Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice.

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice. Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.

CNP signal pathway up-regulated in rectum of depressed rats and the interventional effect of Xiaoyaosan.

AIM: To investigate the distribution and expression of C-type natriuretic peptide (CNP)/natriuretic peptide receptor B (NPR-B) in the rectum of a rodent depression model and the interventional effect of Xiaoyaosan (XYS). METHODS: Male rats (n = 45) of clean grade (200 ± 20 g) were divided into five groups after one week of adaptive feeding: primary control, depression model, low dose XYS, middle dose XYS, and high dose XYS. The animal experiment continued for 3 wk. Primary controls were fed normally ad libitum. The rats of all other groups were raised in solitary and exposed to classic chronic mild unpredictable stimulation each day. XYS groups were perfused intragastrically with low dose, middle dose, and high dose XYS one hour before stimulation. Primary control and depression model groups were perfused intragastrically with normal saline under similar conditions as the XYS groups. Three weeks later, all rats were sacrificed, and the expression levels of CNP and NPR-B in rectum tissues were analyzed by immunohistochemistry, real-time polymerase chain reaction, and Western blotting. RESULTS: CNP and NPR-B were both expressed in the rectum tissues of all rats. However, the expression levels of CNP and NPR-B at both gene and protein levels in the depression model group were significantly higher when compared to the primary control group (n = 9; P < 0.01). XYS intervention markedly inhibited the expression levels of CNP and NPR-B in depressed rats. The expression levels of CNP and NPR-B in the high dose XYS group did not significantly differ from the expression levels in the primary control group. Additionally, the high and middle dose XYS groups (but not the low dose group) significantly exhibited lower CNP and NPR-B expression levels in the rectum tissues of the respectively treated rats compared to the untreated depression model cohort (n = 9; P < 0.01). CONCLUSION: The CNP/NPR-B pathway is upregulated in the rectum of depressed rats and may be one mechanism for depression-associated digestive disorders. XYS antagonizes this pathway at least partially.

Identification of small molecule NPR-B antagonists by high throughput screening--potential use in heart failure.

We found previously that stimulation of natriuretic peptide receptor (NPR)-B by C-type natriuretic peptide (CNP) in failing rat ventricle potentiates ß1-adrenoceptor (ß1-AR)-mediated inotropic response to noradrenaline through cGMP-mediated inhibition of phosphodiesterase (PDE) 3, thereby enhancing cAMP-mediated signalling. Increased cAMP-mediated signalling is deleterious in chronic heart failure (HF; basis for the use of ß-blockers in HF) and we propose to consider NPR-B antagonists as new HF treatment in addition to conventional therapy. Since there is no NPR-B-selective antagonist available for clinical studies, we aimed at identifying a novel small molecule (non-peptide) NPR-B antagonist. An assay was developed and high throughput screening performed on a chemical library of about 20,000 small molecule compounds (<500 Da) to identify NPR-B antagonists based on inhibition of CNP-stimulated cGMP production in NPR-B-expressing HEK293 cells. The screen revealed several potential NPR-B antagonists, of which six were selected for further studies. Three showed selective NPR-B vs NPR-A inhibition and three were partially selective. The compounds mediated reversible, noncompetitive inhibition and most likely act as allosteric modulators binding outside the agonist binding site of NPR-B. In rat ventricular muscle strips, the potentiating effect of CNP upon ß1-AR-evoked inotropic effects could be attenuated by at least one of these compounds. We identified several small molecule NPR-B antagonists by high throughput screening and show in a functional heart preparation that blocking NPR-B stimulation with a small molecule compound can reduce the potentiating effect of CNP on the ß1-AR-mediated inotropic response to noradrenaline.

Phenotypic screening with human iPS cell-derived cardiomyocytes: HTS-compatible assays for interrogating cardiac hypertrophy.

A major hurdle for cardiovascular disease researchers has been the lack of robust and physiologically relevant cell-based assays for drug discovery. Derivation of cardiomyocytes from human-induced pluripotent stem (iPS) cells at high purity, quality, and quantity enables the development of relevant models of human cardiac disease with source material that meets the demands of high-throughput screening (HTS). Here we demonstrate the utility of iPS cell-derived cardiomyocytes as an in vitro model of cardiac hypertrophy. Exposure of cardiomyocytes to endothelin 1 (ET-1) leads to reactivation of fetal genes, increased cell size, and robust expression of B-type natriuretic peptide (BNP). Using this system, we developed a suite of assays focused on BNP detection, most notably a high-content imaging-based assay designed for phenotypic screening. Miniaturization of this assay to a 384-well format enabled the profiling of a small set of tool compounds known to modulate the hypertrophic response. The assays described here provide consistent and reliable results and have the potential to increase our understanding of the many mechanisms underlying this complex cardiac condition. Moreover, the HTS-compatible workflow allows for the incorporation of human biology into early phases of drug discovery and development.

Regulation of C-type natriuretic peptides and natriuretic peptide receptor-B expression in diabetic rats renal treated by Tongluo Recipe.

OBJECTIVE: To investigate the expression of C-type natriuretic peptides (CNP) and natriuretic peptide receptor-B (NPR-B) receptor in diabetic rats renal cortex, and the regulation by Tongluo Recipe (TLR). METHODS: Sixty male SD rats were divided into 3 groups: the normal control group, diabetic model group and diabetic TLR group. Each group was further divided into two subgroups of ten in each, according to 4-week or 12-week observation period. Streptozotocin (STZ)-induced diabetic rats were treated with TLR (1.0 g·kg(-1)·d(-1)) for 4 and 12 weeks, respectively. (1) The essential information was collected for comparing renal mass, serum creatinine and 24 h urine albumen on each group was calculated. (2) CNP mRNA and NPR-B mRNA were detected by realtime-polymerase chain reaction (PCR) on rats renal cortex. (3) Concentration of CNP on renal cortex or serum were analyzed by enzyme-linked immunosorbent assay (ELISA). (4) Pathological evaluation and NPR-B immunostaining for renal tissue were also performed. RESULTS: (1) CNP and NPR-B mRNA levels were detected in each treated or untreated group, with slight elevated in untreated diabetes rats administrated with STZ after 4-week and CNP mRNA level remarkable elevated at 39.21 times higher than normal control group after 12 weeks, but NPR-B mRNA level showed a remarkably down-regulation at 98.07% after 12 weeks. CNP mRNA of TLR-treated group was also elevated after 12-week treatment, but less than untreated group. (2) Concentrations of CNP in renal cortex were obviously increased in treated or untreated diabetes rats, within these groups the treatment of TLR was found more significantly on prompting CNP concentration. Comparing to normal group, serum concentrations of CNP were also increased in treated or untreated diabetic groups, but there was no difference between these diabetic groups. (3) Renal lesions like glomerular volume increased are observed mostly in the relative early stage after 4 weeks. Although TLR treated group had no significant difference in their glomerular volume, the degrees of injury of glomerulus were ameliorated, as well as the NPR-B immunostaining enhanced in glomerulus. Weakly positive immunostaining of NPR-B are observed in glomerulus of normal control, and negative in glomerulus of untreated diabetes rats administrated with STZ after 12 weeks, whereas TLR-treatment groups showed a little enhancement. CONCLUSION: CNP and NPR-B showed different characteristic on renal cortex at different pathological period in diabetes rats, and TLR regulated their expression.

In vitro-targeted gene identification in patients with hepatitis C using a genome-wide microarray technology.

UNLABELLED: Iron in association with reactive oxygen species (ROS) is highly toxic, aggravating oxidative stress reactions. Increased iron not only plays an important role in the progression of hereditary hemochromatosis (HH) but also in common liver diseases such as chronic hepatitis C. The underlying mechanisms of hepatitis C virus (HCV)-mediated iron accumulation, however, are poorly understood. We introduce an in vitro-targeted approach to identify ROS/iron-regulated genes in patients with HCV using a genome-wide DNA microarray. The sensitivity of the 32,231 complementary DNA clone-carrying microarray was approximately 20% as estimated by detecting target genes of the genome-wide transcription factor hypoxia inducible factor 1alpha. Upon in vitro challenge to iron and oxidative stress, 265 iron-related and 1326 ROS-related genes could be identified in HepG2 cells; 233 significantly regulated genes were found in patients with mild (HCV) or severe (HH) iron deposition. Notably, 17 of the in vitro-selected genes corresponded to the genes identified in patients with HCV or HH. Among them, natriuretic peptide precursor B (NPPB) was the only iron-regulated gene identified in vitro that was differentially regulated between HCV and HH. Reverse-transcription polymerase chain reaction confirmed most of the microarray-identified genes in an even larger group of patients (n = 12). In patients with HCV, these included genes that are associated with RNA processing (MED9/NFAT, NSUN2), proliferation, differentiation, hypoxia, or iron metabolism (ISG20, MIG6, HIG2, CA9, NDRG1), whereas none of the nine known iron-related genes showed significant differences between HCV and HH. CONCLUSION: Although high-density microarray technology is less suitable for routine liver diagnosis, its use in combination with prior in vitro selection is a powerful approach to identify candidate genes relevant for liver disease.

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Evidence of a guanylyl cyclase natriuretic peptide receptor in the gills of the new zealand hagfish Eptatretus cirrhatus (Class Agnatha).

Natriuretic peptide binding sites were examined in the gills of the hagfish Eptatretus cirrhatus (Class Agnatha, subfamily Eptatretinae) using radio-ligand binding techniques, molecular cloning and guanylyl cyclase assays. Iodinated rat atrial natriuretic peptide ((125)I-rANP) and iodinated porcine C-type natriuretic peptide ((125)I-pCNP) bound specifically to the lamellar folds and cavernous tissue of E. cirrhatus gills, and 0.3 nmol l(-1) rat ANP competed for 50 % of specific (125)I-rANP binding sites. Affinity cross-linking of (125)I-rANP to gill membranes followed by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single binding site of 150 kDa. In the presence of Mn(2+), 0.1 nmol l(-1) rANP inhibited cGMP production, whereas 1 micromol l(-1) rANP stimulated cGMP production rates. At 1 micromol l(-1), pCNP also stimulated cGMP production. The production of cGMP was also measured in the presence and absence of ATP with either Mn(2+) or Mg(2+). Reverse transcriptase polymerase chain reaction (RT-PCR) of hagfish gill RNA, followed by cloning and sequencing of PCR products, produced a partial cDNA sequence of a natriuretic peptide guanylyl cyclase receptor. The deduced amino acid sequence indicated 87-91 % homology with other natriuretic peptide guanylyl cyclase receptors. This study indicates the presence of a natriuretic peptide guanylyl cyclase receptor in the gills of E. cirrhatus that is similar to the natriuretic peptide guanylyl cyclase receptors in higher vertebrates. These observations demonstrate that the coupling of natriuretic peptide receptors with guanylyl cyclase has a long evolutionary history.

Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes. Dissociation of Ras and Rho pathways.

G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gq and Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in alpha1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by alpha1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Galphaq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Galphaq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

Natriuretic peptide system in the rat submaxillary gland.

Natriuretic peptides and their receptors were characterized in rat submaxillary glands (SGs). Reverse phase-high performance liquid chromatography (HPLC) of rat SGs extracts revealed the presence of the 28-amino-acid (AA) circulating peptide ANP (Ser99-Tyr126) and the 126-AA prohormone (Asn1-Tyr126). The presence of ANP prohormone indicated that SGs are a site of ANP synthesis. Indeed, ANP mRNAs were demonstrated. ANP mRNA was 10 times lower than in the lung and only about 7 times lower than in the hypothalamus. ANP content in SG was determined as 30 +/- 8 ng/mg of protein (n = 7). In addition the presence of another member of the natriuretic peptide family, C-type natriuretic peptide (CNP), was found in SG. The CNP level of 293 +/- 38 pg/mg protein was significantly higher than in the lungs (44 +/- 6 pg/mg protein, P < 0.001, n = 5), but about 15 times lower than in hypothalamus (4.5 +/- 0.6 ng/mg protein, P < 0.001, n = 6). Both guanylyl cyclase and clearance receptors were expressed in SG. The presence of natriuretic peptide transcripts and their receptors suggests a role in rat SG functions.

The C-type natriuretic peptide receptor is the predominant natriuretic peptide receptor mRNA expressed in rat hypothalamus.

The natriuretic peptide receptors (NPR) are membrane-bound guanylate cyclases with extracellular binding domains specific for particular members of the natriuretic peptide family. NPR-A binds atrial natriuretic peptide (ANP) with high affinity, whereas the NPR-B appears to be specific for C-type natriuretic peptide (CNP). Previous data indicating extensive overlap between localization of ANP and CNP in hypothalamic neuroendocrine circuits suggest the importance of determining whether specificity of natriuretic peptide action may be conferred via receptor type present on target cells. To address this issue, we used in situ hybridization histochemistry to localize NPR-A and NPR-B mRNA in the hypothalamus. NPR-A mRNA was not found in substantial abundance in any hypothalamic nucleus; however, detectable NPR-A signal was observed in other brain regions, including the subfornical organ and medial habenula. In contrast, NPR-B mRNA was expressed throughout the hypothalamus, including neurons of the magnocellular and parvocellular paraventricular, the arcuate, and the supraoptic nuclei. Expression was also seen in other nuclei essential to neuroendocrine control, including the median preoptic, anteroventral periventricular, tuberomammilary, ventromedial and suprachiasmatic nuclei. NPR-B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes. The results suggest that NPR-B is the primary natriuretic peptide receptor in hypothalamus, and by inference indicate that CNP is the primary active natriuretic peptide in neuroendocrine regulation.