The purposes of the proposed studies are 1) to generate a comprehensive understanding of the role of the cardiac hormone ANF in the long-term maintenance of fetal cardiovascular function under normal and stress conditions, 2) to examine the endocrine and paracrine factors which regulate the release of ANF under normal and stress conditions, and their site of action at the cellular and molecular levels, and 3) to explore the developmental pattern of ANF gene expression in fetal atria and ventricles, and the effects of stress in altering this pattern of expression. The stress conditions to be studied are prolonged hypoxia and volume expansion. The approach taken is to first utilize the whole animal model to define the long-term cardiovascular and renal effects of ANF. Secondly, whole animal and cell culture experiments will be used in combination to delineate the factors which mediate ANF release and expression. Thirdly, molecular techniques will be used to determine ANF gene expression. These studies will be carried out in ovine fetuses at 105 to 115 days and 130 to 140 days gestation (term = 145 days) in order to determine the developmental changes in these regulatory mechanisms. The cardiovascular variables to be measured are arterial and venous pressures, heart rate, cardiac output, blood gases and pH, hematocrit and plasma protein concentration, urine flow rate and urinary excretion of electrolytes. In the first series of experiments, the effects of a long- term infusion of ANF on fetal cardiovascular variables will be determined in chronically catheterized fetuses. The involvement of ANF in returning arterial pressure, blood volume and urine flow to normal during prolonged hypoxia and volume loading will be investigated by the use of a specific antagonist for ANF. The second series of experiments will be carried out in chronically catheterized fetuses to delineate the effects of endothelin, norepinephrine and epinephrine in mediating ANF release, and in cultured fetal atrial cardiocytes to examine the direct effects of these factors in stimulating ANF secretion and expression of ANF messenger RNA. The third series of experiments will be carried out in fetal heart tissues to determine the pattern of expression of ANF messenger RNA in the atria and ventricles, and the effects of prolonged hypoxia or volume expansion in altering this expression. We will test the following hypotheses in the fetus: 1) ANF is an important hormone in the longterm maintenance of cardiovascular function under normal conditions and during the stress of hypoxia or volume expansion, 2) endothelin and the catecholamines are mediators for ANF release, and they act directly on fetal atrial cardiocytes to enhance ANF secretion and induce the expression of ANF messenger RNA, and 3) the level of ANF gene expression in the atria and ventricles of the fetus during the latter half of gestation can be induced by prolonged stress of hypoxia or volume expansion. These studies are important because they will expand our knowledge of the control of the fetal abnormalities in utero and stress at birth.
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