ATP-independent activation of natriuretic peptide receptors

Natriuretic peptide receptor A (NPR-A) is an essential cardiovascular regulator that is stimulated by atrial natriuretic peptide and B-type natriuretic peptide, whereas natriuretic peptide receptor B (NPR-B) stimulates long bone growth in a C-type natriuretic peptide-dependent manner. Many reports indicate that ATP is essential for NPR-A and NPR-B activation. Current models suggest that natriuretic peptide binding to receptor extracellular domains causes ATP binding to intracellular kinase homology domains, which derepresses adjacent catalytic domains. Here, we report 100-fold activations of natriuretic peptide receptors in the absence of ATP. The addition of a nonhydrolyzable ATP analog had no effect at early time periods (measured in seconds) but increased cGMP production about 2-fold after longer incubations (measured in minutes), consistent with a stabilization, not activation, mechanism. These data indicate that ATP does not activate natriuretic peptide receptors as has been repeatedly reported. Instead, ATP increases activity primarily by maintaining proper receptor phosphorylation status but also serves a previously unappreciated enzyme stabilizing function.     

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.     

A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signal was weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as but more specific than the (32)PO(4) labeling method. The two approaches were highly correlated (R(2) = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levels are best detected by Western blot analysis, not SYPRO Ruby staining.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

BNP-induced activation of cGMP in human cardiac fibroblasts: interactions with fibronectin and natriuretic peptide receptors

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin (FN). FN contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the natriuretic peptide receptor A (NPR-A) sequence contains both RGD and DDX sequences. The goal of the current investigation was to determine potential interactions between FN and NPR-A on BNP induction of cGMP in cultured human cardiac fibroblasts (CFs). Further, we sought to determine whether a Mayo designed NPR-A specific RGD peptide could modify this interaction. Here we reconfirm the presence of all three natriuretic peptide receptors (NPR) in CFs. CFs plated on FN demonstrated a pronounced increase in cGMP production to BNP compared to non-coated plates. This production was also enhanced by the NPR-A specific RGD peptide, which further augmented FN associated cGMP production. Addition of HS-142-1, a NPR-A/B antagonist, abrogated the responses of BNP to both FN and the NPR-A specific RGD peptide. Finally, we defined a possible role for the NPR-C through non-cGMP mechanisms in mediating the anti-proliferative actions of BNP in CFs where the NPR-C antagonist cANF 4-28 but not HS-142-1 blocked BNP-mediated inhibition of proliferation of CFs. We conclude that NPR-A interacts with components of the ECM such as FN to enhance BNP activation of cGMP and that a small NPR-A specific RGD peptide augments this action of BNP with possible therapeutic implications. Lastly, the NPR-C may also have a role in mediating anti-proliferative actions of BNP in CFs.     

Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression

A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.     

Novel bifunctional natriuretic peptides as potential therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.     

Immunolocalization of natriuretic peptides and their receptors in goat (Capra hircus) heart

Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors.     

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

Natriuretic peptides--their receptors and role in cardiovascular system

Natriuretic peptides belong to a family of small proteins that play a major role in modulation of natriuresis, diuresis and vasodilatation. They counteract the activity of renin-angiotensin-aldosterone system. They are also involved in the regulation of homeostasis, fat metabolism and long bone growth. Natriuretic peptides family in mammals consists of three main members: atrial natriuretic peptide (ANP) - secreted by the atrial myocardium; brain natriuretic peptide (BNP)--secreted mainly by the ventricular myocardium, and C-type natriuretic peptide (CNP)--produced and released by endothelial cells. Secretion of these peptides is stimulated by atrial and ventricular distension, increased blood pressure, hypoxia or renal dysfunction. Natriuretic peptides play their roles via interactions with NPR-A and NPR-B receptors which are transmembrane guanylyl cyclases. Their local concentrations, regulated by internalization and degradation, are mediated by the NPR-C receptor and by neutral endopeptidase. The paper presents the current knowledge of structure and biological function of natriuretic peptides.     

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of ¹²⁵I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED₅₀ = 0.6 nM) than rANP (ED₅₀ = SnM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms.

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.     

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.     

Activation of protein kinase C stimulates the dephosphorylation of natriuretic peptide receptor-B at a single serine residue: a possible mechanism of heterologous desensitization

The binding of atrial natriuretic peptide and C-type natriuretic peptide (CNP) to the guanylyl cyclase-linked natriuretic peptide receptors A and B (NPR-A and -B), respectively, stimulates increases in intracellular cGMP concentrations. The vasoactive peptides vasopressin, angiotensin II, and endothelin inhibit natriuretic peptide-dependent cGMP elevations by activating protein kinase C (PKC). Recently, we identified six in vivo phosphorylation sites for NPR-A and five sites for NPR-B and demonstrated that the phosphorylation of these sites is required for ligand-dependent receptor activation. Here, we show that phorbol 12-myristate 13-acetate, a direct activator of PKC, causes the dephosphorylation and desensitization of NPR-B. In contrast to the CNP-dependent desensitization process, which results in coordinate dephosphorylation of all five sites in the receptor, phorbol 12-myristate 13-acetate treatment causes the dephosphorylation of only one site, which we have identified as Ser(523). The conversion of this residue to alanine or glutamate did not reduce the amount of mature receptor protein as indicated by detergent-dependent guanylyl cyclase activities or Western blot analysis but completely blocked the ability of PKC to induce the dephosphorylation and desensitization of NPR-B. Thus, in contrast to previous reports suggesting that PKC directly phosphorylates and inhibits guanylyl cyclase-linked natriuretic peptide receptors, we show that PKC-dependent dephosphorylation of NPR-B at Ser(523) provides a possible molecular explanation for how pressor hormones inhibit CNP signaling.     

G protein-dependent activation of smooth muscle eNOS via natriuretic peptide clearance receptor

In gastrointestinal smooth muscle, the neuropeptides vasoactiveintestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting withVIP₂/PACAP₃ receptors coupled via G_s toadenylyl cyclase and with distinct receptors coupled viaG_i1 and/orG_i2 to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptoras the single-transmembrane natriuretic peptide clearance receptor(NPR-C). RT-PCR and Northern analysis demonstrated expression of thenatriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbitgastric muscle cells. In binding studies using¹²⁵I-labeled atrial natriureticpeptide (¹²⁵I-ANP) and¹²⁵I-VIP as radioligands, VIP,ANP, and the selective NPR-C ligand cANP(4-23) bound with highaffinity to NPR-C. ANP, cANP-(4-23), and VIP initiated identicalsignaling cascades consisting ofCa²⁺ influx, activation of eNOSvia G_i1 andG_i2, stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation wereabolished (93 ± 3 to 96 ± 2% inhibition) by nifedipine,pertussis toxin, the NOS inhibitor,N^G-nitro-L-arginine,and the antagonists ANP-(1-11) and VIP-(10-28). NOS activitystimulated by all three ligands in muscle membranes was additivelyinhibited by G_i1 andG_i2 antibodies (82 ± 2 to 84 ± 1%). In reconstitution studies, VIP, cANP-(4-23), and guanosine 5'-O-(3-thiotriphosphate) stimulated NOS activity inmembranes of COS-1 cells cotransfected with NPR-C and eNOS. Theresults establish a unique mechanism for G protein-dependent activation of a constitutive NOS expressed in gastrointestinal smooth muscle involving interaction of the relaxant neuropeptides VIP and PACAP with a single-transmembrane natriuretic peptide receptor, NPR-C.

     

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

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.