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Type 2 diabetes (T2D) and obesity are strongly associated with low natriuretic peptide (NP) plasma levels and a down-regulation of NP guanylyl cyclase receptor-A (GCA) in skeletal muscle and adipose tissue. However, no study has so far provided evidence for a causal link between atrial NP (ANP)/GCA deficiency and T2D pathogenesis. Here, we show that both systemic and skeletal muscle ANP/GCA deficiencies in mice promote metabolic disturbances and prediabetes. Skeletal muscle insulin resistance is further associated with altered mitochondrial function and impaired endurance running capacity. ANP/GCA-deficient mice exhibit increased proton leak and reduced content of mitochondrial oxidative phosphorylation proteins. We further show that GCA is related to several metabolic traits in T2D and positively correlates with markers of oxidative capacity in human skeletal muscle. Together, these results indicate that ANP/GCA signaling controls muscle mitochondrial integrity and oxidative capacity in vivo and plays a causal role in the development of prediabetes.

Introduction: Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart., Aim: We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of β-adrenergic receptor (β-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied., Main Methods: Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and β-MHC), β-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]., Results: A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of β-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells., Conclusion: Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-β-AR-ROS and NPR-A signaling., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/ Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.

Pericyte dysfunction, with excessive migration, hyperproliferation, and differentiation into smooth muscle-like cells contributes to vascular remodeling in Pulmonary Arterial Hypertension (PAH). Augmented expression and action of growth factors trigger these pathological changes. Endogenous factors opposing such alterations are barely known. Here, we examine whether and how the endothelial hormone C-type natriuretic peptide (CNP), signaling through the cyclic guanosine monophosphate (cGMP) -producing guanylyl cyclase B (GC-B) receptor, attenuates the pericyte dysfunction observed in PAH. The results demonstrate that CNP/GC-B/cGMP signaling is preserved in lung pericytes from patients with PAH and prevents their growth factor-induced proliferation, migration, and transdifferentiation. The anti-proliferative effect of CNP is mediated by cGMP-dependent protein kinase I and inhibition of the Phosphoinositide 3-kinase (PI3K)/AKT pathway, ultimately leading to the nuclear stabilization and activation of the Forkhead Box O 3 (FoxO3) transcription factor. Augmentation of the CNP/GC-B/cGMP/FoxO3 signaling pathway might be a target for novel therapeutics in the field of PAH., (© 2024. The Author(s).)

Histamine-dependent and -independent itch is conveyed by parallel peripheral neural pathways that express gastrin-releasing peptide (GRP) and neuromedin B (NMB), respectively, to the spinal cord of mice. B-type natriuretic peptide (BNP) has been proposed to transmit both types of itch via its receptor NPRA encoded by Npr1 . However, BNP also binds to its cognate receptor, NPRC encoded by Npr3 with equal potency. Moreover, natriuretic peptides (NP) signal through the G i -couped inhibitory cGMP pathway that is supposed to inhibit neuronal activity, raising the question of how BNP may transmit itch information. Here, we report that Npr3 expression in laminae I-II of the dorsal horn partially overlaps with NMB receptor (NMBR) that transmits histaminergic itch via G q -couped PLCβ-Ca 2+ signaling pathway. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not GRP. Consistently, BNP evoked Ca 2+ responses in NMBR/NPRC HEK 293 cells and NMBR/NPRC dorsal horn neurons. These results reveal a previously unknown mechanism by which BNP facilitates NMB-encoded itch through a novel NPRC-NMBR cross-signaling in mice. Our studies uncover distinct modes of action for neuropeptides in transmission and modulation of itch in mice., Competing Interests: QM, XL, XL, JL, AM, DB, BL, HJ, YS, QY, FG, LW, JP, JJ, KS, RK, JY, AT, ZC No competing interests declared, (© 2021, Meng et al.)

C-type natriuretic peptide (CNP) is an important regulator of the male reproductive process. Our previous investigations showed that CNP can significantly stimulate the mRNA expression of androgen-binding protein (Abp) and transferrin (Trf) in the rat Sertoli cells, but the pathways responsible for this process remain to be elucidated. We predict that CNP binds the natriuretic peptide receptor B (NPR-B) to regulate expression of ABP and TRF through the intracellular cyclic guanosine monophosphate (cGMP) pathway. To address this question, in this study, we first confirmed the expression and localization of CNP and NPR-B in rat testes by immunohistochemistry and western blotting. Then, ELISA and real-time PCR were performed to investigate the signaling pathway of CNP in Sertoli cells in rat testes. Our results showed that CNP was mainly localized in the germ cells and Leydig cells, and its receptor, NPR-B, was mostly expressed in the Sertoli cells and vascular endothelial cells. CNP supplementation in the Sertoli cell medium was accompanied by an increase in the amount of intracellular cGMP and in the production of Abp and Trf mRNA, whereas inhibition of PKG with KT5823 led to a decrease in the expression of Abp and Trf mRNA. Moreover, Abp and Trf mRNA were no longer elevated when we used liposome-mediated RNA interference technology to silence the NPR-B gene in a mouse Sertoli cell line (TM4). These results suggest that CNP contributes to the regulation of ABP and TRF in the Sertoli cells through the NPR-B/cGMP/PKG signaling pathways.

Axon bifurcation - a specific form of branching of somatosensory axons characterized by the splitting of the growth cone - is mediated by a cGMP-dependent signaling cascade composed of the extracellular ligand CNP (C-type natriuretic peptide), the transmembrane receptor guanylyl cyclase Npr2 (natriuretic peptide receptor 2), and the kinase cGKI (cGMP-dependent protein kinase I). In the absence of any one of these components, the formation of T-shaped axonal branches is impaired in neurons from DRGs (dorsal root ganglia), CSGs (cranial sensory ganglia) and MTNs (mesencephalic trigeminal neurons) in the murine spinal cord or hindbrain. Instead, axons from DRGs or from CSGs extend only either in an ascending or descending direction, while axons from MTNs either elongate within the hindbrain or extend via the trigeminal ganglion to the masseter muscles. Collateral formation from non-bifurcating stem axons is not affected by impaired cGMP signaling. Activation of Npr2 requires both binding of the ligand CNP as well as phosphorylation of serine and threonine residues at the juxtamembrane regions of the receptor. The absence of bifurcation results in an altered shape of termination fields of sensory afferents in the spinal cord and resulted in impaired noxious heat sensation and nociception whereas motor coordination appeared normal.

Endocytosis is a prominent clathrin-mediated mechanism for concentrated uptake and internalization of ligand-receptor complexes, also known as cargo. Internalization of cargo is the fundamental mechanism for receptor-dependent regulation of cell membrane function, intracellular signal transduction, and neurotransmission, as well as other biological and physiological activities. However, the intrinsic mechanisms of receptor endocytosis and contemporaneous intracellular signaling are not well understood. We review emerging concepts of receptor endocytosis with concurrent intracellular signaling, using a typical example of guanylyl cyclase/natriuretic peptide receptor-A (NPRA) internalization, subcellular trafficking, and simultaneous generation of second-messenger cGMP and signaling in intact cells. We highlight the role of short-signal motifs located in the carboxyl-terminal regions of membrane receptors during their internalization and subsequent receptor trafficking in organelles that are not traditionally studied in this context, including nuclei and mitochondria. This review sheds light on the importance of future investigations of receptor endocytosis and trafficking in live cells and intact animals in vivo in physiological context., (Copyright © 2019. Published by Elsevier Inc.)

Idiopathic Pulmonary Arterial Hypertension (IPAH) is a deadly and disabling disease characterized by severe vascular remodeling of small pulmonary vessels by fibroblasts, myofibroblasts and vascular smooth muscle cell proliferation. Recent studies suggest that the Natriuretic Peptide Clearance Receptor (NPR-C) signaling pathways may play a crucial role in the development of IPAH. Reduced expression or function of NPR-C signaling in pulmonary artery smooth muscle cells may contribute to the pulmonary vascular remodeling, which is characteristic of this disease. The likely mechanisms may involve an impaired interaction between NPR-C, specific growth factors and other signal transduction pathways including but not limited to Gqα/mitogen-activated protein kinase (MAPK)/PI3K and AKT signaling. The resulting failure of growth suppression in pulmonary artery smooth muscle cells provides critical clues to the cellular pathobiology of IPAH. The reciprocal regulation of NPR-C signaling in models of tissue remodeling may thus provide new insights to our understanding of IPAH.

The follicle must fulfill the following criteria if it is to survive the period between early embryonic life and the luteinizing hormone (LH) peak. It should (i) be surrounded by pregranulosa cells; (ii) complete the first meiotic division and become dormant; and (iii) continue metabolism during the dormant stage. Interaction between the natriuretic peptide precursor type C (Nppc) and its receptor, natriuretic peptide receptor 2 (Npr2), affects female fertility through the production of oocytes with developmental capacity and maintain oocyte meiotic arrest. While Nppc is expressed in mural cells, cumulus cells express Npr2. Nppc/Npr2 system exerts its biological function on developing follicles by increasing the production of intracellular cyclic guanosine monophosphate (cGMP). This pathway not only contributes to the development of ovary and the uterus, but aids the formation of healthy eggs in terms of their morphological and genetic aspects. A defect in this pathway leads to asmall ovarian size, string-like uterine horns, and thin endometrium and myometrium. Disorganized chromosomes, abnormal cumulus expansion and early meiotic resumption occur in animals with defective Nppc/Npr2 signaling. The types and number of oocytes also decrease when there is incompetent Nppc/Npr2 signaling. This paper extends on most recent and relevant experimental evidence regarding Nppc/Npr2/cGMP signaling with regard to its crucial role in maintaining oocyte meiotic arrest and the production of oocytes with developmental capacity. We further discuss whether the agonist or antagonist forms of the members of this exciting pathway can be usedfor triggering final oocyte maturation.

Research Question: What is the effect of C-type natriuretic peptide (CNP) on human sperm capacitation in vitro and what is the mechanism of this effect?, Design: CNP/NPR-B expression in the female rat genital tract was examined by immunohistochemistry and western blot assay, and then the role of CNP in human sperm capacitation was determined. The signal transduction pathway of CNP in the process was determined to elucidate the regulation mechanism of CNP by enzyme-linked immunosorbent assay and flow cytometry., Results: Both CNP and NPR-B were expressed in the genital tract of female rats, especially in the mucosa epithelium cell of the oviduct; the CNP level in the rat oviduct was higher than that in the cervix. Both CNP and NPR-B level in the rat oviduct varied during the oestrus cycle, maximal expression being observed at proestrus. Furthermore, intracellular cGMP level in spermatozoa was significantly enhanced by CNP (P < 0.01). PKG activity was detected in the spermatozoa, and it can be activated by the CNP and 8-Br-cGMP (cGMP analogue). The PKG inhibitor KT5823 inhibited the effect of CNP on sperm hyperactivation and the acrosome reaction. Finally, Ca 2+ and tyrosine phosphorylation levels in spermatozoa were markedly improved by CNP and 8-Br-cGMP but significantly inhibited by the addition of KT5823 (P < 0.05)., Conclusions: CNP secreted by the female genital tract might bind to NPR-B on the spermatozoa. It successively stimulated intracellular cGMP/PKG signalling, increased Ca 2+ and tyrosine-phosphorylated proteins, promoted hyperactivation and induced the acrosome reaction, which ultimately facilitated sperm capacitation., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Natriuretic peptide type C (NPPC) and its receptor, natriuretic peptide receptor 2 (NPR2), have essential roles in maintaining meiotic arrest of oocytes in several mammalian species. However, it is not known if a similar mechanism exists in non-mammalian vertebrates. Using zebrafish as a model, we show that Nppc is expressed in ovarian follicle cells, whereas Npr2 is mainly detected in oocytes. Treatment of intact and defolliculated oocytes with 100 nM NPPC for 6 h caused a large increase in cGMP concentrations, and a significant decrease in oocyte maturation (OM), an effect that was mimicked by treatment with 8-Br-cGMP. Treatment with E2 and G-1, the specific GPER agonist, also increased cGMP levels. Cyclic AMP levels were also increased by treatments with 8-Br-cGMP, E2 and G1. The estrogen upregulation of cAMP levels was blocked by co-treatment with AG1478, an inhibitor of EGFR activation. Gene expression of npr2, but not nppc, was significantly upregulated in intact oocytes by 6 h treatments with 20 nM E2 and G-1. Both cilostamide, a phosphodiesterase 3 (PDE3) inhibitor, and rolipram, a PDE4 inhibitor, significantly decreased OM of intact and defolliculated oocytes, and enhanced the inhibitory effects of E2 and G-1 on OM. These findings indicate the presence of a Nppc/Npr2/cGMP pathway maintaining meiotic arrest in zebrafish oocytes that is upregulated by estrogen activation of Gper. Collectively, the results suggest that Nppc through Npr2 cooperates with E2 through Gper in upregulation of cGMP levels to inhibit phosphodiesterase activity resulting in maintenance of oocyte meiotic arrest in zebrafish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

C-type natriuretic peptide (CNP) is regarded as a local, paracrine hormone to regulate vascular tone and cell proliferation. Although several in vivo studies have documented that CNP exerts the inhibitory effects on mesangial cells (MCs) proliferation and collagen production, a limited number of studies exist about the resistance of CNP to MCs proliferation in vitro. Besides, whether its receptor signaling and neutral endopeptidase (NEP) are involved remains unclear. In the present study, human MCs were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 hours, respectively. CNP administration significantly suppresses MCs proliferation and collagen-IV (Col-IV) expression in a time-dependent and dose-dependent manner. As a down-stream signal molecule of CNP activation, the expressions of natriuretic peptide receptor (NPR)-B, cyclic guanosine monophosphate-dependent protein kinases II and NPR-C were obviously augmented, whereas NEP expression was significantly decreased after CNP treatment. In conclusion, receptor signaling and NEP are involved in the resistance of CNP to human mesangial proliferation and Col-IV expression., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching&mdash;the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system&mdash;is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase I&alpha; (cGKI&alpha;). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). Recently, we showed the implication of enhanced expression of Gqα and PLCβ1 proteins in hypertrophy of VSMCs from 16-week-old spontaneously hypertensive rats (SHR). The aim of this study was to investigate whether C-ANP 4-23 , a natriuretic peptide receptor-C (NPR-C) ligand that was shown to inhibit vasoactive peptide-induced enhanced protein synthesis in A10 VSMC could also attenuate hypertrophy of VSMC isolated from rat model of cardiac hypertrophy and to further explore the possible involvement of Gqα/PLCβ1 proteins and ROS-mediated signaling in this effect. The protein synthesis and cell volume, markers of hypertrophy were significantly enhanced in VSMC from 16-week-old SHR compared with age-matched WKY rats and C-ANP 4-23 treatment attenuated both to WKY levels. In addition, C-ANP 4-23 treatment also attenuated the enhanced expression of AT1 receptor, Gqα, PLCβ1, Nox4, and p47 phox proteins, the enhanced activation of EGFR, PDGFR, IGF-1R, enhanced phosphorylation of ERK1/2/AKT and c-Src in VSMC from SHR. Furthermore, the enhanced levels of superoxide anion and NADPH oxidase activity exhibited by VSMC from SHR were also attenuated to control levels by C-ANP 4-23 treatment. These results indicate that C-ANP 4-23 via the activation of NPR-C attenuates VSMC hypertrophy through decreasing the overexpression of Gqα/PLCβ1 proteins, enhanced oxidative stress, increased activation of growth factor receptors, and enhanced phosphorylation of MAPK/AKT signaling pathways. Thus, it can be suggested that C-ANP 4-23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis., (© 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

In addition to controlling blood pressure, cardiac natriuretic peptides (NPs) can stimulate lipolysis in adipocytes and promote the "browning" of white adipose tissue. NPs may also increase the oxidative capacity of skeletal muscle. To unravel the contribution of NP-stimulated metabolism in adipose tissue compared to that in muscle in vivo, we generated mice with tissue-specific deletion of the NP clearance receptor, NPRC, in adipose tissue ( Nprc AKO ) or in skeletal muscle ( Nprc MKO ). We showed that, similar to Nprc null mice, Nprc AKO mice, but not Nprc MKO mice, were resistant to obesity induced by a high-fat diet. Nprc AKO mice exhibited increased energy expenditure, improved insulin sensitivity, and increased glucose uptake into brown fat. These mice were also protected from diet-induced hepatic steatosis and visceral fat inflammation. These findings support the conclusion that NPRC in adipose tissue is a critical regulator of energy metabolism and suggest that inhibiting this receptor may be an important avenue to explore for combating metabolic disease., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. Via its receptor, guanylyl cyclase-A (GC-A), ANP maintains cardiovascular homeostasis by exerting diuretic, natriuretic, and hypotensive effects mediated, in part, by endothelial cells. Both in vivo and in vitro , ANP enhances endothelial barrier function by reducing RhoA activity and reorganizing the actin cytoskeleton. We established mouse endothelial cells that stably express GC-A and used them to analyze the molecular mechanisms responsible for actin reorganization. Stimulation by ANP resulted in phosphorylation of myosin light chain (MLC) and promotion of cell spreading. p21-activated kinase 4 (PAK4) and cerebral cavernous malformations 2 (CCM2), a scaffold protein involved in a cerebrovascular disease, were required for the phosphorylation of MLC and promotion of cell spreading by ANP. Finally, in addition to the GC domain, the kinase homology domain of GC-A was also required for ANP/GC-A signaling. Our results indicate that CCM2 and PAK4 are important downstream mediators of ANP/GC-A signaling involved in cell spreading, an important initial step in the enhancement of endothelial barrier function., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

It is well known that natriuretic peptides (NPs) are involved in the regulation of gastrointestinal motility. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal motility and gastrointestinal dyskinesia is one of the important digestive tract symptoms of depression. However, it is unclear whether they are involved in depression-induced loss of ICC. The aim of the present study was to investigate the relationship between the natriuretic peptide signaling pathway and depression-induced loss of gastric ICC in depressed rats. These results showed that the expression of c-kit and stem cell factor (SCF) in smooth muscle layers of stomach were down-regulated in depressed rats at the mRNA and protein levels. The expression of natriuretic peptide receptor (NPR)-A, B and C were up-regulated in the stomach of depressed rats at the mRNA and protein levels. NPR-A, B and C can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells (GSMCs) obtained from normal rats with different concentrations of C-type natriuretic peptide (CNP). Pretreatment of cultured GSMCs with 8-Brom-cGMP (8-Br-cGMP, a membrane permeable cGMP analog), cANF (a specific NPR-C agonist) and CNP (10-6 mol/L) demonstrated that 8-Br-cGMP had a similar effect as CNP, but treatment with cANF did not. The results of the methyl thiazolyl tetrazolium bromide (MTT) assay indicated that high concentrations of cANF (10-6 mol/L) restrained the proliferation of cultured GSMCs. Taken together, these results indicate that the up-regulation of the NPs/NPR-C and NPs/NPR-A, B/cGMP signaling pathways may be involved in depression-induced loss of gastric ICC.

Circulating natriuretic peptide (NP) levels are reduced in obesity and predict the risk of type 2 diabetes (T2D). Since skeletal muscle was recently shown as a key target tissue of NP, we aimed to investigate muscle NP receptor (NPR) expression in the context of obesity and T2D. Muscle NPRA correlated positively with whole-body insulin sensitivity in humans and was strikingly downregulated in obese subjects and recovered in response to diet-induced weight loss. In addition, muscle NP clearance receptor (NPRC) increased in individuals with impaired glucose tolerance and T2D. Similar results were found in obese diabetic mice. Although no acute effect of brain NP (BNP) on insulin sensitivity was observed in lean mice, chronic BNP infusion improved blood glucose control and insulin sensitivity in skeletal muscle of obese and diabetic mice. This occurred in parallel with a reduced lipotoxic pressure in skeletal muscle due to an upregulation of lipid oxidative capacity. In addition, chronic NP treatment in human primary myotubes increased lipid oxidation in a PGC1α-dependent manner and reduced palmitate-induced lipotoxicity. Collectively, our data show that activation of NPRA signaling in skeletal muscle is important for the maintenance of long-term insulin sensitivity and has the potential to treat obesity-related metabolic disorders., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

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.

The epidermal growth factor (EGF) network, induced by luteinizing hormone (LH), plays an essential role during the regulation of oocyte maturation, cumulus expansion, and ovulation. Binding of brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) to natriuretic receptor 2 (NPR2) generates cyclic guanosine monophosphate (cGMP), a key inhibitor that sustains porcine oocyte meiotic arrest. This correlation suggests that LH interacts with natriuretic-peptide signaling, possibly via the EGF network, to promote porcine meiotic resumption. In testing this hypothesis, we found that the majority of porcine oocytes remain arrested in the germinal-vesicle stage after 44 hr of co-culturing cumulus-oocyte complexes with 10(7) granulosa cells, which secreted active BNP and CNP; these natriuretic peptides associate with NPR2 on cumulus cells, thereby inhibiting porcine oocyte maturation. This inhibitory effect of BNP and CNP was relieved by EGF-like growth factors, whose expression naturally increases in granulosa cells 18 hr after human chorionic gonadotropin injection. LH and the EGF-like peptide amphiregulin (AREG) decreased BNP and CNP production in granulosa cells and down-regulated NPR2 expression in cumulus cells, which together decreased oocyte cGMP to levels that permit meiotic resumption. The effects of AREG on the gene expression of natriuretic-peptide signaling components and on oocyte maturation were completely blocked by the EGF receptor kinase inhibitor AG1478; the effect of LH, however, was only partially reversed by AG1478. Based on these results, LH regulates natriuretic-peptide signaling, although other pathways also cooperate with the EGF network to induce porcine oocyte maturation., (© 2014 Wiley Periodicals, Inc.)

Aims: C-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery., Main Methods: Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry., Key Findings: CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas., Significance: These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.

Background: A recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science 340:968-971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb-/- mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims., Findings: We were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia., Conclusions: Accumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified.

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca(2+)](i) increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca(2+) levels. This pathway involves the activation of Ca(2+)-permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca(2+) channels and ultimately increases myocyte Ca(2)(+)(i) levels. These observations reveal a dual role of the ANP/GC-A-signaling pathway in the regulation of cardiac myocyte Ca(2+)(i) homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca(2+)(i)-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca(2+)](i) might increase the propensity to cardiac hypertrophy and arrhythmias.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Oligodendrocyte precursor cells (OPCs) express NMDA receptors (NMDARs) and form synapses with glutamatergic neurons throughout the CNS. Although glutamate influences the proliferation and maturation of these progenitors in vitro, the role of NMDAR signaling in oligodendrogenesis and myelination in vivo is not known. Here, we investigated the consequences of genetically deleting the obligatory NMDAR subunit NR1 from OPCs and their oligodendrocyte progeny in the CNS of developing and mature mice. NMDAR-deficient OPCs proliferated normally, achieved appropriate densities in gray and white matter, and differentiated to form major white matter tracts without delay. OPCs also retained their characteristic physiological and morphological properties in the absence of NMDAR signaling and were able to form synapses with glutamatergic axons. However, expression of calcium-permeable AMPA receptors (AMPARs) was enhanced in NMDAR-deficient OPCs. These results suggest that NMDAR signaling is not used to control OPC development but to regulate AMPAR-dependent signaling with surrounding axons, pointing to additional functions for these ubiquitous glial cells.

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G(s)α to C2 and the ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity., (© 2011 The Authors Journal compilation © 2011 FEBS.)

B-type natriuretic peptide (BNP) has been known to be secreted from cardiac myocytes and activate its receptor, natriuretic peptide receptor-A (NPR-A), to reduce ventricular fibrosis. However, the function of BNP/NPR-A pathway in the somatic sensory system has been unknown. In the present study, we report a novel function of BNP in pain modulation. Using microarray and immunoblot analyses, we found that BNP and NPR-A were expressed in the dorsal root ganglion (DRG) of rats and upregulated after intraplantar injection of complete Freund's adjuvant (CFA). Immunohistochemistry showed that BNP was expressed in calcitonin gene-related peptide (CGRP)-containing small neurons and IB4 (isolectin B4)-positive neurons, whereas NPR-A was present in CGRP-containing neurons. Application of BNP reduced the firing frequency of small DRG neurons in the presence of glutamate through opening large-conductance Ca2+-activated K+ channels (BKCa channels). Furthermore, intrathecal injection of BNP yielded inhibitory effects on formalin-induced flinching behavior and CFA-induced thermal hyperalgesia in rats. Blockade of BNP signaling by BNP antibodies or cGMP-dependent protein kinase (PKG) inhibitor KT5823 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester] impaired the recovery from CFA-induced thermal hyperalgesia. Thus, BNP negatively regulates nociceptive transmission through presynaptic receptor NPR-A, and activation of the BNP/NPR-A/PKG/BKCa channel pathway in nociceptive afferent neurons could be a potential strategy for inflammatory pain therapy.

Rationale: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized., Objective: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca(2+) channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling., Methods and Results: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca(2+) influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II-induced cardiac hypertrophy in mice., Conclusions: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC's (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (alpha/beta) beta1 isoform of the beta subunit of NOGC (NOGCbeta1) was specifically focused. NOGCbeta1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCbeta1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCbeta1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 +/- 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 +/- 3.4 vs. 1.2 +/- 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCbeta1 abundance was reduced using specific siRNA to NOGCbeta1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCbeta1 (530.2 +/- 141.4 vs. 26.1 +/- 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCbeta1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 +/- 22 vs. 92 +/- 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCbeta1 and GC-A interact and that NOGCbeta1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.

Aim: To investigate atrial natriuretic peptide (ANP) secretion from gastric mucosa and the relationship between the ANP/natriuretic peptide receptor type A (NPR-A) pathway and diabetic gastroparesis., Methods: Male imprinting control region (ICR) mice (4 wk old) were divided into two groups: control mice, and streptozotocin-induced diabetic mice. Eight weeks after injection, spontaneous gastric contraction was recorded by using physiography in control and streptozotocin-induced diabetic mice. The ANP-positive cells in gastric mucosa and among dispersed gastric epithelial cells were detected by using immunohistochemistry and flow cytometry, respectively. ANP and natriuretic peptide receptor type A (NPR-A) gene expression in gastric tissue was observed by using the reverse transcriptase polymerase chain reaction., Results: The frequency of spontaneous gastric contraction was reduced from 12.9 +/- 0.8 cycles/min in the control group to 8.4 +/- 0.6 cycles/min in the diabetic mice (n = 8, P < 0.05). However, the amplitude of contraction was not significantly affected in the diabetic group. The depletion of interstitial cells of Cajal in the gastric muscle layer was observed in the diabetic mice. ANP-positive cells were distributed in the gastric mucosal layer and the density index of ANP-positive cells was increased from 20.9 +/- 2.2 cells/field in control mice to 51.8 +/- 2.9 cells/field in diabetic mice (n = 8, P < 0.05). The percentage of ANP-positive cells among the dispersed gastric epithelial cells was increased from 10.0% +/- 0.9% in the control mice to 41.2% +/- 1.0% in the diabetic mice (n = 3, P < 0.05). ANP and NPR-A genes were both expressed in mouse stomach, and the expression was significantly increased in the diabetic mice., Conclusion: These results suggest that the ANP/NPR-A signaling pathway is upregulated in streptozotocin-induced diabetic mice, and contributes to the development of diabetic gastroparesis.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC), like the other members of the membrane guanylate cyclase family, is a single transmembrane-spanning protein. The transmembrane domain separates the protein into two regions, extracellular and intracellular. The extracellular region contains the ANF-binding domain and the intracellular region the catalytic domain located at the C-terminus of the protein. Preceding the catalytic domain, the intracellular region is comprised of the following functional domains: juxtaposed 40 amino acids to the transmembrane domain is the ATP-regulated module (ARM) domain [also termed the kinase homology domain (KHD)], and the putative dimerization domain. The ANF-RGC signaling is initiated by hormone, ANF, binding to its extracellular binding site. The binding signal is transduced through the transmembrane domain to the intracellular portion where ATP binding to the ARM domain partially activates the cyclase and prepares it for subsequent steps involving phosphorylation and attaining the fully activated state. This chapter reviews the signaling modules of ANF-RGC.

The adult differentiated insulin-secreting pancreatic islet beta-cell experiences slow growth. This study shows that atrial natriuretic peptide (ANP) stimulates cell proliferation and [(3)H]thymidine incorporation in INS-1E glucose-sensitive rat beta-cell line cells and isolated rat islet DNA. In addition, cGMP, the second messenger of natriuretic peptide receptors (NPR) A and B, stimulated islet DNA biosynthesis. The NPR-A receptor was expressed in INS-1E cells and islets. ANP-stimulated INS-1E cell DNA biosynthesis was blocked by preincubation with LY294002 (50 microM), an inhibitor of phosphatidylinositol 3'-kinase (PI3K). An indicator of cell cycle progression, cyclin D2 mRNA was increased by 2- to 3-fold in ANP- or 8-Br-cGMP-treated INS-1E cells and islets, and these responses were inhibited by LY294002. ANP and 8-Br-cGMP stimulated the phosphorylation of Akt and Foxo1a in INS-1E cells and islets, and LY294002 inhibited these responses. In contrast, ANP reduced the levels of phospho-ERK in INS-1E cells. Pancreas duodenum homeobox-1 (PDX-1) is essential for pancreas development, insulin production, and glucose homeostasis, and ANP increased PDX-1 mRNA levels by 2- to 3-fold in INS-1E cells and islets. The levels of glucokinase mRNA in islets and INS-1E cells were also increased in response to ANP. The evidence suggests that pancreatic beta-cell NPR-A stimulation results in activation of a growth-promoting signaling pathway that includes PI3K/Akt/Foxo1a/cyclin D2. These data support the conclusion that the activation of Akt by ANP or 8-Br-cGMP promotes cyclin D2, PDX-1, and glucokinase transcription by phosphorylating and restricting Foxo1a activity.

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.

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM's structural motif, (669)WTAPELL(675). This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the (669)WTAPELL(675) motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.

Objective: Recent studies have suggested the abundant expression of natriuretic peptide receptor in adipose tissue. This study was designed to investigate the levels of natriuretic receptor-A (NPR-A) and NPR-C gene expression during the process of preadipocyte differentiation and its role in adipogenesis and lipid metabolism., Methods: We measured mRNA levels of NPR-A and NPR-C during the process of rat preadipocyte differentiation in vitro. We also measured the effects of ANP and C-ANP, a ligand for NPR-C, on preadipocyte differentiation. In addition, we assessed the effects of ANP and C-ANP on lipolysis and the cellular mechanism., Results: The mRNA levels of NPR-A and NPR-C on day 3, 6, 10 are (-26%, +226%), (+6%, +568%), and (+207%, +3232%) respectively as compared with day 1. ANP (10(-)(7) M) and 8-bromo-cGMP (10(-)(4) M) significantly increased Oil Red positive area and cell number of matured-adipocytes. ANP and 8-bromo-cGMP also increased the mRNA levels of adipocyte-related genes such as PPARgamma, leptin, and adiponectin on day 3, whereas C-ANP did not change these parameters. ANP (10(-)(9)-10(-)(6) M) increased intracellular cGMP levels and promoted lipolysis in adipocytes and the effects were abolished by HS-142-1, and KT5823. Conversely C-ANP (10(-)(6) M) decreased intracellular cAMP levels and lipolysis and its effect was inhibited by PTX., Conclusion: Results suggest that ANP may promote adipocyte differentiation and lipolysis via the NPR-A/cGMP/PKG pathway. Direct action of ANP via NPR-C in adipogenesis may be either absent or barely present, but ANP may play a counter regulatory role in lipolysis via NPR-C/Gi pathway.

Isatin is an endogenous indole, which is increased in mammalian brain and peripheral tissues under conditions of stress. Physiological concentrations of isatin inhibit natriuretic peptide (NPR) receptor binding and NPR-dependent signalling. The inhibition of NPR signalling by isatin is attenuated by a nonhydrolyzable ATP analogue. In this study we have demonstrated that short term incubation of rat brain synaptosomes with a physiological concentration of isatin caused a rapid 3-fold accumulation of ATP. The additional increase of ATP in the presence of tyrphostin, an inhibitor of tyrosine kinase, which uses ATP for phosphorylation of some proteins, suggests the dependence of this phenomenon on the activity of ATP-consuming systems. ATP inhibited binding of [3H]isatin to both particulate and soluble fractions of the rat brain. These results suggest that isatin induces accumulation of ATP, which in turn may displace isatin from both membrane-bound and soluble binding sites. Since natriuretic peptides are known to decrease stress hormone release this regulatory loop may be involved in the maintenance of natriuretic peptide signalling under conditions of stress and thus contribute to the control of stress responses.

During the transition at birth to air breathing, regulation of surfactant release from alveolar type II (ATII) cells is critical. Atrial natriuretic peptide (ANP) stimulates natriuretic peptide receptor-A (NPR-A) and increases intracellular cGMP. We examined the changes in ANP and NPR-A in respiratory epithelium during the perinatal period using immunohistochemistry and studied the effect of ANP on surfactant release from ATII cells isolated from fetal and newborn lambs. NPR-A mRNA was detected in the fetal lung by Northern Blot and RT-PCR. At 100 d gestation (term 145 d), ANP staining was absent and NPR-A staining was weak in cuboidal epithelial cells. ANP and NPR-A staining was prominent in ATII cells at 136 d gestation and was undetectable postnatally. ANP stimulated (maximal effect at 10(-10)M) surfactant release from both late gestation fetal and neonatal ATII cells. Protein kinase G inhibition significantly blocked this release. We conclude that ANP stimulates surfactant release in isolated perinatal ATII cells by a cGMP-dependent mechanism. ANP and NPR-A expression in ATII cells is greatest in late gestation and declines sharply postnatally. We speculate that increased activity of the ANP/NPR-A pathway in late gestation may prime the surfactant system, preparing the lung for air breathing.

Natriuretic peptides (NP) represent a family of structurally homologous but genetically distinct peptide hormones involved in regulation of fluid and electrolyte balance, blood pressure, fat metabolism, cell proliferation, and long bone growth. Recent work suggests a role for natriuretic peptide receptor B (NPR-B) signaling in regulation of cardiac growth by either a direct effect on cardiomyocytes or by modulation of other signaling pathways including the autonomic nervous system. The research links NPR-B for the first time to a cardiac phenotype in vivo and underlines the importance of the NP in the cardiovascular system. This manuscript will focus on the role of NPR-B and its ligand C-type natriuretic peptide in cardiovascular physiology and disease and will evaluate these new findings in the context of the known function of this receptor, with a perspective on how future research might further elucidate NPR-B function.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single transmembrane spanning modular protein. It binds ANF to its extracellular module and activates its intracellular catalytic module located at its carboxyl end. This results in the accelerated production of cyclic GMP, which acts as a critical second messenger in decreasing blood pressure. Two mechanistic models have been proposed for the ANF signaling of ANF-RGC. One is ATP-dependent and the other ATP-independent. In the former, ATP works through the ARM (ATP-regulated transduction module) of ANF-RGC. This model has recently been challenged [Antos et al. (2005) J Biol Chem 280:26928-26932] in support of the ATP-independent model. The present in-depth study analyzes the major principles of this challenge and concludes that the challenge lacks merit. The study then moves on to dissect the ATP mechanism of ANF signaling of ANF-RGC. It shows that the ATP photoaffinity probe, [gamma(32)P]-8-azido-ATP, reacts with Cys(634) residue in the ATP-binding pocket of ARM, and also signals the ANF-dependent activation of ANF-RGC. The target site of the 8-azido (nitrene) group is between the Cys(634) and Val(635) bond of the ATP-binding pocket. Thus, the study experimentally validates the ARM model-predicted role of Val(635) in the folding pattern of the ATP-binding pocket. And, it also identifies another residue Cys(634) that along with eight already identified residues is a part of the fold around the adenine ring of the ATP pocket. This information establishes the direct role of ATP in ANF signal transduction model of ANF-RGC, and provides a significant advancement on the mechanism by which the ATP-dependent transduction model operates.

Dopamine (DA) and atrial natriuretic factor (ANF) share a number of physiological effects. We hypothesized that ANF and the renal dopaminergic system could interact and enhance the natriuretic and diuretic effects of the peptide. We have previously reported that the ANF-stimulated DA uptake in renal tubular cells is mediated by the natriuretic peptide type-A receptor (NPR-A). Our aim was to investigate the signaling pathways that mediate ANF effects on renal 3H-DA uptake. Methylene blue (10 microM), an unspecific inhibitor of guanylate cyclase (GC), blunted ANF elicited increase of DA uptake. ODQ (10 microM) a specific inhibitor of soluble GC, did not modify DA uptake and did not reverse ANF-induced increase of DA uptake; then the participation of nitric oxide-dependent pathways must be discarded. The second messenger was the cGMP since the analogous 125 microM 8-Br-cGMP mimicked ANF effects. The specific inhibitor of the protein kinase G (PKG), KT 5823 (1 microM) blocked ANF effects indicating that PKG is involved. We examined if ANF effects on DA uptake were able to modify Na+, K+ -adenosine triphosphatase (Na+, K+ -ATPase) activity. The experiments were designed by means of inhibition of renal DA synthesis by carbidopa and neuronal DA uptake blocked by nomifensine. In these conditions renal Na+, K+ -ATPase activity was increased, in agreement with the decrease of DA availability. When in similar conditions, exogenous DA was added to the incubation medium, the activity of the enzyme tended to decrease, following to the restored availability of DA. The addition of ANF alone had similar effects to the addition of DA on the sodium pump, but when both were added together, the activity of Na(+), K(+)-ATPase was decreased. Moreover, the extraneuronal uptake blocker, hydrocortisone, inhibited the latter effect. In conclusion, ANF stimulates extraneuronal DA uptake in external cortex tissues by activation of NPR-A receptors coupled to GC and it signals through cGMP as second messenger and PKG. Dopamine and ANF may achieve their effects through a common pathway that involves reversible deactivation of renal tubular Na+, K+ -ATPase activity. This mechanism demonstrates a DA-ANF relationship involved in the modulation of both decreased sodium reabsorption and increased natriuresis.

Atrial natriuretic peptide (ANP) and the closely-related peptides BNP and CNP are highly conserved cardiovascular hormones. They bind to single transmembrane-spanning receptors, triggering receptor-intrinsic guanylyl cyclase activity. The "truncated" type-C natriuretic peptide receptor (NPR-C) has long been called a clearance receptor because it lacks the intracellular guanylyl cyclase domain, though data suggest it might negatively couple to adenylyl cyclase via G(i). Here we report the molecular cloning and characterization of the Xenopus laevis type-C natriuretic peptide receptor (XNPR-C). Analysis confirms the presence of a short intracellular C-terminus, as well as a high similarity to fish and mammalian NPR-C. Injection of XNPR-C mRNA into Xenopus oocytes resulted in expression of high affinity [(125)I]ANP binding sites that were competitively and completely displaced by natriuretic analogs and the unrelated neuropeptide vasoactive intestinal peptide (VIP). Measurement of cAMP levels in mRNA-injected oocytes revealed that XNPR-C is negatively coupled to adenylyl cyclase in a pertussis toxin-sensitive manner. When XNPR-C was co-expressed with PAC(1) receptors for pituitary adenylyl cyclase-activating polypeptide (PACAP), VIP and natriuretic peptides counteracted the cAMP induction by PACAP. These results suggest that VIP and natriuretic peptides can potentially modulate the action of PACAP in cells where these receptors are co-expressed.

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.

Atrial natriuretic peptide (ANP) and brain (B-type) natriuretic peptide (BNP) are circulating hormones of cardiac origin that play an important role in the regulation of intravascular blood volume and vascular tone. The plasma concentrations of ANP and BNP are elevated in heart failure, and they are considered to compensate for heart failure because of their diuretic, natriuretic, and vasodilating actions and inhibitory effects on renin and aldosterone secretion. Evidence is also accumulating from recent work that ANP and BNP exert their cardioprotective functions not only as circulating hormones but also as local autocrine and/or paracrine factors. In studies using cultured neonatal myocytes and fibroblasts, exogenous administration of both ANP and ANP antagonists demonstrated that ANP has antihypertrophic and antifibrotic functions. Corroborating these in vitro results, mice lacking natriuretic receptor-A (NPR-A), the receptor for ANP and BNP, develop cardiac hypertrophy and fibrosis independent of their blood pressure. Recent studies also suggest that the intracardiac natriuretic peptides/cGMP system plays a counter-regulatory role against the intracardiac renin-angiotensin-aldosterone system and TGF-beta mediated pathway. In a clinical setting, human recombinant ANP and BNP may be used for a therapy of heart failure; however, further evaluation is required in the future.

Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own and as enhancers for the endogenous agonists in both increasing agonist potency and providing additive efficacy-superagonism. The additive efficacy can also be observed with agonists, which are neutral or even negative modulators of the potency of the endogenous ligand. Based on the prevailing dimeric concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but-importantly-in the "other" or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric modulators can act through binding in the "other" protomer. It is suggested that for the in vivo, clinical setting a positive ago-allosteric modulator should be the preferred agonist drug.