Diabetes is associated with increased risk of cardiovascular diseases, which arise in part due to abnormalities in vascular smooth muscle (VSM) contraction resulting from an abnormal vascular tone. The overall goal of this project is to examine how insulin regulates the intracellular signal transduction pathways, which mediate relaxation in normal VSM and to identify potential pathway defects in diabetes. Contraction and relaxation of VSM is mediated primarily by phosphorylation and dephosphorylation of the regulatory myosin light chain (MLC20) by myosin light chain kinase and myosin bound phosphatase (MBP) respectively. The proposed studies will test the hypotheses that 1) insulin-induced vasorelaxation is mediated by MBP activation, and 2) cellular sensitivity and responsiveness to insulin and other vasoactive hormones are controlled by the factors which regulate the site-specific phosphorylation status of the myosin-bound subunit (MBS) of the phosphatase, MBP. Using aortic tissue, the proposed studies will examine 1) the role of MBP in insulin-mediated relaxation of VSM and to identify the mechanism whereby MBS regulates the MBP enzymatic activity. 2) Define the roles of PI3-kinase/Akt and RhoA/Rho kinase in insulin-mediated MBP activation and identify potential defects in these pathways in diabetes, and 3) Investigate potential interactions between angiotensin II, endothelin I and interleukin-1 [3/IL-6 and insulin signaling system and its impact on Rho signaling, MBS phosphorylation and MBP enzymatic activity. The functions of the key regulatory components on myosin dephosphorylation/relaxation will be elucidated by transfecting the gene of interest (activated MBS, PI3-kinase, iNOS, Akt, etc) into aortic tissue using adenovirus mediated gene transfer technique. Cumulatively, this work will lead to define the role of MBS in insulin activation of MBP, and the molecular basis of MBP regulation by insulin and potential cross-talk between insulin signaling pathways, cytokines and other vasoactive hormones. Thus, in a therapeutic context, the activation of MBP could be of value in preventing excessive contractility of VSM and thereby will have an important effect on reducing and/or preventing stroke, cardiovascular disease and renal failure in diabetes.
