The physiological and pathophysiological modulation of the endocrine function of the heart.

Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascular homeostasis through the polypeptide hormones ANF and BNP, which are members of the natriuretic peptide (NP) family. Given that NPs are of interest from the basic and clinical points of view, the genetic expression and secretion of ANF and BNP as well as the nature of the interaction of these hormones with their receptors has been the subject of extensive studies since the discovery of ANF in 1980. Following hemodynamic overload, increased secretion of NPs by the heart can be seen. This change may occur without an increase in gene expression as observed for atrial NPs following acute volume expansion, or it can occur with an increase in both ANF and BNP gene expression in atria only as seen in mineralocorticoid escape during which it is obvious that a critical decrease in hormone stores must be reached before transcriptional activation occurs. Chronic hemodynamic pressure or volume overload results in increased expression of NPs in atria and ventricles. Under these circumstances, the increased production of BNP by hypertrophic ventricles changes the normal plasma concentration ratio of ANF to BNP, a fact that has clinical diagnostic and prognostic implications. There are exceptions to this rule: chronic, severe L-NAME hypertension, which may occur without left ventricular hypertrophy, does not cause this effect and increased ventricular NP gene expression can occur in mineralocorticoid hypertension before detectable ventricular hypertrophy. Atrial and ventricular NP gene expression appears to be under different transcriptional control because pharmacological treatments such as chronic ACE inhibition or ET(A) receptor blockade can reverse the increased ventricular NP expression but has no detectable effect on atrial NP gene expression. This is not unlike the myosin heavy chain switch that is observed in certain pathologies, and can be pharmacologically reversed in a manner similar to NPs in the ventricles but it does not occur in atrial muscle. These observations made in vivo or using isolated adult atria often differ strikingly from results obtained using the mixed phenotype afforded by cardiocytes in culture, indicating that the kinds of questions addressed by each approach must be judiciously chosen. G-protein coupled receptor-mediated actions of neurohumors such as endothelin and phenylephrine are normally used to stimulate NP gene expression and release in different in vitro models. The main physiological stimulus for increased ANF release, atrial muscle stretch, also appears to rely on G-protein-coupled mechanisms. Alternative agonists and receptor types at play are suggested by the finding that circulating levels of BNP are selectively increased before and during overt cardiac allograft rejection episodes in human patients. The data suggest that enhanced BNP plasma levels could form a basis for a noninvasive test for cardiac allograft rejection. However, the molecular mechanism by which expression of NPs are regulated in the transplanted heart is not well understood. Conditioned medium from mixed lymphocyte reaction cultures, considered an in vitro model of transplantation immunity, induces specific upregulation of BNP as do individual pro-inflammatory cytokines. Findings such as these suggest that the study of NPs will continue to produce a wealth of information relevant to basic and clinical scientists.

Modulation of cardiac natriuretic peptide gene expression following endothelin type A receptor blockade in renovascular hypertension.

OBJECTIVE: Increased expression of the cardiac natriuretic peptides (NP), atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) is observed during chronic hemodynamic overload. The mechanisms underlying this process are not fully understood. In vitro, endothelin 1 (ET-1) is a powerful stimulator of cardiac NP and, therefore, has been assumed to be one possible mediator of increased NP gene expression following chronic pressure or volume overload. In the present work we investigated the possible role of ET-1 in mediating the observed upregulation of cardiac NP in two kidney-one clip (2K-1C) Goldblatt hypertensive rats treated for 6 weeks with the ET-1 type A (ET(A)) receptor subtype receptor antagonist ABT-627. METHODS: 2K-1C hypertension was induced in male Sprague-Dawley rats weighing 100-125 g by placing a silver clip (internal diameter 0.25 mm) around the left renal artery through a flank incision. The right kidney was left undisturbed. Sham operated rats underwent the same experimental procedures but no clip was placed on the left renal artery. ABT-627 was administered (10 mg/kg per day) in the drinking water for 6 weeks. RESULTS: In hypertensive rats, ABT-627 prevented a further rise in blood pressure beginning at 3 weeks after clipping and reduced the ventricular hypertrophy observed at the end of the experiment. ET(A) blockade prevented enhanced NP gene expression in the right ventricle and partially prevented it in the left ventricle. No modifications in atrial NP gene expression were observed in either control or 2K-1C animals. ET(A) blockade decreased BNP circulating levels but did not affect ANF plasma levels in clipped rats. ABT-627 increased alpha-myosin heavy chain gene expression and decreased the abundance of the beta isoform transcript. CONCLUSION: The results obtained in the present investigation show the participation of ET-1 in the increased expression of ventricular NP in 2K-1C renovascular hypertension and an apparent lack of effect of ET(A) blockade on atrial NP gene expression in both control and hypertensive animals thus showing that in vivo, atrial and ventricular NP gene expression are differentially regulated.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Effect of selective ET(A) receptor blockade on natriuretic peptide gene expression in DOCA-salt hypertension.

To determine the role of endothelin-1 (ET-1) in the upregulation of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) observed in deoxycorticosterone acetate (DOCA)-salt hypertension, the selective ET-1 type-A receptor (ET(A)) antagonist ABT-627 was chronically administered to normal controls and hypertensive rats. Chronic ET(A) blockade in DOCA-salt-treated rats prevented the increase in blood pressure and circulating natriuretic protein (NP) levels and partially prevented left ventricular hypertrophy. The changes observed in NP gene expression in the atria were not affected by ABT-627. In the ventricles, ABT-627 reduced NP gene expression. Rats receiving the ET(A) antagonist alone showed reduced left ventricular NP gene expression. ABT-627 did not affect ventricular collagen III gene expression but enhanced left ventricular alpha-myosin heavy chain expression. These findings suggest that in vivo, ventricular but not atrial NP production is regulated by ET-1. This difference in response between atrial and ventricular NP gene expression to ET(A) receptor blockade is similar to that observed by us after applying angiotensin-converting enzyme inhibitors in other hypertensive models. In general therefore, atrial NP gene expression may not be as sensitive to the endocrine environment as is ventricular NP gene expression.

[Competitiveness in science. Today, tomorrow, and forever]. / Competitividad en ciencia. Hoy, mañana y siempre.

Paternalistic governments and highly bureaucratized administrations produce mediocre science policy decisions that often allow for the co-existence of potentially competitive scientists alongside with those that are not. This invariably results in failure to produce significant research. It seems apparent therefore, that policy change aiming at improving science and technology must begin with intensification of the level of individual competitiveness. Nations that have internationally competitive levels of technical and scientific activity such as Japan, USA and Canada, share in common certain features that foster individual competitiveness despite the fact that their socioeconomic basis are vastly different. These common features include administrative continuity, very high academic standards and a highly educated work force. The scientist's emotional cost in competitive environments is high but there seems to be no alternative given the sophistication of the topics that are dealt with in formerly purely descriptive sciences such as biomedicine, and given the enormous speed of electronic communications. The role of governments in fostering science and technology should be mainly concerned with conducting a sound fiscal policy in order to provide for the needs of education and scientific activity. Governments can also play a key role in insuring that science remains competitive through the delineation of rules that increase individual competitiveness rather than with policy schemes that fail to directly address the responsibility of the individual. Policies to increase individuals' performance may prove costly to politicians given that these adjustments imply unpopular decisions regarding an increase in academic performance expectation beginning in high school and the re-assignment of functions of individuals or institutions that do not meet international productivity criteria.

Competitividad en ciencia. Hoy, mañana y siempre. / [Competitiveness in science. Today, tomorrow, and forever]

Paternalistic governments and highly bureaucratized administrations produce mediocre science policy decisions that often allow for the co-existence of potentially competitive scientists alongside with those that are not. This invariably results in failure to produce significant research. It seems apparent therefore, that policy change aiming at improving science and technology must begin with intensification of the level of individual competitiveness. Nations that have internationally competitive levels of technical and scientific activity such as Japan, USA and Canada, share in common certain features that foster individual competitiveness despite the fact that their socioeconomic basis are vastly different. These common features include administrative continuity, very high academic standards and a highly educated work force. The scientists emotional cost in competitive environments is high but there seems to be no alternative given the sophistication of the topics that are dealt with in formerly purely descriptive sciences such as biomedicine, and given the enormous speed of electronic communications. The role of governments in fostering science and technology should be mainly concerned with conducting a sound fiscal policy in order to provide for the needs of education and scientific activity. Governments can also play a key role in insuring that science remains competitive through the delineation of rules that increase individual competitiveness rather than with policy schemes that fail to directly address the responsibility of the individual. Policies to increase individuals performance may prove costly to politicians given that these adjustments imply unpopular decisions regarding an increase in academic performance expectation beginning in high school and the re-assignment of functions of individuals or institutions that do not meet international productivity criteria.

Discoordinate modulation of natriuretic peptides during acute cardiac allograft rejection in humans.

BACKGROUND: Increased circulating levels of the cardiac polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) may be observed after orthotopic cardiac transplantation. Both the hypertrophic and inflammatory processes in the allograft may contribute to this increase, but no mechanistic explanation has been suggested for this observation. METHODS AND RESULTS: Plasma immunoreactive ANF and BNP determinations were performed in 10 consecutive transplant patients. These were correlated with degree of rejection as reflected by histopathological findings at serial endomyocardial biopsies. Three patients had associated hemodynamic measurements and blood samples 24 hours before and after transplantation. All rejection episodes that received treatment were accompanied by a marked increase in BNP plasma levels to > approximately 400 pg/mL. Steadily increasing BNP levels preceded overt rejection as assessed by histopathological criteria. The increase in plasma BNP was not always accompanied by an increase in ANF, which suggests the specific upregulation of BNP gene expression during acute rejection episodes. Treatment of the acute rejection episodes led to a substantial decrease of BNP plasma levels. CONCLUSIONS: The significant selective increase in plasma BNP levels found in the present study has not been previously described. This finding provides a new insight into the mechanism of allograft rejection and the modulation of natriuretic peptide synthesis and release. Furthermore, although preliminary, the data suggest that BNP plasma levels could form the basis for a new, noninvasive screening test to predict acute cardiac allograft rejection. Because treatment with the antilymphocyte monoclonal antibody OKT3 (murine monoclonal antibody to the CD3 antigen of the human T-cell) decreased BNP plasma levels, cytokine production by T-cells may mediate the selective increase in circulating BNP.

Variable renal atrial natriuretic factor gene expression in hypertension.

We have previously established the existence of atrial natriuretic factor (ANF) gene expression within the renal parenchyma. Neither the role nor the regulation of this extracardiac source of ANF is clearly defined. To determine whether renal ANF gene expression, similar to cardiac expression, is linked to the activity of the renin-angiotensin system (RAS), we compared renal ANF gene expression in rats after suprarenal aortic banding, a hypertension model associated with activation of RAS, and in the deoxycorticosterone acetate (DOCA)-salt model, which is characterized by depression of RAS. Renal ANF mRNA was measured with a quantitative competitive reverse transcription polymerase chain reaction method. DOCA-salt hypertension significantly reduced the expression of renal ANF. In contrast, aortic banding significantly increased renal ANF expression. In both cases, ANF gene expression in the heart increased. Ramipril treatment at 10 micrograms/kg of aortic-banded rats, a treatment that specifically affects local RAS but maintains hypertension, normalized renal ANF mRNA levels. Altogether, these results suggest that renal ANF gene expression is modulated by local RAS and is independent of circulating RAS and hypertension per se. The marked decrease of renal ANF mRNA in DOCA-salt hypertension suggests a pathogenic role for renal ANF gene downregulation by decreasing the sodium excretory mechanism mediated by the local expression of ANF acting on receptors found in the inner medullary collecting ducts. In aortic banding, renal ANF gene expression upregulation suggests a local compensatory function consistent with the consensus role of natriuretic peptides in the modulation of RAS, thus ameliorating the sodium-retaining effects of renal underperfusion.

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 alpha1-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.

Regulation of aortic atrial natriuretic factor and angiotensinogen in experimental hypertension.

We investigated the relation between atrial natriuretic factor (ANF) gene expression and the status of the renin-angiotensin system (RAS) in aortic tissue in rats made hypertensive by either aortic banding or by deoxycorticosterone acetate (DOCA)-salt administration. These experimental models of hypertension are known to have differences in terms of the status of RAS. ANF messenger RNA (mRNA) levels were measured in aortic tissue by using a newly developed quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) technique. Changes in the proportions of alpha1 and alpha2 isoforms of Na+K+-adenosine triphosphatase (ATPase) mRNA levels were used as indicators of aortic hypertrophy. Treatment with DOCA alone, salt alone, or DOCA-salt for 5 weeks increased aortic-weight/body-weight ratio and aortic angiotensinogen mRNA levels, but did not change alpha1 or alpha2 Na+K+-ATPase mRNA levels. Aortic ANF mRNA levels had a tendency to increase after treatment with DOCA, salt, or DOCA-salt, but this change did not reach statistical significance. Suprarenal aortic banding for 6 weeks or 12 weeks increased aortic-weight/body-weight ratio (12 weeks), decreased alpha2 Na+K+-ATPase and angiotensinogen mRNA levels, but did not affect alpha1 Na+K+-ATPase mRNA levels or ANF mRNA levels. Treatment with ramipril, an angiotensin-converting enzyme (ACE) inhibitor was carried out for 6 weeks just after aortic banding (prevention experiment) or after 6 weeks in rats that were banded for the previous 6 weeks (regression experiment). High-dose ramipril (1 mg/kg)--a treatment known to inhibit both tissue and circulating RAS--normalized aortic-weight/body-weight ratio, and also normalized alpha2 Na+K+-ATPase mRNA levels. Aortic angiotensinogen mRNA levels of banded rats treated with high-dose ramipril was higher than those of the normal control, sham operated, and banded rats. Treatment with high-dose ramipril did not affect alpha1 Na+K+-ATPase mRNA levels or ANF mRNA levels. Low-dose ramipril (10 microg/kg)--a treatment that selectively inhibits tissue RAS--normalized aortic-weight/body-weight ratio but did not normalize alpha2 Na+K+-ATPase mRNA levels (regression experiment) or angiotensinogen mRNA levels (prevention experiment) and did not change either alpha1 Na+K+-ATPase mRNA levels or ANF mRNA levels. The results suggest that, in contrast to previous findings in heart and kidney, the regulation of ANF mRNA levels in aortic tissue is largely independent of pressure load, volume load, and plasma or tissue RAS. It is suggested that any antihypertrophic actions of ANF may be mediated by the increased circulating ANF levels and its interaction with its receptor or through CNP.

Tissue-specific regulation of renal and cardiac atrial natriuretic factor gene expression in deoxycorticosterone acetate-salt rats.

Atrial natriuretic factor (ANF) is expressed in several noncardiac tissues where it may have an autocrine or paracrine function. Such function may be expected of locally synthesized ANF in the renal parenchyma. Previous investigations of the existence of ANF mRNA in the renal parenchyma have yielded conflicting results. The investigations reported here were designed to detect and measure ANF mRNA in normal rats and in rats subjected to a deoxycorticosterone acetate (DOCA)-salt treatment schedule known to strongly activate cardiac ANF gene expression. The expression of the renal ANF gene was measured using a newly developed quantitative competitive reverse transcription-polymerase chain reaction (QC-RT-PCR). This method uses an internal competitor that serves as an internal standard and makes the procedure independent of measurement relative to housekeeping genes. It was found that renal ANF mRNA levels were 10(7) times lower than those found in left or right atria, but immunoreactive (ir) renal ANF concentration by specific radioimmunoassay was 10(4) times lower than that of atrial irANF levels. Reverse-phase high-performance liquid chromatography analysis revealed that more than 99% of renal irANF is processed ANF(99-126). This finding suggests that most of the irANF measured in kidney extracts likely originates from atrial sources. Left atrial ANF mRNA levels after 1 week of DOCA-salt treatment was significantly higher than that of control rats ([21.06+/-2.99] x 10(-l5) mol/microg total RNAversus [8.59 +/-1.26] x 10(-5) mol/microg total RNA, P<.05). However, renal ANF mRNA levels in DOCA-salt rats were significantly decreased compared with those of control rats ([1.64+/-0.34] x 10(-22) mol/microg total RNA versus [3.96+/-0.61]x 10(-22) mol/microg total RNA, P<.05). These results indicate that (1) renal ANF mRNA can be consistently and specifically demonstrated after reverse transcription and PCR amplification; (2) renal and cardiac ANF synthesis are regulated in a tissue-specific, opposite manner during DOCA-salt treatment; and (3) the finding that renal ANF mRNA is downregulated by DOCA-salt treatment together with previous findings suggest the need for further investigation into the role of renal ANF mRNA downregulation in the pathogenetic mechanism that leads to volume expansion and hypertension after chronic DOCA-salt treatment.

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 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.

Evidence for load-dependent and load-independent determinants of cardiac natriuretic peptide production.

BACKGROUND: In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix. METHODS AND RESULTS: Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the following 6 weeks. Banding caused an increase in blood pressure, left ventricular weight-to-body weight ratio, plasma and ventricular NP, ventricular NP mRNA, collagen III, and beta-MHC mRNA. Ramipril at 1 mg/kg normalized all these parameters while ramipril at 10 micrograms/kg normalized left ventricular weight-to-body weight ratio but not blood pressure. Plasma and ventricular NP content and mRNA levels were partially normalized by ramipril (10 micrograms/kg). Ramipril (10 micrograms/kg) prevented increased collagen III mRNA levels but did not affect beta-MHC mRNA levels. CONCLUSIONS: (1) NP production and secretion in aortic-banded rats are independently related to increased blood pressure and hypertrophy. (2) A load-dependent component is more important than a load-independent component in regulating left ventricular NP production. (3) ANF production is more sensitive than BNP production to the load-independent component. (4) Low-dose ramipril treatment reverses hypertrophy and the increased collagen III expression but does not reverse the increased beta-MHC isoform expression, suggesting that these are independently regulated processes. (5) Aortic banding and ACE inhibition do not affect atrial NP production and content.

Alpha 1-adrenergic stimulation of isolated rat atria results in discoordinate increases in natriuretic peptide secretion and gene expression and enhances Egr-1 and c-Myc expression.

We studied the effects of alpha1-adrenergic stimulation on atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) secretion and gene expression in isolated right atria. The early-response genes Egr-1 and c-myc were also studied as potential markers of transcriptional activation after alpha1-adrenergic stimulation. Isolated right atria from rats were stimulated for up to 8 h by the alpha1-adrenergic agonist phenylephrine (PE). PE at 10, 50, or 100 microM stimulated the secretion of immunoreactive (ir) ANF, beginning at 0.5 h and peaking after 1.5 h, IrANF secretion remained significantly elevated for 8 h with 100 microM PE, reached control levels after 5 h with 10 microM PE, and after 6 h microM PE with 50 microM PE, PE at 50 or 100 microM stimulated irBNP secretion after 15 min, which peaked at 1 h, and thereafter remained above control levels. Calculation of irANF/irBNP ratios revealed that their stimulated secretion was not coregulated. PE caused significant changes in steady state transcript levels for the genes studied. After 6 h, 50 microM PE caused a 49% increase in ANF messenger RNA (mRNA) levels. BNP mRNA levels were increased by 135% after 6 h and by 77% after 8 h. Egr-1 mRNA levels were increased by 81% after 4 h, 167 after 6 h, and 40% after 8 h of treatment, mRNA levels of c-myc were increased by 49% after 4 h and 53% after 6 h. PE-induced increases in secretion and gene expression were inhibited by the alpha1-adrenergic receptor antagonist prozosin (10 microM). We conclude that both ANF and BNP secretion from atria can be stimulated by PE, and that their secretion is not coregulated. The kinetics of enhanced natriuretic peptide gene expression and secretion did not change in parallel, suggesting that these processes are not acutely coordinated. The enhanced expression of Egr-1 and c-myc suggests that they may be involved in the modulation of atrial gene expression in response to alpha1-adrenergic stimulation. The results presented suggest that compensatory adrenergic activation such as those seen in several clinical entities may be one of the factors that provide long-term enhanced natriuretic peptide production, thus contributing to the maintenance of cardiovascular homeostasis.

Mechanical and neuroendocrine regulation of the endocrine heart.

The cardiac natriuretic peptides (NP) -- atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) -- are polypeptide hormones produced by cardiocytes in the atria of mammals. ANF and BNP are continuously released from the heart, but appropriate mechanical or neuroendocrine stimuli increase their rate of release with or without a concomitant increase in synthesis. The results of our investigations lead us to propose that the endocrine response of the heart to pressure or volume load varies in relation to whether the challenge is acute, subacute or chronic. The acute response to stretch is based on a phenomenon referred to as "stretch-secretion coupling" which results in enhanced secretion of NP stored in the atria. NP release following stretch is made at the expense of a depletable NP pool with no apparent effect on synthesis. The stimulation of NP production that is seen during mineralcorticoid escape is referred to as "subacute" and is characterized by stimulation of atrial ANF and BNP gene transcription secondary to volume overload in which plasma ANF, but not plasma BNP, is significantly elevated. With chronic stimulation, as seen in DOCA-salt treatment at the hypertensive stage, activation of the cardiac fetal program in ventricle is seen together with a stimulation of ANF and BNP production in both atria and ventricles. However, the activation of NP gene expression in the atria is not necessarily associated with fetal isogene expression even though the ventricular hypertrophic process is characterized by the expression of fetal isogenes, including ANF and BNP, that are normally expressed in the fetal ventricle. It seems likely that the acute stimulation of NP release is based on an electromechanical coupling. However, protracted stimulation of release is seen in situations in which profound neuroendocrine changes have taken place, thus suggesting that the primary stimulus for chronically enhanced NP gene expression and NP release is based on changes in the hormonal environment of the atrial cardiocyte. It is concluded that the endocrine heart responds to changes in hemodynamic load with specific changes in translational, post-translational and storage processes for ANF and BNP following acute or chronic stimulation. As a result, plasma levels of ANF and BNP may be used as indicators of the degree of atrial hemodynamic overload and ventricular hypertrophy, respectively. It may be advanced that the endocrine heart differentiates and responds to different hemodynamic challenges in either acute or chronic conditions with specific changes in transcription, translation, post-translational processing, storage, and release of ANF and BNP. We propose that this differentiation is part of the reason for the heart to produce two hormones with similar spectra of activity. This paradigm warrants further investigation.

Regulation of alpha-smooth muscle actin expression in adult cardiomyocytes through a tyrosine kinase signal transduction pathway.

Adult rat ventricular myocytes assume after 2 weeks in culture a flattened spread morphology and a loss in organized myofibrils. This sequence of phenotypic changes is accompanied by the reexpression of the fetal gene program. Although different signal transduction pathways were recently shown to be involved in cell growth and differentiation, not much is known about tyrosine kinase activation and cardiac myocyte differentiation. We investigated whether the tyrosine kinase signal transduction pathway is involved in the dedifferentiation of adult rat ventricular myocytes in long-term culture using a specific inhibitor of tyrosine phosphorylation, genistein. For this experiment, adult rat ventricular myocytes were cultured as previously described and incubated in culture medium containing different concentrations of genistein (10-250 microM). After 24 hr of incubation and in a concentration-dependent manner genistein prevented cell spreading. However, at high concentration, cells detached from the plates (10% to 100 microM and 95% at 250 microM). The effect of genistein on adult rat ventricular myocyte phenotype in culture was investigated by examining the expression of total actins and alpha-smooth muscle actin and alpha-sarcomeric actin in cells after 6 days of incubation with and without genistein. Myofibrillar proteins were extracted and separated by gel electrophoresis. Expression of alpha-smooth muscle actin and alpha-sarcomeric actin was determined by Western blotting using specific antibodies. While there was an increase in the amount of total actins and no change in the amount of alpha-sarcomeric actin in the cells exposed to genistein, the amount of alpha-smooth muscle actin decreased with increasing concentrations of genistein reaching undetectable levels at 100 microM. These results demonstrate that genistein inhibits cell spreading and the reexpression of alpha-smooth muscle actin in adult rat ventricular myocytes in culture in a dose-dependent manner, therefore, inhibiting the process of dedifferentiation.

Dissociation of cardiac hypertrophy, myosin heavy chain isoform expression, and natriuretic peptide production in DOCA-salt rats.

We examined the relationship between cardiac hypertrophy, myosin heavy chain (MHC) isoform expression, and production of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) before and after the development of DOCA-salt hypertension. DOCA-salt rats exhibited significant left ventricular hypertrophy at the prehypertensive stage (1 week of treatment), without MHC isoform switch or change in natriuretic peptide gene expression. In the hypertensive stage (5 weeks of treatment), pronounced left ventricular hypertrophy was observed, and this was characterized by an increase in beta-MHC protein, resulting in a switch from 90% alpha-MHC to 51% alpha-MHC and 49% beta-MHC. ANF and BNP mRNA levels and peptide content were significantly increased at this stage. Unexpectedly, the MHC isoform switch was evident in the non-hypertrophied right ventricle to the same degree as in the left ventricle. Natriuretic peptide production was also increased in the right ventricle at 5 weeks of treatment, but to a lesser degree than in the left ventricle. In contrast, in the hypertrophied left atrium there was no MHC isoform switch, while ANF and BNP mRNA levels were augmented. Plasma ANF was significantly increased in the prehypertensive stage; this was accompanied by a partial depletion of atrial ANF stores. Plasma BNP was increased only in the hypertensive stage, reflecting an increase in ventricular BNP synthesis and secretion. These results suggest that 1) cardiac hypertrophy, MHC isoform expression, and stimulation of natriuretic peptide production are processes that may be dissociated from each other; 2) increases in plasma ANF without a concomitant increase in plasma BNP reflect atrial hemodynamic overload, while increases in both ANF and BNP in plasma are associated with ventricular hypertrophy; and 3) there exist differences in the storage, secretion, and processing patterns of ANF and BNP in the atria.

Epicardial mesothelial cells synthesize and release endothelin.

Adult rat ventricular cardiocytes, when cocultured with epicardial mesothelial cells (EMC), demonstrate remarkable plasticity of phenotype accompanied by a significant increase in cardiocyte contractile protein content, suggesting that a factor with growth-promoting properties may take part in EMC-adult rat ventricular cardiocyte interactions. Endothelin (ET) has been shown to induce cell hypertrophy, including enhancement of expression of muscle-specific genes. We investigated the ability of EMC to synthesize and release ET. By light microscopy, specific immunostaining, with either ET-1 or Big ET-1 antibodies, was visualized in EMC as a fine punctate distributed throughout the cytoplasm. Reverse phase-high performance liquid chromatography (HPLC) of epicardial mesothelial cells conditioned medium showed several peaks of immunoreactive ET. The major peak eluted with the same retention time as that of ET-1. By Northern blot analysis, a specific 2.3-kilobase (kb) mRNA species was detected by hybridization to a cDNA insert encoding for rat prepro-ET-1. ET accumulated in the culture medium in a time-dependent manner, whereas cell content remained comparatively low. Angiotensin II (AII) dose-dependently stimulated release of immunoreactive ET into the culture medium.

Atrial natriuretic factor significantly contributes to the mineralocorticoid escape phenomenon. Evidence for a guanylate cyclase-mediated pathway.

The mechanism underlying the mineralocorticoid escape phenomenon remains unknown. To assess the possible contribution of natriuretic peptides to mineralocorticoid escape, rats were injected with 5 mg deoxycorticosterone acetate for 3 d. Plasma atrial natriuretic factor (ANF) rose to twice basal levels and atrial ANF content decreased significantly by 24 h of treatment. This coincided with renal escape and with a significant increase in urinary cGMP excretion. Plasma ANF remained elevated and atrial ANF content continued to decline by 48 and 72 h while atrial ANF mRNA levels increased significantly only at 72 h. Plasma brain natriuretic peptide did not increase during escape although atrial brain natriuretic peptide mRNA levels increased significantly. Chronically administered HS-142-1 (HS), a specific antagonist of the guanylate cyclase-coupled natriuretic peptide receptors, significantly and dose-dependently impaired the escape phenomenon. The highest dose of HS completely suppressed the increase in urinary cGMP. Despite the continued suppression, partial escape was observed by the end of the observation period. HS alone influenced neither plasma nor tissue or urine parameters. These findings show that despite activation of atrial ANF, blockade of the guanylate cyclase-coupled natriuretic peptide receptors impairs the ability of the kidney to escape the Na+ retaining effect of excess mineralocorticoid in a dose-dependent fashion. Later-acting, unknown mechanisms eventually come into play to mediate the escape phenomenon through a guanylate cyclase-independent pathway. Therefore, ANF of cardiac origin appears to be a major factor initiating mineralocorticoid escape through a guanylate cyclase-dependent pathway.

Selective changes in natriuretic peptide and early response gene expression in isolated rat atria following stimulation by stretch or endothelin-1.

OBJECTIVE: Important physiological and pathophysiological conditions are associated with changes in secretion and synthesis of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). The aim of this study was to examine the effects of mechanical stretch and endothelin-1 on ANF and BNP secretion and gene expression in isolated adult rat atria, as paradigms for mechanical and endocrine stimulation of atrial tissue. The expression of the early response genes c-fos, c-jun, Egr-1, and c-myc was also studied, since their protein products may be involved in controlling natriuretic peptide gene expression. METHODS: Isolated rat atria were stimulated by stretch (5 g) or endothelin-1 (10(-7) M) for 30 min, 2 h, or 4 h. ANF and BNP secretion was measured by radioimmunoassay, and relative mRNA levels were determined by northern blotting. RESULTS: Atrial stretch resulted in an immediate 1.8-fold increase in ANF release, which returned to basal levels after 160 min. Endothelin-1 caused a gradual increase in ANF release, up to 2.3 times basal levels, and thereafter returned towards basal levels. BNP secretion was increased threefold by endothelin-1, and remained significantly raised for 90 min. BNP mRNA levels were transiently increased by 33% after 2 h of endothelin-1 stimulation. Stretch increased c-fos mRNA levels (+55%) and Egr-1 mRNA levels (+70%) after 2 h, and increased c-myc mRNA levels (+69%) after 4 h. Endothelin-1 increased Egr-1 mRNA levels up to +767% after 4 h. CONCLUSIONS: Endothelin-1 stimulates BNP secretion from rat atria; this is followed by an increase in BNP mRNA levels. Conversely, acute secretion of ANF by stretch or endothelin-1 is not accompanied by changes in ANF mRNA levels. Atrial stretch results in changes in the expression of the early response genes c-fos, Egr-1, and c-myc, while endothelin-1 stimulates Egr-1 expression. The specific changes in natriuretic peptide and early response gene expression reveal distinct mechanisms of modulation of atrial gene expression by mechanical and endocrine stimuli.