This proposal evaluates the effects of endothelium-dependent vasodilators (EDVs) on pulmonary artery (PA) endothelial cell cytosolic calcium concentration ([Ca2+]i) and nitric oxide synthesis. The importance of endothelial nitric oxide production in determining PA pressure and responsiveness to vasodilators is assessed in normal and hypertensive rats. EDRF has previously been defined by its bioassay properties. Several laboratories have proposed that EDRF bioactivity is due to the synthesis of nitric oxide from L-arginine in endothelial cells. Nitric oxide then diffuses to the adjacent vascular smooth muscle where it stimulates guanylate cyclase. It is uncertain how an EDV initiates nitric oxide synthesis; however the increase in endothelial [Ca2+]i, caused by most EDVs has been implicated as a possible signal. This """"""""nitric oxide hypothesis"""""""" requires validation in the lung. Few investigators have correlated PA vasodilation with direct measurement of nitric oxide; most have relied on EDRF bioassays or the use of nonspecific EDRF-inhibitors. HYPOTHESIS: 1 Endothelium-dependent vasodilation in the lung is not solely the result of nitric oxide synthesis. Preliminary data showing that bradykinin (BK)- and A23187-induced PA vasodilation is associated with nitric oxide synthesis whereas that caused by acetylcholine (ACH) is not. A chemiluminescence assay will be used to determine whether the magnitude and time course of nitric oxide synthesis explains the vasodilation caused by a battery of EDVs (in normal and monocrotaline PHT rats). The specificity of putative inhibitors of nitric oxide synthesis, L-monomethyl arginine (L-NMMA) and L- nitroarginine (Nitro-Arg) will be studied using a new nitric oxide assay. 2. Nitric oxide synthesis in response to EDVs is preceded by and results from a transient rise in [Ca2+]i in PA endothelial cells. Preliminary data show that BK and A23187 increase whereas ACH reduces [Ca2+]i. The spatial and temporal characteristics of changes in [Ca2+]i which occur in response to EDVs will be measured using an imaging fluorescent microscopy system. The cytosolic """"""""Ca2+ maps"""""""" created by this system will reveal the intracellular location and temporal dynamics of the various Ca2+ pools which account for the net change in [Ca2+]i caused by EDVs. It may be that change in a specific intracellular Ca2+ pool is responsible for EDV-induced nitric oxide synthesis. The importance of extracellular Ca2+ versus intracellular Ca2+ in increasing [Ca2+]i and initiating nitric oxide synthesis will be addressed. 3. Endothelial damage may alter the lung's capacity to generate nitric oxide and contribute to the development of pulmonary hypertension (PHT). The relationship of vascular structure to the ability of PA rings to dilate and synthesize nitric oxide will be studied in lungs from rats with monocrotaline-induced PHT.
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