Identification and solubilization of atrial natriuretic factor receptors in human placenta

Specific high affinity 125I-atrial natriuretic factor binding sites have been identified in human placental membranes. Using the nonionic detergent, Triton X-100, these binding sites were quantitatively solubilized and retained binding activity. In the solubilized preparation, the macromolecular component that binds atrial natriuretic factor is a 160,000 dalton protein as shown by covalently cross-linking it to 125I-atrial natriuretic factor with the bifunctional chemical crosslinker, disuccinimidyl suberate, followed by gel electrophoresis and autoradiography. On Sephadex G-200 gel filtration in the presence of detergent, the hormone-receptor complex elutes in the molecular weight range of 140,000. These observations suggest strongly that a 140- 160,000 dalton protein present in human placental membranes is the receptor for specific recognition of atrial natriuretic factor.     

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.     

Relationship between arginine vasotocin-like and natriuretic peptide-like immunoreactive structures in the brain of the toad Bufo marinus

The distribution of natriuretic peptide-like immunoreactivity was investigated in the brain of Bufo marinus and compared with arginine vasotocin-like immunoreactivity using fluorescence immunohistochemistry. The antisera used were rabbit anti-porcine brain natriuretic peptide, which recognises the three main structural forms of natriuretic peptides, and guinea-pig antivasopressin, which recognises arginine vasotocin. Natriuretic peptide-like immunoreactive fibres were observed in many regions of the brain, being densest in the preoptic/hypothalamic region of the diencephalon and the interpeduncular nucleus of the mesencephalon. Natriuretic peptide-like immunoreactive cell bodies were observed in the dorsal and medial pallium, the medial amygdala, the preoptic nucleus, the ventral hypothalamus, the nucleus posterodorsalis tegmenti mesencephali, and the interpeduncular nucleus. No natriuretic peptide-like immunoreactivity was seen in the pituitary gland. The distribution of arginine vasotocin-like immunoreactivity was similar to that described previously for other amphibian species. Numerous immunoreactive cell bodies were present in the preoptic nucleus whilst immunoreactive fibres were observed in the preoptic/hypothalamic region as well as in extrahypothalamic regions such as the medial amygdala and the medial pallium. Double-labelling immunohistochemistry revealed no colocalisation of arginine vasotocin-like and natriuretic peptide-like immunoreactivities in the same neural elements. The results suggest that natriuretic peptides and arginine vasotocin have distinct distributions in the brain but that natriuretic peptide-like immunoreactive fibres in the hypothalamus could influence the activity of arginine vasotocin-like immunoreactive cell bodies.     

The guanylyl cyclase receptor family

Cyclic GMP (cGMP) signals through protein kinases, ion channels, and possibly other effector systems as a second messenger. Its synthesis is regulated by guanylyl cyclase, whose activity is found in various cellular compartments including the plasma membrane and cytosol. A soluble form of guanylyl cyclase, which occurs as a heterodimer, appears to serve as a receptor for nitric oxide or nitrosothiols, or both. Recent research suggests the presence of multiple subtypes of the soluble form of guanylyl cyclase and tissue-specific expression of the different forms. At least two different forms of the plasma membrane guanylyl cyclase are known to occur in various mammalian tissues. One form, GC-A, is a receptor for atrial natriuretic peptide, and the binding of ligand causes marked increases in cGMP production. The other form, GC-B, is stimulated more effectively by a brain natriuretic peptide than by atrial natriuretic peptide, but its natural ligand remains in question. Both plasma membrane forms of the enzyme contain a single, putative transmembrane domain. The intracellular region of both forms contains a protein kinase-like domain just within the transmembrane domain. The protein kinase-like domain is followed by a cyclase catalytic region near the carboxyl terminus that is homologous to two internally homologous domains found in a bovine brain adenylyl cyclase. The possibility that other guanylyl cyclase receptor subtypes exist is now being explored. If they do, we may subsequently find that a diversity of specific ligands signals through cGMP.     

Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor

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

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype)

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED₅₀ of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a K_d of 13±1 pM, and natriuretic peptides compete for [¹²⁵I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.Abbreviations BSA bovine serum albumin - dNTP deoxynucleotide triphosphate - SDS sodium dodecyl sulfate - DEAE-dextran diethylaminoethyl-dextran - EDTA ethylenediamine tetraacetic acid - Tris Tris(hydroxymethyl)aminomethane - DMSO dimethyl sulfoxide - RP-HPLC reverse phase-high performance liquid chromatography - AMV avian myeloblastosis virus - Arg arginine - Lys lysine     

HS-142-1, a novel polysaccharide of microbial origin, specifically recognizes guanylyl cyclase-linked ANP receptor in rat glomeruli.

HS-142-1, a novel polysaccharide, of microbial origin had been characterized as a specific antagonist of guanylyl cyclase-linked atrial natriuretic peptide (ANP) receptors (ANP-GC receptor) in bovine adrenal cortex. The effect of HS-142-1 on ANP receptors of rat glomeruli were examined. HS-142-1 blocked rat ANP (r-ANP)-stimulated cGMP production in a concentration-dependent manner, although it caused only slight inhibition in the specific binding of [125I]-rANP to the glomeruli where only a small portion of the binding sites are coupled to guanylyl cyclase. HS-142-1 recognized the 135K ANP receptor which is thought to be ANP-GC receptors but did not recognized 60K receptor, guanylyl cyclase-free type from affinity cross-linking studies with glomerular membranes. These results indicate that HS-142-1 is a specific antagonist for the ANP-GC receptor in rat glomeruli, and that it will be a powerful tool for understanding the physiological roles of ANP in renal responses.     

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.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Brain natriuretic peptide receptors in the rat peripheral sympathetic ganglia

High affinity binding sites for brain natriuretic peptide were characterized in the rat superior cervical ganglia by quantitative autoradiography. In addition, the peptide increased the formation of cyclic GMP in the ganglia in vitro. Brain natriuretic peptide displaced atrial natriuretic peptide from its binding sites. Our results suggest that brain natriuretic peptide and atrial natriuretic peptide may share physiologically active receptors in sympathetic ganglia. Brain natriuretic peptide may modulate the synaptic transmission in sympathetic ganglia, in addition or in conjunction with atrial natriuretic peptide.     

The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.     

Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain.

Quantitative autoradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA-salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111-126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.     

The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide [PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version] to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. [125I]PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited [125I]PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.     

Primary structure and functional expression of the human receptor for Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (STa) produced by Escherichia coli induces intestinal secretion in mammals by binding to the brush border membrane of the small intestine and activating guanylyl cyclase. We report here the cloning and expression of a cDNA encoding the human receptor for STa. The receptor contains both an extracellular ligand binding site and a cytoplasmic guanylyl cyclase catalytic domain, making it a member of the same receptor family as the natriuretic peptide receptors. Stable mammalian cell lines over-expressing the STa receptor specifically bind 125I-STa (Kd approximately 1.0 nM) and respond to STa by dramatically increasing (approximately 50-fold) cellular cGMP levels. Sequence comparisons between the human and the rat STa receptors show less conservation in the extracellular domain than similar comparisons of natriuretic peptide receptors. This divergence may indicate important species differences in ligand-receptor interaction.     

Atrial natriuretic peptide-dependent photolabeling of a regulatory ATP-binding site on the natriuretic peptide receptor-A

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (DeltaGC). We demonstrate that this receptor (NPR-A-DeltaGC) can be directly labeled by 8-azido-3'-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor DeltaKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-DeltaGC is reduced by 50% in the presence of 550 microm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently.     

Renal actions of dendroaspis natriuretic peptide in rabbits

Dendroaspis natriuretic peptide (DNP) is one of four members of the natriuretic peptide family sharing functional and structural properties. The purpose of the present study was to elucidate the physiological role of DNP on renal functions and its cellular mechanism in the rabbit kidney. DNP (5 μg/kg/min) infused intravenously increased urine volume and urinary excretion of electrolytes. These renal actions induced by DNP were more pronounced than those caused by atrial natriuretic peptide (ANP). We compared profiles of (125)I-ANP and (125)I-DNP by reverse-phase HPLC during incubation in rabbit plasma at 37°C for 1, 2, and 4h. While (125)I-ANP was quickly degraded within 1h, (125)I-DNP was still stable in plasma for 4h. DNP induced the greatest cyclic guanosine monophosphate (cGMP) production in the glomeruli in a dose-dependent manner, when compared to other renal structures including cortical tubules, outer medullary tubules, and inner medullary tubules. Affinity cross-linking analysis revealed NPR-A is selective receptor for DNP in glomeruli. Forskolin, a stimulator of adenylyl cyclase, significantly decreased cGMP production in the renal glomeruli but not in the renal medulla. In summary, DNP is a more effective activator of renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. These findings suggest that DNP plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

Urodilatin attenuates sympathetic neurotransmission in the rabbit isolated vas deferens without activating guanylyl cyclase.

Natriuretic peptides are produced in cardiovascular, renal and neural tissues and are believed to reduce arterial blood pressure by augmenting sodium and water loss in the urine. Another potential antihypertensive action of these peptides involves a suppression of adrenergic neurotransmission. Atrial, brain and C-type natriuretic peptides suppress sympathetic neurotransmission but no data are available on neuromodulatory actions of urodilatin. This study investigates the hypothesis that urodilatin and brain natriuretic peptide inhibit sympathetic neurotransmission by elevating guanylyl cyclase activity. Both brain natriuretic peptide and urodilatin suppressed force generation in response to electrical stimulation of the vas deferens. Brain natriuretic peptide accelerated the production of cyclic guanosine monophosphate equipotently with its effects on neurotransmission. However, urodilatin failed to increase guanylyl cyclase activity, thus dissociating its effects on neurotransmission from guanylyl cyclase stimulation. None of the natriuretic peptides altered contractile effects of either adenosine triphosphate or norepinephrine, the two putative neurotransmitters secreted from adrenergic nerves in the vas deferens. These data are consistent with the following conclusions: 1) all of the known endogenous natriuretic peptides suppress adrenergic neurotransmission; 2) guanylyl cyclase activation is not required for the inhibition of sympathetic neurotransmission by natriuretic peptides; and 3) inhibitory effects of the natriuretic peptides on neurotransmission result from a suppression of neurotransmitter exocytosis. The novel findings of this study include both the suppression of sympathetic neurotransmission by urodilatin and its biological activity in the absence of guanylyl cyclase activation.