Atrial Natriuretic Peptide Receptor Research Articles

Regulation of atrial natriuretic peptide (ANP) and its role in blood pressure

Atrial natriuretic peptide (ANP) a powerful vasodilator, and a protein (28-amino acid peptide) hormone secreted by heart muscle cells. It is released in response to atrial distention, stretching of the vessel walls, sympathetic stimulation of ?-adrenoceptors, raised sodium concentration, angiotensin-II and endothelin. ANP binds to three cell surface receptors called ANP receptors. The overall effect of ANP on the body is to counter increases in blood pressure and volume caused by the renin-angiotensin system. It has also been reported to increase the release of free fatty acids from adipose tissue. Regulation of its effects is achieved through gradual degradation of the peptide by the enzyme neutral endopeptidase (NEP). Inhibitors of NEP are currently being developed to treat disorders ranging from hypertension to heart failure. Synthetic analogs of ANP have been investigated as potential therapies for the treatment of decompensated heart failure and other diseases.DOI: http://dx.doi.org/10.3329/icpj.v1i7.10812International Current Pharmaceutical Journal 2012, 1(7): 176-179

P-0033 Natriuretic Peptide Receptor a as a Novel Target for Gastric Cancer

IntroductionThe receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), have been reported to express on lung cancer, prostate cancer, ovarian cancer, NPRA expression and signaling is important for tumor growth, its deficiency protect C57BL/6 mice from lung, skin, and ovarian cancers, and these result suggest that NPRA is a new target for cancer therapy. Recently, NPRA has been demonstrated express in pre-implantation embryos and in ES cells, it has a novel role in the maintenance of self-renewal and pluripotency of ES cells. However, the direct role of NPRA signaling in gastric cancer remains unclear. MethodsNPRA expression was examined by western blotting, immunofluorescence and immunohistochemistry. NPRA was downregulated by transfection of siRNA and shRNA. Resultswe demonstrated that NPRA is abundantly expressed on human gastric cancer AGS cells, but not in nontumorigenic epithelial cells. NPRA expression showed positive correlation with clinical staging and prognosis in gastric cancer patient. Down-regulation of NPRA by siNPRA induced apoptosis in AGS cells. The mechanism of siNPRA-induced anti-AGS effects was linked to NPRA-induced expression of KCNQ1, a K+ channel overexpressed in AGS and significantly reduced by siNPRA. ConclusionCollectively, these results suggest that NPRA promotes gastric cancer development in part by regulating KCNQ1. Our findings also suggest that NPRA is a potential prognostic marker and a target for gastric cancer therapy.

Stimulation of Aquaporin-Mediated Fluid Transport by Cyclic GMP in Human Retinal Pigment Epithelium In Vitro

The retinal pigment epithelium (RPE) expresses aquaporin-1 (AQP1) and components of the natriuretic peptide signaling pathway. We hypothesized that stimulation of the natriuretic signaling pathway in RPE with atrial natriuretic peptide (ANP) and with membrane-permeable analogs of cGMP would induce a net apical-to-basal transport of fluid. The hypothesis was tested using human RPE cultures that retain properties seen in vivo. Confluent monolayers were treated with ANP or membrane-permeable cGMP analogs in the presence of anantin, H-8, and an AQP1 inhibitor, AqB013. Fluid movement from the apical to basal chambers was measured by weight and used to calculate net fluid transport. Our results demonstrated a 40% increase in net apical-to-basal fluid transport by ANP (5 μM) that was inhibited completely by the ANP receptor antagonist anantin and a 60% increase in net apical-to-basal fluid transport in response to the extracellularly applied membrane-permeable cGMP analog pCPT-cGMP (50 μM), which was not affected by the protein kinase G inhibitor H-8. The aquaporin antagonist AqB013 (20 μM) inhibited the cGMP-stimulated RPE fluid flux. The effect of cGMP is consistent with an enhancement of the net fluid flux in RPE mediated by AQP1 channels. Pharmacologic activation of cGMP signaling and concomitant stimulation of fluid uptake from the subretinal space could offer insights into a new approach to treating or reducing the risk of retinal detachment.

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume‐expanded mice

We tested the hypothesis that inhibition of phosphodiesterase 4 (PDE4) with rolipram to increase vascular endothelial cAMP and stabilize the endothelial barrier would attenuate the action of endogenous atrial natriuretic peptide (ANP) to increase vascular permeability to the plasma protein albumin after an acute plasma volume expansion. After rolipram pretreatment (8 mg (kg body wt)(-1), intraperitoneal, 30 min) more than 95% of the peak increase in plasma volume after volume expansion (4.5% bovine serum albumin, 114 μl (g body wt)(-1) h(-1), 15 min) remained in the vascular space 75 min after the end of infusion, whereas only 67% of the fluid was retained in volume-expanded animals with no rolipram pretreatment. Rolipram significantly decreased 30 min fluorescently labelled albumin clearance (μl (g dry wt)(-1)) relative to untreated volume-expanded controls in skin (e.g. back, 10.4 ± 1.6 vs. 19.5 ± 3.6, P = 0.04), muscle (e.g. hamstring, 15.0 ± 1.9 vs. 20.8 ± 1.4, P = 0.04) and in colon, caecum, and rectum (average reduction close to 50%). The mass of muscle and skin tissue accounted for 70% of volume-expansion-dependent albumin shifts from plasma to interstitium. The results are consistent with observations that the PDE4 inhibitor rolipram attenuates ANP-induced increases in vascular permeability after infusion of exogenous ANP and observations of elevated central venous pressure after a similar volume expansion in mice with selective deletion of the endothelial ANP receptor. These observations may form the basis for new strategies to retain intravenous fluid containing macromolecules.

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

Plasma soluble corin and N-terminal pro-atrial natriuretic peptide levels in pregnancy induced hypertension

Objective One of the theories involved in the pathogenesis of pregnancy induced hypertension involves salt and water retention. We aimed to measure the proenzyme convertase corin, responsible for pro-atrial natriuretic peptide (ANP) cleavage to active ANP, in plasma of hypertensive pregnant females. Study design Sixty pregnant females suffering from pregnancy induced hypertension in second and third trimesters of pregnancy were compared to twenty eight healthy pregnant females of the same gestational period. Concomitant urine and plasma samples were collected for the determination of some biochemical parameters. Plasma soluble corin and N-terminal (NT) pro-ANP (1–98) values were determined in both groups using enzyme immunoassays. Results Plasma soluble corin mean value was significantly higher in the patient group compared to the control group. Upon dividing the patient group according to blood pressure, plasma NT pro-ANP showed significantly higher mean value in the group with blood pressure ⩾ 140/90 mmHg compared to the group with blood pressure < 140/90 mmHg and control group. Conclusions High plasma soluble corin and NT pro-ANP values in hypertensive pregnant females particularly those with blood pressure ⩾ 140/90 mmHg speculates an ANP receptor/ post receptor signaling defect, which would aggravate the pregnancy induced hypertensive state.

Opposite effects of ANP receptors in attenuation of LPS-induced endothelial permeability and lung injury

Atrial natriuretic peptide (ANP) has been recently identified as a modulator of acute lung injury (ALI) induced by pro-inflammatory agonists. While previous studies tested effects of exogenous ANP administration, the role of endogenous ANP in the course of ALI remains unexplored. This study examined regulation of ANP and its receptors NPR-A, NPR-B and NPR-C by LPS and involvement of ANP receptors in the modulation of LPS-induced lung injury. Primary cultures of human pulmonary endothelial cells (EC) were used in the in vitro tests. Expression of ANP and its receptors was determined by quantitative RT-PCR analysis. Agonist-induced cytoskeletal remodeling was evaluated by immunofluorescence staining, and EC barrier function was characterized by measurements of transendothelial electrical resistance. In the murine model of ALI, LPS-induced lung injury was assessed by measurements of protein concentration and cell count in bronchoalveolar lavage fluid (BAL). LPS stimulation significantly increased mRNA expression levels of ANP and NPR-A in pulmonary EC. Pharmacological inhibition of NPR-A augmented LPS-induced EC permeability and blocked barrier protective effects of exogenous ANP on LPS-induced intercellular gap formation. In contrast, pharmacological inhibition of ANP clearance receptor NPR-C significantly attenuated LPS-induced barrier disruptive effects. Administration of NPR-A inhibitor in vivo exacerbated LPS-induced lung injury, whereas inhibition of NPR-C suppressed LPS-induced increases in BAL cell count and protein content. These results demonstrate for the first time opposite effects of NPR-A and NPR-C in the modulation of ALI and suggest a compensatory protective mechanism of endogenous ANP in the maintenance of lung vascular permeability in ALI.

Preparation and Characterization of Molecularly Imprinted Polymeric Nanoparticles for Atrial Natriuretic Peptide (ANP).

Natriuretic peptide receptor A (NPRA), the receptor for the cardiac hormone atrial natriuretic peptide (ANP), is expressed abundantly on cancer cells and disruption of ANP-NPRA signaling inhibits tumor burden and metastasis. Since antagonists of NPRA signaling have not provided reproducible results, we reasoned that a synthetic neutralizing antibody to ANP, which has high selectivity and affinity for ANP, could be used to regulate ANP levels and attenuate NPRA signaling. In this study, we prepared molecularly imprinted polymer nanoparticles (MIPNPs) for ANP using a short peptide of ANP as the template and determined their binding affinity and selectivity. The MIPNPs were prepared by precipitation polymerization using NH2-SLRRSS-CONH2, which is a short peptide from ANP as template, methacrylic acid (MAA) and N-isopropylacrylamide (NIPAm) as functional monomers, bis-acrylamide (BIS) as crosslinker. The average diameter of MIPNPs and non-imprinted nanoparticles (NIPNPs) in water is 215.8 ±4.6 nm and 197.7±3.1 nm respectively. The binding isotherm analysis showed that MIPNPs have a much higher binding affinity for template peptide and ANP than NIPNPs. Scatchard analysis gave an equilibrium dissociation constant, Kd of 7.3 μM with a binding capacity 106.7 μmol/g for template peptide and Kd of 7.9 μM with a binding capacity of 36.0 μmol/g for ANP. Measurements of binding kinetics revealed that MIPNPs reach protein adsorption equilibrium in 30 min. MIPNPs found to have high specificity for ANP with little affinity for BSA or scrambled ANP peptide. MIPNPs also recognized and adsorbed ANP in cell culture media spiked with ANP and human plasma. Taken together, these results indicate that MIPNPs have high affinity and selectivity for ANP and can be used as a synthetic antibody for modulating ANP-NPRA signaling in cancers.

Atrial natriuretic peptide/natriuretic peptide receptor A (ANP/NPRA) signaling pathway: A potential therapeutic target for allergic asthma

Allergic asthma is a chronic inflammatory disease of airway and immune disorder is an acknowledged mechanism. Numerous data demonstrate Th1/Th2 cells play an important role in the development of allergic asthma. Atrial natriuretic peptide (ANP) is a multifunctional hormone secreted by cardiac atria, lung, and so forth, which has been recognized for several decades due to its general effects on cardiovascular system, and natriuretic peptide receptor A (NPRA) is the major effecting receptor for ANP. In recent years, more and more studies suggest that ANP/NPRA signaling pathway is implicated in modulation of immnue and inflammatory reaction. Moreover, there are some reports about significant changes of ANP production in peripheral blood from asthmatics in acute exacerbation compared with patients during the remission and the healthy. Nevertheless, it is unknown that why ANP shows an observable change and what role ANP plays in asthma until now. We propose that ANP/NPRA signaling pathway is involved in immune dysfunction and airway inflammation of allergic asthma based on our experimental results, which suggests ANP/NPRA signaling pathway may be a potential therapeutic target for allergic asthma.

Tissue- and development-specific glycosylation states of the natriuretic peptide receptors guanylyl cyclase-A (GC-A) and GC-B

Background The natriuretic peptides (NPs) atrial NP (ANP), B-type NP (BNP) and C-type NP (CNP) elicit cellular effects by binding to transmembrane proteins with guanylyl cyclase (GC) activity, referred to as GC-A (representing the common receptor for ANP and BNP) and GC-B (the CNP receptor). GC-A and GC-B are structurally closely related proteins with very similar cDNA-deduced molecular masses in the range of 115 kDa, but their native molecular weights are significantly (by up to 20 kDa) higher due to glycosylation. Differential glycosylation of cell surface receptors can affect folding, trafficking, ligand binding and/or agonistinduced signal transduction. Moreover, specific temporal expression patterns of carbohydrate structures are frequently involved in developmental processes. Hitherto, however, the glycosylation states of GC-A and GC-B in vivo are largely unknown, since currently available data are based to a great extent on studies with receptor-transfected cell lines. This investigation aimed to characterize the sizes and posttranslational modifications of native GC-A and GCB in different tissues of adult rats and mice. Based on increasing evidence for specific activities of NPs and their receptors in the developing brain (1-3), we examined the glycosylation of GC-A and GC-B during postnatal brain development. Methods and results Western blot (as well as receptor/ligand crosslinking) experiments and treatments with carbohydrate-digesting enzymes served to investigate the sizes and posttranslational modifications of the two receptors. Due to marked tissue-specific differences in N-linked glycosylation, mean apparent molecular masses of both rat and mouse GC-A range from 119 kDa in brain to values between 124 (lung) and 132 (heart) kDa in peripheral tissues. The reduced glycosylation in brain seems to be central nervous system (CNS)-specific, since the pituitary, which contains both CNS and CNS-unrelated tissue, co-expresses the CNS-typical (119 kDa, reference: olfactory bulb) and the peripheral (up to 130 Da) forms of GC-A. GC-B sizes differed in the same manner between peripheral organs, indicating equal tissue-dependent influences on oligosaccharide processing at GC-A and GC-B. A higher (by 3 kDa) total amount of N-linked carbohydrates suggests usage of an additional consensus N-glycosylation site (7 instead of 6 in the case of GC-A). The existence of region-specific GC-B size variants in cerebellum and olfactory bulb discriminates this receptor from GC-A in the adult brain. Both GC-A and GC-B (but not two other glycosylated membrane proteins examined) are hyperglycosylated at N-linked sites during early postnatal brain development. At postnatal day 1, the vast majority of GC-B (but not GC-A) contain additionally an O-linked carbohydrate modification.

Identification of a possible role for atrial natiuretic peptide in MDMA-induced hyperthermia

MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to (1) determine if ANP is released following MDMA administration, and (2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60min following MDMA (40mg/kg, sc) administration in 16 male Sprague–Dawley rats. A robust increase in ANP was seen within 10min of MDMA administration. ANP levels returned to baseline at 20min and then gradually rose over the 60min monitoring period. The administration of Anantin (40mg, ip), 15min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA.

Epithelium integrity is crucial for the relaxant activity of brain natriuretic peptide in human isolated bronchi

BACKGROUND AND PURPOSE Brain natriuretic peptide (BNP) plays an important role in several biological functions, including bronchial relaxation. Here, we have investigated the role of BNP and its cognate receptors in human bronchial tone. EXPERIMENTAL APPROACH Effects of BNP on responses to carbachol and histamine were evaluated in non-sensitized, passively sensitized, epithelium-intact or denuded isolated bronchi and in the presence of methoctramine, N(ω) -nitro-L-arginine methyl ester (L-NAME) and aminoguanidine. Natriuretic peptide receptors (NPRs) were investigated by immunohistochemistry, RT-PCR and real-time PCR. Release of NO and acetylcholine from bronchial tissues and cultured BEAS-2B bronchial epithelial cells was also investigated. KEY RESULTS BNP reduced contractions mediated by carbachol and histamine, with decreased E(max) (carbachol: 22.7 ± 4.7%; histamine: 59.3 ± 1.8%) and increased EC(50) (carbachol: control 3.33 ± 0.88 µM, BNP 100 ± 52.9 µM; histamine: control 16.7 ± 1.7 µM, BNP 90 ± 30.6 µM); BNP was ineffective in epithelium-denuded bronchi. Among NPRs, only atrial NPR (NPR1) transcripts were detected in bronchial tissue. Bronchial NPR1 immunoreactivity was detected in epithelium and inflammatory cells but faint or absent in airway smooth muscle cells. NPR1 transcripts in bronchi increased after incubation with BNP, but not after sensitization. Methoctramine and quinine abolished BNP-induced relaxant activity. The latter was associated with increased bronchial mRNA for NO synthase and NO release, inhibited by L-NAME and aminoguanidine. In vitro, BNP increased acetylcholine release from bronchial epithelial cells, whereas NO release was unchanged. CONCLUSIONS AND IMPLICATIONS Epithelial cells mediate the BNP-induced relaxant activity in human isolated bronchi.

Expression of mRNA of vasopressin, ANP and aldosterone receptors in the rat inner ear

Expression of mRNA of vasopressin, ANP and aldosterone receptors in the rat inner ear

Atrial natriuretic peptide ameliorates peritoneal fibrosis in rat peritonitis model

Atrial natriuretic peptide (ANP) was recently reported to ameliorate fibrosis in the heart and experimental renal diseases and vascular thickening after balloon injury. Peritoneal fibrosis is an important complication of long-term peritoneal dialysis, and peritonitis is a factor in its onset. In the present study, we investigated the effects of ANP in a rat peritonitis-induced peritoneal fibrosis model. As pretreatment, an osmotic pump containing vehicle (saline) or ANP (0.15 or 0.3 μg/min) was inserted through the carotid vein in male Sprague-Dawley rats. ANP or saline was continuously infused using the osmotic pump. Three days after administration of ANP or saline, rats underwent peritoneal scraping in a blind manner and were sacrificed on Day 14. The effects of ANP were evaluated based on peritoneal thickness, immunohistochemistry and real-time polymerase chain reaction. In each experiment, we evaluated messenger RNA (mRNA) expression of the ANP receptor natriuretic peptide receptor A (NPR-A) in the peritoneum after scraping. The effects of ANP were also studied in cultured peritoneal fibroblasts and mesothelial cells. We observed a significant increase in NPR-A mRNA in the peritoneum. Peritoneal thickness increased with time and peaked on Day 14, but ANP significantly reduced peritoneal thickness. Parameters such as number of macrophages and CD-31-positive vessels and expression of type III collagen/transforming growth factor-β/plasminogen activator inhibitor-1 (PAI-1)/connective tissue growth factor (CTGF) were significantly suppressed by ANP. In cultured peritoneal fibroblasts and mesothelial cells, ANP suppressed angiotensin II-induced upregulation of CTGF and PAI-1. Our results suggest that ANP is useful in preventing inflammation-induced peritoneal fibrosis.

Natriuretic Peptide Receptor A as a Novel Target for Prostate Cancer

BackgroundThe receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in prostate cancer remains unclear.ResultsNPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine over-expressed in PCa and significantly reduced by siNPRA. Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression.ConclusionsCollectively, these results suggest that NPRA promotes PCa development in part by regulating MIF. Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.

Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C

Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A (GC-A) and natriuretic peptide receptor-C (NPR-C). Internalization of GC-A and NPR-C is poorly understood, in part, because previous studies used (125)I-ANP binding to track these receptors, which are expressed in the same cell. Here, we evaluated GC-A and NPR-C internalization using traditional and novel approaches. Although HeLa cells endogenously express GC-A, (125)I-ANP binding and cross-linking studies only detected NPR-C, raising the possibility that past studies ascribed NPR-C-mediated processes to GC-A. To specifically measure internalization of a single receptor, we developed an (125)I-IgG-binding assay that tracks extracellular FLAG-tagged versions of GC-A and NPR-C independently of each other and ligand for the first time. FLAG-GC-A bound ANP identically with wild-type GC-A and was internalized slowly (0.5%/min), whereas FLAG-NPR-C was internalized rapidly (2.5%/min) in HeLa cells. In 293 cells, (125)I-ANP and (125)I-IgG uptake curves were superimposable because these cells only express a single ANP receptor. Basal internalization of both receptors was 8-fold higher in 293 compared with HeLa cells and ANP did not increase internalization of FLAG-GC-A. For FLAG-NPR-C, neither ANP, BNP, nor CNP increased its internalization in either cell line. Prolonged ANP exposure concomitantly reduced surface and total GC-A levels, consistent with rapid exchange of extracellular and intracellular receptor pools. We conclude that ligand binding does not stimulate natriuretic peptide receptor internalization and that cellular environment determines the rate of this process. We further deduce that NPR-C is internalized faster than GC-A and that increased internalization is not required for GC-A down-regulation.

Natriuretic peptide receptor A knock out restricts tumorigenesis through ablation of myeloid derived suppressor cells expansion (66.3)

Abstract Recent data from our group and a number of others have demonstrated that expansion and activation of Myeloid Derived Suppressor Cells(MDSC) play a pivotal role in tumorigenesis. The receptor for atrial natriuretic peptide, natriuretic peptide receptor A (NPRA) has been implicated in cancers. We have shown that NPRA expression and signaling is important for tumor growth. We hypothesize a novel link between NPRA expression, altered differentiation of myeloid cells and tumor formation. MDSC population consists of two major subsets of Ly6G+ granulocytic and Ly6C+ monocytic cells. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. MDSC isolated from tumor bearing mice expressed high levels of NPRA. Next we hypothesized that NPRA knock out would result in restricted tumor growth. Using NPRA deficient mice, we report that these mice have altered differentiation of myeloid cells. As a consequence tumor cells injected subcutaneously to NPRA KO mice failed to grow. We investigated the nature of cells responsible for tumorigenesis and proved that ablation of a subset of MDSC cells restricts tumor formation. This work may indicate key roles for NPRA in myeloid cell differentiation and cancer formation.

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

Phosphodiesterase 4 inhibition attenuates atrial natriuretic peptide‐induced vascular hyperpermeability and loss of plasma volume

Inhibition of phosphodiesterase 4 (PDE4) to increase endothelial cAMP and stabilize the endothelial barrier attenuates acute inflammatory increases in vascular permeability.We extended this approach to attenuate physiological increases in vascular permeability in response to atrial natriuretic peptide (ANP), which acts with the kidney to regulate plasma volume. We measured blood-to-tissue albumin clearance and changes in plasma volume in isoflurane-anaesthetized mice (C57BL/6J) pre-treated with rolipram (8 mg kg(-1) I.P., 30 min). Rolipram significantly reduced albumin permeability, measured using a dual-label fluorescence method, in skin and skeletal muscle compared with ANP alone (500 ng kg(-1) min(-1)). Skin and muscle tissue accounted for 70% of the reduction in whole body albumin clearance taking into account albumin clearance in gastrointestinal (GI) tissue, heart and kidney. The action of ANP and rolipram to modify albumin clearances in duodenum and jejunum could be accounted for by local increases in vascular perfusion to increase surface area for exchange. ANP increased haematocrit from 40.6% to 46.8%, corresponding to an average loss of 22% plasma fluid volume (227 μl), and this was almost completely reversed with rolipram. Renal water excretion accounted for less than 30% of plasma fluid loss indicating that reduced albumin permeability and reduced filtration into vasodilated GI tissue were the predominant actions of PDE4 inhibition. Similar fluid retention was measured in mice with endothelial-restricted deletion of the guanylyl cyclase-A receptor for ANP. Stabilizing the endothelial barrier to offset ANP-induced increases in vascular permeability may be part of a strategy to maintain plasma volume.