CONTROL OF ANF PRODUCTION IN HYPERTENSION

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Mammalian cardiac atria contain a group of biologically active peptides called atrial natriuretic factor (ANF). Injection of atrial extract or pure ANF to animals causes a rapid onset of natriuresis, diuresis and lowering of blood pressure. The mammalian ANF gene contains three exons separated by two introns. The primary transcript is precursor ANF mRNA (pre- ANF mRNA) and is processed to ANF mRNA, which in turn encodes a 152 amino acid precursor (pre-pro-ANF) containing a 24 amino acid signal sequence. The pre-pro ANF is rapidly cleaved to produce pro-ANF which is processed to ANF and released into circulation. The experiments described herein will attempt to identify the factors that may be important in regulating the transcription of ANF gene, post-transcriptional processing, synthesis of pro-ANF and secretion of ANF from atria using the in vitro heart perfusion, techniques. Rates of pre-ANF mRNA synthesis will be based on the incorporation of (3H) uridine into pre-ANF mRNA during the period when labeling is rising linearly, and the specific activity of UTP, and, when a steady state is reached, (3H) UMP in pre-ANF mRNA. Rates of processing of ANF mRNA will be based on the radioactivity incorporated into ANF mRNA and the specific activity of (3H) UMP in pre-ANF mRNA. The relative rates of pre-ANF mRNA synthesis will also be estimated by the in vitro run-off transcription of the isolated nuclei. Rates of pro-ANF synthesis will be based on the incorporation of (3H)-phenylalanine(phe) into pro-ANF, the specific activity of (3H)-phe-tRNA during the period when labeling of pro-ANF is rising linearly and the steady-state relationship between specific activities of (3H)-phe-tRNA and (3H)-phe in pro-ANF. Rates of secretion will be estimated by the appearance of ANF in the perfusate. The effects of insulin, amino acid availability, left-atrial filling pressure, acetylcholine, vasopressin, norepinephrine and dexamethasone on the transcription, synthesis of pro-ANF and secretion of ANF will be examined. Finally, total contents of atrial ANF mRNA, and plasma level of ANF, as well as rates of synthesis of pre-ANF mRNA and pro-ANF and secretion of ANF will be measured in atria from salt-loading rats and volume-dependent hypertensive rats. These quantitative methods will help to understand more completely how the atrial and plasma levels of ANF are controlled under normal and hypertensive conditions.