Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture

Abstract

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/μg DNA) and released mostly (83.3 ± 6.7%) low molecular weight ANF, at an average rate of 97 ± 18 fmol∙min−1∙μg−1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 ± 0.4 pmoi/μg DNA and 1.7 ± 0.4 fmol∙min−1∙μg−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. The present investigation shows that adult rat atrial cardiocytes, maintained superfused in microcarrier culture and in a serum-supplemented medium for up to 15 days, retain phenotypic and biochemical characteristics normally associated with the dual contractile–endocrine nature of mammalian atrial cardiocytes in vivo. The results obtained in the present work strongly support the view that ANF post-translational processing is an intrinsic property of the atrial cardiocytes and is independent of any other cell type.Key words: atrial natriuretic factor, post-translation processing, cardyocytes, adult rats, cell cultures.