Cardiac natriuretic peptides.

Investigations into the mixed muscle-secretory phenotype of cardiomyocytes from the atrial appendages of the heart led to the discovery that these cells produce, in a regulated manner, two polypeptide hormones - the natriuretic peptides - referred to as atrial natriuretic factor or atrial natriuretic peptide (ANP) and brain or B-type natriuretic peptide (BNP), thereby demonstrating an endocrine function for the heart. Studies on the gene encoding ANP (NPPA) initiated the field of modern research into gene regulation in the cardiovascular system. Additionally, ANP and BNP were found to be the natural ligands for cell membrane-bound guanylyl cyclase receptors that mediate the effects of natriuretic peptides through the generation of intracellular cGMP, which interacts with specific enzymes and ion channels. Natriuretic peptides have many physiological actions and participate in numerous pathophysiological processes. Important clinical entities associated with natriuretic peptide research include heart failure, obesity and systemic hypertension. Plasma levels of natriuretic peptides have proven to be powerful diagnostic and prognostic biomarkers of heart disease. Development of pharmacological agents that are based on natriuretic peptides is an area of active research, with vast potential benefits for the treatment of cardiovascular disease.

Techniques for the Evaluation of the Genetic Expression, Intracellular Storage, and Secretion of Polypeptide Hormones with Special Reference to the Natriuretic Peptides (NPs).

Techniques developed over the years in our laboratory for the study of tissue expression, storage, and secretion of the cardiac hormones ANF, BNP, and CNP are described below. They have proven highly reliable in our hands when the steps outlined are followed as described. Given the generic nature of the procedures, these should be applicable to other polypeptides.

INTERACTING DISCIPLINES: Cardiac natriuretic peptides and obesity: perspectives from an endocrinologist and a cardiologist.

Since their discovery in 1981, the cardiac natriuretic peptides (cNP) atrial natriuretic peptide (also referred to as atrial natriuretic factor) and brain natriuretic peptide have been well characterised in terms of their renal and cardiovascular actions. In addition, it has been shown that cNP plasma levels are strong predictors of cardiovascular events and mortality in populations with no apparent heart disease as well as in patients with established cardiac pathology. cNP secretion from the heart is increased by humoral and mechanical stimuli. The clinical significance of cNP plasma levels has been shown to differ in obese and non-obese subjects. Recent lines of evidence suggest important metabolic effects of the cNP system, which has been shown to activate lipolysis, enhance lipid oxidation and mitochondrial respiration. Clinically, these properties lead to browning of white adipose tissue and to increased muscular oxidative capacity. In human association studies in patients without heart disease higher cNP concentrations were observed in lean, insulin-sensitive subjects. Highly elevated cNP levels are generally observed in patients with systolic heart failure or high blood pressure, while obese and type-2 diabetics display reduced cNP levels. Together, these observations suggest that the cNP system plays a role in the pathophysiology of metabolic vascular disease. Understanding this role should help define novel principles in the treatment of cardiometabolic disease.

Go protein subunit Goα and the secretory process of the natriuretic peptide hormones ANF and BNP.

Expression of the G protein subunit Goα has been shown to be prominent in the atria of the rat heart and to be significantly associated with atrial natriuretic factor (ANF)-containing atrial-specific secretory granules by immunocytochemistry. In addition, differential expression profile analysis using oligonucleotide arrays has shown that the Goα isoform 1 (Goα1) is 2.3-fold more abundant in the atria than it is in the ventricles. In the present report, we show protein-protein interaction between Goα and ANF by yeast two-hybrid and by immunoprecipitation. A cardiac conditional Goα knockout model developed for the present study showed a 90% decrease in Goα expression and decreased atrial expression and ANF and brain natriuretic peptides (BNP) content. Expression of chromogranin A, a specific atrial granule core constituent, was not affected. Morphometric assessment of atrial tissue showed a very significant decrease in atrial-specific granule density as well as granule core electron density. Atrial electrical activity was not affected. The results obtained are compatible with the suggestion that Goα plays a role in ANF sorting during intracellular vectorial transport and with the presence of a mechanism that preserves the molar relationship between cellular ANF and BNP stores in the face of the decreased production of these hormones.

The heart as an endocrine organ.

The concept of the heart as an endocrine organ arises from the observation that the atrial cardiomyocytes in the mammalian heart display a phenotype that is partly that of endocrine cells. Investigations carried out between 1971 and 1983 characterised, by virtue of its natriuretic properties, a polypeptide referred to atrial natriuretic factor (ANF). Another polypeptide isolated from brain in 1988, brain natriuretic peptide (BNP), was subsequently characterised as a second hormone produced by the mammalian heart atria. These peptides were associated with the maintenance of extracellular fluid volume and blood pressure. Later work demonstrated a plethora of other properties for ANF and BNP, now designated cardiac natriuretic peptides (cNPs). In addition to the cNPs, other polypeptide hormones are expressed in the heart that likely act upon the myocardium in a paracrine or autocrine fashion. These include the C-type natriuretic peptide, adrenomedullin, proadrenomedullin N-terminal peptide and endothelin-1. Expression and secretion of ANF and BNP are increased in various cardiovascular pathologies and their levels in blood are used in the diagnosis and prognosis of cardiovascular disease. In addition, therapeutic uses for these peptides or related substances have been found. In all, the discovery of the endocrine heart provided a shift from the classical functional paradigm of the heart that regarded this organ solely as a blood pump to one that regards this organ as self-regulating its workload humorally and that also influences the function of several other organs that control cardiovascular function.

[The endocrine heart and inflammation]. / El corazón endocrino y el proceso inflamatorio.

The endocrine heart produces the polypeptide hormones Atrial Natriuretic Factor (ANF or ANP) and Brain Natriuretic Peptide (BNP). Through the peripheral actions of these hormones the heart contributes to the regulation of the cardiac preload and afterload. More recently, new functions for these hormones have been described including the modulation of the immune response. Plasma levels of BNP but not those of ANF, increase following an acute rejection episode of a cardiac allotransplant but return to levels pre-rejection with successful treatment. This observation constitutes the first observation leading to characterizing the interactions of BNP with the immune response. Several other pathologies with an inflammatory component are now known to be associated with an increase in the production of BNP. Such an increase is due to an increase in the transcriptional activity of the BNP gene induced by cytokines and related substances. In vitro investigations have shown that an increase in BNP directly modulates immunological activity. Inflammation and hemodynamic changes co-exist in several cardiovascular diseases and therefore it may be beneficial to measure circulating levels of both ANF and BNP as biomarkers of changes in intravascular volume and of changes in intravascular volume plus inflammation, respectively. Changes in plasma ANF, that are relatively larger than those of BNP, might be an indication of hemodynamic deterioration while important changes in circulating BNP could indicate a worsening of the inflammatory process.

El corazón endocrino y el proceso inflamatorio / [The endocrine heart and inflammation].

The endocrine heart produces the polypeptide hormones Atrial Natriuretic Factor (ANF or ANP) and Brain Natriuretic Peptide (BNP). Through the peripheral actions of these hormones the heart contributes to the regulation of the cardiac preload and afterload. More recently, new functions for these hormones have been described including the modulation of the immune response. Plasma levels of BNP but not those of ANF, increase following an acute rejection episode of a cardiac allotransplant but return to levels pre-rejection with successful treatment. This observation constitutes the first observation leading to characterizing the interactions of BNP with the immune response. Several other pathologies with an inflammatory component are now known to be associated with an increase in the production of BNP. Such an increase is due to an increase in the transcriptional activity of the BNP gene induced by cytokines and related substances. In vitro investigations have shown that an increase in BNP directly modulates immunological activity. Inflammation and hemodynamic changes co-exist in several cardiovascular diseases and therefore it may be beneficial to measure circulating levels of both ANF and BNP as biomarkers of changes in intravascular volume and of changes in intravascular volume plus inflammation, respectively. Changes in plasma ANF, that are relatively larger than those of BNP, might be an indication of hemodynamic deterioration while important changes in circulating BNP could indicate a worsening of the inflammatory process.

Phospholipase C signaling tonically represses basal atrial natriuretic factor secretion from the atria of the heart.

The cardiac hormone atrial natriuretic factor (ANF or ANP) plays significant, well-established roles in a large number of physiological and pathophysiological processes, including water and electrolyte balance, blood pressure regulation, and cardiovascular growth. Understanding the regulation of its production and secretion by atrial cardiomyocytes is incomplete. We have previously established a significant role of G(i/o) protein signaling in modulating ANF secretion as promoted by stretch of the atrial myocardium. In the present study, we investigated the role of G(q) protein signaling and its relationship to G(i/o) protein signaling using pharmacological manipulation of proximal effectors of G(αq) in an ex vivo model of spontaneously beating rat atria. Phospholipase C (PLC) and protein kinase C (PKC) inhibitors dramatically increased basal secretion of ANF. Furthermore, although atrial wall stretch is a potent stimulus for secretion, stretch unexpectedly reduced ANF secretion to basal levels under PLC and PKC inhibitory conditions. Inhibition of the inositol triphosphate receptor did not appear to affect basal secretion but dose-dependently blocked stretch-secretion coupling. The results obtained demonstrate that the PLC and PKC signaling cascades play important albeit unexpected roles in the regulation of basal and stimulated ANF secretion and suggest interplay between the G(q) and G(i/o) protein signaling pathways.

El corazón endocrino y el proceso inflamatorio. / [The endocrine heart and inflammation].

The endocrine heart produces the polypeptide hormones Atrial Natriuretic Factor (ANF or ANP) and Brain Natriuretic Peptide (BNP). Through the peripheral actions of these hormones the heart contributes to the regulation of the cardiac preload and afterload. More recently, new functions for these hormones have been described including the modulation of the immune response. Plasma levels of BNP but not those of ANF, increase following an acute rejection episode of a cardiac allotransplant but return to levels pre-rejection with successful treatment. This observation constitutes the first observation leading to characterizing the interactions of BNP with the immune response. Several other pathologies with an inflammatory component are now known to be associated with an increase in the production of BNP. Such an increase is due to an increase in the transcriptional activity of the BNP gene induced by cytokines and related substances. In vitro investigations have shown that an increase in BNP directly modulates immunological activity. Inflammation and hemodynamic changes co-exist in several cardiovascular diseases and therefore it may be beneficial to measure circulating levels of both ANF and BNP as biomarkers of changes in intravascular volume and of changes in intravascular volume plus inflammation, respectively. Changes in plasma ANF, that are relatively larger than those of BNP, might be an indication of hemodynamic deterioration while important changes in circulating BNP could indicate a worsening of the inflammatory process.

Brain natriuretic Peptide production and secretion in inflammation.

Gene expression and secretion of the cardiac polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are simultaneously upregulated in various cardiac disorders such as congestive heart failure, ischemic heart disease, and hypertensive heart disease, in which hemodynamic or neuroendocrine changes are key components in the progression of disease. However, during acute cardiac allograft rejection, plasma BNP levels are increased but not those of ANF. Successful treatment of the rejection episode decreases the elevated plasma BNP to prerejection values suggesting that substances related to inflammation may selectively influence BNP gene expression. Indeed, cytokines such as TNFα and IL-1ß selectively stimulate cardiac BNP at the transcriptional and translational levels in cardiomyocyte cultures without affecting ANF. This selective BNP increase is seen in vivo, in addition to acute cardiac allograft rejection, in several circumstances where inflammation significantly contributes to the pathogenesis of disease such as in sepsis and in acute myocarditis.

Ras dexamethasone-induced protein 1 is a modulator of hormone secretion in the volume overloaded heart.

Because of the crucial role of the endocrine heart in maintaining homeostasis, considerable effort has been focused on the elucidation of the mechanistic underlying gene expression and secretion of the cardiac hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). However, much remains to be determined regarding specific molecular events involved in cardiocyte secretory function. In this work, we identified genes involved in the transcriptional response of the endocrine heart to volume overload (VO) and signaling pathways involved in its regulation. To this end, the cardiac atrial and ventricular transcriptomes were analyzed in the heart of rats subjected to experimentally induced aorto-caval shunt VO. Pathway analysis revealed unique gene expression profiles in the VO atria for G-protein signaling, notably a significant downregulation of Ras dexamethasone-induced protein 1 (RASD1). In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion. Concurrent knockdown of RASD1 and its effectors Gα(o1) or Gß(1)γ(2) abrogated the endocrine response, demonstrating a previously unknown negative modulator role for RASD1. RASD1 thus emerges as a tonic inhibitor of ANF secretion and illustrates for the first time the concept of inhibitory protein regulators of ANF release. The novel molecular function identified herein for RASD1 is of considerable importance given its therapeutic implications for cardiovascular disease.

Characterization of a long-acting recombinant human serum albumin-atrial natriuretic factor (ANF) expressed in Pichia pastoris.

The cardiac hormone atrial natriuretic factor (ANF) combines pharmacological properties of drugs used to treat essential hypertension (EH), congestive heart failure (CHF) and acute myocardial infarction (AMI). Treatment of CHF or AMI patients with an intravenous (iv) infusion of the circulating form of ANF (ANF(99-126)) produces significant clinical improvement. The short half-life (5 min) and peptide nature of ANF impose logistic restrictions for chronic administration. To increase its half-life, we fused ANF and human serum albumin (HSA) mini-genes by recombination in Pichia pastoris. The activity of three configurations of the fusion protein was tested in vitro and in vivo. The fusion protein that comprised of C-terminus HSA connected to N-terminus ANF via a hexaglycine linker showed the best outcome; it increased cGMP production in vitro. In vivo an iv bolus of HSA-ANF into mice increased significantly plasma cGMP levels and lowered blood pressure (BP) for up to 6 h hence successfully extended ANF half-life in plasma while retaining its biological activity. HSA-ANF represents the basis for development in the chronic therapeutic use of ANF.

Uncoordinated regulation of atrial natriuretic factor and brain natriuretic peptide in lipopolysaccharide-treated rats.

We investigated the expression and secretion of the natriuretic peptides (NPs) ANF and BNP in lipopolysaccharide (LPS)-induced sepsis and its association with cytokines and other biologically active substances. LPS treatment increased plasma levels of ANF and BNP. The latter increase was larger than the increase in plasma ANF. LPS also increased cardiac content and gene expression of BNP but not of ANF. LPS treatment significantly increased gene expression cytokines, chemokines and proteases, which significantly correlated with BNP gene expression. SB203580, a p38 MAP kinase inhibitor, inhibited the elevation of BNP in plasma. The present work suggests that during inflammation, BNP gene expression and secretion is uniquely related to changes in gene expression in the absence of hemodynamic changes and hence differentiates ANF and BNP as biomarkers of cardiac disease.

Thirty years of research on atrial natriuretic factor: historical background and emerging concepts.

The discovery of the natriuretic properties of atrial muscle extracts pointed to the existence of an endocrine function of the heart that is now known to be mediated by the polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). On the basis of such a finding, approximately 27 000 publications to date have described a wide variety of biological properties of the heart hormones as well as their application as therapeutic agents and biomarkers of cardiac disease. Stimulation of secretion of ANF and BNP from the atria is mediated through mechanisms involving G proteins of the G(q) or G(o) types. We showed that the latter type underlies the transduction of muscle stretch into stimulated secretion and that it is more highly abundant in atria than in ventricles. The Gα(o)()-1 subunit appears to play a key role in the biogenesis of atrial granules and in the intracellular targeting of their contents. Protein interaction studies using a yeast two-hybrid approach showed interactions between Gα(o)()-1, proANF, and the intermediate conductance, calcium-activated K(+) channel SK4. Pharmacological inhibition of this channel decreases ANF secretion. Unpublished studies using in vitro knockdowns suggest interdependency in granule protein expression levels. These studies suggest previously unknown mechanisms of intracellular targeting and secretion control of the heart hormones that may find an application in the therapeutic manipulation of circulating ANF and BNP.

Cardiac hormones ANF and BNP modulate proliferation in the unidirectional mixed lymphocyte reaction.

BACKGROUND: Previous investigations have shown that the plasma levels of the cardiac hormone brain natriuretic peptide (BNP) increase during acute cardiac allograft rejection as diagnosed by endomyocardial biopsy. Successful immunosuppressant treatment decreased plasma BNP levels, suggesting a role for BNP in transplantation immunity. We tested a possible immunomodulatory effect of the natriuretic peptides (NPs) BNP, atrial natriuretic factor (ANF), and C-type NP (CNP) using the unidirectional mixed lymphocyte reaction (MLR). METHODS: Lymphocytes were isolated from the lymph nodes of Brown Norway (BN) and Lewis (L) rats. BN lymphocytes were gamma-irradiated to inhibit DNA synthesis. Lymphocytes at 2.5 x 10(6) cell/ml were mixed (at an L:BN ratio of 4:1) and incubated. On Days 2 and 3, ANF (10(-6) to 10(-11) mol/liter), BNP (10(-5) to 10(-11) mol/liter), or CNP (10(-6) to 10(-12) mol/liter) were added. Cell proliferation was measured on Day 4. RESULTS: Reverse transcript-polymerase chain reaction (RT-PCR) analysis of BN and L lymphocytes detected NP receptor (NPR) mRNA amplicons of the expected size. MLR induced an increase in relative receptor abundance as follows: NPRA > NPRB > NPRC. ANF and BNP significantly inhibited up to approximately 50% lymphocyte proliferation in a dose-dependent manner in the range of 10(-11) to 10(-6) mol/liter, whereas CNP significantly decreased lymphocyte proliferation only modestly (approximately 20%) at 10(-8) mol/liter and at 10(-6) mol/liter. CONCLUSIONS: Both ANF and BNP have immunomodulatory functions, although the response to cardiac rejection observed clinically involves increases in plasma levels of BNP only. This is likely related to BNP gene promoter sequences previously reported to be responsive to specific cytokines and related substances. The modulation of the MLR by NP suggests a possible clinical use of these peptides in transplantation immunity.