Secretion of atrial natriuretic factor-(1-98) by primary cardiac myocytes.
Previous studies have demonstrated that primary cultures of cardiac myocytes maintained in a complete serum-free medium contain a precursor to atrial natriuretic factor (ANF-(1-126]. The cultured cells secrete this precursor unless maintained in the presence of glucocorticoids wherein the known circulating form derived from the C-terminal of ANF (ANF-(99-126] is secreted. The present study was designed to determine the fate of the N-terminal region of the ANF precursor during secretion from myocytes maintained in glucocorticoids. A radioimmunoassay (RIA) was developed using synthetic ANF-(1-16); the antiserum demonstrated cross-reactivity toward ANF-(1-126) and ANF-(1-98)-like peptides but did not cross-react with ANF-(99-126). Coupling this RIA with an ANF-(99-126)-specific RIA and reversed phase, size exclusion, and ion exchange high performance liquid chromatography (HPLC), it was shown that primary cultures of atrial myocytes maintained in dexamethasone contained ANF-(1-126) and secreted ANF-(99-126) and a peptide that was chromatographically indistinguishable from ANF-(1-98). Isolated perfused rat hearts were also shown by RIA and HPLC to secrete similar peptides. The primary cells were labeled with [35S]methionine, and the secreted N-terminal ANF-related material was immunoprecipitated with the ANF-(1-16) antiserum. HPLC, tryptic peptide mapping, and radiosequencing demonstrated that this peptide possessed an N-terminal structure identical to that of ANF-(1-126). When the cells were labeled with [3H] leucine and the secreted N-terminal ANF-related material was immunoprecipitated and analyzed by tryptic mapping, it was shown to possess labeled tryptic peptides consistent with the structure of ANF-(1-98). Tryptic mapping of [3H]arginine-labeled N-terminal ANF-related material demonstrated the presence of all peptides consistent with the ANF-(1-98) structure, including ANF-(92-98). These studies demonstrate that primary atrial myocytes contain ANF-(1-126) and in the presence of dexamethasone secrete both ANF-(1-98) and ANF-(99-126), the two major circulating forms of the hormone.
Glembotski
C C
CC
Biology Department, San Diego State University, California 92182.
Dixon
J E
JE
Gibson
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006325:Heart Atria / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011498:Protein Precursors; D011863:Radioimmunoassay; D051381:Rats; D013917:Thrombin; D014357:Trypsin
D010446:Peptide Fragments; D011498:Protein Precursors; C055659:atrial natriuretic factor prohormone (1-98); D009320:Atrial Natriuretic Factor; D014357:Trypsin; D013917:Thrombin