Effect of phorbol esters on cytosolic Ca2+ level, myosin phosphorylation and muscle tension in high K+-stimulated bovine tracheal smooth muscle

To determine the role of protein kinase C (PKC) in bovine tracheal smooth muscle contractility, we examined the effects of phorbol esters on cytosolic Ca2+ level ([Ca2+](i)), myosin light chain (MLC) phosphorylation and contractile force in intact muscle and contraction in a permeabilized preparation. In intact muscle, 12-deoxyphorbol 13-isobutyrate (DPB, 1 mu M) increased the force without changing [Ca2+](i). High K+ (72.7 mM) induced sustained contraction with sustained increase in [Ca2+](i). In the muscle stimulated by high K+, 50 nM DPB increased the contractile force without changing [Ca2+](i), and 1 mu M DPB increased the contractile force with decreasing [Ca2+](i). Thus DPB shifted the [Ca2+](i)/force relationship for high K+ to the lower [Ca2+](i) in a concentration-dependent manner. In permeabilized muscle, DPB did not induce contraction in the absence of Ca2+ (much less than 0 nM), but shifted the Ca2+/force relationship to the lower Ca2+ levels. In the muscle stimulated with high K+, DPB (50 nM and 1 mu M) increased MLC phosphorylation and force without changing the MLC phosphorylation/force relationship. DPB (1 mu M) increased PKC activity estimated by the translocation from the cytoplasm to the membrane. These results suggest that DPB increases the Ca2+ sensitivity of MLC phosphorylation via the activation of PKC. Furthermore, DPB at higher concentration has an inhibitory effect on stimulated [Ca2+](i).