Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca²⁺ sensitization of myosin light chain phosphorylation in smooth muscle

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.