C-type Natriuretic Peptide Binding Research Articles

Abstract 16716: Intravascular Injection of Sendai Virus Vector Carrying Continuously cGMP Producing Mutant of Type B Natriuretic Peptide Receptor Can Ameliorate Pulmonary Arterial Hypertension

Introduction: Type B natriuretic peptide receptor (NPR-B) is one of the transmembrane guanylyl cyclases and synthesizes cGMP on C-type natriuretic peptide binding. We have found a novel constitutively active mutation in the NPR-B gene in a family case showing overgrowth and bone anomalies. Previously, we constructed adenovirus vectors carrying this mutant NPR-B and administered to Sugen PAH rats intratracheally to investigate its therapeutic effect. However, the use of adenovirus vectors actually caused inflammatory host response in the lung. Objective: Sendai virus vector (SeV) is well known for its high transduction efficiency as well as the low toxicity. Recently, the results of a Phase I/IIa clinical study of SeV in human peripheral arterial disease have been reported. The objective of this study was to investigate the therapeutic effect of SeV carrying constitutively active mutant of NPR-B for PAH in rats. Methods and Results: We constructed SeV carrying wild-type (WT), and mutant (Mut) NPR-B. Control SeV carrying Azami-Green was purchased from MBL (Medical & Biological laboratories, Nagoya, Japan). The overexpression of the mutant in human pulmonary artery smooth muscle cells by SeV induced significant increase in cGMP levels (control, 0.193±0.115 pmol/mL; WT-NPR-B, 38.3±29.6 pmol/mL; Mut-NPR-B, 2460±577 pmol/mL; n=3; p<0.01), and suppressed proliferation as assessed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation (EdU positive ratio: control, 0.18±0.017; WT, 0.12±0.023; Mut, 0.085±0.010; n=6; P < 0.05). In Sugen PAH rats, intravascular administration of SeV carrying Mut-NPR-B significantly decreased right ventricular systolic pressure (Control, 61.4±8.4 mmHg; WT, 61.5±5.4 mmHg; Mut, 46.9±4.9mmHg; n=6; p<0.01) and the ratio of right ventricular/left ventricular pressure (Control, 0.67±0.05; WT, 0.70±0.03; Mut, 0.53±0.09; n=6; p<0.01). Furthermore, the proportion of occluded arteries was lower in Mut transduced rats than in control or WT transduced rats histologically. Conclusions: The CNP/NPR-B signaling pathway is an additional therapeutic target for the treatment of PAH. The enhancement of CNP/NPR-B pathway could ameliorate both intimal and medial remodeling of pulmonary artery in PAH.

The role of particulate guanylyl cyclase B (GC-B) in auditory function in adult mice

Background cGMP signaling triggered by the binding of C-type natriuretic peptide (CNP) to its receptor guanylyl cyclase B (GC-B; NPR2; NPRB) has been linked by genetic evidence to a remarkable variety of physiological functions like skeletal bone growth, female fertility, cardiac growth, fat metabolism and gastrointestinal function. For the nervous system it has been recently demonstrated that the CNP/GC-B/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway is essential for sensory axon branching at the dorsal root entry zone of the spinal cord and at the rhombomeres of the hindbrain during embryonic development [1]. Also in the auditory system, distinct auditory nerve fiber (ANF) types that differ in their discharge rate and sound sensitivity bifurcate in the cochlear nucleus (CN), sending collaterals to the anteroventral, posteroventral, and dorsal subdivisions. The lack of GC-B has been shown to lead to a central phenotype [2].

Catalytically active guanylyl cyclase-B requires glycosylation and mutations that inhibit this process cause dwarfism

Background C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth, axonal path finding and inhibits meiosis in the oocyte [1]. The biologic signaling receptor for CNP is guanylyl cyclase (GC)-B, also known at NPRB or NPR2 [2]. GC-B is a homo-oligomer, possibly a dimer, containing a glycosylated extracellular ligand-binding domain, a single membrane-spanning region, and intracellular kinase homology domain (KHD), dimerization domain and C-terminal GC catalytic domain. Phosphorylation of the region leading into and at the beginning of the kinase homology domain is required for CNP activation of GC-B and dephosphorylation inactivates the enzyme [3]. Homozygous inactivating mutations in GC-B result in Acromesomelic Dysplasia, Type Maroteaux (AMDM) dwarfism [4-6], and heterozygous inactivating mutations in GC-B cause non-pathological reductions in stature [7]. Conversely, genetic mutations that increase GC-B activity result in skeletal overgrowth [8-10]. More than fifteen inactivating missense mutations in GC-B have been identified in humans. These mutations are randomly distributed from the N-terminus (P32) to the C-terminus (G959A) of the enzyme, consistent with two potential mechanisms. The first involves multiple processes like disruption of CNP or GTP binding to the extracellular or catalytic domains, respectively. The second more general mechanism involves conformational changes in secondary, tertiary or quaternary structure that preclude catalytic domain formation or activation. Previous investigators reported that 11 out of 12 [1], 2 out of 3 [2] or 1 out of 2 [3] missense mutations inhibited the transport of GC-B to the cell surface due to defective cellular trafficking and retention in the ER as indicated by reduced immunofluorescence imaging. Thus, the current hypothesis is that AMDM mutations inactivate GCB by disrupting intracellular trafficking. Here, we report that four intracellular GC-B mutants known to cause AMDM dwarfism bind CNP on the cell surface but have dramatically reduced catalytic activity.

Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation.

ABSTRACTConsiderable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression.

Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line

Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260–1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca 2+] i ) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca 2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1 h, 5 mM NH 4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca 2+] i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca 2+ accumulation in the absence of Na +. In cells incubated with ammonia in the absence of Ca 2+ a slight CNP-dependent increase of [Ca 2+] i was observed, most likely representing Ca 2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca 2+] i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

A Loss-of-Function Mutation in Natriuretic Peptide Receptor 2 (Npr2) Gene Is Responsible for Disproportionate Dwarfism in cn/cn Mouse

The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F2 progeny mapped the cn locus on an approximately 0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in +/+ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-of-function mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal.

Guanylyl cyclase-B represents the predominant natriuretic peptide receptor expressed at exceptionally high levels in the pineal gland

The generation and function(s) of the signalling molecule cyclic GMP (cGMP) in brain are still poorly understood. One mechanism to raise intracellular cGMP levels is binding of C-type natriuretic peptide (CNP) to a membrane guanylyl cyclase (GC), termed GC-B. Here, we demonstrate an exceptionally strong expression of GC-B in the pineal gland. Crosslinking experiments performed with 125 I-Tyr 0-CNP and membranes from various rat tissues identified the receptor as a 130-kDa protein, expressed at highest levels in pineal membranes. Receptor autoradiography on brain sections confirmed a striking density of CNP binding sites in pineal tissue, whereas binding sites for the related atrial natriuretic peptide (ANP) predominate in other regions of the brain. Incubations of freshly dissected whole pineal glands in either the absence or presence of natriuretic peptides followed by immunohistochemical analyses of cGMP revealed strong accumulations of cGMP in response to CNP but not to ANP in the majority of pinealocytes. Stimulation of soluble GC (sGC) activity by use of sodium nitroprusside (SNP) resulted in a very similar pattern of cGMP immunostaining, indicating a co-expression at high levels of particulate and soluble forms of GC. These findings point to a major role of cGMP signalling in pinealocytes and suggest an important regulatory function for CNP.

Regulation of Natriuretic Peptide Receptors by Thyrotropin in FRTL-5 Rat Thyroid Cells: Evidence for Nonguanylate Cyclase Atrial Natriuretic Factor-Binding Sites in Cells Lacking the Natriuretic Peptide Receptor C

Natriuretic peptide receptors (NPR) are expressed in thyroid-derived cells, including the rat FRTL-5 thyroid cell line. We have previously demonstrated that atrial natriuretic factor (ANF) binding consistent with the NPR-A receptor is significantly increased in FRTL-5 cells cultured in the presence of TSH. The purpose of the present study was to determine whether TSH treatment, therefore, results in higher levels of ANF-induced intracellular cGMP, and whether TSH elicits similar effects on cGMP signaling through the NPR-B receptor. We now show that contrary to expectation, long term exposure to 1 mIU/ml bovine TSH (6H medium) does not significantly alter maximal ANF-induced cGMP formation. Moreover, TSH treatment decreased C-type natriuretic peptide (CNP)-induced cGMP generation in FRTL-5 cells, suggesting a down-regulation of NPR-B. A similar effect of TSH on ANF- and CNP-induced cGMP was observed in FRTL cells, the precursor of the FRTL-5 cell line. Scatchard analysis of [125I]ANF binding in TSH-treated (6H) FRTL-5 cultures indicated a 5.6-fold increase in high affinity ANF-binding sites compared with TSH-deficient (5H) cultures [binding capacity (Bmax) of 6H cells, 227.2 +/- 33.7 fmol/mg protein; Bmax of 5H cells, 40.2 +/- 4.7 fmol/mg protein]. The effect of TSH on [125I]ANF binding was mimicked by forskolin and (Bu)2cAMP, indicating receptor up-regulation via a cAMP pathway. High affinity [125I]CNP-binding sites were present in much lower abundance (Bmax of 5H, 0.80 +/- 0.06 fmol/mg protein), and no effect of TSH treatment on them could be demonstrated. However, low affinity [125I]CNP binding was increased by TSH. RT-PCR confirmed the presence of both NPR-A and NPR-B transcripts in FRTL-5 cells and showed that TSH treatment significantly decreased NPR-B, but not NPR-A. NPR-C transcript was not detectable by RT-PCR in FRTL-5 cells cultured in high TSH medium, suggesting that the ANF-binding sites increased by TSH are not NPR-C. Both CNP and ANF transcript were also expressed in FRTL-5 cells, and CNP was increased by TSH. Together the data support the down-regulation of functional NPR-B and no change in functional NPR-A by TSH. The vast majority of ANF-binding sites in FRTL-5 cells, therefore, are not coupled to cGMP production and may represent a novel or altered form of NPR that is regulated by TSH independently of NPR-A and NPR-B.

Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland.

In the shark, C-type natriuretic peptide (CNP) is the only cardiac natriuretic hormone identified and is a potent activator of Cl- secretion in the rectal gland, an epithelial organ of this species that contains cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. We have cloned an ancestral CNP receptor (NPR-B) from the shark rectal gland that has an overall amino acid identity to the human homologue of 67%. The shark sequence maintains six extracellular Cys present in other NPR-B but lacks a glycosylation site and a Glu residue previously considered important for CNP binding. When shark NPR-B and human CFTR were coexpressed in Xenopus oocytes, CNP increased the cGMP content of oocytes (EC50 12 nM) and activated CFTR Cl- channels (EC50 8 nM). Oocyte cGMP increased 36-fold (from 0.11 +/- 0.03 to 4.03 +/- 0.45 pmol/oocyte) and Cl- current increased 37-fold (from -34 +/- 14 to -1,226 +/- 151 nA) in the presence of 50 nM CNP. These findings identify the specific natriuretic peptide receptor responsible for Cl- secretion in the shark rectal gland and provide the first evidence for activation of CFTR Cl- channels by a cloned NPR-B receptor.

Characterisation of C-type natriuretic peptide receptors in the gill of dogfish Triakis scyllia.

Only C-type natriuretic peptide (CNP) has been identified in primitive elasmobranch fish. CNP is the most conserved molecule in the natriuretic peptide family, suggesting that it is the ancestral type. As a first step to investigating the ancestral type of natriuretic peptide receptors, CNP receptors were characterised in an elasmobranch (dogfish, Triakis scyllia) by radioligand-binding analysis using 125I-[Tyr0]-dogfish (df)CNP. None of the modifications of the CNP molecule that occur at the time of iodination (addition of a Tyr residue at the N-terminus, introduction of iodine into Tyr0 or oxidation of Met17) affect the affinity of dfCNP for the receptors. Neither did oxidation of Met17 decrease the ability of CNP to stimulate cGMP production. In the tissues examined, CNP receptors were densest in the gill cells followed by the intestine, interrenal gland and rectal gland, all of which are involved in osmoregulation in elasmobranchs. CNP-stimulated guanylate cyclase (GC) activity was highest in the interrenal gland, intestine, brain and rectal gland, followed by the gill cells. Since the gill cells seem to contain both GC-coupled and uncoupled receptors, this tissue was used to characterise dogfish CNP receptors. Scatchard analysis of the saturation isotherm revealed two classes of binding site: one has a Kd of 24.0 pM and Bmax of 59.9 fmol/mg protein, and the other has low affinity (Kd > 1 nM) and high capacity (Bmax > 200 fmol/mg protein). The higher-affinity binding sites may represent GC-uncoupled receptors, because C-ANF, a specific ligand for GC-uncoupled receptors, almost completely displaced CNP binding. Affinity-labelling experiments showed that dogfish receptors have molecular masses of about 90, 170 and 340 kDa, and CNP binding to the former two receptors is inhibited by C-ANF. After reduction with 2-mercaptoethanol, most 170 kDa labelling was shifted to 90 kDa. It is concluded that GC-uncoupled receptors in the dogfish gill have higher molecular mass than those of mammals and eel (about 65 kDa), and are present mostly as monomers even in non-reducing conditions. However, a small population of GC-coupled receptors is also present, as demonstrated by an increase in cGMP production.

Production and characterization of monoclonal antibodies against human natriuretic peptide receptor-A or -B

Monoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in human.

Natriuretic Peptide Receptor-B (Guanylyl Cyclase-B) Mediates C-type Natriuretic Peptide Relaxation of Precontracted Rat Aorta

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.

Characterization of C-type natriuretic peptide receptors in human mesangial cells

Our aim was to examine whether the human glomerulus was a target for C-type natriuretic peptide (CNP) and how A, B and C receptors of natriuretic peptides (ANPR-A, ANPR-B, ANPR-C) were distributed in glomerular mesangial and epithelial cells. CNP stimulated cyclic GMP production in cultured human mesangial and epithelial cells with similar threshold concentrations (1 nM) and maximum effects (basal value x 30 at 1 microM). In contrast, atrial natriuretic peptide (ANP) was only stimulatory in epithelial cells. [125I] CNP bound specifically to mesangial cells with a Kd of 0.47 nM and Bmax of 42 fmol/mg. Equilibrium of binding was obtained after four to five hours at +4 degrees C and nonspecific binding represented 10 to 20% of total binding. HS142-1 (100 micrograms/ml), a specific inhibitor of ANPR-A and ANPR-B, suppressed 90% of CNP-dependent cyclic GMP production whereas it had little effect on [125I]-CNP binding, suggesting that C receptors were largely predominant in mesangial cells. No biological effect of CNP on mesangial cells, including change in basal or angiotensin II-induced contractility and inhibition of basal or serum-dependent proliferation, could be demonstrated. Similar results were obtained with 8-bromo-cyclic GMP and sodium nitroprusside. Intraglomerular localization of ANPR-A, ANPR-B and ANPR-C mRNA was studied using reverse transcriptase-polymerase chain reaction with amplification of their corresponding cDNA by different primers. Amplification products were identified on gel electrophoresis by their predicted sizes and sequencing. ANPR-A, ANPR-B and ANPR-C mRNA were present in epithelial cells whereas only ANPR-B and ANPR-C mRNA were detected in mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat renal preglomerular vessels, glomeruli and papillae do not express detectable quantities of B-type natriuretic peptide receptor

Atrial natriuretic factor (ANF) receptor subtypes were quantitated by radioligand studies in three different rat renal isolated tissues: preglomerular vessels, glomeruli and papillae. In preglomerular vessels 100% of [125I]-ANF binding was displaced with high affinity by ANF. C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP(4- 23)) a specific ligand for ANP-C receptors, displaced 30% of total binding with a lower affinity than ANF. C-type natriuretic peptide (CNP) displaced [125I]-ANF binding in a biphasic manner, indicating that it binds to two sites with affinities three orders of magnitude apart. When CNP was incubated in the presence of 0.1 mumol/l C-ANF to saturate ANP-C receptors, the high-affinity binding site vanished and maximum binding decreased to 70%, suggesting that CNP binds with high affinity to ANP-C receptors. Since the ANP-A receptor has little or no avidity for CNP, it is probably the low-affinity binding site. CNP and C-ANF displaced most [125I]-[Tyr]CNP(1-22) binding with very close affinities, indicating that CNP binds primarily preglomerular vascular ANP-C receptors. In glomeruli CNP behaved similarly to C-ANF in its ability to displace approximately 85% of [125I]-ANF binding; the remaining 15% was completely displaced by ANF. C-ANF and CNP inhibited 100% of [125I]-[Tyr]CNP(1-22) binding. Both findings suggest that [125I]-[Tyr]CNP(1-22) binds ANP-C receptors exclusively. In renal papillae no displacement of [125I]-ANF by C-ANF was observed, and CNP was bound to ANP-A receptors with the same very low affinity as in preglomerular vessels. The absence of [125I]-[Tyr]CNP(1-22) binding to papillary membranes indicates that ANP-B receptors are not expressed in that tissue. Unlike ANF, CNP stimulated cGMP production in glomeruli and papillae only at extremely high, supraphysiological concentrations. The present results suggest that ANP-B receptors either are absent or are present in undetectable amounts in rat renal preglomerular vessels, glomeruli and papillae.

Effects of C‐Type Natriuretic Peptide on Rat Astrocytes: Regional Differences and Characterization of Receptors

We have compared the effects of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on the accumulation of cyclic GMP (cGMP) in secondary cultures of rat astrocytes. The order of potency of these peptides was CNP > ANP > BNP, which would be compatible with a predominance of guanylate cyclase B (GC-B)- versus guanylate cyclase A (GC-A)-type receptors in these cells. Accordingly, we found by northern blot analysis that the mRNA transcripts of GC-B were much more abundant in astrocytes than the transcripts of GC-A. In addition, astrocytes from diencephalon accumulated two times more cGMP in response to CNP than astrocytes from cortex. Binding experiments with 125I-labeled ANP or [Tyro]-CNP established that these ligands recognized only clearance-type receptors on astrocytes. However, the number of binding sites was approximately 100 times higher in astrocytes from cortex than in astrocytes from diencephalon and thus was inversely correlated to the amplitude of the cGMP response in the same cells. We found no further evidence for differences in the levels of GC-B receptors in astrocytes from the two regions because (a) the abundance of GC-B mRNA was similar and (b) there was no difference in particulate guanylate cyclase activity in astrocytes from each region. In addition, occupancy of clearance receptors with C-ANP4-23 did not affect the accumulation of cGMP in response to CNP; this makes it unlikely that the differences in cGMP responsiveness can be accounted for by binding and sequestration of CNP to the clearance receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of C-type natriuretic peptide receptors in hamster glomeruli.

The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

Receptors for C-type natriuretic peptide in cultured rat glial cells

To characterize sites of action of C-type natriuretic peptide (CNP) in the glial cells, the effect of CNP on cGMP accumulation and the binding of [ 125I]CNP in rat astrocyte RCR-1 cells were studied. CNP stimulated cGMP accumulation in the cells from 10 −9 M in a dose-dependent manner, but ANP (atrial natriuretic peptide) had a negligible effect on cGMP accumulation in the cells. [ 125I]CNP was bound to the cells and its K d value was 2 orders of magnitude lower than that of the ED 50 value for stimulation of cGMP accumulation in the cells. Not only CNP but also ANP displaced [ 125I]CNP binding to the cells. These results suggest that RCR-1 cells have a B-receptor which contains a guanylate cyclase domain and is preferentially activated by CNP, and that they also have a C-receptor which does not contain a guanylate cyclase domain that reacts with both ANP and CNP.

Autoradiographic visualization of the natriuretic peptide receptor-B in rat tissues

Natriuretic peptide receptor-B (NPRB) was visualized in rat tissues by in vitro autoradiography, using its putative physiological agonist C-type natriuretic peptide (CNP). In initial studies, we determined that atrial natriuretic peptide (ANP) is not a suitable ligand for labeling the NPRB: in tissues reported to contain NPRB transcripts, CNP did not inhibit [ 125I]ANP binding except to NPRC sites. Therefore, to visualize the NPRB we used 125I[Tyr o]-CNP as a radioligand with an excess of NPRC-blocking peptide: C-ANP. With this approach we detected the highest number of NPRB-like sites in the pars intermedia of the pituitary gland. A large number of these sites were present in pituitary neural and anterior lobes, area postrema, adrenal medulla and cortex. A moderate NPRB population was observed in the subfornical organ, plexiform layer of the olfactory bulb and kidney. Low concentrations of NPRB were noted in the cerebellum and cerebrum but not in the choroid plexus and pia-arachnoid. Saturation experiments performed on cerebellum sections revealed a very low concentration ( B max 4.8 fmol/mg protein) of high affinity ( K d 1.2 nM) NPRB-like sites. This study is the first demonstration of 125I[Tyr o]-CNP binding sites with characteristics of the NPRB in intact tissues.