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Genistein, an isoflavonoid that can inhibit protein tyrosine kinase (PTK) phosphorylation, has been shown to play pivotal roles in the signal transduction pathways of hypoxic disorders. In this study, we established a rat model of isolated beating atrium and investigated the regulator role of genistein and its downstream signaling pathways in acute hypoxia-induced atrial natriuretic peptide (ANP) secretion. Radioimmunoassay was used to detect the ANP content in the atrial perfusates. Western blot analysis was used to determine the protein level of hypoxia-inducible factor 1[alpha] (HIF-1[alpha]), and GATA4 in the atrial tissue. The results showed that acute hypoxia substantially promoted ANP secretion, whereas this effect was partly attenuated by the PTKs inhibitor genistein (3 [micro]M). By Western blotting analysis, we found that hypoxia-induced increase in phosphorylation of Akt and transcriptional factors, including HIF-1[alpha], were also reversed by genistein. The perfused HIF-1[alpha] inhibitors rotenone (0.5 [micro]M) or CAY10585 (10 [micro]M) plus genistein significantly abolished the enhanced ANP section induced by hypoxia. Additionally, the perfused PI3K/Akt agonist insulin-like growth factor 1 (30 [micro]M) also abolished ANP secretion induced by genistein and inhibited expression of HIF-1[alpha]. In summary, our data suggested that acute hypoxia markedly increased ANP secretion by PTKs through the phosphoinositide-3 kinase (PI3K)/HIF-1[alpha] dependent pathway. Key words: atrial natriuretic peptide, hypoxia, hypoxia inducible factor-1a, protein tyrosine kinases, genistein. On a montre que la genisteine, un isoflavonoide pouvant entrainer une inhibition de la phosphorylation des PTK (<< protein tyrosine kinases >>), joue un role central dans les voies de transduction des signaux dans les troubles hypoxiques. Dans cette etude, nous avons mis au point un modele d'atrium contractile isole chez le rat, et etudie le role regulateur de la genisteine et ses voies de signalisation en aval dans la secretion de peptide natriuretique atrial (ANP) engendree par l'hypoxie aigue. A l'aide d'un radioimmunoessai, nous avons evalue les taux d'ANP dans les perfusats atriaux. A l'aide de l'immunobuvardage de Western, nous avons etabli les taux en proteines d'HIF-1[alpha] (<< hypoxia-inducible factor-1a >>) et de GATA4 dans le tissu atrial. Les resultats ont montre que l'hypoxie aigue favorise substantiellement la secretion d'ANP, tandis que cet effet s'attenuait partiellement avec l'inhibiteur des PTK genisteine (a 3 [micro]M). A l'aide de l'immunobuvardage de Western, nous avons observe que l'augmentation de la phosphorylation d'Akt et de facteurs de la transcription, y compris HIF-1[alpha], engendree par l'hypoxie etait aussi inversee avec la genisteine. La perfusion des inhibiteurs de l'HIF-1[alpha] rotenone (a 0,5 [micro]M) ou CAY10585 (a 10 [micro]M) avec la genisteine permettait clairement d'inhiber completement l'augmentation de la secretion d'ANP engendree par l'hypoxie. De plus, la perfusion de l'agoniste de l'axe PI3K/Akt IGF-1 (a 30 [micro]M) a aussi permis d'inhiber completement la secretion d'ANP engendree par la genisteine, ainsi que d'inhiber l'expression de l'HIF-1[alpha]. En resume, nos donnees laissaient entendre que l'hypoxie aigue entrainerait une augmentation marquee de la secretion d'ANP engendree par les PTK par l'intermediate de la voie de signalisation dependante de PI3K/HIF-1[alpha]. [Traduit par la Redaction] Mots-cles: peptide natriuretique atrial, hypoxie, HIF-1[alpha], PTK, genisteine., Introduction Atrial natriuretic peptide (ANP) is one of the cardiac hormones synthesized and primarily stored in the cardiac atria with multiple functions on cardio-related disease (Cannone et al. 2019; Zhou [...]

To assess the physiological and clinical implications of the C-type natriuretic peptide (CNP)/guanylyl cyclase B (GC-B) system in the human vasculature, we have examined gene expressions of CNP and its receptor, GC-B, in human vascular endothelial cells (ECs) and smooth muscle cells (SMCs) and have also compared the endothelin-1(ET-1)/endothelin receptor-A (ETR-A) and endothelin receptor-B (ETR-B) system in human aortic ECs (HAECs) and vascular SMCs (HSMCs) in vitro. We also examined these gene expressions in human embryonic stem (ES)/induced pluripotent stem cell (iPS)-derived ECs and mural cells (MCs). A little but significant amount of mRNA encoding CNP was detected in both human ES-derived ECs and HAECs. A substantial amount of GC-B was expressed in both ECs (iPS-derived ECs and HAECs) and SMCs (iPS-derived MCs and HSMCs). ET-1 was expressed solely in ECs. ETR-A was expressed in SMCs, while ETR-B was expressed in ECs. These results indicate the existence of a vascular CNP/GC-B system in the human vascular wall, indicating the evidence for clinical implication of the CNP/GC-B system in concert with the ET-1/ETR-A and ETR-B system in the human vasculature. Key words: natriuretic peptide family, C-type natriuretic peptide, guanylyl cyclase B, iPS cells, embryogenic stem cells, endothelin, endothelial cell, vascular hormone. En vue d'evaluer les implications physiologiques et cliniques du systeme peptide natriuretique de type C (PNC)/guanylyl cyclase B (GC-B) dans les vaisseaux sanguins humains, nous avons etudie l'expression genique du PNC et de la GC-B, son recepteur, dans les cellules endotheliales (CE) vasculaires humaines ainsi que dans les cellules musculaires lisses (CML) vasculaires, et compare le systeme endotheline 1 (ET-1)/recepteurs de l'endotheline de type A (RET-A) et recepteurs de l'endotheline de type B (RET-B) dans des CE aortiques humaines (CEAH) et des CML vasculaires humaines (CMLH) in vitro. Nous avons aussi etudie l'expression de ces genes dans des CE derivees de cellules souches embryonnaires (CSE)/cellules souches pluripotentes induites (CSPi) humaines, ainsi que dans des cellules parietales (CP). Nous avons decele une quantite faible, mais notable, d'ARNm codant le PNC dans les CE humaines derivees de CSE, ainsi que dans les CEAH. Une quantite substantielle de GC-B s'exprimait dans les CE (CE derivees de CSPi et CEAH) comme dans les CML (CP derivees de CSPi et CMLH). L'ET-1 s'exprimait uniquement dans les CE. Les RET-A s'exprimaient dans les CML, tandis que les RET-B s'exprimaient dans les CE. Ces resultats sont un signe de la presence d'un systeme PNC/GC-B vasculaire dans la paroi vasculaire chez l'homme, ce qui montre l'existence d'une implication clinique du systeme PNC/GC-B de concert avec le systeme ET-1/RET-A et RET-B dans les vaisseaux sanguins chez l'homme. [Traduit par la Redaction] Mots-cles: famille des peptides natriuretiques, peptide natriuretique de type C, guanylyl cyclase B, cellules souches pluripotentes induites, cellules souches embryonnaires, endotheline, cellule endotheliale, hormone vasculaire., Introduction Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), have been characterized as cardiac or vascular hormones that elicit their biological [...]

Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPAR γ coactivator-1 α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1 α and mitochondrial OXPHOS gene expression in a cyclic GMP-dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training-induced improvement in skeletal muscle fat oxidative capacity in humans., Introduction The cardiac hormones, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), play a major role in the regulation of fluid homeostasis and cardiac physiology (1). Natriuretic peptide-mediated (NP-mediated) [...]

The aim of this study was to prospectively evaluate the prevalence of pulmonary hypertension (PH) in patients with idiopathic pulmonary fibrosis (IPF). One hundred thirty-nine patients (101 male, mean age = 68.6 ± 9 years), with confirmed IPF and who were admitted to eight Pulmonary Departments in Greece between November 2005 and December 2006 were included in the study. Pulmonary artery systolic pressure (PASP) was estimated by echocardiography, and PH was defined as PASP > 36 mmHg. We compared demographics, pulmonary function tests, NYHA functional status, 6-min walk distance (6MWD), B-type natriuretic peptide (BNP), Pa[O.sub.2], and P(A-a)[O.sub.2] at rest data between patients with PH and without PH (PASP < 36 mmHg). Increased estimated right ventricular systolic pressure was present in 55% of patients (mean PASP = 47.1 ± 11.2 mmHg vs. 30.3 ± 3.8 mmHg, respectively). Patients with PH had a lower but not statistically significant [DL.sub.CO] (47.1 ± 18.8 vs. 52.5 ± 20.1), lower Pa[O.sub.2] at rest (64.6 ± 12.2 vs. 71.1 ± 11.3, P = 0.004), and lower mean 6MWD (282 ± 118 vs. 338 ± 91, P = 0.007). Significant differences were also observed in the NYHA functional status between the two groups (P = 0.02). Statistically significant correlations were observed between PASP and Pa[O.sub.2] at rest (r = -0.331, P = 0.00), P(A-a)[O.sub.2] at rest (r = 0.494, P = 0.00X 6MWD (r =-0.264, P = 0.01), Sp[O.sub.2] at rest (r = -0.293, P = 0.00), Sp[O.sub.2] at the end of exercise (r = -0.364, P = 0.00), and also BNP values (r = 0.319, P = 0.01). Moreover, Pa[O.sub.2] (P = 0.02), P(A-a)[O.sub.2] (P = 0.005), and Sp[O.sub.2] at the end of exercise (P = 0.023) were independent predictors of the presence of estimated PH. Using Doppler echocardiography as a screening tool for the estimation of PH, we found that PH is common in patients with IPF. Gas exchange parameters at rest and exercise desaturation might indicate underlying PH in IPF. Keywords Pulmonary hypertension * Idiopathic pulmonary fibrosis * Diffusing capacity of the lung for carbon monoxide * Alveolar-arterial oxygen gradient * 6-min walk distance * Resting room air pulse oximetry, Introduction Pulmonary hypertension (PH) is a well-recognized complication of idiopathic pulmonary fibrosis (IPF). Pathogenetic mechanisms implicated in the development of PH include vascular destruction, pulmonary hypoxic vasoconstriction, and vascular remodeling [...]

Urotensin II (UII) is a vasoactive peptide with potent cardiovascular effects through a G protein-coupled receptor. Hypoxia stimulates the secretion of UII and atrial natriuretic peptide (ANP). However, the effect of UII on hypoxia-induced cardiac hypertrophy is still controversial. The present study was conducted to determine whether human UII (hUII)-mediated ANP secretion influences hypoxia-induced cardiac hypertrophy using in vitro and in vivo models. Hypoxia caused an increase in ANP secretion and a decrease in atrial contractility in isolated perfused beating rat atria, hUII (0.01 and 0.1 nM) attenuated hypoxia-induced ANP secretion without changing the atrial contractility, and the hUII effect was mediated by the UII receptor signaling involving phospholipase C, inositol 1,3,4 trisphosphate receptor, and protein kinase C. Rats treated with monocrotaline (MCT, 60 mg/kg) showed fight ventricular hypertrophy with increases in pulmonary arterial pressure and its diameter and plasma levels of UII and ANP that were attenuated by the pretreatment with an UII receptor antagonist, urantide. An acute administration of hUII (5 [micro]M injection plus 2.5 [micro]M infusion for 15 min) decreased the plasma ANP level in MCT-treated rats but increased the plasma ANP level in MCT plus urantide-treated and sham-operated rats. These results suggest that hUII may deteriorate MCT-induced cardiac hypertrophy mainly through a vasoconstriction of the pulmonary artery and partly through the suppression of ANP secretion. atrial natriuetic peptide; hypoxia; pulmonary hypertension doi: 10.1152/ajpheart.00438.2010.

The aim of this study was to evaluate the relative contributions of various hormones involved in the regulation of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise and to assess the impact of obesity on this regulation. Eight lean and eight obese men performed a 60-min cycle exercise bout at 50% of their peak oxygen uptake on two occasions: during intravenous infusion of octreotide (a somatostatin analog) or physiological saline (control condition). Lipolysis in SCAT was evaluated using in situ microdialysis. One microdialysis probe was perfused with the adrenergic blockers phentolamine and propranolol while another probe was perfused with the phosphodiesterase and adenosine receptor inhibitor aminophylline. Compared with the control condition, infusion of octreotide reduced plasma insulin levels in lean (from ~3.5 to 0.5 [micro]U/ml) and in obese (from ~9 to 2 [micro]U/ml), blunted the exercise-induced rise in plasma GH and epinephrine levels in both groups, and enhanced the exercise-induced natriuretic peptide (NP) levels in lean but not in obese subjects. In both groups, octreotide infusion resulted in higher exercise-induced increases in dialysate glycerol concentrations in the phentolamine-containing probe while no difference in lipolytic response was found in the aminophylline-containing probe. The results suggest that insulin antilipolytic action plays a role in the regulation of lipolysis during exercise in lean as well as in obese subjects. The octreotide-induced enhancement of exercise lipolysis in lean subjects was associated with an increased exercise-induced plasma NP response. Adenosine may contribute to the inhibition of basal lipolysis in both subject groups. octreotide; adenosine: catecholamines; microdialysis; exercise-induced lipolysis doi: 10.1152/ajpendo.00767.2009.

The natriuretic peptide (NP) family consists of seven paralogs [atrial NP (ANP), brain NP (BNP), ventriclar NP (VNP), and C-type NP 1-4 (CNP1-4)] in teleosts, but relative biological activity of the seven NPs has not been comprehensively examined using homologous peptides. In this study, we newly identified CNP3 and CNP4 in eels to use homologous peptides, but the CNP2 gene may have been silenced in this species. The CNP4 gene was expressed exclusively in the brain as CNP1, but the CNP3 gene, from which cardiac ANP, BNP, and VNP were generated by tandem duplication, was most abundantly expressed in the pituitary, suggesting its local action. All NPs induced hypotension dose dependently after intra-arterial injection with a potency order of ANP > VNP > BNP > CNP4 > CNP1 = CNP3. The degree of hypotension was similar at the ventral and dorsal aorta, indicating similar actions on the branchial and systemic circulation. The hypotension induced by cardiac NPs was longer lasting than CNPs, probably because of the difference in preferential receptors. Among cardiac NPs, the hypotensive effect of VNP lasted much longer than those of ANP and BNP, even though VNP disappeared from the blood more quickly than ANP. To analyze the unique effect of VNP, we examined possible involvement of the autonomic nervous system using ANP, VNP, and CNP3. [beta]-adrenergic blockade diminished hypotensive effects of all three NPs, but [alpha]-adrenergic and cholinergic blockade enhanced only the effect of VNP, suggesting a specific mechanism for the VNP action. The NP-induced tachycardia was diminished by all blockers examined. Furthermore, the cardiovascular action of VNP was not impaired by a blocker of NP receptor, HS-142-1. Taken together, the homologous NPs exhibit diverse cardiovascular actions in eels partially through the autonomic nervous system, and the unique VNP action may be mediated by a novel receptor that has not been identified in teleosts. natriuretic peptide family; hypotension; atropine; phentolamine; propranolol doi: 10.1152/ajpregu.00789.2009.

Angiotensin-(1-7) [ANG-(1-7)], one of the bioactive peptides produced in the renin-angiotensin system, plays a pivotal role in cardiovascular physiology by providing a counterbalance to the function of ANG II. Recently, it has been considered as a potential candidate for therapeutic use in the treatment of various types of cardiovascular diseases. The aim of the present study is to explain the modulatory role of ANG-(1-7) in atrial natriuretic peptide (ANP) secretion and investigate the functional relationship between two peptides to induce cardiovascular effects using isolated perfused beating rat atria and a cardiac hypertrophied rat model. ANG-(1-7) (0.01, 0.1, and 1 [micro]M) increased ANP secretion and ANP concentration in a dose-dependent manner at high atrial pacing (6.0 Hz) with increased cGMP production. However, at low atrial pacing (1.2 Hz), ANG-(1-7) did not cause changes in atrial parameters. Pretreatment with an antagonist of the Mas receptor or with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), or nitric oxide synthase blocked the augmentation of high atrial pacing-induced ANP secretion by ANG-(1-7). A similar result was observed with the inhibition of the [Na.sup.+]/[H.sup.+] exchanger-1 and [Ca2.sup.+]/ calmodulin-dependent kinase II (CaMKII). ANG-(1-7) did not show basal intracellular [Ca2.sup.+] signaling in quiescent atrial myocytes. In an in vivo study using an isoproterenol-induced cardiac hypertrophy animal model, an acute infusion of ANG-(1-7) increased the plasma concentration of ANP by twofold without changes in blood pressure and heart rate. A chronic administration of ANG-(1-7) increased the plasma ANP level and attenuated isoproterenol-induced cardiac hypertrophy. The antihypertrophic effect was abrogated by a cotreatment with the natriuretic peptide receptor-A antagonist. These results suggest that 1) ANG-(1-7) increased ANP secretion at high atrial pacing via the Mas/PI3K/Akt pathway and the activation of [Na.sup.+]/[H.sup.+] exchanger-1 and CaMKII and 2) ANG-(1-7) decreased cardiac hypertrophy which might be mediated by ANP. atrial natriuretic peptide; cardiac hypertrophy; atrial myocyte; Mas; signal transduction: phosphatidylinositol 3-kinase doi: 10.1152/ajpheart.00608.2009.

Leptin is a circulating adipocyte-derived hormone that influences blood pressure (BP) and metabolism. This study was designed to define the possible role of leptin in regulation of the atrial natriuretic peptide (ANP) system using acute and chronic experiments. Intravenous infusion of rat leptin (250 [micro]g/kg injection plus 2 [micro]g*[kg.sup.-1]*[min.sup.-1] for 20 min) into Sprague-Dawley rats increased BP by 25 mmHg and decreased plasma level of ANP from 80.3 [+ or -] 3.45 to 51.8 [+ or -] 3.3 pg/ml. Reserpinization attenuated the rise in BP, but not the reduction of plasma ANP during lepfin infusion. [N.sup.[omega]]-nitro-L-arginine methyl ester prevented the effects of leptin on the reduction of ANP level. In hyperleptinemic rats that received adenovirus containing rat leptin cDNA (AdCMV-leptin), BP increased during first 2 days and then recovered to control value. Plasma concentration of ANP and expression of ANP mRNA, but not of atrial ANP, in hyperleptinemic rats were lower than in the control groups on the first and second week after administration of AdCMV-leptin. These effects were not observed by the pretreatment with [N.sup.[omega]]-nitro-L-arginine methyl ester. No differences in renal function and ANP receptor density in the kidney were found between hyperleptinemic and control rats. Basal ANP secretion and isoproterenol-induced suppression of ANP secretion from isolated, perfused atria of hyperleptinemic rats were not different from those of other control groups. These data suggest that leptin inhibits ANP secretion indirectly through nitric oxide without changing basal or isoproterenol-induced ANP secretion. sympathetic nervous system; atrial natriuretic peptide; adenosine 3',5'-cyclic monophosphate; hypertension; receptor doi: 10.1152/ajpregu.00598.2009.

The incretin hormone glucagon-like peptide-1 (GLP-1) and its analogs are currently emerging as antidiabetic medications. GLP-1 improves left ventricular ejection fraction (LVEF) in dogs with heart failure (HF) and in patients with acute myocardial infarction. We studied metabolic and cardiovascular effects of 48-h GLP-1 infusions in patients with congestive HF. In a randomized, double-blind crossover design, 20 patients without diabetes and with HF with ischemic heart disease, EF of 30 [+ or -] 2%, New York Heart Association II and III (n = 14 and 6) received 48-h GLP-1 (0.7 pmol x [kg.sup.-1] x [min.sup.-1]) and placebo infusion. At 0 and 48 h, LVEF, diastolic function, tissue Doppler regional myocardial function, exercise testing, noninvasive cardiac output, and brain natriuretic peptide (BNP) were measured. Blood pressure, heart rate, and metabolic parameters were recorded. Fifteen patients completed the protocol. GLP-1 increased insulin (90 [+ or -] 17 pmol/1 vs. 69 [+ or -] 12 pmol/1; P = 0.025) and lowered glucose levels (5.2 [+ or -] 0.1 mmol/1 vs. 5.6 [+ or -] 0.1 mmol/1; P < 0.01). Heart rate (67 [+ or -] 2 beats/min vs. 65 [+ or -] 2 beats/min; P = 0.016) and diastolic blood pressure (71 [+ or -] 2 mmHg vs. 68 [+ or -] 2 mmHg; P = 0.008) increased during GLP-1 treatment. Cardiac index (1.5 [+ or -] 0.1 l x [min.sup.-1] x [m.sup.-2] vs. 1.7 [+ or -] 0.2 1 x [min.sup.-1] x [m.sup.-2]; P = 0.54) and LVEF (30 [+ or -] 2% vs. 30 [+ or -] 2%; P = 0.93), tissue Doppler indexes, body weight, and BNP remained unchanged. Hypoglycemic events related to GLP-1 treatment were observed in eight patients. GLP-1 infusion increased circulating insulin levels and reduced plasma glucose concentration but had no major cardiovascular effects in patients without diabetes but with compensated HF. The impact of minor increases in heart rate and diastolic blood pressure during GLP-1 infusion requires further studies. Hypoglycemia was frequent and calls for caution in patients without diabetes but with HF. metabolism; nondiabetics; insulin resistance doi:10.1152/ajpheart.00930.2009

The objective was to study atrial natriuretic peptide (ANP) effects on mean arterial pressure (MAP) and cardiovascular nitric oxide (NO) system in spontaneously hypertensive rats (SHRs), investigating the receptors and signaling pathways involved. In vivo, SHRs and Wistar-Kyoto (WKY) rats were infused with saline (0.05 ml/min) or ANP (0.2 [micro]g x [kg.sup.-1] x [min.sup.-1]) for 1 h. MAP and nitrites and nitrates excretion (NOx) were determined. NO synthase (NOS) activity and endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) NOS expression were measured in the heart and aorta. In vitro, heart and aortic NOS activity induced by ANP was determined in the presence of iNOS and nNOS inhibitors, natriuretic peptide receptor (NPR)-A/B blocker, [G.sub.i] protein, and calmodulin inhibitors. As a result, ANP diminished MAP and increased NOx in both groups. Cardiovascular NOS activity was higher in SHRs than in WKY rats. ANP increased NOS activity, but the activation was lower in SHRs than in WKY rats. ANP had no effect on NOS isoform expression. NOS activity induced by ANP was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in ventricle and aorta but not in atria. Cardiovascular NOS response to ANP was reduced by [G.sub.i] protein and calmodulin inhibitors in both groups. In conclusion, in atria, ventricle, and aorta, ANP interacts with NPR-C receptors, activating [Ca.sup.2+]-calmodulin eNOS through [G.sub.i] protein. In ventricle and aorta, NOS activation also involves NPR-A/B. The NOS response to ANP was impaired in heart and aorta of SHRs. The impaired NO-system response to ANP in hypertensive animals, involving alterations in the signaling pathway, could participate in the maintenance of high blood pressure in this model of hypertension. cardiovascular nitric oxide synthase; heart; natriuretic peptide receptors; hypertension; aortic artery doi:10.1152/ajpheart.00488.2009

Am J Physiol Renal Physiol 298: F103-F108, 2010. First published November 11, 2009; doi:10.1152/ajprenal.00479.2009.--Atrial natriuretic peptide (ANP) elicits natriuresis; however, the relative contributions of proximal and distal nephron segments to the overall ANP-induced natriuresis have remained uncertain. This study was performed to characterize the effects of ANP on distal nephron sodium reabsorption determined after blockade of the two major distal nephron sodium transporters with amiloride (5 [micro]g/g body wt) plus bendroflumethiazide (12 [micro]g/g body wt) in male anesthetized C57/BL6 and natriuretic peptide receptor-A gene (Npr1) targeted four-copy mice. The lower dose of ANP (0.1 ng x g body [wt.sup.-1] x [min.sup.-1], n = 6) increased distal sodium delivery (DSD, 2.4 [+ or -] 0.4 vs. 1.6 [+ or -] 0.2 [micro]eq/min, P < 0.05) but did not change fractional reabsorption of DSD compared with control (86.3 [+ or -] 2.0 vs. 83.9 [+ or -] 3.6%, P > 0.05), thus limiting the magnitude of the natriuresis. In contrast, the higher dose (0.2 ng x g body [wt.sup.-1] x min-1, n = 6) increased DSD (2.8 [+ or -] 0.3 [micro]eq/min, P < 0.01) and also decreased fractional reabsorption of DSD (67.4 [+ or -] 4.5%, P < 0.01), which markedly augmented the natriuresis. In Npr1 gene-duplicated four-copy mice (n = 6), the lower dose of ANP increased urinary sodium excretion (0.6 [+ or -] 0.1 vs. 0.3 [+ or -] 0.1 [micro]eq/min, P < 0.05) and decreased fractional reabsorption of DSD compared with control (72.2 [+ or -] 3.4%, P < 0.05) at similar mean arterial pressures (91 [+ or -] 6 vs. 92 [+ or -] 3 mmHg, P > 0.05). These results provide in vivo evidence that ANP-mediated increases in DSD alone exert modest effects on sodium excretion and that inhibition of fractional reabsorption of distal sodium delivery is requisite for the augmented natriuresis in response to the higher dose of ANP or in Npr1 gene-duplicated mice. atrial natriuretic peptide; renal sodium reabsorption; distal nephron segments; amiloride; bendroflumethiazide

Am J Physiol Heart Circ Physiol 298: H136-H143, 2010. First published October 30, 2009; doi: 10.1152/ajpheart.00251.2009.--We have previously reported that 1) inhibition of cyclooxygenase-2 and [PGE.sub.2] production reduces hypertrophy after myocardial infarction in mice and 2) [PGE.sub.2] acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with [PGE.sub.2] activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced [PGE.sub.2] activation of ERK1/2. [PGE.sub.2] activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in [PGE.sub.2] regulation of gene expression. [PGE.sub.2] stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. [PGE.sub.2] stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in [PGE.sub.2] regulation of early growth response- 1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos. prostaglandins; myocytes; prostaglandin E receptor 4

Nesher M, Dvela M, Igbokwe VU, Rosen H, Lichtstein D. Physiological roles of endogenous ouabain in normal rats. Am J Physiol Heart Circ Physiol 297: H2026-H2034, 2009. First published October 16, 2009; doi: 10.1152/ajpheart.00734.2009.--Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats. vascular reactivity; sodium homestasis; cardiac glycosides doi: 10.1152/ajpheart.00734.2009

Kake T, Kitamura H, Adachi Y, Yoshioka T, Watanabe T, Matsushita H, Fujii T, Kondo E, Tachibe T, Kawase Y, Jishage K, Yasoda A, Mukoyama M, Nakao K. Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice. Am J Physiol Endocrinol Metab 297: E1339-E1348, 2009. First published October 6, 2009; doi: 10.1152/ajpendo.00272.2009.--C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia. blood level elevation doi: 10.1152/ajpendo.00272.2009

Wang X, McLennan SV, Allen T J, Tsoutsman T, Semsarian C, Twigg SM. Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor. Am J Physiol Cell Physiol 297: C1490-C1500, 2009. First published July 22, 2009; doi: 10.1152/ajpcell.00049.2009.--Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and [alpha]-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures, small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes. diabetic cardiomyopathy; apoptosis; hypertrophy doi: 10.1152/ajpcell.00049.2009

Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C [gamma]1 (PLC[gamma]1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLC[gamma]1 at the plasma membrane. Here, we identify the C-type natriuretic peptide receptor (NPR-C), an atypical G protein-coupled receptor, as a protein binding partner for AHNAK1 in various cell types. Mass spectrometry and MASCOT analysis of excised bands from NPR-C immunoprecipitation studies revealed multiple signature peptides corresponding to AHNAK1. Glutathione S-transferase (GST) pulldown assays using GST-AHNAK1 fusion proteins corresponding to each of the distinct domains of AHNAK1 showed the C1 domain of AHNAK1 associates with NPR-C. The role of NPR-C in mediating AA-dependent AHNAK1 calcium signaling was explored in various cell types, including 3T3-L1 preadipocytes during the early stages of differentiation. Sucrose density gradient centrifugation studies showed AHNAK1 resides in the nucleus, cytoplasm, and at the plasma membrane, but small interfering RNA (siRNA)-mediated knockdown of NPR-C resulted in AHNAK1 accumulation in the nucleus. Overexpression of a portion of AHNAK1 resulted in augmentation of intracellular calcium mobilization, whereas siRNA-mediated knockdown of NPR-C or AHNAK1 protein resulted in attenuation of intracellular calcium mobilization in response to phorbol 12-myristate 13-acetate. We characterize the novel association between AHNAK1 and NPR-C and provide evidence that this association potentiates the AA-induced mobilization of intracellular calcium. We address the role of intracellular calcium in the various cell types that AHNAK1 and NPR-C were found to associate. 3T3-L1; smooth muscle cells; rat gastric muscosa cell 1; phospholipase doi: 10.1152/ajpcell.00219.2009.

Natriuretic peptides (NPs) are a family of cardiac- and vascular-derived hormones that are well known for regulating blood pressure, but their expression in the brain poses an intriguing yet unanswered question concerning their roles in the nervous system. Here, we report several unique activities of these hormones in regulating axonal development of dorsal root ganglion (DRG) neurons in the spinal cord. First, the C-type NP (CNP) is expressed in a restricted area of the dorsal spinal cord and provides a cue that is necessary for bifurcation of central sensory afferents. Second, in the culture of embryonic DRG neurons, CNP stimulates branch formation, induces axon outgrowth, and attracts growth cones. Furthermore, these activities are mediated by cyclic guanosine-3',5'-monophosphate (cGMP) signaling and can be elicited by other members of NP hormones. Thus, NPs represent a new class of extracellular factors that regulate key axonal processes during development. Because their receptors are present in many regions of the embryonic and adult brain, we propose that these hormones have wide influence on the development and function of the nervous system. axon branching | guidance | outgrowth | sensory neurons | cGMP doi/ 10.1073/pnas.0906880106

Neuronal circuits are shaped during development by the coordinated action of guidance factors and signals that regulate axonal branching. Unlike guidance cues, the molecules and signaling cascades that underlie axonal branching remain to be resolved. Here we show that the secreted molecule C-type natriuretic peptide (CNP) induces a cGMP signaling cascade via its receptor particulate guanylyl cyclase Npr2 which is essential for sensory axon bifurcation at the dorsal root entry zone (DREZ) of the spinal cord. In contrast, another form of sensory axon branching--collateral formation--is not affected by this pathway. We also demonstrate that cGMP signaling via the nitric oxide-stimulated soluble guanylyl cyclase system (NO-GC) is dispensable for sensory axon branching. Functionally, the bifurcation error in CNP mutant mice is maintained at mature stages and results in a reduced input on secondary neurons as detected by patch-clamp recordings. axon branching and pathfinding | cGKI | cGMP signaling | Npr2

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG. AQP2; cell volume kinetics; cGMP-generating signaling pathways; natriuretic peptides; water homeostasis; uroguanylin, phosphodiesterase-5

Renal dopamine and nitric oxide contribute to natriuresis during high-salt intake which maintains sodium and blood pressure homeostasis. We wanted to determine whether concurrent inhibition of these natriuretic factors increases blood pressure during high-sodium intake. Male Sprague-Dawley rats were divided into the following groups: 1) vehicle (V)-tap water, 2) NaC1-1% NaCl drinking water, 3) 30 mM L-buthionine sulfoximine (BSO), an oxidant, 4) BSO plus NaCl, and 5) BSO plus NaCl with 1 mM tempol (antioxidant). Compared with V, NaCl intake for 10 days doubled sodium intake and increased urinary dopamine level but reduced urinary nitric oxide content. NaCl intake also reduced basal renal proximal tubular NaK-ATPase activity with no effect on blood pressure. However, NaCl intake in BSO-treated rats failed to reduce basal Na-K-ATPase activity despite higher urinary dopamine levels. Also, dopamine failed to inhibit proximal tubular Na-K-ATPase activity and these rats exhibited reduced urinary nitric oxide levels and high blood pressure. Tempol supplementation in NaCl plus BSO-treated rats reduced blood pressure. BSO treatment alone did not affect the urinary nitric oxide and dopamine levels or blood pressure. However, dopamine failed to inhibit proximal tubular Na-K-ATPase activity in BSO-treated rats. BSO treatment also increased basal protein kinase C activity, DI receptor serine phosphorylation, and oxidative markers like malondialdehyde and 8-isoprostane. We suggest that NaCl-mediated reduction in nitric oxide does not increase blood pressure due to activation of D 1 receptor signaling. Conversely, oxidative stress-provoked inhibition of D1 receptor signaling fails to elevate blood pressure due to presence of normal nitric oxide. However, simultaneously decreasing nitric oxide levels with NaCl and inhibiting D1 receptor signaling with BSO elevated blood pressure. dopamine; G proteins; Na-K-ATPase; e-buthionine sulfoximine

B-type natriuretic peptide (BNP) is an established first-line therapy for acute decompensated heart failure (HF), but its efficacy in preventing left ventricular (LV) remodeling after myocardial injury is unknown. The goal of this study was to evaluate the effects of BNP therapy on remodeling after ischemic injury in an awake canine model. Dogs were chronically instrumented for hemodynamics. Ischemia was created by daily coronary embolization (Embo; 3.1 x [10.sup.4] beads/day) for 3 wk; 60 min after the first embolization, BNP (100 ng x [kg.sup.-1] x [min.sup.-1]; n = 6) or saline (control; n = 6) was continuously infused via a left atrial catheter for 3 wk. Hemodynamics and echocardiography were performed in an awake state at baseline, 3 wk after Embo + BNP infusion, and 4 wk after stopping Embo + BNP infusion. End-systolic elastance ([E.sub.es]) and LV change in pressure over time (dP/dt) were preserved throughout Embo + BNP therapy versus control therapy ([E.sub.es]: 3.76 [+ or -] 1.01 vs. 1.41 [+ or -] 0.16 mmHg/ml; LV dP/dt: 2,417 [+ or -] 96 vs. 2,068 [+ or -] 95 mmHg/s; both P < 0.05 vs. control). LV end-diastolic dimension was significantly smaller in BNP-treated dogs compared with control dogs (4.29 [+ or -] 0.10 vs. 4.77 [+ or -] 0.17 cm), and ejection fraction was maintained in treated dogs vs. control dogs (53 [+ or -] 1% vs. 46 [+ or -] 2%) (both P < 0.05 vs. control). Cyclooxygenase (COX)-2 expression in terminal LV tissue was significantly reduced after BNP therapy. Treatment with continuous infusion of BNP preserved LV geometry, improved systolic function, and prevented the progression of systolic HF after persistent ischemic injury. heart failure; myocardial function

B-type natriuretic peptide (BNP) is a peptide hormone of myocardial origin with significant cardioprotective properties. Patients with myocardial ischemia present with high levels of BNP in plasma and elevated expression in the myocardium. However, the molecular mechanisms of BNP induction in the ischemic myocardium are not well understood. The aim of the investigation was to assess whether myocardial hypoxia induces the production of BNP in human ventricular myocytes. To test the hypothesis that reduced oxygen tension can directly stimulate BNP gene expression and release in the absence of hemodynamic or neurohormonal stimuli, we used an in vitro model system of cultured human ventricular myocytes (AC16 cells). Cells were cultured under normoxic (21% [O.sub.2]) or hypoxic (5% [O.sub.2]) conditions for up to 48 h. The accumulation of BNP, atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF) was then measured. Hypoxia stimulated the protein release of BNP and VEGF but not ANP. In concordance, the increased mRNA levels of BNP and VEGF but not ANP were found on culturing AC 16 cells under hypoxic conditions. The analysis of the transcriptional activity of the hypoxia-inducible factor 1 (HIF-1) in nuclear extracts showed that HIF-1 activity was induced under hypoxic conditions. Finally, the treatment of AC16 cells with the HIF-1 inhibitor rotenone in hypoxia inhibited BNP and VEGF release. In conclusion, these data indicate that hypoxia induces the synthesis and secretion of BNP in human ventricular myocytes, likely through HIF-1-enhanced transcriptional activity. B-type natriuretic peptides; hypoxia-inducible factor 1; myocardial ischemia; heart failure; vascular endothelial growth factor