Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction

Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG). Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG. Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase. While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA. PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported. Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation. Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor. PKG translocation is ANP-dependent but not nitric oxide-dependent. Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects. As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.     

Atrial natriuretic peptide in hypoxia

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. Among them, atrial natriuretic peptide (ANP) synthesis and secretion is increased during hypoxic exposure and plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases, including chronic hypoxia-induced pulmonary hypertension and vascular remodeling, and right ventricular hypertrophy and right heart failure. This review discusses the roles of ANP and its receptors in hypoxia-induced pulmonary hypertension. We and other investigators have demonstrated that ANP gene expression is enhanced by exposure to hypoxia and that the ANP so generated protects against the development of hypoxic pulmonary hypertension. Results also show that hypoxia directly stimulates ANP gene expression and ANP release in cardiac myocytes in vitro. Several cis-responsive elements of the ANP promoter are involved in the response to changes in oxygen tension. Further, the ANP clearance receptor NPR-C, but not the biological active NPR-A and NPR-B receptors, is downregulated in hypoxia adapted lung. Hypoxia-sensitive tyrosine kinase receptor-associated growth factors, including fibroblast growth factor (FGF) and platelet derived growth factor (PDGF)-BB, but not hypoxia per se, inhibit NPR-C gene expression in pulmonary arterial smooth muscle cells in vitro. The reductions in NPR-C in the hypoxic lung retard the clearance of ANP and allow more ANP to bind to biological active NPR-A and NPR-B in the pulmonary circulation, relaxing preconstricted pulmonary vessels, reducing pulmonary arterial pressure, and attenuating the development of hypoxia-induced pulmonary hypertension and vascular remodeling.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Role of EDHF in type 2 diabetes-induced endothelial dysfunction

Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when nitric oxide-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K+), and hydrogen peroxide (H2O2) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild-type (WT, normal), db/db (advanced type 2 diabetic) mice, and db/db mice null for TNF (dbTNF-/dbTNF-). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 10 micromol/l) and prostaglandin synthase inhibitor indomethacin (Indo, 10 mumol/l)] was diminished after the administration of catalase (an enzyme that selectively dismutates H2O2 to water and oxygen, 1,000 U/ml); administration of the combination of charybdotoxin (a nonselective blocker of intermediate-conductance Ca2+-activated K+ channels, 10 micromol/l) and apamin (a selective blocker of small-conductance Ca2+-activated K+ channels, 50 micromol/l) also attenuated EDHF-induced vasodilation, but the inhibition of EETs synthesis [14,15-epoxyeicosa-5(Z)-enoic acid; 10 mumol/l] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after an inhibition of K+ channel, EETs synthesis, or H2O2 production. Our molecular results indicate that mRNA and protein expression of interleukin-6 (IL-6) were greater in db/db versus WT and dbTNF-/dbTNF- mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg.ml(-1).kg(-1) ip for 3 days) attenuated IL-6 expression in db/db mice. The incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of l-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of L-NAME and Indo in db/db mice. In db(TNF-)/db(TNF-) mice, EDHF-induced vasodilation was greater and comparable with controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6-induced endothelial dysfunction by the activation of H2O2 or a K+ channel in type 2 diabetes.     

Atrial natriuretic peptide in infants and children

Atrial natriuretic peptide (ANP) was measured in the plasma of 192 normal infants and children aged 1 day to 18 years. Plasma ANP was high during postnatal adaptation, particularly in premature infants. In 96 infants and children aged 4 months to 18 years, plasma ANP was similar to values obtained in 7 healthy adult volunteers (23.9 +/- 11.9 vs. 25.7 +/- 4.6 fmol/ml). There was no significant relationship between ANP and age. ANP is elevated about twofold in full-term neonates being 3-4 days of age, and returned to normal thereafter. It is concluded that ANP is raised during the postnatal adaptation. This hormone is possibly involved in the postnatal volume contraction and may antagonize vasoconstrictor hormones that are elevated during the postnatal period.     

Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.     

Atrial natriuretic peptide deposited as atrial amyloid fibrils

Deposition of amyloid in the atria is exceedingly common in the aging heart. We have extracted amyloid fibrils from atria and purified a major protein which had N-terminal amino acid sequence identical to that of atrial natriuretic peptide (ANP). Antisera to ANP and to the amyloid fibril protein both labelled atrial muscle cells and atrial amyloid in an identical way.     

Atrial natriuretic peptide hormonal system in plants.

To determine if atrial natriuretic peptides are present in plants as well as animals, where they are important for water and sodium metabolism, the leaves and stems of the Florida Beauty (Dracena godseffiana) were examined. The N-terminus consisting of amino acids (a.a.) 1-98 (i.e., pro ANF 1-98), the mid portion of the N-terminus (a.a. 31-67; pro ANF 31-67), and C-terminus (a.a. 99-126; ANF) of the 126 a.a. atrial natriuretic factor (ANF) prohormone were all present in the leaves and stems of this plant. The concentrations of pro ANF 1-98, pro ANF 31-67 and ANF-like peptides of 120 +/- 20, 123 +/- 21, and 129 +/- 20 ng/g of plant tissue in leaves and 109 +/- 20, 96 +/- 21, and 124 +/- 18 ng/g of tissue, respectively, in the stems were lower (P less than 0.05) than their concentrations in rat (Rattus norvegicus) heart atria of 196 +/- 40, 192 +/- 28, and 189 +/- 15 ng/g of tissue respectively, but higher (P less than 0.001) than their respective concentrations of 4.3 +/- 1.4, 4.1 +/- 1.2, and 3.9 +/- 1 ng/g of rat heart ventricular tissue. We conclude that the atrial natriuretic peptide-like hormonal system is present in the plant kingdom as well as in the animal kingdom.     

Atrial natriuretic peptide induces shedding of endothelial glycocalyx in coronary vascular bed of guinea pig hearts

Atrial natriuretic peptide (ANP) is reported to enhance vascular permeability in vivo. Our aim was to evaluate the impact of ANP on coronary extravasation of fluids and macromolecules and on the integrity of the endothelial glycocalyx. Isolated guinea pig hearts (n = 6/group) were perfused with Krebs-Henseleit buffer in a Langendorff mode. A 6% hydroxyethyl starch (HES) solution was infused into the coronary system for 20 min without (Control group) and simultaneously with (ANP group) ANP at 10(-9) M. In two further series, the glycocalyx was enzymatically degraded by means of heparinase (Hep) application (10 IU over 15 min), followed again by the infusion of HES in the absence (Hep group) and presence (ANP+Hep group) of ANP. Net fluid filtration, extravasation of HES, electron microscopic visualization of the glycocalyx, and quantification of shedding of syndecan-1, a component of the glycocalyx, were determined. An increase in fluid leak was observed in ANP, ANP+Hep, and Hep hearts [+29%, +31%, +14%, respectively; a decrease was observed in Control hearts (-13%)]. Similarly, an accelerated extravasation of colloid was observed in these three groups. Coronary release of syndecan-1 increased 9- to 18-fold during infusion of ANP. Electron microscopy revealed a dramatic degradation of the glycocalyx after ANP. These results indicate that the endothelial glycocalyx serves as a barrier to transmural exchange of fluid and colloid in the coronary vascular system. ANP causes rapid shedding of individual components of the glycocalyx and histologically detectable degradation. Thus the permeability-increasing effect of ANP may be at least partially related to changes in the integrity of the endothelial glycocalyx.     

Atrial natriuretic peptide in lymphoid organs of various species

1. Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (1-126) as well the physiologically active ANP (99-126), were identified by chromatographic analysis and RIA in extracts of thymus, spleen and lymph nodes of rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Atrial natriuretic peptide, B-type natriuretic peptide, and serum collagen markers after acute myocardial infarction.

Experimental data suggest that atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) act locally as antifibrotic factors in heart. We investigated the interrelationships of natriuretic peptides and collagen markers in 93 patients receiving thrombolytic treatment for their first acute myocardial infarction (AMI). Collagen formation following AMI, evaluated as serum levels of amino terminal propeptide of type III procollagen, correlated with NH(2)-terminal proANP (r = 0.45, P < 0.001), BNP (r = 0.55, P < 0.001) and NH(2)-terminal proBNP (r = 0.50, P < 0.01) on day 4 after thrombolysis. Levels of intact amino terminal propeptide of type I procollagen decreased by 34% (P < 0.001), and levels of carboxy terminal cross-linked telopeptide of type I collagen (ICTP) increased by 65% (P < 0.001). ICTP levels correlated with NH(2)-terminal proBNP (r = 0.25, P < 0.05) and BNP (r = 0.28, P < 0.05) on day 4. Our results suggest that ANP and BNP may act as regulators of collagen scar formation and left ventricular remodeling after AMI in humans. Furthermore, degradation of type I collagen is increased after AMI and may be regulated by BNP.     

Atrial natriuretic peptide messenger RNA and peptide in rats with aortic valve insufficiency

To investigate ANP gene expression in diseased hearts of animals without genetic defects, immunoreactive ANP (IR-ANP) and ANP mRNA were measured in a rat with aortic valve insufficiency (AI), which was produced by puncturing one of the aortic valve leaflets with a plastic rod. Plasma IR-ANP concentration was higher in AI rats than in sham rats (p less than 0.05). Decreased atrial concentration of IR-ANP (p less than 0.05) and unchanged atrial ANP mRNA concentration were shown in AI rats. The ventricular concentrations of IR-ANP and ANP mRNA in AI rats were 5.4 and 2.4 times higher than those in sham rats (p less than 0.05, respectively). These results demonstrate that gene expression of ventricular ANP is markedly increased in AI rats while that of atrial ANP is not changed.