In vascular smooth muscle (VSM), a rise in the cytosolic free Ca2+ concentration, [Ca2+]cyt, is """"""""necessary and sufficient"""""""" to trigger contraction. The overall goal of this project is to elucidate the factors that regulate intracellular Ca2+ in VSM, and to determine how they influence physiological and pathophysiological processes. The main hypothesis is that the plasmalemmal Na+ gradient helps regulate, via Na/Ca exchange (NCX), not only [Ca2+]cyt, but the intracellular stores of Ca2+ as well. Consequently, the Na+ gradient plays a critical role in the control of cell responsiveness. The emphasis will be on the NCX and its functional and spatial relationships to other, related transporters and to intracellular Ca2+ stores in VSM cells. The following specific aims are proposed: 1. To determine the contribution of NCX to regulation of intracellular Ca2+ storage in primary cultured rat arterial myocytes. Three spatially and functionally-distinct Ca2+ stores in VSM cells will be identified pharmacologically and their regulation by the Na+ gradient and NCX will be examined. Digital imaging methods with Ca2+- and Na+-sensitive dyes will be used to measure the Ca2+ content of the stores as well as cytoplasmic Na+ and Ca2+. These imaging studies will be correlated with contractile responses measured in arterial rings. 2. To determine the spatial distribution of key Na+ and Ca2+ transporters (NCX, Na+ and Ca2+ pumps) and relate them to sites of Ca2+ storage. These morphological data will be correlated with functional data from #1. Organelles (mitochondria and sarcoplasmic reticulum, SR) will be stained with fluorochromes [e.g., furaptra, DiOCb(3)]. Fluorescently labeled antibodies will be used to identify the NCX, Na+ pump, and plasmalemmal (PM) and SR Ca2+ pumps. Deconvolution and 3-D image restoration, as well as double-labeling [e.g., DiOCb(3) plus furaptra or a fluorescently labeled antibody], will be used to localize stores and transporters. Spatial correlation of the distribution of these organelles and transporters, to the PM NCX will be determined in freshly isolated as well as in cultured rat arterial myocytes. 3. To relate the physiological role of the NCX to other Ca2+ homeostasis in response to physiological stimuli and pharmacological perturbations will be characterized with digital imaging in fura-2 and furaptra-loaded cells. The NCX will be knocked down with antisense oligonucleotides, the PM Ca2+ pump will be inhibited with La3+, and intracellular stores will be emptied with selective agents. Standard molecular biological methods will be used for antisense studies.
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