Signal Transduction Of Agonist Induced Contraction In Smooth Muscle

The purpose of this research was two-fold: 1) To study signal transduction of agonist induced Ca²⁺-sensitization in smooth muscle. 2).To identify the role of MLC20 phosphatase in determining the different contractile behavior of tonic and phasic smooth muscle. Isometric tension, MLC₂₀ phosphorylation and free Ca²⁺ concentration were monitored in chemically permeabilized smooth muscle strips in which the receptor- G-protein coupling were retained.First, the major two components of pharmacomechanical coupling in smooth muscle, Ca²⁺ -release and Ca²⁺-sensitization were dissociated by Ca²⁺ channel blockers verapamil and nifedipine at G-protein/phospholipase C (PLC) level. In permeabilized portal vein smooth muscle, verapamil and nifedipine inhibited Ca²⁺-release induced by an α1-adrenergic agonist (phenylephrine) and by GTPγS, but not that induced by InsP3. These Ca²⁺ channel blockers also did not block the phenylephrine- or GTPyS- induced force development at constant cytoplasmic Ca²⁺: Ca²⁺-sensitization. An α1-blocker (prazosin) inhibited both the Ca²⁺-release and Ca²⁺ -sensitizing effects of phenylephrine, but not those of GTPγS, nor did it block InsP3-induced Ca²⁺-release. Ca²⁺ channel blockers selectively uncouple the Ca²⁺-release, but not the Ca²⁺-sensitizing, component of pharmacomechanical coupling indicating the divergence at G-protein/PLC level on their signal transduction pathway.The observation that signal transduction pathway of Ca²⁺-release and Ca²⁺-sensitization divergence at very upstream (G-protein/PLC level) provide the necessity for searching of second messenger of Ca²⁺-sensitization. The fact that free arachidonic acid level is increased upon agonist stimulation in many cell system, including smooth muscle, leads us to test the Ca²⁺-sensitizing effect of free arachidonic acid in permeabilized smooth muscle to explore its possible second messenger role in mediating agonist induced Ca²⁺ -sensitization. Arachidonic acid (AA) increased, at...