Absence Of Atrial Natriuretic Peptide Research Articles

Valporic acid enhances the Atrial Natriuretic Peptide (ANP) mediated anti-hypertrophic activity by modulating the Npr1 gene transcription in H9c2 cells in vitro

The present study was aimed to determine whether stimulating Npr1 gene activity using Valporic acid (VA), a small short chain fatty acid molecule can enhance ANP mediated anti-hypertrophic activity in isoproterenol (ISO) – treated H9c2 cells in vitro. H9c2 cells were treated with ISO (10−5 M) and co-treated with VA (10−5 M) in the presence and absence of ANP (10−8M), for 48h. ATRA (10−5 M) was used as a positive inducer of Npr1 gene transcription. The mRNA expression of Npr1 and PKG-I genes, proto-oncogenes (c-fos, c-jun and c-myc) and hypertrophic markers (ANP, BNP, α-sk and β-MyHC), genes were determined by quantitative PCR (qPCR). The protein profiling of NPR-A, PKG-I and cGMP were evaluated by Western blot, immunofluorescence and ELISA respectively. A marked reduction in the level of expression of Npr1 (3- fold) and PKG-I (2.5-fold) genes and increased expression of proto-oncogenes (p< 0.001, respectively) and hypertrophic marker genes (p<0.001, respectively) were noticed in the ISO-treated H9c2 cells as compared with control cells. In contrast, the VA treated cells showed maximal Npr1 gene expression (3.5-fold) as compared with ATRA treated cells (2 fold), which is well correlated with the intracellular cGMP levels (80% vs 60%) and reduced (2.5-fold) HDAC −1&−2 mRNA expression. Furthermore, VA or ATRA treatment effectively reversed the ISO-induced altered expression of Npr1 and PKG-I genes, proto-oncogenes, and hypertrophic markers genes. Interestingly, the results of the present study suggest that ANP mediated anti-hypertrophic activity was enhanced with either VA (p<0.001) or ATRA (p<0.01) co-treatment. Together, we conclude that VA in combination with ANP can be a novel therapeutical approach for the treatment and management of left ventricular cardiac hypertrophy.

Suppression of atrial natriuretic peptide/natriuretic peptide receptor-A-mediated signaling upregulates angiotensin-II-induced collagen synthesis in adult cardiac fibroblasts

Cardiac hormone atrial natriuretic peptide (ANP) and its receptor natriuretic peptide receptor-A (NPR-A) system acts as an intrinsic negative regulator of abnormal extracellular matrix (ECM) remodeling in the heart. However, the underlying mechanism by which ANP/NPR-A system opposes the ECM remodeling in the diseased heart is not well understood. Here, we investigated the anti-fibrotic mechanism of ANP/NPR-A in fibrotic agonist Angiotensin- II (ANG II)-treated adult cardiac fibroblast (CF) cells. Normal and NPR-A-suppressed adult CF cells were treated with ANG II (10(-7) M) in the presence and absence of ANP (10(-8) M) for 24 h. Total collagen concentration, activity and expression of MMP-2 and MMP-9, and nuclear translocation of Nuclear factor-kappaB (NF-κB-p50) were studied. NPR-A-suppressed adult CF cells exhibited a more pronounced increase in collagen production, ROS generation, and NF-κB-p50 nuclear translocation as compared to adult CF cells treated with agonist alone. ANP co-treatment significantly reverses the agonist-induced above changes in normal adult CF cells, while it failed to reverse the agonist-induced collagen synthesis in the NPR-A-suppressed adult CF cells. The cGMP analog (8-bromo-cGMP) treatment significantly attenuated the agonist-induced collagen synthesis both in normal and NPR-A-suppressed adult cells. The results of this study suggest that ANP/NPR-A signaling system antagonizes the agonist-induced collagen synthesis via suppressing the activities of MMP-2, MMP-9, ROS generation, and NF-κB nuclear translocation mechanism.

Atrial Natriuretic Peptide Reduces Vascular Leakage and Choroidal Neovascularization

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANP's effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANP's TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.

Altered regulation of renal interstitial hydrostatic pressure and the renal renin–angiotensin system in the absence of atrial natriuretic peptide

Although it has been well established that atrial natriuretic peptide gene-disrupted (ANP-/-) mice are a useful model of salt-sensitive hypertension, surprisingly little is known about the control of their intrarenal renin-angiotensin system (RAS) and pressure-natriuresis mechanism, key components in blood pressure, fluid and electrolyte regulation. The aim of this study was to determine whether ANP disruption results in changes in the renal and adrenal local RAS and the acute pressure-natriuresis mechanism. Renal and adrenal renin, angiotensin type 1 (AT1)(A and B) and angiotensin type 2 (AT2) receptor messenger RNA expression levels were determined by northern blotting or real time reverse transcriptase-polymerase chain reaction. Plasma aldosterone and renal and adrenal angiotensin II peptide levels were determined by radioimmunoassay. To examine the acute pressure-natriuresis response, changes in renal interstitial hydrostatic pressure (RIHP) were assessed after manipulations of renal arterial pressure (RAP) in anaesthetized mice. Renal and adrenal renin mRNA and angiotensin II levels were lower in ANP-/- and +/- mice compared with +/+mice. ANP-/- mice also had greater renal AT1A and adrenal AT2 mRNA levels compared with the other genotypes. RAP and RIHP were significantly higher in -/-mice compared with +/+mice. Furthermore, there was a blunted slope of the RAP-RIHP relationship after increases in RAP in ANP-/- mice. These data indicate that ANP disruption results in a blunting of the dynamic properties of the acute pressure-natriuresis mechanism at increased levels of RAP, as well as a reduced expression of renal and adrenal local RAS.

Antioxidant actions contribute to the antihypertrophic effects of atrial natriuretic peptide in neonatal rat cardiomyocytes

Reactive oxygen species (ROS) such as superoxide have been linked to the hypertrophic response of the heart to stimuli including angiotensin II (AngII), mechanical stretch, and pressure overload. We have previously demonstrated that cGMP and protein kinase G mediate the antihypertrophic actions of the natriuretic peptides in rat cardiomyocytes and isolated whole hearts. The impact of natriuretic peptides on cardiac ROS generation, however, has not been investigated. We tested the hypothesis that reduced superoxide accumulation contributes to the antihypertrophic action of atrial natriuretic peptide (ANP). Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or endothelin-1 (ET(1), 60 nmol/L) in the presence and absence of ANP (1 micromol/L) or tempol (100 micromol/L). Hypertrophic responses, cardiomyocyte superoxide generation, and cardiomyocyte expression of NADPH oxidase were determined. AngII induced increases in cardiomyocyte size (to 176 +/- 9% n = 8 p < 0.001, at 48 h), beta-myosin heavy chain expression (to 4.0 +/- 1.6-fold n = 6 p < 0.05, at 48 h), c-fos expression (to 1.9 +/- 0.5-fold n = 7 p < 0.01, at 6 h), superoxide generation (to 181+/-21% n = 8 p < 0.005, at 24 h), and expression of the gp91phox subunit of NADPH oxidase (to 2.4 +/- 0.5-fold n = 7 p < 0.05, at 48 h). These effects were all significantly inhibited by ANP: cardiomyocyte size, beta-myosin heavy chain expression, c-fos expression, superoxide generation and gp91phox expression were reduced to 107 +/- 5% (n = 5 p < 0.05), 1.2 +/- 0.2-fold (n = 6 p < 0.05), 0.9 +/- 0.2-fold (n = 7 p < 0.05), 141 +/- 21% (n = 8 p < 0.05), and to 1.0 +/- 0.5-fold (n = 7 p < 0.05), respectively. These effects were mimicked by tempol. ANP and tempol also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation, but had no effect on markers of hypertrophy when studied alone. This data indicates that the antihypertrophic actions of ANP are accompanied by reduced levels of superoxide, suggesting an antioxidant action contributes to the antihypertrophic actions of ANP.

Atrial natriuretic peptide induces an acrosome reaction in giant panda spermatozoa and enhances their penetration of salt-stored porcine oocytes

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5 h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1 nM ANP. Treatment with C-ANP-(4–23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 5′-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted in a higher proportion ( P < 0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte ( P < 0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine salt-stored oocytes by frozen-thawed giant panda spermatozoa.

Direct ANP inhibition of hypoxia-induced inflammatory pathways in pulmonary microvascular and macrovascular endothelial monolayers

Atrial natriuretic peptide (ANP) has been shown to reduce hypoxia-induced pulmonary vascular leak in vivo, but no explanation of a mechanism has been offered other than its vasodilatory and natriuretic actions. Recently, data have shown that ANP can protect endothelial barrier functions in TNF-alpha-stimulated human umbilical vein endothelial cells. Therefore, we hypothesized that ANP actions would inhibit pulmonary vascular leak by inhibition of TNF-alpha secretion and F-actin formation. Bovine pulmonary microvascular (MVEC) and macrovascular endothelial cell (LEC) monolayers were stimulated with hypoxia, TNF-alpha, or bacterial endotoxin (LPS) in the presence or absence of ANP, and albumin flux, NF-kappa B activation, TNF-alpha secretion, p38 mitogen-activated protein kinase (MAPK), and F-actin (stress fiber) formation were assessed. In Transwell cultures, ANP reduced hypoxia-induced permeability in MVEC and TNF-alpha-induced permeability in MVEC and LEC. ANP inhibited hypoxia and LPS increased NF-kappa B activation and TNF-alpha synthesis in MVEC and LEC. Hypoxia decreased activation of p38 MAPK in MVEC but increased activation of p38 MAPK and stress fiber formation in LEC; TNF-alpha had the opposite effect. ANP inhibited an activation of p38 MAPK in MVEC or LEC. These data indicate that in endothelial cell monolayers, hypoxia activates a signal cascade analogous to that initiated by inflammatory agents, and ANP has a direct cytoprotective effect on the pulmonary endothelium other than its vasodilatory and natriuretic properties. Furthermore, our data show that MVEC and LEC respond differently to hypoxia, TNF-alpha-stimulation, and ANP treatment.

Effects of pressure overload on extracellular matrix expression in the heart of the atrial natriuretic peptide-null mouse.

This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition.

The atrial natriuretic peptide as a regulator of Kupffer cell functions.

Kupffer cells (KCs), the resident macrophages of the liver, contribute prominently to liver injury by inflammatory mediators. Pre-conditioning with the atrial natriuretic peptide (ANP), known also as a regulator of macrophage functions, attenuates hepatic ischemia-reperfusion injury. Therefore, the aim of this study was to determine the presence of functional ANP receptors on isolated KCs and to investigate whether this hepatoprotective hormone influences the activation of KCs. KCs were isolated by collagenase/pronase digestion followed by elutrial centrifugation and cultured for 1 to 3 days. Intracellular cyclic guanosine 3'5'-monophosphate (cGMP) concentrations were measured by radioimmunoassay after treating the cells with sodium nitroprusside or ANP. KCs were stimulated with bacterial lipopolysaccharide in the presence or absence of ANP, and inflammatory mediators were determined. Phagocytosis was assayed using Coumarin-labeled latex particles and flow cytometric analysis. Treatment of KCs with ANP but not with sodium nitroprusside resulted in a significant elevation of intracellular cGMP levels indicating functional type A natriuretic peptide receptors (NPR-As). ANP significantly reduced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFalpha) secretion, paralleled by an increased cell-associated TNFalpha. LPS-induced TNFalpha mRNA expression was not affected. ANP significantly increased phagocytotic activity of KCs via NPR-A. No effect of ANP on LPS-activated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 protein levels, iNOS mRNA expression, nitric oxide, and PGE2-production was observed. We demonstrated functional cGMP-dependent ANP receptors in isolated rat KCs. ANP reduced TNFalpha release possibly by influencing post-translational processing of TNFalpha in LPS-activated KCs. In addition, we demonstrated that ANP enhances phagocytosis in KCs. These effects may contribute to the hepatoprotective actions of ANP.

Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability.

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.

Agonistic Induction of a Covalent Dimer in a Mutant of Natriuretic Peptide Receptor-A Documents a Juxtamembrane Interaction That Accompanies Receptor Activation

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.

Hypoxia reduces atrial natriuretic peptide clearance receptor gene expression in ANP knockout mice.

We tested the hypotheses that hypoxic exposure is associated with exacerbated pulmonary hypertension and right ventricular (RV) enlargement, reduced atrial natriuretic peptide (ANP) clearance receptor (NPR-C) expression, and enhanced B-type natriuretic peptide (BNP) expression in the absence of ANP. Male wild-type [ANP(+/+)], heterozygous [ANP(+/-)], and homozygous [ANP(-/-)] mice were studied after a 5-wk hypoxic exposure (10% O(2)). Hypoxia increased RV ANP mRNA and plasma ANP levels only in ANP(+/+) and ANP(+/-) mice. Hypoxia-induced increases in RV pressure were significantly greater in ANP(-/-) than in ANP(+/+) or ANP(+/-) mice (104 +/- 17 vs. 45 +/- 10 and 63 +/- 7%, respectively) as were increases in RV mass (38 +/- 4 vs. 26 +/- 5 and 29 +/- 4%, respectively). NPR-C mRNA levels were greatly reduced in the kidney, lung, and brain by hypoxia in all three genotypes. RV BNP mRNA and lung and kidney cGMP levels were increased in hypoxic mice. These findings indicate that disrupted ANP expression worsens hypoxic pulmonary hypertension and RV enlargement but does not alter hypoxia-induced decreases in NPR-C and suggest that compensatory increases in BNP expression occur in the absence of ANP.

Natriuretic Peptides Inhibit G Protein Activation: MEDIATION THROUGH CROSS-TALK BETWEEN CYCLIC GMP-DEPENDENT PROTEIN KINASE AND REGULATORS OF G PROTEIN-SIGNALING PROTEINS

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.

Efficacy of isatin analogues as antagonists of rat brain and heart atrial natriuretic peptide receptors coupled to particulate guanylyl cyclase

Isatin is an endogenous indole and an inhibitor of atrial natriuretic peptide (ANP) receptors coupled with particulate guanylyl cyclase (GC). In this study, several isatin analogues were tested as inhibitors of ANP-stimulated GC in rat brain and heart membranes. None of these analogues affected activity in the absence of ANP, or stimulated ANP-induced activity. In both tissues, some 5-substituted isatins (5-hydroxyisatin, 5-methylisatin, and 5-aminoisatin) exhibited more effective inhibitory activity than isatin itself, with ic 50 values in the range 1.3–20 μM. The efficacy of other analogues varied and was not consistent between the two tissues, raising the possibility of receptor heterogeneity and relative selectivity of inhibition. Some substituted isatins may have a role as pharmacological tools for investigating the physiological roles of natriuretic peptides and their receptors.

Chronic regulation of arterial blood pressure by ANP: role of endogenous vasoactive endothelial factors.

Atrial natriuretic peptide (ANP) exerts a chronic hypotensive effect due to a decrease in total peripheral resistance (TPR). This study examines if chronic ANP-dependent vasodilation is attributable to differences in the cardiovascular regulatory activity of vascular endothelium (VE), based on evidence that ANP affects synthesis/release and target cardiovascular effects of endothelin-1 (ET-1), C-type natriuretic peptide (CNP), and nitric oxide (NO). To determine if the synthetic activity of resistance vasculature VE is chronically altered by plasma ANP activity, we measured ET-1, CNP, and endothelial constitutive NO synthase (ecNOS) concentration and total NOS enzyme activity in homogenates of kidney, heart, lung, hindquarter skeletal muscle, and brain from hypotensive transgenic mice with elevated plasma ANP, hypertensive knockout mice (-/-) characterized by the absence of ANP, and the corresponding normotensive wild-type (NT, +/+) mice. Tissue distribution and abundance patterns of ET-1, CNP, ecNOS, and NOS enzyme activity were comparable between the different genotypes and did not differ significantly between mutant and control mice. Antagonism of ETA/B receptors in -/- and +/+ mice in vivo with SB-209670 reduced arterial blood pressure (ABP) significantly and comparably in both genotypes (-27 +/- 4 and -25 +/- 2% change for -/- and +/+ mice, respectively) independent of any significant changes in heart rate (HR) (-6 +/- 8 and -4 +/- 4% change for -/- and +/+ mice, respectively). Immunoneutralization of CNP-specific guanylate cyclase-linked receptors (GC-B) with monoclonal antibodies (3G12) increased ABP slightly, but not significantly, by similar relative amounts in both -/- (10 +/- 6% change) and +/+ mice (8 +/- 3% change), without changing HR significantly (4 +/- 1% change for both +/+ and -/- mice). Inhibition of NOS activity (by NG-nitro-L-arginine methyl ester) significantly increased ABP, but the changes were comparable between -/- (53 +/- 5% change) and +/+ mice (50 +/- 6% change) and occurred in the absence of significant changes in HR (-1 +/- 5 and 7 +/- 5% change for -/- and +/+ mice, respectively). We conclude that the differences in ABP associated with chronic variations in endogenous ANP activity are not due to alterations in synthesis or responsiveness of the cardiovascular system to the effects of ET-1, CNP, or NO.

Human natriuretic peptide receptor-A guanylyl cyclase is self-associated prior to hormone binding.

The human natriuretic peptide receptor-A (NPR-A) guanylyl cyclase is specifically activated to synthesize cGMP by binding of atrial natriuretic peptide (ANP) to the receptor's extracellular domain. In this report, NPR-A monoclonal and polyclonal antibodies were used to assess the aggregation status of wild-type NPR-A and a truncation mutant lacking most of the NPR-A cytoplasmic domain. On intact human embryonic kidney 293 cells, in the absence of ANP, recombinant human NPR-A is self-aggregated through disulfide bonds in an M(r) > 500,000, possibly tetrameric, complex. Under nonreducing conditions, truncated NPR-A was a monomer, indicating that the cytoplasmic domain is necessary for NPR-A self-association. In the presence of the homobifunctional cross-linker dithiobis(succinimidyl propionate), or disuccimidyl suberate, truncated NPR-A could be cross-linked as a dimer and trimer only in the presence of ANP. Wild-type NPR-A was cross-linked with disuccinimidyl suberate to an M(r) > 500,000 species in the absence of ANP, and with ANP, a smaller, M(r) approximately 400,000 receptor trimer cross-linking product was observed, together with the larger, possibly tetrameric complex. When whole cell stimulation of cGMP production by ANP was tested on the low level of endogenous 293 cell NPR-A, maximal stimulation was observed regardless of truncated NPR-A overexpression. The absence of a dominant negative effect by the truncated NPR-A, together with the cross-linking data, demonstrates that preassociated NPR-A is the functionally relevant form of this receptor.

HS-142-1, a Novel Non-peptide Antagonist for Atrial Natriuretic Peptide Receptor, Selectively Inhibits Particulate Guanylyl Cyclase and Lowers Cyclic GMP in LLC-PK1Cells

HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK1 cells. At concentrations from 0.1 to 100 μg/ml (= 2.5 × 10(-8) - 2.5 × 10(-5) M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10(-8) M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10(-5) M sodium nitroprusside. HS-142-1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulation of cyclic GMP in LLC-PK1 cells through interacting with guanylyl cyclase-linked receptors.

Neutral Endopeptidase 24.11 Inhibition Reduces Pulmonary Vascular Remodeling in Rats Exposed to Chronic Hypoxia

Inhibition of the metabolism of endogenous atrial natriuretic peptide (ANP), by continuous infusion of a specific inhibitor of neutral endopeptidase (membrane metalloendopeptidase E.C. 3.4.24.11), UK 73,967 (candoxatrilat), was undertaken in rats, in which chronic hypoxia was used as a stimulus to induce pulmonary hypertension and right ventricular hypertrophy. Inhibition of neutral endopeptidase 24.11 with low-dose and high-dose UK 73,967 (NEI) increased endogenous plasma ANP by greater than 155% during the development of pulmonary hypertension. NEI treatment reduced mean pulmonary arterial pressure in hypoxia as follows: vehicle 26.6 +/- 4.0 mm Hg; low-dose NEI 22.7 +/- 1.9 mm Hg, and high-dose NEI 22.6 +/- 2.5 mm Hg (both p less than 0.01 compared with hypoxic vehicle); however, it was without effect on pulmonary arterial pressure in normoxia (17.6 +/- 2.2 mm Hg) or on systemic blood pressure. The development of right ventricular hypertrophy was also reduced in both groups treated with NEI (right ventricular weight/left ventricular weight: 0.43 +/- 0.03 vehicle; 0.40 +/- 0.02 low-dose NEI and 0.40 +/- 0.02 high-dose NEI, both p less than 0.05 compared with vehicle). Remodelling of the pulmonary vasculature, characterized by extension of the muscle within the small pulmonary arteries toward the periphery of the lung, was reduced by NEI treatment (percentage of thick-walled peripheral vessels; 19.2 +/- 3.1% vehicle; 10.4 +/- 2.3% low-dose NEI and 8.1 +/- 1.8% high-dose NEI, both p less than 0.001 compared with vehicle). In the isolated blood perfused rat lung pulsed doses of NEI had no effect on pulmonary vascular tone in the absence of ANP. Specific inhibition of the enzyme neutral endopeptidase reduces vascular remodelling, the development of pulmonary hypertension, and right ventricular hypertrophy. Endogenous ANP modulates vascular remodelling in vivo. Retarding the metabolism of endogenous ANP through inhibition of neutral endopeptidase 24.11 represents a potential approach toward therapy. g

Effects of atrial natriuretic peptide on endothelin-induced vasoconstriction and intracellular calcium mobilization

Mechanisms for the antagonistic action of atrial natriuretic peptide (ANP) toward endothelin were studied. We examined the effects of ANP on endothelin-induced vasoconstriction in isolated perfused kidney and the elevation of intracellular free calcium concentration using Fura-2 in cultured rat vascular smooth muscle cells (VSMC). Endothelin produced an increase in renal vascular resistance in isolated perfused kidney (2 x 10(-10) mol/l, +109%; P less than 0.01). ANP completely abolished renal vasoconstriction induced by endothelin. Endothelin produced a biphasic elevation in intracellular calcium concentration in the VSMC. Ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetr aac etic acid (EGTA; 3 mmol/l) or nicardipine (10(-8) mol/l) decreased the sustained elevation of intracellular calcium concentration by 10(-8) mol/l endothelin to the basal level (endothelin, +15% versus EGTA + endothelin, -1%; P less than 0.05; endothelin, +18% versus nicardipine + endothelin, +5%; P less than 0.01). Pretreatment with 10(-6) mol/l ANP suppressed neither the peak phase of intracellular calcium concentration elevation (endothelin, 10(-8) mol/l, +35% versus endothelin + ANP, +31%; not significant) nor the sustained phase of intracellular calcium concentration elevation (endothelin, 10(-8) mol/l, +21% versus endothelin + ANP, +16%; not significant). ANP markedly increased the production of cyclic 3',5'-guanosine monophosphate (cGMP) in the VSMC (ANP, 10(-6) mol/l, +1100%; P less than 0.01). However, endothelin did not influence cGMP production in the presence or absence of ANP. ANP may antagonize the vasoconstrictive action of endothelin through an effect on the steps subsequent to the mobilization of intracellular calcium in the pathway of vascular smooth muscle contraction.

Impaired Natriuretic Response to Atrial Natriuretic Peptide in the Isolated Kidney of Rats with Experimental Cirrhosis

1. Sodium retention in cirrhosis may be partly attributable to resistance to a putative circulating natriuretic factor. In cirrhosis, plasma concentrations of atrial natriuretic peptide are often increased in the presence of sodium retention. 2. In order to determine whether the kidney of cirrhotic animals may be insensitive to atrial natriuretic peptide, isolated perfused kidneys from six cirrhotic and five control rats were exposed to increasing concentrations of atrial natriuretic peptide. Cirrhosis had been induced by carbon tetrachloride administration. 3. Excretion in vivo of a 2 mmol sodium load, administered by gavage, was impaired in cirrhotic animal for up to 4 h as compared with control animals (4.2 +/- 1.9 vs 34.9 +/- 13.4% P less than 0.05). 4. During perfusion at 110 mmHg in the absence of atrial natriuretic peptide, sodium excretion by isolated kidneys of cirrhotic animals tended to be lower than in control animals, but the difference was not significant (4.93 +/- 1.01 vs 8.41 +/- 1.48 mumol min-1 g-1 kidney weight, P = 0.09). There was a smaller increase in urinary sodium excretion by the kidneys of cirrhotic rats compared with control rats in the presence of atrial natriuretic peptide at 10, 50 and 200 pmol/l (respectively: 0.06 +/- 0.08 vs 1.29 +/- 0.35 mumol/min-1 g-1 kidney weight, P less than 0.02; 0.49 +/- 0.08 vs 1.82 +/- 0.42 mumol min-1 g-1 kidney weight, P less than 0.03; 1.42 +/- 0.16 vs 3.23 +/- 0.73 mumol min-1 g-1 kidney weight, P less than 0.05), but not at 1000 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)