew P. SOMLYO; Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle. Biochem J 1 September 1996; 318 (2): 469–475. doi: https://doi.org/10.1042/bj3180469
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