Characterization of natriuretic peptide production by adult heart atria

The cardiac polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are synthesized and costored by atrial cardiocytes and share receptors and many biologic properties. Although some aspects of their synthesis and release are specific for each peptide, it is not clear whether they share intracellular sorting and secretory mechanisms. In the present work we take advantage of a stable isolated rat atrial preparation that allows, for the first time, long-term study of synthesis, trafficking, targeting, and secretion of ANF and BNP by adult atrial muscle. Three model stimuli of secretion were used: increased intra-atrial pressure, endothelin-1 (ET-1), and phenylephrine (PE), representing mechanical, hormonal, and α1-adrenergic stimuli, respectively. To gain further insight into the secretory process under basal and agonist-induced secretion, we employed agents known to inhibit protein synthesis (cycloheximide) or to interfere with the vectorial transport of protein targeted for secretion (brefeldin A and monensin). All these agents induced significant changes in ANF and BNP release. Cycloheximide decreased natriuretic peptide secretion under basal and stimulated conditions. Brefeldin A dramatically increased basal as well as stimulated secretion of ANF and BNP. Monensin partially decreased basal ANF and BNP secretion and completely blocked stimulated secretion. None of these agents modified proteolytic processing as assessed by reverse-phase HPLC analysis. Double-label pulse-chase experiments using [3H]- and [14C]leucine demonstrated that the secretory response to ET-1, in contrast to the response to muscle stretch, is based on peptide other than newly synthesized or relatively newly stored ANF. It is concluded that, in adult atrial cardiocytes, ANF and BNP are sorted to constitutive and regulated pathways in a manner that is substantially unique for atrial cardiocytes. In particular, it appears that basal and stimulated ANF and BNP secretion may have a large “constitutive-like” component, as previously defined in other endocrine systems. This type of secretion is based on the preferential release of hormone through vesicles arising from immature secretory granules. The capacity of the atria to release ANF and BNP in response to stimuli, therefore, may depend more on stimulation of the rate of formation of immature granules than on the amount of stored hormone.

BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria

We have assessed the effects of stretch or endothelin-1 (ET-1) on atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) secretion and gene expression using a new model of isolated right atria from the rat. This model allows for comparatively long-term in vitro study of adult tissue while retaining the anatomic conformation of the atrium. Stretch and ET-1 resulted in a transient stimulation of ANF and BNP secretion, with an initially larger proportional increase in ANF release. Stretch and ET-1 induced a marked increase in BNP gene expression after 1.5 and 4 h, respectively; the increase in BNP mRNA levels was maintained throughout the 8-h experimental period. Stretch and ET-1 also stimulated c- myc and Egr-1 mRNA levels, two markers of mechanical and receptor-mediated transcriptional activation. The selective response of the BNP gene to stretch and ET-1 and the distinct responses of ANF and BNP secretion indicate that the atrial cardiocytes have the capability to individually regulate the synthesis of its endocrine products. This suggests that each hormone plays a specific role in the response of the heart to hemodynamic or neuroendocrine imbalances.

Proportions of rat ANP-secreting cells that are cardiomyocytes and that synthesize the hormone

Atrial natriuretic peptide (ANP) is released from the heart and participates in regulating blood pressure and volume. We recently developed a reverse hemolytic plaque assay to measure the release of ANP from individual rat atrial cardiocytes. This assay determines the total proportion of atrial cardiocytes committed to releasing ANP. We combined the plaque assay with immunocytochemistry (IC) and in situ hybridization (IS). Combining the plaque assay with IC for myosin revealed that 13.5 +/- 0.9% (%myosin+ and plaque forming; mean +/- SE, n = 4) of atrial cardiocytes are cardiomyocytes that release ANP. Combination of the plaque assay with IS for ANP mRNA showed that 16.6 +/- 0.6% (%in situ+ and plaque forming; mean +/- SE, n = 4) of the cardiocytes in the rat atria synthesize and release the hormone. Incubation of atrial cardiocytes with dexamethasone to stimulate ANP gene expression did not alter the total proportion of in situ-positive ANP-secreting cells. These data suggest that, within the total ANP-secreting population of the rat atria, only 33% of the secreting cells are cardiomyocytes. In addition, 68% of the ANP-secreting cells do not appear to synthesize the hormone. These results imply that muscle and nonmuscle cells are involved in secreting ANP and that cardiomyocytes synthesize the hormone.

EVIDENCE FOR A NOVEL SOURCE OF RELAXIN: ATRIAL CARDIOCYTES

ABSTRACT Antibody-directed, complement-induced erthrocyte lysis (reverse hemolytic plaque assay) around atrial cardiocytes was used to determine whether this cell type possesses the capacity to secrete the insulin-like hormone relaxin. After 2h of incubation, 33 ± 4% (n = 3) of cardiocytes derived from the atria of neonatal rats secreted detectable amounts of immunoreactive relaxin (i.e. formed plaques) when cultured in monolayers. Increased culture time of cardiocytes failed to increase the fraction of cardiocytes that secreted relaxin. The cumulative amount of relaxin secreted after 3h of incubation (plaque area) was 31% greater (P < 0.05) than the amount of hormone present after lh of incubation, evidence of sustained peptide secretion by cultured cardiocytes. These data suggest that the source of the endogenous ligand for the specific and high-affinity relaxin receptors located in rat atria is the atrial cardiocyte itself. Therefore, relaxin may act via autocrine and/or paracrine routes to regulate cardiovascular structure and/or function.

Cardiac and plasma atrial natriuretic peptide after 9-day hindlimb suspension in rats

To determine atrial natriuretic peptide (ANP) adaptation to simulated weightlessness, immunoreactive plasma (ir-NH2- and ir-COOH-terminals) and atrial (ir-COOH-terminal) ANP levels, atrial mRNA expression, immunoreactive cardiocyte ANP levels (ir-NH2- and ir-COOH-terminals), and ultrastructural observations of granules in atrial cardiocytes were assessed in male Wistar rats after a 9-day hindlimb suspension. Plasma ir-NH2- and ir-COOH-terminal ANP concentrations decreased by 17 (P < 0.05) and 37% (P < 0.05), respectively, in suspended rats. A concomitant ir-COOH-terminal ANP content reduction was also observed in left (31%; P < 0.01) and right atria (25%; P < 0.05). Atrial ANP mRNA expression was severely depleted in the right atrium and less so in the left atrium after 9 days of hindlimb suspension. Immunocytochemistry observations demonstrated lowered NH2- and COOH-terminal ANP immunoreactivities in left and right atria from suspended rats. A reduced number of storage granules (dense granules) in both atria was also noted on ultrastructural analysis. It was concluded that ANP biosynthesis, storage, and release were decreased after a 9-day hindlimb suspension.

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/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.