Inhibitory Effect of Forskolin on Myosin Phosphorylation Dependent and Independent Contractions in Bovine Tracheal Smooth Muscle

Abstract

In bovine tracheal smooth muscle, carbachol (CCh, 1μM) and high K⁺ (72.7mM) induced sustained increases in cytosolic Ca²⁺level ([Ca²⁺] i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10μM) inhibited the CCh-induced increase in [Ca²⁺MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K⁺-induced contraction and MLC phosphorylation without changing [Ca²⁺]In the absence of extracellular Ca²⁺ (with 0.5mM EGTA), CCh (10μM) and caffeine (20mM) induced transient increase in [Ca2+] i and contractile force by releasing Ca²⁺from cellular store. FK strongly inhibited the CCh-induced Ca²⁺transient, but failed to inhibit the caffeine-induced Ca2+transient. In the absence of external Ca²⁺, 12-deoxyphorbol 13-isobutylate (DPB, 1μM) induced sustained contraction without increase in [Ca²⁺] and MLC phosphorylation. FK inhibited this contraction without changing [Ca²⁺] In permeabilized muscle, Ca²⁺induced contraction in a concentration-dependent manner. FK (10μM) and cAMP (1-100μM) shifted the Ca²⁺-force curve to the higher Ca²⁺levels. CCh with GTP, GTPyS or DPB enhanced contraction in the presence of constant level of Ca²⁺. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca²⁺. cAMP (300μM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca²⁺influx and Ca²⁺release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.