(Verbatim from the application): Recent studies from our laboratory and by others have indicated that cyclic adenosine diphosphate-ribose (cADPR) is produced and hydrolyzed in coronary arterial smooth muscle cells, and that this nucleotide serves as a second messenger to stimulate Ca24 release from the sarcoplasmic reticulum (SR). This proposal will examine the hypothesis that endogenous cADPR-induced Ca2+ release contributes to the control of [Ca2+], in vascular smooth muscle cells from small coronary arteries and that NO decreases intracellular cADPR production and consequently lowers [Ca2+], thereby resulting in vasodilation. We will first examine the role of cADPR in the regulation of ryanodine receptor/Ca2+ release channel activity using SR Ca2+ channel reconstitution and lipid bilayer clamp techniques. Then, we will further determine the role of cADPR-mediated activation of ryanodine receptors in the control of intracellular [Ca2+] in coronary arterial smooth muscle cells and the contribution of this signaling pathway to the inhibitory effect of NO on [Ca2+]i using single cell fluorescence microscopic spectrometry. Since cADPR has been reported to act through Ca2+-induced Ca2+ release (CICR), we will address whether the cADPR-mediated effects of NO are associated with inhibition of CICR or ryanodine receptors. We will also examine the effects of NO on the production or metabolism of cADPR by reverse phase-HPLC analysis of ADP-ribosylcyclase and cADPR hydrolase activities and to explore the mechanisms by which NO modulates these enzyme activities in coronary arterial smooth muscle. The involvement of cGMP and nitrosylation-mediated dimerization of ADP-ribosylcyclase in the effect of NO will be determined. Using videomicroscopy of isolated pressurized small coronary arteries, we will determine the role of cADPR, CICR and ryanodine receptors in the development of coronary tension and in mediating the vasodilator response to NO. Finally, we will explore the precise mechanisms by which cADPR binds or activates ryanodine receptors and produces Ca2+ release from the SR using radioligand binding or ADP ribosylation assays. These studies will define a new signaling mechanism regulating [Ca2+]i and vasomotor response in coronary resistance arteries and increase our understanding of the cellular mechanism mediating the vasodilator effect of NO.
Showing the most recent 10 out of 88 publications
