(Verbatim from the application): The research will focus on the role and mechanisms of local Ca2+ in adrenergic control of the diameter of resistance arteries. The term 'local Ca2+' means [Ca2+] oscillations, waves, sparks and possible inter-cellular Ca2+ movements. The research is motivated by two recent experimental results: 1) [Ca2+] within individual smooth muscle cells (SMC) of intact pressurized arteries oscillates during adrenergic stimulation, in a pattern quite different from average (wall) [Ca2+], and 2) in adjacent SMC, the [Ca2+] oscillations may be synchronized at the same frequency, but are often asynchronous, at different frequencies. The relationship between these events and oscillations of membrane potential (Vm), vasoconstriction, and vasomotion induced by adrenergic stimulation are not known. The experiments will exploit the recent ability to image and control Ca2+ and other signaling molecules within individual SMC and endothelial cells (EC) within the wall of an intact, pressurized rat mesenteric small artery. Specific goals to be achieved are: 1) Determine the cellular mechanisms of the [Ca2+]-oscillations in SMC and determine whether endothelium modulates these mechanisms in pressurized arteries, 2) Determine the effectiveness, in situ, of SMC gap junctions and myo-endothelial gap junctions in transmitting Ca2+ and inositol tris-phosphate (InsP3) between cells, 3) Determine how activation of alpha1-receptors on SMC affects the frequency of Ca2+ sparks, 4) Test hypotheses on the mechanisms of synchronized [Ca2+] oscillations, Vm oscillations, and vasomotion. The hypotheses are distinguished by the putative roles of Vm, EC, gap-junctions, L-type Ca2+-channels, Ca2+-activated K+ and Cl- channels, ryanodine receptors and InsP3 receptors. The research will utilize fluorescent Ca2+ indicators and a combined confocal and multi-photon microscope for local [Ca2+] measurements and diffraction-limited photolysis of caged compounds within cells of the arterial wall. The role of gap junctions in intercellular signaling will be investigated using i) two-photon photolysis of caged Ca2+ and caged InsP3 in individual SMC, ii) specific (peptide) blockers of gap-junction channels, and iii) electrical measurements. A multi-focal multi-photon microscope will be used for fast optical sectioning of the arterial wall. Arterial diameter will be measured. Arteries will be de-endothelialized and motion blocked chemically, as appropriate. Local activation of alpha1-receptors on individual SMC will be achieved by two-photon photolysis of caged norepinephrine (NE). Whole-artery activation will be achieved by exposure to alpha1-receptor agonists and neuronal stimulation. By providing high-resolution images of molecular messengers (Ca2+) within the walls of intact pressurized arteries, the research will provide a new, more integrated view of the cellular and inter-cellular mechanisms that control vascular resistance.
