REGULATION OF PULMONARY ENDOTHELIAL PERMEABILITY

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In humans and animals with chronic heart failure, the lung undergoes important protective adaptations as evidenced by the ability of these patients to frequently tolerate the accompanying pulmonary hypertension without the development of alveolar edema and respiratory distress. To optimally maintain pulmonary function in heart failure, it is critical that therapies which are directed at improving cardiac function do not adversely interfere with these protective lung adaptations. This means that first we must understand the mechanisms by which heart failure results in the tolerance to pulmonary hypertension and the increased resistance to acute lung injury. Since the pulmonary endothelial barrier plays a key role in regulating movement of fluid into lung parenchyma, our inquiry is directed at understanding adaptations in function of this barrier in heart failure. Our working HYPOTHESIS is that heart failure results in a the functional "down-regulation" of pulmonary endothelial sensitivity to injury, due to alterations in P450-mediated signaling regulating Ca2+ entry subsequent to depletion of intracellular Ca2+ stores and to alterations in endothelial cell cAMP content and turnover. The Specific Aims are to determine if 1) P450-mediated signaling between store depletion and Ca2+ entry in pulmonary endothelium is deficient following the development of heart failure; 2) after heart failure, resistance to increases in lung endothelial permeability is due to altered regulation of plasma membrane ion channels and resultant decreases in Ca2+ entry; and 3) an elevation in cAMP in lung endothelium is a critical element which contributes to maintenance of an intact endothelial barrier in heart failure. This proposal involves the collaborative efforts of several investigators with complementary expertise in order to investigate this problem in a multidisciplinary fashion. To address this hypothesis, pulmonary endothelial permeability will be compared in isolated lung and in primary isolates of macro- and microvascular endothelial cells from normal dogs and dogs with heart failure induced by rapid ventricular pacing. Biochemical approaches will include measurement of microsomal P450 metabolism and turnover of cAMP. Immunoblotting will be used to evaluate protein expression for specific P450 epoxygenase, adenylyl cyclase and phosphodiesterase isoforms in lung and lung endothelium. Fura PE3, a ratiometric dye, will be used to measure Ca2+ transients, while patch clamp techniques will allow direct measurement of store operated Ca2+ channel activity.