| Appropriate control of vascular tone is critical for normal physiologic function, and involves both Ca2+-dependent and Ca2+ -independent processes. Although the general processes involved in regulation of vascular smooth muscle (VSM) contractility are well-established, many of the molecular mechanisms underlying these responses are poorly understood. The work presented here focused on elucidation of several molecular mechanisms that control VSM contractility, and involved three major areas of study. First, the molecular composition of 4-aminopyridine-sensitive delayed rectifier potassium (KDR) channels in mesenteric resistance arteries was determined and found to consist of heteromultimers of Kv1.2, Kv1.5, Kv1.6 and Kv beta subunits. Complementary studies demonstrated that these heteromultimeric channels are important in negative feedback control of myogenic depolarization and constriction, highlighting an important role for these channels in control of VSM tone. This work was extended in a study that identified a novel cAMP-dependent protein kinase (PKA) phosphorylation site, serine-449, on the KDR channel alpha-subunit, Kv1.2. Additional studies provided evidence for the role of Rho-associated kinase (ROK)-dependent Ca2+ sensitization pathways, involving phosphorylation of the myosin phosphatase targeting subunit, MYPT1, in myogenic control of arterial diameter in the cerebral vasculature. This study involved the development of a highly sensitive western blot detection method to permit, for the first time, measurement of MYPT1 phosphorylation in very small, pressurized, myogenic blood vessels. These findings are significant because the potential role of Ca2+ sensitization in control of myogenic tone has been an outstanding question for some time. Therefore, the work of this thesis provides unique insight into the basic molecular mechanisms that contribute to the precise regulation of VSM contractility. |
