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.     

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.     

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.     

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.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

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.     

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.     

Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation

Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field.     

Cardiac and intestinal natriuretic peptides: insights from genetically modified mice

Since the original discovery of atrial natriuretic peptide (ANP) more than two decades ago, the application of gene targeting technology in mice has provided new insights into the diverse physiological functions of natriuretic peptides and their membrane guanylyl cyclase (GC) receptors. Disruption of the genes for ANP or its receptor, GC-A, demonstrated that this system is not only essential for the maintenance of normal blood pressure and volume, but in addition exerts local antihypertrophic effects in the heart. Disruption of the genes encoding B-type (BNP) or C-type natriuretic peptides (CNP) or the CNP-receptor, GC-B, demonstrated that these "natriuretic" peptides are in fact unlikely to physiologically regulate renal sodium excretion but instead exert important autocrine/paracrine cGMP-mediated effects on cellular proliferation and differentiation in various tissues. Notably, the intestinal peptide uroguanylin, which activates a third guanylyl cyclase receptor (GC-C), exerts diuretic/natriuretic activity and links the intestine and kidney in an endocrine way to modulate renal function in response to oral salt load. Reviewed here is the physiology of cardiac and intestinal natriuretic peptides and their guanylyl cyclase receptors, with special focus on the information gained to date from genetically modified mice.     

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.     

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.     

HS-142-1, a novel antagonist for natriuretic peptides,has no effect on the third member of membrane bound guanylate cyclases (GC-C) in T84 cells

HS-142-1, a novel non-peptide antagonist for natriuretic peptide, exerts antagonistic actions almost equally on two similar guanylate cyclase-linked natriuretic peptide receptors (GC-A and GC-B), but has little or no effect on the binding of natriuretic peptides to a membrane protein, the so-called “clearance receptor”, which binds all natriuretic peptides. The third mammalian form of membrane bound guanylate cyclases (GC-C) was identified not as a natriuretic peptide receptor, but as a receptor for heat-stable enterotoxins (STa). In this study, we examined effects of HS-142-1 on GC-C (STaR) in T84 cells and showed that HS-142-1 exerts neither agonistic nor antagonistic activity for GC-C, indicating that HS-142-1 is not a common antagonist for a family of membrane bound guanylate cyclase receptors, but a specific antagonist for the guanylate cyclase-linked natriuretic peptide receptors.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

Rationale

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

Objective

We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.

Methods and Results

Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.

Conclusion

These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natriuretic peptide receptors in cultured rat diencephalon

To characterize the type of cell expressing natriuretic peptide receptors in the brain and the nature of these receptors, we conducted studies in primary cultured glial and neuronal cells derived from fetal rat diencephalon. The glial predominant cultures (95% of total cells and glial fibrillary acidic protein positive) expressed nearly a 10-fold greater specific binding of the natriuretic peptides to cell surface receptors compared with the neuron-predominant cultures. Scatchard analysis of binding studies with 125I-atrial natriuretic peptide (ANP) and 125I-brain natriuretic peptide (BNP) revealed a single class of receptors with dissimilar affinities (0.25 +/- 0.09 and 0.74 +/- 0.07 nM, respectively, n = 3 experiments p less than 0.01) but similar numbers of binding sites for both peptides (93 and 88 fmol/mg of protein, respectively). Cross-linking of 125I-ANP and BNP to cultured glia followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography identified distinct bands at either approximate Mr 130,000, or 102,000 and 66,000, corresponding to two high molecular weight (B) receptors and one low molecular weight (C) receptor described in other tissues. Different subtypes of astrocytes appeared to express different B receptors. Binding and cross-linking of radiolabeled ANP or BNP were competitively inhibited equally by unlabeled ANP or BNP, indicating that ANP and BNP probably bind the same receptors. The glial cultures functionally expressed a receptor(s) with guanylate cyclase activity; BNP was less potent than ANP in stimulating cGMP at lower concentrations. These results indicate that both high and low molecular weight natriuretic peptide receptors are expressed in astrocyte-predominant cultures from the fetal diencephalon and suggest that glia participate in several actions of ANP which are probably mediated through this area of the brain.     

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.