Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture.
Post-translational processing of the cardiac polypeptide hormone atrial natriuretic factor (ANF) was studied using primary cultures of cardiocytes derived from adult rat atria. Atrial cardiocytes attached to microcarrier beads were maintained for up to 15 days under continuous superfusion in minichromatographic columns. The cultures were characterized for their ability to store, process, and release ANF and by immunofluorescence microscopy for ANF, desmin, and myosin. Nuclear staining using the fluorescent DNA stain Hoechst 33258 was carried out to determine the total number of cells in culture. Column eluates were assayed for ANF by radioimmunoassay and analyzed by reverse phase high-performance liquid chromatography. For comparison purposes, superfusion experiments using freshly isolated cardiocytes supported in Bio-Gel P2 were carried out. Freshly isolated atrial cardiocytes stored high molecular weight ANF (5.2 +/- 1.9 pmol/micrograms DNA) and released mostly (83.3 +/- 6.7%) low molecular weight ANF, at an average rate of 97 +/- 18 fmol.min-1 x micrograms-1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 +/- 0.4 pmol/micrograms DNA and 1.7 +/- 0.4 fmol.min-1 x micrograms-1 DNA, respectively, and 62.7 +/- 6.3% of the released peptide was of a low molecular weight. There was no correlation between changes in cell population and the extent of processing. Cultures of noncardiocytes, superfused with exogenous proANF, did not significantly process proANF to a lower molecular weight peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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University of Ottawa Heart Institute, Ottawa Civic Hospital, ON, Canada.
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D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism* / Q000494:pharmacology; D006690:Bisbenzimidazole; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016895:Culture Media, Serum-Free; D004247:DNA / Q000032:analysis / Q000378:metabolism; D003893:Desmin; D005455:Fluorescent Antibody Technique; D006325:Heart Atria / Q000166:cytology / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D016895:Culture Media, Serum-Free; D003893:Desmin; D010446:Peptide Fragments; D011498:Protein Precursors; C472610:atrial natriuretic peptide, rat; D009320:Atrial Natriuretic Factor; D004247:DNA; D006690:Bisbenzimidazole