Hypertension induced by nitric oxide synthase inhibition activates the atrial natriuretic peptide (ANP) system

We assessed the effect of nitric oxide (NO) synthase inhibition on plasma atrial natriuretic peptide (ANP) concentration and content in some brain structures [neurohypophysis (NH), adenohypophysis (AH), medial basal hypothalamus (MHB) and olfactory bulb (OB)] in rats before and after blood volume expansion (BVE). Male Wistar rats were injected i.p. with N(pi)-nitro-L-arginine (L-NNA), 25 mg/kg of body weight, 40 min before the experiment (acute treatment) or L-NNA at a dose of 25 mg/kg body weight, twice a day, for 4 days (chronic treatment). The acute treatment caused an increase in the blood pressure and plasma ANP concentration in rats under basal conditions and after BVE. A decrease in ANP content was observed in the OB and NH, whereas no significant changes were found in the AH or MBH. In chronically treated rats, we also found an increase in blood pressure and in plasma ANP concentration under basal conditions and after BVE. The ANP content increased in the OB, NH and AH. These results indicate that systemic NO synthase inhibition increases ANP concentration in plasma and in areas of the central nervous system. We hypothesize that ANP participates in the hypertension-induced by NO synthesis blockade acting by baroreceptors input to the brain to stimulate ANP release and synthesis that reduces NO prival hypertension.     

Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to alpha-human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of alpha-hANP. 125I-pBNP bound to the cells in a time-dependent manner similar to that of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of alpha-hANP. pBNP and alpha-hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.     

Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 μM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.     

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.     

Superactive analogs of the atrial natriuretic peptide (ANP)

Three analogs of the atrial natriuretic peptide ANP(105-126), lacking the N-terminal exocyclic peptide segment and containing 2-mercaptoacetic acid, 3-mercaptopropionic acid or 4-mercaptobutyric acid in place of the cysteine residue in position 105 of the peptide sequence, were synthesized by the solid-phase method. The resulting des-amino analogs showed 2 to 4 times higher diuretic/natriuretic activity than the most active natural ANP and displayed a potent hypotensive effect as well. All three analogs were relatively less potent in various in vitro bioassays and in a binding assay, indicating that their high activities in vivo may be due to resistance to enzymatic degradation and to reduced non-specific tissue adsorption. These compounds not only will serve as useful pharmacologic tools but also represent prototypes for the development of further reduced-size ANP analogs.     

Elevation of intracellular calcium levels contributes to the inhibition of nitric oxide production by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) attenuates LPS-induced inducible nitric oxide synthase (iNOS) expression in murine macrophages by destabilizing iNOS mRNA. Because elevated intracellular free Ca2+ levels [Ca2+]i reduce iNOS mRNA stability, the aim of the present study was to determine whether inhibition of iNOS by ANP is due to alterations in intracellular calcium. As determined by fluorescence photometry, ANP (10(-7) and 10(-6) mol/L) was shown to elevate intracellular calcium levels in bone marrow-derived macrophages. This effect seemed to be mediated via the guanylate cyclase-coupled A receptor, because dibutyryl-cGMP mimicked and the A-receptor antagonist HS-142-1 partially abrogated the effect of ANP. Because the Ca2+ increase was also observed in Ca2+-free buffer, it is suggested that the liberation of intracellular calcium pools contributes to the elevation of [Ca2+]i by ANP. The B-receptor ligand C-type natriuretic peptide (CNP), which does not alter iNOS expression, had no effect on [Ca2+]i. The Ca2+-ionophore 4-Br-A23187 and thapsigargin, a compound known to liberate Ca2+ from intracellular stores, were further demonstrated to reduce LPS-induced NO production in macrophages (Griess assay), confirming a functional link for elevated [Ca2+]i and iNOS inhibition. These effects were abrogated by coincubation with extra- as well as intracellular Ca2+ chelators (EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)). The inhibitory effect of ANP on NO production was also abrogated by Ca2+ chelation. These findings support a causal relationship between reduced iNOS induction and elevation of [Ca2+]i. Taken together, the data indicate that intracellular Ca2+ elevation by ANP is involved in the inhibition of LPS-induced nitric oxide production in macrophages.     

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.     

Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.     

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Plasma and atrial levels of atrial natriuretic peptide (ANP) in pulmonary hypertensive rats

Immunoreactive atrial natriuretic peptide (IR-ANP) was measured in plasma and atrium of normal and monocrotaline induced pulmonary hypertensive rats (PH rats). In these animals, there was right ventricular hypertrophy and right ventricular systolic pressure was elevated. Fourteen days after a single dose of monocrotaline (40 mg/kg), plasma IR-ANP concentrations were significantly elevated (964.3 +/- 63.0 pg/ml vs. 521.0 +/- 81.9 pg/ml in controls, p less than 0.001). Tissue levels of IR-ANP in the right atrium in PH rats was significantly lower than those in the controls (45.1 +/- 3.9 ng/mg vs. 240.5 +/- 10.4 ng/mg, p less than 0.001), while there was no significant difference in tissue levels of atrial IR-ANP in the left atrium between the two groups. Thus, development of pulmonary hypertension led to an increase in release of ANP from the right atrium.     

Locally different role of atrial natriuretic peptide (ANP) in the pericardial fluid

Pericardial fluid (PF) contains several vasoactive agents in higher concentrations than venous plasma (VP). However, with human atrial natriuretic peptide (ANP) controversial data have been reported in earlier studies performed on a limited number of patients (less than 20). The present study was designed to characterize the ANP levels in human PF and cardiac tissues, and to ascertain whether myocardial ischemic state is a major factor in determining ANP production of the human heart. In a total of 316 consecutive patients undergoing open heart surgery ANP levels in VP, PF, atrial and ventricular tissues were measured by radioimmunoassay and analyzed by high-performance liquid chromatography (HPLC). The data are presented as median and 25th-75th percentiles. Our results showed ANP concentration [ANP] of PF significantly exceeded that of VP and [ANP] in the atrial tissue was significantly higher than in the ventricular tissue (p < 0.001). In patients without myocardial ischemia (valvular heart disease) [ANP] in the PF was 258.3 (189.9-342.5) pg/ml, in the VP 28.4 (11.7-57.6) pg/ml and 151.7 (78.4-447.6) ng/mg in the atrial, 0.4 (0.2-1.6) ng/mg in the ventricular tissue. The corresponding values for patients with coronary artery disease were 208.1 (153.8-318.9) pg/ml in the PF, 19.8 (9.4-27.9) pg/ml in the VP, 129.6 (66.5-455.0) ng/mg in the atrial and 1.0 (0.1-1.8) ng/mg in the ventricular tissue. The ventricular tissue levels correlated to the atrial tissue levels (r = 0.317; p < 0.05). Great difference (p < 0.001) was found in the atrial tissue levels between females [414.6 (119.7-734.4) ng/mg] and males [105.4 (65.3-204.2) ng/mg]. In HPLC analysis the majority of the pericardial fluid and tissue ir-ANP coeluted with human ANP [99-126]. In conclusion, [ANP] in PF of cardiosurgical patients is higher by an order of magnitude than in VP. Intrapericardial ANP may reflect the peptide concentration in the myocardial interstitium and may represent a paracrine regulatory mechanism, which seems independent of ANP-induced putative antiischemic influences.     

Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain.

Quantitative autoradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA-salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111-126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.     

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.     

Evolutionary implication of the absence of atrial natriuretic peptide (ANP) in euryhaline Oryzias fishes

The genus Oryzias contains nearly 20 species, including the Japanese medaka (Oryzias latipes). Because each species exhibits different adaptability to environmental salinity, Oryzias fishes offer unique opportunities for comparative studies. To understand the mechanisms of osmotic adaptation, we are studying the functional evolution of the natriuretic peptide (NP) family??a group of small peptide hormones involved in body fluid regulation??by using Oryzias fishes. Analysis of the Japanese medaka genome revealed that 7 NP subtypes, namely, Atrial NP (ANP), B-type NP (BNP), Ventricular NP (VNP), and 4?C-type NPs (CNP-1 through CNP-4) were generated from a CNP-4-like ancestral gene discovered in the cyclostomes before the ray-finned fish/lobe-finned fish divergence. This evolutionary history has been confirmed by the discovery of hidden NP genes in tetrapods. Through analyses of phylogenetic distribution of NP subtypes, we also found that specific losses of subtypes have occurred in each vertebrate lineage. For example, ANP is absent in the Japanese and Indian medaka and the flying fish, suggesting that loss of the ANP gene occurred after the divergence of Beloniformes from Cyprinodontiformes. This fact also supports the inclusion of Oryzias into Beloniformes as suggested by phylogenetic analysis using whole mitochondrial genome sequences. How Oryzias fishes have retained their euryhalinity with a reduced number of NPs is an interesting question. CNP-3, which is functionally flexible, may be a substitute for the lost cardiac NPs.     

Calcitonin gene-related peptide(CGRP) stimulates the release of atrial natriuretic peptide(ANP) from isolated rat atria

The effect of calcitonin gene-related peptide(CGRP) on the release of atrial natriuretic peptide(ANP) was studied in spontaneously beating, isolated rat atria. CGRP stimulated the ANP release in a dose-dependent manner. When the atria were incubated with a combination of phentolamine, propranolol, and atropine, these antagonists blocked neither the rise in ANP release nor the positive chronotropic and inotropic effects of CGRP. Therefore, we conclude that CGRP stimulates ANP release as well as cardiac contractility independently of adrenergic and cholinergic receptors.     

A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.     

Binding of synthetic beta-human atrial natriuretic peptide to cultured rat vascular smooth muscle cells

We have studied the effects of synthetic beta-human atrial natriuretic peptide (beta-hANP), an antiparallel dimer of alpha-hANP, on receptor binding and cGMP generation in cultured rat vascular smooth muscle cells and compared the effects with those of alpha-hANP. Characteristics of temperature-dependent binding and degradation of 125I-beta-hANP were similar to those of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for beta-hANP with a maximal binding capacity one-half that of alpha-hANP. Parallel and antiparallel dimers were equipotent in inhibiting the binding and stimulating intracellular cGMP formation, of which the maximal effect was about one-half that of alpha-hANP. Reverse-phase high performance liquid chromatography revealed that most of beta-hANP added to cells was converted to a small molecular mass component corresponding to alpha-hANP after incubation. These data suggest that the less potent effect of beta-hANP in receptor binding and cGMP generation may be partly accounted for by the possible conversion of beta-hANP to alpha-hANP at the site of target cells.