This figure demonstrates the role of the small GTPase, RhoA in smooth muscle contraction. The kinetics of Ca2+ release and force development following photolysis of an agonist, caged phenylephrine, using a 50nsec pulse of 347nm light in an intact 100mm diameter strip of portal vein smooth muscle loaded with a Ca2+ indicator, fluo-3, with one end of the muscle attached to a tension transducer and mounted in a muscle trough. The changes in Ca2+ and tension are shown. Subsequently, the muscle membranes are broken open to allow access of a RhoA/RhoGDI complex with bound caged GTPgS under conditions where the Ca2+ concentration is held constant. The caged GTPgS complex is without effect but following photolysis the GTPgS bound RhoA dissociates from RhoGDI and signals through Rho kinase to inhibit myosin phosphatase leading to an increase in phosphorylation of the regulatory light chains of myosin resulting in an increase in force, a mechanism known as Ca2+-sensitization. This experiments demonstrates that in intact muscle the initial force reflects Ca2+ activation, while the secondary rise in force at a time when Ca2+ (has fallen) is consistent with activation of the small GTPase RhoA.
 |
