REGULATION OF NITRIC OXIDE SYNTASE IN CARDIAC MYOCYTES

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Atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) have vasodepressor effects, while in the kidney they have potent natriuretic and diuretic activities. Although ANF and BNP have similar biological activities, there are distinct features associated with each hormone. Differences include tissue distribution, susceptibility to degradation by endopeptidase, circulating plasma half-life, affinity for biological and clearance receptors, form of the peptide stored in secretory granules, and possible DNA regulatory sequences involved in gene expression. In adult hearts, the gene for ANF is preferentially expressed in the atria, and ventricular expression is less than 5% of that of the whole heart. In contrast, ventricular BNP mRNA represents greater than 70% of whole heart levels. ANF and BNP mRNA levels are augmented in the ventricle subsequent to a number of different pathophysiological conditions of volume and pressure overload, including hypertension and heart failure. Given that the BNP gene is expressed primarily in the ventricles and that the ratio of BNP to ANF is different in atria and ventricles, we hypothesize that there are clear differences in the regulation of synthesis of ANF and BNP. The objective of this proposal is to define transcriptional and post- transcriptional mechanisms involved in the differential regulation of the genes encoding ANF and BNP in pathophysiological conditions resulting in cardiac hypertrophy. To do this, we will make use of a new model of cardiac hypertrophy, utilizing cultured adult feline cardiocytes stimulated to beat with isoproterenol. Specifically, the hypotheses to be tested are: 1) that there are differences in the rates of transcription of the ANF and BNP genes, and furthermore that there are cis-acting regulatory elements in the 5' flanking sequences (FS) of the BNP gene which are distinct from those characterized for the ANF gene in neonatal cardiocytes; 2) that during cardiac hypertrophy reexpression of the ANF and BNP genes involves cis-acting regulatory regions distinct from those involved in normal tissue-specific expression of the genes; 3) that there are different trans- acting regulatory proteins affecting ANF and BNP gene expression in quiescent vs beating adult feline cardiocytes (a model of hypertrophic growth); and 4) that there are differences in the stabilities of ANF and BNP mRNAs in normal and hypertrophied ventricles. Thus, these studies will provide a detailed analysis of the differences in regulation of the ANF and BNP genes, and provide a basis for understanding the relevance of each hormone in disease states, such as hypertension and hypertrophy.