The lung as a target organ for thyroxine

The isolated perfused in situ rat lung preparation was used to investigate the chronic effect of thyroxine on the intermediary metabolism in the mammalian lung. Treatment with thyroxine caused stimulation of the rate of glucose utilization (91 +/- 11 mumol/g dry weight/hr versus 54 +/- 5 mumol/g dry weight/hr). The increase in the rate of glucose uptake was not accompanied by a similar increase in lactate output. Alanine and pyruvate release were also similar in both groups. The implication is that oxidative metabolism of glucose was increased. This study provides the first unequivocal evidence that the mammalian lung is a target organ for thyroxine.     

Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.     

The lung as a possible target for the immune reaction in myositis

Interstitial lung disease is a common manifestation of autoimmune myositis that confers significant morbidity and mortality. The vulnerability of the lung may offer insight into the etiology of this autoimmune disease. The frequency and patterns of lung injury vary based on the autoantibody. Antibodies against the aminoacyl-tRNA synthetases and melanoma differentiation-induced gene-5 are frequently associated with interstitial lung disease. Although the mechanisms underlying these associations have not been fully elucidated, emerging data highlight the importance of autoantigen expression and conformation in the target tissue (lung and muscle, in this case), as well as identifying relevant amplifying pathways (such as regeneration).     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against alpha-human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Summary Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against -human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

The conformation of alpha-human atrial natriuretic polypeptide in solution

The three-dimensional structure of alpha-human ANP in solution was determined through the combined use of nuclear magnetic resonance spectroscopy and distance geometry. The results are based on distance constraints determined by nuclear Overhauser effect measurements and one disulfide bond. The structure is as follows. Three separate regions, which are Ser1-Cys7, Arg11-Ile15, and Gln18-Tyr28 each have some ordered structure. The remaining parts in the sequences of Gly9-Gly10 and Gly16-Ala17 act as hinges. And the C-terminal part is folded back toward the cyclic moiety. The conformation of alpha-hANP reported here is expected to give a better understanding of the relationships between its biological activities and three-dimensional structure.     

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Specific receptors for atrial natriuretic polypeptide on basolateral membranes isolated from rat renal cortex

The binding of alpha-human atrial natriuretic polypeptide (alpha-hANP) to brush border and basolateral membranes isolated from the rat renal cortex was studied at 0 degree C by a rapid filtration technique. Specific binding of 125I-alpha-hANP to basolateral membranes reached a steady state at 4 hr. The binding to brush border membranes was maximal at 5-15 min and then rapidly decreased. The analysis of incubation mixtures with basolateral membranes revealed little degradation of 125I-alpha-hANP during the 4-hr incubation, while there was extensive degradation of the ligand with brush border membranes during the 30-min incubation. High affinity binding of 125I-alpha-hANP was demonstrated on basolateral membranes but not on brush border membranes. These data suggest that specific receptors for alpha-hANP are localized on basolateral membranes of the renal cortex.     

Secretory form of atrial natriuretic polypeptide as cardiac hormone in humans and rats

To elucidate the secretory form of atrial natriuretic polypeptide from the atrium, the molecular form of atrial natriuretic polypeptide in the perfusate from the isolated beating rat heart and in plasma taken at the coronary sinus of 10 patients during cardiac catheterization has been investigated using high performance gel permeation chromatography and reverse phase high performance liquid chromatography coupled with radioimmunoassay for atrial natriuretic polypeptide. Atrial natriuretic polypeptide in the perfusate from the rat heart showed a single peak eluting at the position of a low molecular weight form of atrial natriuretic polypeptide, without any detectable amounts of atrial natriuretic polypeptide with high molecular weights. The major component of atrial natriuretic polypeptide in the rat heart perfusate co-migrated with rat alpha-atrial natriuretic polypeptide in reverse phase high performance liquid chromatography. In 9 out of 10 patients atrial natriuretic polypeptide in plasma taken at the coronary sinus revealed a single peak of atrial natriuretic polypeptide emerging at the position of human alpha-atrial natriuretic polypeptide in gel filtration. Only one plasma sample had a small quantity of high molecular weight forms with the predominant low molecular weight form of atrial natriuretic polypeptide. The major component of atrial natriuretic polypeptide in the plasma extract from the coronary sinus was identified with human alpha-atrial natriuretic polypeptide. These results indicate that alpha-ANP, a 28-amino acid polypeptide, is secreted as a cardiac hormone into the coronary blood stream from the atrium.     

The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium.

The recently described NK2 family of homeodomain proteins are key developmental regulators. In Drosophila melanogaster, two members of this family, bagpipe and tinman, are required for visceral and cardiac mesoderm formation, respectively. In vertebrates, tinman appears to represent a family of closely related NK2 genes, including Nkx-2.5, that are expressed at an early stage in precardiac cells. Consistent with a role for Nkx-2.5 in heart development, inactivation of the Nkx-2.5 gene in mice causes severe cardiac malformations and embryonic lethality. However, little is known about the molecular action of Nkx-2.5 and its targets in cardiac muscle. In this paper, we report the identification and characterization of a functional and highly conserved Nkx-2.5 response element, termed the NKE, in the proximal region of the cardiac atrial natriuretic factor (ANF) promoter. The NKE is composed of two near-consensus NK2 binding sites that are each able to bind purified Nkx-2.5. The NKE is sufficient to confer cardiac cell-specific activity to a minimal TATA-containing promoter and is required for Nkx-2.5 activation of the ANF promoter in heterologous cells. Interestingly, in primary cardiocyte cultures, the NKE contributes to ANF promoter activity in a chamber- and developmental stage-specific manner, suggesting that Nkx-2.5 and/or other related cardiac proteins may play a role in chamber specification. This work provides the identification of a direct target for NK2 homeoproteins in the heart and lays the foundation for further molecular analyses of the role of Nkx-2.5 and other NK2 proteins in cardiac development.     

The cockroach heart as a bioassay organ

Detailed procedures are presented for denervation of the American cockroach heart, Periplaneta americana L., by removal of the lateral cardiac nerve cords. Results of bioassay with the innervated heart are also presented and compared to responses obtained from identical assay conditions with the denervated heart.The response of the innervated heart to 10^-3 M sodium azide, slight changes in the concentration of sodium ion, reduced glutathione, saline dilutions, and low amounts of ethanol and acetone was characterized by the immediate appearance of irregularities in the heartbeat. In contrast, the responses of the denervated heart to the first three of these compounds were always characterized by a smooth even heartbeat changing gradually in amplitude and/or rate and/or contractile state of the alary muscles. All assay conditions examined caused qualitatively less response in the beat of the denervated heart.Irregularities in the beat of the innervated heart which were induced in bioassay were found to be due to extreme sensitivity of the spontaneously active cardiac neurons.

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Zusammenfassung Das isolierte abdominale Herz von Periplaneta americana L. reagiert auf Durchspülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung mit einem unmittelbaren Anstieg des Tonus bis zu fast systolischer Hemmung. Danach verringert das Herz den Tonus allmählich, schlägt unregelmäßig und tritt in eine kurze Periode diastolischer Pause ein, bevor es in Diastole stehen bleibt. Die Herzschlagaktivität wurde wieder aufgenommen, wenn das Natriumazid durch Spülung mit physiologischer Kochsalzlösung ersetzt wird.Nach Entfernung beider Paare der Herzseitennerven gab das Herz von P. americana auf Bespülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung keine Initialreaktion. Statt dessen stieg der Herzschlag nach 2 min allmählich leicht an und die Herzschlagamplitude nahm im Verlaufe von 6 min sehr allmählich ab. Nach 6 min blieb das Herz in Diastole stehen.Die Herzreaktionen der amerikanischen Küchenschabe waren bei Auftropf-Tests mit anormaler Natriumchloridkonzentration, herabgesetztem Glutathion und Lösungsmitteln ähnlich den für Natriumazid beschriebenen. Die Reaktion des denervierten Herzens ist durch das Fehlen von Unregelmäßigkeiten im Herzschlagmuster gekennzeichnet.Sowohl das denervierte wie das innervierte Herz reagieren auf Testtropfen von nur etwas anormalen Calciumchloridkonzentrationen.

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Supported by USPHS Training grant PHS GM 1076.     

Central interaction of the brain atrial natriuretic polypeptide (ANP) system and the brain renin-angiotensin system in ANP secretion from heart--evidence for possible brain-heart axis

To elucidate the involvement of the brain renin-angiotensin system and the brain atrial natriuretic polypeptide (ANP) system in the regulation of ANP secretion from the heart, the effects of intracerebroventricular administration of angiotensin II and ANP on the plasma ANP level were examined in conscious unrestrained rats. The intracerebroventricular administration of angiotensin II at doses of 100 ng and 1 microgram significantly enhanced ANP secretion induced by volume-loading with 3-mL saline infusion (peak values of the plasma ANP level: control, 220 +/- 57 pg/mL; 100 ng angiotensin II, 1110 +/- 320 pg/mL, p less than 0.01; 1 microgram angiotensin II, 1055 +/- 60 pg/mL, p less than 0.01). The intracerebroventricular injection of angiotensin II at the same doses alone had no significant effect on the basal plasma ANP level. The enhancing effect of central angiotensin II on ANP secretion induced by volume-loading was significantly attenuated by pretreatment with the intravenous administration of the V1-receptor antagonist of vasopressin or with the intracerebroventricular administration of phentolamine. The intracerebroventricular administration of alpha-rANP(4-28) (5 micrograms) had no significant influence on the basal plasma ANP level; however, it significantly attenuated central angiotensin II potentiating effect of volume-loading induced ANP secretion. These results indicate that the brain renin-angiotensin system regulates ANP secretion via the stimulation of vasopressin secretion and (or) via the activation of the central alpha-adrenergic neural pathway, and that the brain ANP system interacts with the brain renin-angiotensin system in the central modulation of ANP secretion from the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Where and when natriuretic peptides are secreted in the heart

This review presents recent data on the structure, synthesis, and secretion of cardiac natriuretic peptides. It is known that these hormones have a broad spectrum of activity, but they remain the least studied and poorly understood link in the regulation of the water-salt homeostasis. Emphasis is placed on the problem of ontogenetic formation of the heart secretory activity during embryogenesis. We discuss the available scarce and scattered information on the paracrine and autocrine effects of the peptides on intercellular interactions, and on the division, growth and differentiation of the heart cells. These issues are hardly addressed in Russian literature.     

Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure

The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.     

Alpha-human atrial natriuretic polypeptide (alpha-hANP) in normal volunteers and patients with heart failure or hypertension

The presence of alpha-hANP immunoreactive material in human heart and plasma was investigated with a specific and sensitive radioimmunoassay and immunohistochemical method. It was found that alpha-hANP immunoreactive staining of specific atrial granules was located around the nucleus of atrial cardiocytes. No immunoreactive staining was found in the ventricle. The content of immunoreactive hANP was 0.5 pmol/mg protein in the atria and 0.11 +/- 0.01 pmol/ml in the plasma of 26 normal volunteers. In 16 patients with congestive heart failure and 26 patients with essential hypertension, the plasma level of immunoreactive alpha-hANP was significantly lower than that in normal humans. The above evidence indicate that alpha-hANP is a putative hormone secreted by human atrium. A relative shortage of alpha-hANP in the circulatory system may be involved in the mechanism of heart failure and hypertension.     

Presence of atrial natriuretic polypeptide in the pulmonary vein and vena cava

Striated muscle cells and storage granules observed in the atria were found in main branches of the pulmonary veins and superior and inferior venae cavae of the rat, pig, and ox. The presence of atrial natriuretic polypeptide (ANP) in these veins was examined by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for ANP. The veins contained 0.6 to 8.0 ng ANP/mg wet tissue with the major molecular form being gamma-ANP. ANP was detected in the peripheral lung tissue in a small quantity, but was not detected in the pulmonary artery. The identification of gamma-ANP and storage granules stained with an anti-ANP antiserum in the pulmonary vein and vena cava suggest that the veins may participate in regulating volume status, blood pressure, and cardiovascular homeostasis through the release of ANP.     

The heart as an endocrine organ

The discovery of atrial natruietic peptide firmly established that the heart was not just a pump and provided the long-sought-after link between the heart and the kidney in the control of soduim balance. Pharmacological targeting of the natruiretic peptide system is now leading to novel advances in the treatment of hypertension and of heart failure - two of the most common causes of human disability and death.     

Liver aldolase as a possible target for the action of N-methyl-N-nitrosourea

The effect of N-methyl-N-nitrosourea (MNU) on the activity of cytoplasmic and reversibly bound to subcellular structures liver aldolase was studied. In vitro, the activity of aldolase purified from rabbit muscles is inhibited by MNU by 70-80% relative to fructose-1,6-diphosphate and by 50-60% relative to fructose-1-phosphate. These substrates and the competitive inhibitor ATP do not protect the enzyme against the inactivation by MNU. MNU inhibits the activity of cytoplasmic aldolase by 30-40% and 20% 2-24 hours after a single injection (80 mg/kg) in vivo. The enzyme affinity for fructose-1,6-diphosphate is markedly decreased (2-fold). Activation of cytoplasmic aldolase relative to both substrates, which is especially well-pronounced with fructose-1-phosphate after inhibition of the enzyme activity, was observed. The enzyme activity relative to both substrates was found to increase in the mitochondrial and nuclear fractions within 48 hours. MNU has no effect on the activity of aldolase bound to microsomes. MNU influences the aldolase binding to organelle membranes. MNU injections at early periods (2-168 hours) accounts for the differences in the kinetic properties of cytoplasmic and reversibly bound to subcellular structures liver aldolase. These changes persist within 168 hours after MNU administration and may result in disturbances in cell metabolism as well as in the regulation of metabolic pathways, such as glycolysis and gluconeogenesis.     

The heart as an endocrine organ

Modern data on atrial natriuretic factor (ANF) are presented. Synthesis of this factor, its storage and release from cardiac atria are described. The role of ANF in the body fluid volume regulation and blood pressure homeostasis is discussed. ANF is regarded as a circulating hormone.