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.

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.

Estrogen, natriuretic peptides and the renin-angiotensin system.

There are significant gender-specific differences in the incidence of hypertension and the clinical outcome of cardiovascular disease between premenopausal women and age-matched men, suggesting that sex hormones such as estrogen (E) might be responsible for the observed cardioprotective effects. This cardioprotective action of E is thought to involve lipoproteins. However, the effect of E on the lipid profile accounts for about 50% of the reduction in cardiovascular disease, indicating that there might be other mechanisms by which E exerts its cardioprotective effects. At present, the underlying mechanism of E action is poorly understood. In this review, the interplay between E, the natriuretic peptides (NP) and the renin-angiotensin system (RAS) is examined. It is hypothesized that E might, through endocrine and/or paracrine action, modulate cardiac NP in females by affecting the RAS either directly or indirectly.

Atrial natriuretic factor and brain natriuretic peptide gene expression in the spontaneous hypertensive rat during postnatal development.

The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production in the spontaneously hypertensive rat (SHR), an experimental model of human hypertension, have produced controversial results and were carried out when hypertension was already established (> 17 weeks). At this time, age-related physiologic and molecular changes in cardiac muscle are difficult to separate from those related to hypertension, ie, increased ANF production and plasma levels. In addition, most of the studies used male rats because the rate of increase in arterial blood pressure--as well as the level to which it rises--is greater in males than in females. Studies of a similar nature using female SHR are not available. The aim of this work was to determine 1) whether ANF and BNP production and secretion increase with the development of hypertension in genetically hypertensive rats; 2) whether a sexual dimorphism in ANF and BNP production and secretion is present in the genetically hypertensive rat during the development of hypertension; and 3) whether the demand for ANF and BNP is the same from each chamber of the heart under these experimental conditions. Age-matched male and female SHR, Wistar-Kyoto (WKY), and Sprague Dawley (SD) rats at 2, 4, and 8 weeks of age were used. The normotensive SD were included to provide a wider basis for baseline findings, as WKY rats are not always a suitable control for SHR due to genetic variations. Natriuretic peptide plasma levels and tissue content were measured by radioimmunoassay. ANF, BNP, as well as alpha- and beta-myosin heavy chain (MHC) mRNA were estimated by Northern blot analysis. Blood pressure (BP) of more than 150 mm Hg was found only in 8-week-old male SHR. Plasma immunoreactive (ir)ANF and irBNP increased significantly at puberty (8 weeks) in both male and female SHR. The earliest molecular change encountered during the development of hypertension was a significant increase in BNP mRNA in the right and left atria from both male and female 8-week-old SHR. In the ventricles from both male and female SHR, there was no increase in the ratio of left ventricular wet weight/body weight, no increase in ventricular ANF mRNA transcripts, and no myosin heavy chain isoform switch (a protein marker of hypertrophy). irBNP ventricular concentration, however, increased significantly in both male and female SHR, but only in female SHR was there a concomitant increase in BNP mRNA. These results suggest that 1) ANF and BNP production are not coordinated in all cardiac compartments during the development of hypertension; 2) upregulation of BNP in the atria from male and female SHR is the earliest event detected at 8 weeks; 3) the prehypertensive stage, in the genetically hypertensive female rats, is associated with an increase in ventricular irBNP concentration and BNP mRNA; 4) there is a dissociation between BP and plasma levels of NP; and 5) as well, there is a dissociation between NP gene expression and MHC isoform switch. The regulation of NP is not coordinated in either gender during the development of hypertension. The activation of the BNP gene in female SHR suggests that BNP might play an important role at the onset of hypertension.

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.

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.

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.

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.

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)

Stretch-secretion coupling in atrial cardiocytes. Dissociation between atrial natriuretic factor release and mechanical activity.

We investigated possible relations between excitation-contraction coupling and atrial natriuretic factor (ANF) release in rat atrial muscle using the isolated perifused atria preparation. To this purpose, the extracellular ionic environment was manipulated by replacement of Ca2+ with the polyvalent cations Ba2+, Sr2+, and La3+. Intracellular Ca2+ was altered by treatment with caffeine and ryanodine and by chemical (49 mM K+) depolarization. Replacement of Ca2+ with Ba2+ or Sr2+ abolished atrial spontaneous mechanical activity but did not prevent ANF release. Chemical depolarization also abolished spontaneous mechanical activity and significantly reduced ANF release in Ca(2+)-free media and in 0.625 mM extracellular Ca2+ but had no effect in the presence of 1.25 or 2.5 mM Ca2+, suggesting that changes in cytosolic Ca2+ levels do not affect ANF release in media containing Ca2+ in the physiological concentration range. The kinetics of ANF release observed during caffeine (10(-6) to 10(-2) M) treatment was similar to that seen in atrial preparations without treatment. At 10(-2) M, caffeine induced an increase in atrial beating rate and resting tension. Ryanodine (10(-4) M) pretreatment reduced stretch-induced ANF release by an average 34%, in addition to inhibiting tension development and beating. These findings clearly show marked differences between atrial cardiocytes and most other endocrine cells in terms of the effect of specific changes in the ionic environment on the secretory response; they also support the view that basal ANF release from atrial cardiocytes is not dependent on contractile atrial activity or Ca2+. In fact, Ca2+ appears to tonically inhibit the rate of basal ANF release. We conclude that, although indispensable for excitation-contraction coupling, intracellular Ca2+ transients by influx or from intracellular stores are not essential for basal ANF release. However, a ryanodine-sensitive compartment appears to be partly responsible for the increased ANF output after muscle stretch.

A decade of atrial natriuretic factor research.

Present views on the biological significance of atrial natriuretic factor (ANF) relate this polypeptide hormone to the regulation of blood pressure and volume through its modulating effects on renal function, on blood vessel tone and permeability, and on the renin-angiotensin-aldosterone system. Although very important advances in the understanding of ANF have been made over the decade since its discovery, some fundamental facts about ANF biosynthesis and release remain to be elucidated. Stretch-induced enhancement of ANF release appears as the most significant mechanism underlying the endocrine response of the atria to acute volume load. This response decays over a period of minutes, indicating that chronic stimulation of ANF release involves mechanisms different from, or in addition to, those acting during acute stretch-stimulated release. In neither acute nor chronic conditions are the cellular or molecular mechanisms underlying ANF release understood. To better understand long-term stimulation of ANF release, we have conducted extensive in vitro testing of several hormones and neurotransmitters to determine their ability to modify ANF release. From these studies, clear-cut evidence of ANF stimulation was obtained with the vasopressor peptide endothelin. Investigations on the cell and molecular biology of cardiac muscle development and hypertrophy have shown that ANF is involved in cardiac growth. The role played by ANF in these processes is now being determined, but this is one line of evidence that suggests that this hormone, together with other natriuretic peptides, may have autocrine or paracrine functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of manipulations of Ca2+ environment on atrial natriuretic factor release.

The effects of Ca2+ on the kinetics of atrial natriuretic factor (ANF) release [measured as immunoreactive cardionatrin (IRC)] were studied on an in vitro, spontaneously beating rat atrial preparation. It was found that ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca2+-free media induced a significant increase in the rate of basal IRC release. Reintroduction of Ca2+ reversed the augmented basal IRC release induced by EGTA and restored mechanical activity. It was also found that the stretch-induced IRC release was independent of extracellular Ca2+ and took place even in the presence of EGTA. The presence of absence of Ca2+ had no apparent effect on ANF processing. In all instances, cardionatrin I (ANF 99-126) was found to be the most abundant peptide released. Morphologically, no obvious differences were observed during either basal or stretch-induced IRC release. These results suggest that, unlike most other endocrine secretory systems, a reduction of cytosolic Ca2+ stimulates basal IRC release. These findings suggest an adaptation of atrial cardiocytes to accomplish their dual role as secretory and contractile cells.

Optimization of atrial natriuretic factor radioimmunoassay.

The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = ANF 99-126) is described. The method was evaluated for accuracy, specificity and precision using different protocols and in various species. The antiserum raised against C I cross-reacts 100% with both human and rat C I and 122% with cardionatrin IV (C IV = ANF 1-126). Dextran-coated charcoal (DCC) and double antibody (DA) disequilibrium protocols were used for the separation of free from antibody-bound radioactivity. The sensitivity, coefficient of variation within assay and between assay for DCC was 4.6 pg/tube, 3.1% and 13.8% respectively, and 1.5 pg/tube, 3.8% and 9.1% for the DA. The mean normal plasma C I levels in human, rat and dog after plasma acidification and extraction using the DA method was 49.7 +/- 4.0; 253.6 +/- 19.6 and 59.9 +/- 3.4 pg/mL respectively.

Functional-morphological studies on in vitro cardionatrin release.

Atrial natriuretic peptides (ANP) are hormones produced by the heart atrial cardiocytes in mammals. The main function of these peptides appears to be that of modulating the actions of the renin-angiotensin-aldosterone system. In the rat ANP are stored within specific atrial granules, mainly as a 126-amino acid peptide (cardionatrin IV) which is processed to a 28-amino acid peptide (cardionatrin I) just prior to or during release from cardiocytes. Development of an isolated perifused rat atria preparation has allowed quantitative and qualitative studies on ANP release. Increasing mechanical load in this preparation gives an increased rate of release of immunoreactive cardionatrin. This finding suggests that there is an intrinsic 'stretch-secretion coupling' in the atria.

Alignment of rat cardionatrin sequences with the preprocardionatrin sequence from complementary DNA.

Mammalian atria contain peptides that promote the excretion of salt and water from the kidney. When rat atrial tissue is extracted under conditions known to inhibit proteolysis, four natriuretic peptides, cardionatrins I to IV, are consistently isolated. These peptides derive from a common precursor, preprocardionatrin, of 152 amino acids, whose sequence was determined by DNA sequencing of a complementary DNA clone. Amino acid sequencing located the start points of cardionatrins I, III, and IV in the overall sequence. Cardionatrin IV most closely resembles procardionatrin because it begins immediately after the signal sequence at residue 25. Cardionatrin III begins at residue 73, and cardionatrin I, sequenced previously, begins at residue 123. Compositional analysis indicated that each of these cardionatrins extends up to tyrosine at position 150 but lacks the terminal two arginine residues.

Demonstration of stellate cells of the pars intermedia of the pituitary gland using a new silver impregnation technique.

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 micron sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.

The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties.

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.

Structural relationships between parenchymal and stromal elements in the pars intermedia of the rat adenohypophysis as demonstrated by extracellular space markers.

The pars intermedia (PI) of the rat adenohypophysis was studied by light and transmission electron microscopy after conventional staining as well as ruthenium red staining, and after systemic injection of horseradish peroxidase. The studies disclose a complex and constant system of two channel types (Type I and Type II channels) formed by PI cells with specific relationships to a very rich nerve supply, to each other, and to a stellate cell type proposed here to represent an element of neuroglia. The channel system could perform a function in the movement of fluids and solutes within the PI which is verturally avascular in the rat as well as in other mammals.