Agonist-induced changes in cytosolic Ca2+ are central to the signal transduction process of vascular endothelial cells (VECs). Stimulation of VECs by the potent vasodilator, bradykinin (BK) produces a bi-phasic change in cytosolic Ca2+. An initial transient component reflects release of Ca2+ from internal stores and a sustained component reflects Ca2+ influx from the extracellular space. The rise in cytosolic Ca2+ stimulates the release of nitrogen monoxide which produces relaxation of the underlying smooth muscle cells and decreases vascular resistance. While it is thought that release of Ca2+ from internal stores reflects activation of Ca2+ channels in the endoplasmic reticulum (ER) by inositol-triphosphate (IP3), the molecular mechanisms associated with Ca2+ influx have not been defined. Ca2+ may enter the cell via specialized junctions between the plasmalemma nd the ER. Such junctions may allow for the direct movement of Ca2+ from extracellular space into the IP3-sensitive pool. In the presence of IP3, Ca2+ would be immediately released into the cytosol. Alternatively, Ca2+ may enter the cytoplasm without prior entry into internal stores. In order to distinguish between these possibilities, the experiments of this proposal will characterize the Ca2+ transport systems thought to be important in signal transduction. The fluorescent indicator, fura-2 will be used to measure cytosolic Ca2+ and radioisotopic flux will be employed to evaluate kinetic parameters of specific Ca2+ flux pathway in bovine aortic endothelial cells.
The specific aims of this proposal are 1) to characterize the BK-stimulated Ca2+ influx pathway with respect to saturability, selectivity, blockade and sensitivity to membrane potential; 2) to characterize ATP-dependent Ca2+ uptake and IP3- and GTP-induced release of Ca2+ from internal stores; and 3) to identify physical and functional junctions between the plasmalemma and the internal Ca2+ storage sites. The results of these studies will provide specific information concerning the molecular mechanisms associated with Ca2+ signalling in VECs and form the test of the hypothesis that vectorial Ca2+ influx occurs from the extracellular space to the cytosol via the ER. This information will form the groundwork for our long term objectives which are to understand the role of Ca2+ homeostatic mechanisms in the response of VECs to 1) hemodynamic shear stress, 2) oxidant-induced cell injury and death, and 3) essential hypertension.
