Atrial natriuretic factor (ANF) is a peptide hormone synthesized in the cardiac myocytes of mammalian atria which exerts natriuretic, diuretic and vasorelaxant activities. ANF elicits multiple physiological responses and has been shown to activate membrane-bound guanylate cyclase and to inhibit adenylate cyclase. However, the precise mechanisms whereby ANF elicits physiological responses is not yet known. Recently we discovered that ANF dramatically affects steroidogenic responsiveness in mouse Leydig cells as well as in human ovarian granulosa cells. In normal mouse Leydig cells, AN stimulates steroidogenic responsiveness without changing the level of CAMP, suggesting that the steroidogenic effect in these cells in response to ANF may be regulated by the mechanism which involves a second messenger other than cAMP. Thus, ANF may emerge as a new intragonadal regulatory factor (i addition to gonadotropins which may control the biochemistry and function of Leydig cells) and, therefore, deserves examination of its cellular mechanism of action in gonadal cell functions. Clonally-derived mouse Leydig tumor (MA-10) cells possess an extraordinarily high density of ANF receptors and show dramatic increases in the accumulation of intracellular cGMP in response to ANF. To study the cellular and molecular mechanisms of ANF receptor will be purified from MA-10 cells and molecular properties determined. Anti-ANF receptor antibodies will be raised and biosynthesis and intracellular sequestration of the ANF receptor will be studied. The partial amino acid sequence of the purified receptor will be determined to prepare oligonucleotide probes. Both antibody and oligonucleotide probes will be used in cloning the cDNA to determine the complete primary structur of testicular ANF receptor molecules. By site-directed mutagenesis, different deletion mutations in ANF receptor cDNA will be constructed to define the structure-function relationships of different domains during signalling process and biological action of ANF. These fundamental, structural, functional and methodological studies of ANF receptors will greatly strengthen our understanding of the mechanisms of ANF action in various physiological function(s) and specially in male gonad.
