Stable expression of natriuretic peptide receptors: effects of HS-142-1, a non-peptide ANP antagonist.

We established clonal cell lines stably expressing each of two subtypes of membrane bound guanylate cyclases (GC-A and GC-B), which are known as natriuretic peptide receptors. Using these cell lines, we showed that GC-A is an ANP/BNP receptor, whereas GC-B is a specific receptor for CNP. Effects of HS-142-1, a novel non-peptide ANP antagonist, on GC-A and GC-B were examined by using these cells. In cells expressing either GC-A or GC-B, HS-142-1 inhibited cGMP production elicited by ANP or CNP with IC50 values of 1.8 micrograms/ml and 1.5 micrograms/ml, respectively, and also competitively blocked specific binding of the natriuretic peptides with IC50 values of 2.2 micrograms/ml and 3.3 micrograms/ml, respectively. These results indicate that HS-142-1 is a potent antagonist of CNP as well as ANP. We also showed that CNP suppressed the growth of cells expressing GC-B by 22% and that HS-142-1 blocked the antiproliferative action of CNP.     

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

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.     

Cloning and characterization of two forms of C-type natriuretic peptide receptor in rat brain.

Two similar membrane bound guanylate cyclases (GC-A and GC-B) are known as natriuretic peptide receptors, but have not been well characterized yet. In this study, we have isolated two forms of GC-B cDNA clones along with GC-A cDNA clones from rat brain. The two forms of rat GC-B differ from each other only by 75bp deletion at 3'-flanking region of the putative transmembrane domain, the shorter form lacking the nucleotide binding site by the deletion. Expression of these cDNAs on mammalian cells revealed that (1) GC-B is a specific receptor for CNP whereas GC-A is stimulated effectively both by ANP and BNP, and (2) the two forms of GC-B possess practically the same high binding affinity for CNP while the shorter form could not induce cGMP production by the binding of CNP. These data indicate that in rat brain is present the non-functional receptor for CNP caused by the short deletion.     

C-type natriuretic peptide and guanylyl cyclase B receptor

Guanylyl cyclases (GC) are widely distributed enzymes that signal via the production of the second messenger cGMP. The particulate guanylyl cyclases share a similar topology: an extracellular ligand binding domain and intracellular regulatory kinase-homology and cyclase catalytic domains. The natriuretic peptide receptors GC-A and -B mediate the effects of a family of peptides, atrial, B- and C-type natriuretic peptide (ANP, BNP and CNP, respectively), with natriuretic, diuretic and vasorelaxant properties. ANP and BNP, through the activation of GC-A, act as endocrine hormones to regulate blood pressure and volume, and inhibit cardiac hypertrophy. CNP, on the other hand, acts in an autocrine/paracrine fashion to induce vasorelaxation and vascular remodeling, and to regulate bone growth through its cognate receptor GC-B. GC-B, like GC-A, is phosphorylated in the basal state, and undergoes both homologous and heterologous desensitization, reflected by dephosphorylation of specific sites in the kinase-homology domain. This review will examine the structure and function of GC-B, and summarize the physiological processes in which this receptor is thought to participate.     

Diverging vasorelaxing effects of C-type natriuretic peptide in renal resistance arteries and aortas of GC-A-deficient mice

This study aimed to characterize the vasorelaxing effects of ANP, BNP and CNP in isolated renal resistance arteries (RRA) from wild-type mice and mice with either systemic (GC-A -/-) or smooth muscle-restricted deletion of GC-A (SMC GC-A KO). In RRA from wild-type (GC-A +/+) mice natriuretic peptides (NP) induced concentration-dependent vasorelaxations with the rank order of potency ANP>BNP>CNP. In RAA obtained from mice with systemic or smooth muscle-restricted deletion of GC-A, the effects of ANP and BNP were abolished. In contrast, CNP induced concentration-dependent vasorelaxations of GC-A -/- and SMC GC-A KO RRA. However, the efficacy of CNP for vasorelaxation was markedly diminished compared with wild-type RRA. Such changes in CNP responsiveness did not affect large arteries as the aorta and they were not due to vascular changes secondary to chronic arterial hypertension in GC-A -/- mice. Unaltered vasorelaxing effects of acetylcholine and sodium nitroprusside demonstrated unaltered function of downstream targets regulated by cGMP in vascular smooth muscle. An increased expression of the clearance receptor (NPR-C) or diminished expression of GC-B were not found to account for the differences in CNP responsiveness. In conclusion, observations in isolated aortic rings do not necessarily allow conclusions concerning the physiology of natriuretic peptides in the smaller resistance size arteries. Changes at the GC-B receptor level are likely to explain the diminished responsiveness of GC-A-deficient RRA to CNP.     

Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases

Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR-1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology.     

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

Introduction

The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.

Methods

Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF^4-23. Nitric oxide (NO) production, prostaglandin E₂ (PGE₂) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.

Results

The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE₂ release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF^4-23 had the opposite effect and reduced NO and PGE₂ release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE₂ release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE₂ release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.

Conclusions

Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.     

The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family

Atrial natriuretic peptide (ANP) binds directly to a plasma membrane form of guanylate cyclase (GC-A), stimulating the production of the second messenger cyclic GMP. We show that a second guanylate cyclase/receptor (GC-B) exists, with distinctly different specificities for various natriuretic peptides. A cDNA clone encoding GC-B was isolated by low-stringency screening of a rat brain cDNA library using GC-A cDNA as a probe. The deduced amino acid sequence of GC-B is 78% identical with GC-A within the intracellular region, but 43% identical within the extracellular domain. Cyclic GMP concentrations in cells transfected with GC-A were half-maximally elevated at 3 nM ANP, 25 nM brain natriuretic peptide (BNP), and 65 nM atriopeptin 1, while 25 microM ANP, 6 microM BNP, and greater than 100 microM atriopeptin 1 were required for half-maximal stimulation of GC-B. The potencies of natriuretic peptides on GC-A and GC-B activity are therefore markedly different; furthermore, despite the specificity of GC-B for BNP, the relatively high BNP concentration required to elicit a response suggests the possible presence of a more potent, unidentified natural ligand.     

Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptors

Natriuretic peptides are a family of hormones/paracrine factors that regulate blood pressure, cardiovascular homeostasis and bone growth. The mammalian family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). A family of three cell surface receptors mediates their physiologic effects. Two are receptor guanylyl cyclases known as NPR-A/GC-A and NPR-B/GC-B. Peptide binding to these enzymes stimulates the synthesis of the intracellular second messenger, cGMP, whereas a third receptor, NPR-C, lacks enzymatic activity and functions primarily as a clearance receptor. Here, we provide a brief review of how various desensitizing agents and/or conditions inhibit NPR-A and NPR-B by decreasing their phosphorylation state.     

Effects of natriuretic peptides (ANP, BNP, CNP) on catecholamine synthesis and TH mRNA levels in PC12 cells

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are present in adrenal chromaffin cells, and are co-secreted with catecholamines suggesting that these natriuretic peptides (NPs) may modulate functions of chromaffin cells in an autocrine and/or paracrine manner. Therefore, we investigated the effects of NPs on tyrosine hydroxylase (TH: a rate-limiting enzyme in biosynthesis of catecholamine) mRNA in rat pheochromocytoma PC12 cells. It was also determined whether the cyclic GMP/cGMP-dependent protein kinase (cGMP/PKG) pathway was involved in theses effects. Finally, we examined the effects of NPs on intracellular catecholamine content to confirm increase of catecholamine synthesis following TH mRNA induction. NPs (0.1 microM) induced significant increases of the TH mRNA (ANP= BNP> CNP). Also, the effects of NPs on TH mRNA were mimicked by 8-bromo cyclic GMP (1mM), and were blocked by KT5823 (1 microM) (inhibitor PKG) or LY83583 (1 microM) (guanylate cyclase inhibitor). Moreover, NPs were shown to induce significant increases of intracellular catecholamine contents (ANP= BNP> CNP). These findings suggest that NPs induced increases of TH mRNA through cGMP/PKG dependent mechanisms, which, in turn, resulted in stimulation of catecholamine synthesis in PC12 cells.     

Csk mediates G-protein-coupled lysophosphatidic acid receptor-induced inhibition of membrane-bound guanylyl cyclase activity

Natriuretic peptides (NPs) are involved in many physiological processes, including the regulation of vascular tone, sodium excretion, pressure-volume homeostasis, inflammatory responses, and cellular growth. The two main receptors of NP, membrane-bound guanylyl cyclases A and B (GC-A and GC-B), mediate the effects of NPs via the generation of cGMP. NP-stimulated generation of cGMP can be modulated by intracellular processes, whose exact nature remains to be elucidated. Thus, serum and lysophosphatidic acid (LPA), by unknown pathways, have been shown to inhibit the NP-induced generation of cGMP. Here we report that the nonreceptor-tyrosine-kinase Csk is an essential component of the intracellular modulation of atrial natriuretic peptide (ANP)-stimulated activation of GC-A. The genetic deletion of Csk (Csk(-)(/)(-)) in mouse embryonic fibroblasts blocked the inhibitory effect of both serum and LPA on the ANP-stimulated generation of cGMP. Moreover, using a chemical rescue approach, we also demonstrate that the catalytic activity of Csk is required for its modulatory function. Our data demonstrate that Csk is involved in the control of cGMP levels and that membrane-bound guanylyl cyclases can be critically modulated by other receptor-initiated intracellular signaling pathways.     

Atrial Natriuretic Peptide Regulates Ca2+ Channel in Early Developmental Cardiomyocytes

Background

Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca²⁺ currents has not been investigated in developmental cardiomyocytes.

Methodology/Principal Findings

We investigated the role of ANP in regulating L-type Ca²⁺ channel current (I_CaL) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I_CaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I_CaL by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I_CaL induced by ANP was not affected by either Nω, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I_CaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested.

Conclusion/Significances

Taken together, these results indicate that ANP induced depression of action potentials and I_CaL is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3′, 5′–cyclic monophophate (cAMP)–cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.     

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

Gene Expression of ANP,BNP and ET-1 in the Heart of Rats during Pulmonary Embolism

Aims

Atrial natriuretic petide (ANP), brain natriuretic peptide (BNP) and endothelin-1 (ET-1) may reflect the severity of right ventricular dysfunction (RVD) in patients with pulmonary embolism (PE). The exact nature and source of BNP, ANP and ET-1 expression and secretion following PE has not previously been studied.

Methods and Results

Polystyrene microparticles were injected to induce PE in rats. Gene expression of BNP, ANP and ET-1 were determined in the 4 cardiac chambers by quantitative real time polymerase chain reaction (QPCR). Plasma levels of ANP, BNP, ET-1 and cardiac troponin I (TNI) were measured in plasma. PE dose-dependently increased gene expression of ANP and BNP in the right ventricle (RV) and increased gene expression of ANP in the right atrium (RA). In contrast PE dose-dependently decreased BNP gene expression in both the left ventricle (LV) and the left atrium (LA). Plasma levels of BNP, TNI and ET-1 levels dose-dependently increased with the degree of PE.

Conclusion

We found a close correlation between PE degree and gene-expression of ANP, and BNP in the cardiac chambers with a selective increase in the right chambers of the heart.The present data supports the idea of natriuretic peptides as valuable biomarkers of RVD in PE.     

Characterization of VIP and PACAP receptors in cultured rat penis corpus cavernosum smooth muscle cells and their interaction with guanylate cyclase-B receptors

Penile corpus cavernosum smooth muscle relaxation can be induced by both cyclic AMP and cyclic GMP-elevating agents, but possible interactions between these two signalling pathways are still poorly understood. Using in vitro cultured rat penile corpus cavernosum smooth muscle (CCSM) cells, we have characterized the local expression and functional activities of receptors for the cAMP-elevating peptides, PACAP and VIP, and for the cGMP-elevating peptides, CNP and ANP. Stimulation of the cells with various concentrations of PACAP(-27/-38) or VIP resulted in rapid and dose-dependent increases in cyclic AMP levels. RT-PCR analyses revealed gene expression of PAC(1) and VPAC(2) but not of VPAC(1) receptors in the cells. The natriuretic peptide, CNP, and the nitric oxide donor, sodium nitroprusside, were capable of enhancing cyclic GMP formation, indicating the presence of membrane-associated in addition to soluble guanylate cyclase (sGC) activities in these cells. Findings that cyclic GMP formation was preferentially activated by CNP but not by the related peptide, ANP, were consistent with RT-PCR analyses, demonstrating gene expression of the CNP receptor, GC-B, but not of the ANP receptor, GC-A, in these cells. Prior exposure of the cells to 10(-8) M PACAP resulted in a marked down-regulation of GC-B activity, whereas sGC was not affected. These findings provide functional and molecular evidence for the presence of three receptors, PAC(1), VPAC(2) and GC-B, involved in cyclic nucleotide signalling in penile CCSM cells. The observed cross-talk of the PACAP/VIP receptors with GC-B but not with sGC may have implications for the therapy of erectile dysfunction.     

Vascular Relaxation Induced by C-Type Natriuretic Peptide Involves the Ca2+/NO-Synthase/NO Pathway

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.