Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

G(i-1)/G(i-2)-dependent signaling by single-transmembrane natriuretic peptide clearance receptor

Single-transmembrane natriuretic peptide clearance receptor (NPR-C), which is devoid of a cytoplasmic guanylyl cyclase domain, interacts with pertussis toxin (PTx)-sensitive G proteins to activate endothelial nitric oxide synthase (eNOS) expressed in gastrointestinal smooth muscle cells. We examined the ability of NPR-C to activate other effector enzymes in eNOS-deficient tenia coli smooth muscle cells; these cells expressed NPR-C and NPR-B but not NPR-A. Atrial natriuretic peptide (ANP), the selective NPR-C ligand cANP-(4-23), and vasoactive intestinal peptide (VIP) inhibited (125)I-ANP and (125)I-VIP binding to muscle membranes in a pattern indicating high-affinity binding to NPR-C. Interaction of VIP with NPR-C was confirmed by its ability to inhibit (125)I-ANP binding to membranes of NPR-C-transfected COS-1 cells. In tenia muscle cells, all ligands selectively activated G(i-1) and G(i-2); VIP also activated G(s) via VIP(2) receptors. All ligands stimulated phosphoinositide hydrolysis, which was inhibited by ANP-(1-11), PTx, and antibodies to phospholipase C-beta3 (PLC-beta3) and Gbeta. cANP-(4-23) contracted tenia muscle cells; contraction was blocked by U-73122 and PTx and by antibodies to PLC-beta3 and Gbeta in intact and permeabilized muscle cells, respectively. VIP and ANP contracted muscle cells only after inhibition of cAMP- and cGMP-dependent protein kinases. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP in a PTx-sensitive fashion. We conclude that NPR-C is coupled to activation of PLC-beta3 via betagamma-subunits of G(i-1) and G(i-2) and to inhibition of adenylyl cyclase via alpha-subunits.     

Natriuretic peptides inhibit adenylyl cyclase activity in dispersed eel gill cells

The effect of natriuretic peptides on forskolin-evoked adenylyl cyclase activity was investigated in dispersed gill cells from the Australian short-finned eel (Anguilla australis). Molecular cloning techniques were employed to identify the putative G-protein-activating motif within the intracellular domain of the eel natriuretic peptide C receptor. Eel ANP, eel CNP and the NPR-C-specific C-ANF inhibited the forskolin-stimulated production of cyclic AMP. This effect was abolished by pretreatment of cells with pertussis toxin. Eel VNP was without effect on adenylyl cyclase activity. PCR and molecular cloning indicated that the intracellular domain of A. australis NPR-C has the same amino acid sequence as Anguilla japonica. Alignment of these sequences with Rattus norvegicus NPR-C indicated conservation of the putative G-protein-activating motif BB...BBXXB (B=basic, X=nonbasic residues). These data suggest that branchially-expressed NPR-C may play a physiological role additional to that of ligand clearance.Abbreviations ANP atrial natriuretic peptide - CNP C-type natriuretic peptide - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - eANP-NH₂ amidated form of eel ANP - GC guanylyl cyclase - G_i inhibitory G-protein - IBMX isobutylmethylxanthine - NP natriuretic peptide - NPR natriuretic peptide receptor - PCR polymerase chain reaction - PTX pertussis toxin - VNP ventricular natriuretic peptideCommunicated by I.D. Hume     

Inhibition of L-type Ca2+ current by C-type natriuretic peptide in bullfrog atrial myocytes: an NPR-C-mediated effect

Single atrial myocytes were isolated from the bullfrog heart and studied under current and voltage clamp conditions to determine the electrophysiological effects of the C-type natriuretic peptide (CNP). CNP (10(-8) M) significantly shortened the action potential and reduced its peak amplitude after the application of isoproteronol (10(-7) M). In voltage clamp studies, CNP inhibited isoproteronol-stimulated L-type Ca2+ current (ICa) without any significant effect on the inward rectifier K+ current. The effects of cANF (10(-8) M), a selective agonist of the natriuretic peptide C receptor (NPR-C), were very similar to those of CNP. Moreover, HS-142-1, an antagonist of the guanylyl cyclase-linked NPR-A and NPR-B receptors did not alter the inhibitory effect of CNP on ICa. Inclusion of cAMP in the recording pipette to stimulate ICa at a point downstream from adenylyl cyclase increased ICa, but this effect was not inhibited by cANF. These results provide the first demonstration that CNP can inhibit ICa after binding to NPR-C, and suggest that this inhibition involves a decrease in adenylyl cyclase activity, which leads to reduced intracellular levels of cAMP.     

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus, were characterized using autoradiographical, molecular, and physiological techniques. Specific 125I-rat ANP binding sites were present in the carotid and pulmonary arteries, the lateral aorta, the pre- and post-cava, and the jugular vein, and generally occurred in each layer of the blood vessel. The 125I-rat ANP binding was partially displaced by the specific natriuretic peptide receptor C ligand, C-ANF, which indicates the presence of two types of natriuretic peptide receptors in the blood vessels. This was confirmed by a RT-PCR study, which demonstrated that guanylyl cyclase receptor (NPR-GC) and NPR-C mRNAs are expressed in arteries and veins. An in vitro guanylyl cyclase assay showed that frog ANP stimulated the production of cGMP in arterial membrane fractions. Physiological recordings from isolated segments of the carotid and pulmonary arteries and the lateral aorta, which had been pre-constricted with arginine vasotocin, showed that rat ANP, frog ANP and porcine CNP relaxed the vascular smooth muscle with relatively similar potency. Together, the data show that the central vasculature contains two types of natriuretic peptide receptors (NPR-C and NPR-GC) and that the vasculature is a target for ANP and CNP.     

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.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

Natriuretic peptides: a new lipolytic pathway in human fat cells

Human fat cell lipolysis was considered until recently to be an exclusive cAMP/protein-kinase A (PKA)-regulated metabolic pathway under the control of catecholamines and insulin. Moreover, exercise-induced lipid mobilization in humans was considered to mainly depend on catecholamine action and interplay between fat cell beta- and alpha2-adrenergic receptors controlling adenylyl cyclase activity and cAMP production. We have recently demonstrated that natriuretic peptides stimulate lipolysis and contribute to the regulation of lipid mobilization in humans. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) stimulate lipolysis in human isolated fat cells. Activation of the adipocyte plasma membrane type A guanylyl cyclase receptor (NPR-A), increase in intracellular guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels and activation of hormone-sensitive lipase mediate the action of ANP. ANP does not modulate cAMP production and PKA activity. Increment of cGMP induces the phosphorylation of hormone-sensitive lipase and perilipin A via the activation of a cGMP dependent protein kinase-I (cGK-I). Plasma concentrations of glycerol and non-esterified fatty acids are increased by i.v. infusion of ANP in humans. Physiological relevance of the ANP-dependent pathway was demonstrated in young subjects performing physical exercise. ANP plays a role in conjunction with catecholamines in the control of exercise-induced lipid mobilization. This pathway becomes of major importance when subjects are submitted to chronic treatment with a beta-blocker. Oral beta-adrenoceptor blockade suppresses the beta-adrenergic component of catecholamine action in fat cells and potentiates exercise-induced ANP release by the heart. These findings may have several implications whenever natriuretic peptide secretion is altered such as in subjects with left ventricular dysfunction, congestive heart failure and obesity.     

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C)

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.     

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.

     

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 is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

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.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

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.     

Natriuretic peptides in fish physiology

Natriuretic peptides exist in the fishes as a family of structurally-related isohormones including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and ventricular natriuretic peptide (VNP); to date, brain natriuretic peptide (or B-type natriuretic peptide, BNP) has not been definitively identified in the fishes. Based on nucleotide and amino acid sequence similarity, the natriuretic peptide family of isohormones may have evolved from a neuromodulatory, CNP-like brain peptide. The primary sites of synthesis for the circulating hormones are the heart and brain; additional extracardiac and extracranial sites, including the intestine, synthesize and release natriuretic peptides locally for paracrine regulation of various physiological functions. Membrane-bound, guanylyl cyclase-coupled natriuretic peptide receptors (A- and B-types) are generally implicated in mediating natriuretic peptide effects via the production of cyclic GMP as the intracellular messenger. C- and D-type natriuretic peptide receptors lacking the guanylyl cyclase domain may influence target cell function through G(i) protein-coupled inhibition of membrane adenylyl cyclase activity, and they likely also act as clearance receptors for circulating hormone. In the few systems examined using homologous or piscine reagents, differential receptor binding and tissue responsiveness to specific natriuretic peptide isohormones is demonstrated. Similar to their acute physiological effects in mammals, natriuretic peptides are vasorelaxant in all fishes examined. In contrast to mammals, where natriuretic peptides act through natriuresis and diuresis to bring about long-term reductions in blood volume and blood pressure, in fishes the primary action appears to be the extrusion of excess salt at the gills and rectal gland, and the limiting of drinking-coupled salt uptake by the alimentary system. In teleosts, both hypernatremia and hypervolemia are effective stimuli for cardiac secretion of natriuretic peptides; in the elasmobranchs, hypervolemia is the predominant physiological stimulus for secretion. Natriuretic peptides may be seawater-adapting hormones with appropriate target organs including the gills, rectal gland, kidney, and intestine, with each regulated via, predominantly, either A- or B-type (or C- or D-type?) natriuretic peptide receptors. Natriuretic peptides act both directly on ion-transporting cells of osmoregulatory tissues, and indirectly through increased vascular flow to osmoregulatory tissues, through inhibition of drinking, and through effects on other endocrine systems.     

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC(1) receptor isoform activation of specific intracellular signaling pathways.

Pituitary adenylate cyclase-activating polypeptides (PACAP) have potent regulatory and neurotrophic activities on superior cervical ganglion (SCG) sympathetic neurons with pharmacological profiles consistent for the PACAP-selective PAC(1) receptor. Multiple PAC(1) receptor isoforms are suggested to determine differential peptide potency and receptor coupling to multiple intracellular signaling pathways. The current studies examined rat SCG PAC(1) receptor splice variant expression and coupling to intracellular signaling pathways mediating PACAP-stimulated peptide release. PAC(1) receptor mRNA was localized in over 90% of SCG neurons, which correlated with the cells expressing receptor protein. The neurons expressed the PAC(1)(short)HOP1 receptor but not VIP/PACAP-nonselective VPAC(1) receptors; low VPAC(2) receptor mRNA levels were restricted to ganglionic nonneuronal cells. PACAP27 and PACAP38 potently and efficaciously stimulated both cAMP and inositol phosphate production; inhibition of phospholipase C augmented PACAP-stimulated cAMP production, but inhibition of adenylyl cyclase did not alter stimulated inositol phosphate production. Phospholipase C inhibition blunted neuron peptide release, suggesting that the phosphatidylinositol pathway was a prominent component of the secretory response. These studies demonstrate preferential sympathetic neuron expression of PACAP-selective receptor variants contributing to regulation of autonomic function.     

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.