Effect of phorbol esters on Ca²⁺ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13- dibutyrate (PDBu) on isometric force at a constant submaximal Ca²⁺ concentration (i.e., the effect on Ca²⁺ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca²⁺ sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19-31) (PSSI), and staurosporine] on PDBu-induced Ca²⁺ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca²⁺ sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca²⁺ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.