It is widely accepted that an increase in [Ca2+] initiates contraction by activation of myosin light chain (MLC) kinase and phosphorylation of the MLC. We have demonstrated that a second parallel, independent pathway exists for the regulation by testing the hypothesis that disinhibition of caldesmon by phosphorylation allow inherently activated myosin crossbridges to interact with actin and produce force. To test this hypothesis, we will first utilize an antisense oligodeoxynucleotide approach to produce a caldesmon-deficient vascular tissue. With this caldesmon """"""""knock-out"""""""" tissue, we can determine with greater precision, the role of this protein in contractile function. We will then determine which endogenous kinases is physiologically relevant for caldesmon phosphorylation. We will distinguish among p38, p42, and p44 members of the mitogen-activated protein kinase family for catalyzing caldesmon phosphorylation. We will also test the alternate the alternate hypothesis that caldesmon is not involved in the regulation of smooth muscle contraction per se, but involved in the structural integrity of the tissue. These hypothesis will be tested using intact and Triton X-100 skinned strips of swine carotid artery. We will determine the Ca2+ dependence of actin-activated myosin ATPase activity, force, crossbridge, cycling rate and attachment, kinase activities, protein content, and protein phosphorylation in control strips containing endogenous caldesmon, in caldesmon-deficient strips, and in caldesmon-deficient strips to which exogenous caldesmon has been re-introduced. The following specific aims will be pursued in this proposal: 1. To use antisense inhibition of h- caldesmon synthesis in cultured smooth muscle strips from porcine carotid arteries; 2. To determine the roles of caldesmon in carotid strips using control and caldesmon-deficient tissues; 3. To determine which specific kinase within the MAP kinase family is the caldesmon kinase; and 4. To determine the upstream pathway(s) leading to activation of the specific kinases identified. We know that pathways exist other than the one in which MLC phosphorylation acts as a simple Ca2+-dependent switch. What these pathways are and how they are regulated is still unknown. Caldesmon has been at the center of this controversy for over a decade. The successful completion of these specific aims should provide a reasonably definitive answer to the question: Is caldesmon involved in contractile regulation.
| Soloviev, Anatoly; Tishkin, Sergey; Ivanova, Irina et al. (2009) Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells. Life Sci 84:164-71 |
| Singh, Kamaljit; Arora, Divya; Falkowski, Danielle et al. (2009) An efficacious protocol for C-4 substituted 3,4-dihydropyrimidinones. Synthesis and calcium channel binding studies. European J Org Chem 2009: |
| Wang, Tanchun; Kendig, Derek M; Smolock, Elaine M et al. (2009) Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase. Am J Physiol Renal Physiol 297:F1534-42 |
| Basha, Maureen; Labelle, Edward F; Northington, Gina M et al. (2009) Functional significance of muscarinic receptor expression within the proximal and distal rat vagina. Am J Physiol Regul Integr Comp Physiol 297:R1486-93 |
