Effects of the calmodulin antagonist, fluphenazine, on phosphorylation of myosin and phosphorylase in intact smooth muscle

The activities of phosphorylase kinase and myosin light-chain kinase are regulated by Ca²⁺ binding to calmodulin. However, differences in the activation properties of the purified enzymes are apparent, since calmodulin binds to phosphorylase kinase in the absence of Ca²⁺ whereas prior formation of a Ca²⁺.calmodulin complex is necessary for calmodulin to bind to and activate myosin light chain kinase. Since the phenothiazines have been implicated as anticalmodulin drugs and inhibit contractile activity in smooth muscle, we examined the effects of the phenothiazine, fluphenazine, on isometric tension development and phosphorylation of phosphorylase and the phosphorylatable light chain (P-light chain) of myosin in intact bovine tracheal smooth muscle. Preincubation with 50 μM fluphenazine for 5 min inhibited the maximal rate and extent of isometric tension development and P-light chain phosphorylation in the presence of 60 mM KCl. Application of fluphenazine after tension and the phosphate content of the P-light chain had reached steady-state levels in response to 60 mM KCl produced little relaxation or dephosphorylation of the P-light chain. KCl-mediated phosphorylase a formation was not inhibited by preincubation with fluphenazine for 5 min. However, long periods of preincubation (30-60 min) produced significant inhibition of phosphorylase a formation and proportionally greater inhibition of tension and P-light chain phosphorylation. Since phosphorylase a formation was not inhibited during short-term preincubation with fluphenazine, KCl-dependent increases in the concentration of free intracellular Ca²⁺ may not have been affected. Moreover, since both isometric tension development and P-light chain phosphorylation were attenuated in a parallel manner, inhibition of contractile activity in intact smooth muscle by anticalmodulin agents may be directly related to inhibition of P-light chain phosphorylation.