Signal transduction and calcium sensitivity of smooth muscle contraction

Abstract: Smooth muscles are components of several physiologically important organs and often involved in disease. Smooth muscle contraction is regulated by variations in intracellular [Ca2+]; increased levels activate the myosin light chain kinase (MLCK) which phosphorylates myosin and initiates contraction. Myosin phosphatase (MLCP) dephosphorylates myosin and promotes relaxation. Several cellular signaling pathways modulate the phosphorylation process by altering the Ca2+ sensitivity, mainly by influencing the MLCP activity. A general objective of this thesis was to study the cellular mechanisms and the physiological role of some of these pathways. In (I) we show, using Fura-2 measurements of intracellular [Ca2+], that the cell permeable cGMP analogue 8-Br-cGMP induced relaxation in intestinal smooth muscle by lowering intracellular [Ca2+] and decreasing Ca2+ sensitivity. Using transgenic mice lacking cGMP activated kinase in smooth muscle, we show that the cGMP relaxant effect was due to activation of cGK. Using tissue permeabilized with staphylococcus aureus ?-toxin, we show that cGMP induced Ca2+ desensitization and relaxation in a nano-molar concentration interval. Cross-activation of cAMP dependent kinase by cGMP was minimal and occurred at ~1000-fold higher cGMP concentrations. In (II) we show that cGK relaxed by reversing Ca2+ sensitization via a novel relaxant pathway involving dephosphorylation of a regulatory protein, CPI-17. In (III) the subunit composition of the MLCP was examined in hypertrophic intestinal tissue. An increase in the ratio of CPI-17/relative to the catalytic subunit of MLCP was associated with increased responsiveness to protein kinase C, Rho kinase and cGK. Hypertrophic tissue had increased responsiveness to contractile agonist, suggesting that altered cellular signaling influences the reactivity of the intestine. In (IV) the role of Ca2+ sensitization mechanisms during agonist induced tonic contractions in vascular tissue was examined. Using replacement of Ca2+ with Sr2+, we show that Ca2+ sensitization was not sufficient to maintain tonic force. Ca2+ dependent steps causing membrane depolarization are needed. Ca2+ sensitization is influenced by cellular targeting of the signaling molecules. Structures of proposed importance are the membrane associated caveolae. In (V) caveolin-1 deficient mice were used. We show that smooth muscle from these animals completely lack caveolae. The receptor mediated responses to endothelin-1 were attenuated in intestinal tissue from these mice. However, Ca2+ sensitization via small G-proteins and protein kinase C was unaffected, suggesting that caveolae primarily affect steps involved in Ca2+ elevation.