Natriuretic peptides (NPs) are peptide hormones that exert their biological actions by binding to three types of cell surface natriuretic peptide receptors (NPRs). The receptor NPR-B binding C-type natriuretic peptide (CNP) acts locally as a paracrine and/or autocrine regulator in a wide variety of tissues. Mutations in the gene NPR2 have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), an autosomal recessive skeletal disproportionate dwarfism disorder in humans.
In the study, presented here, genotyping of six consanguineous families of Pakistani origin with AMDM was carried out using polymorphic microsatellite markers, which are closely linked to the gene NPR2 on chromosome 9p21-p12. To screen for mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected and unaffected individuals of the families and sequenced.
Sequence analysis of the gene NPR2 identified a novel missence mutation (p.T907M) in five families, and a splice donor site mutation c.2986 + 2 T > G in the other family.
We have described two novel mutations in the gene NPR2. The presence of the same mutation (p.T907M) and haplotype in five families (A, B, C, D, E) is suggestive of a founder effect.
Acromesomelic dysplasia-type Maroteaux; gene NPR2; missence mutation (T907M); splice site mutation c.2986 + 2 T > G
The cn/cn dwarf mouse is caused by a loss-of-function mutation in the natriuretic peptide receptor 2 (NPR-2) gene which helps positively regulate endochondral longitudinal bone growth. The gene mutation corresponds to that in the human skeletal dysplasia Acromesomelic Dysplasia Maroteaux type (AMDM). This study assesses histomorphometric, ultrastructural and radiographic correlates of the growth abnormality.
Ten litters of cn/cn and cn/+littermates at ages ranging from 2.5 to 6.5 weeks were studied by skeletal radiographs, histomorphometry and physeal ultrastructure. Skeletal radiographs were done on 2 cn/cn and 2 cn/+littermates at 5 weeks of age. Humeral, femoral, and tibial lengths were measured from 34 intact bones (17 cn/cn, 17 cn/+) at 2.5 to 6.5 weeks. Growth plate histomorphometry in 50 bones (26 cn/cn and 24 cn/+) determined the hypertrophic zone/entire physeal cartilage ratios in 204 sections (87 cn/+, 117 cn/cn) at 3 time periods (2.5-3, 4–4.5, and 6–6.5 weeks). Electron microscopy assessed 6 cn/cn and 6 cn/+age and site-matched physeal cartilage.
Cn/cn mice were two thirds the size of the cn/+. Cn/cn bones were normal in shape or only minimally deformed except for the radius with mid-diaphyseal bowing. Length ratios of cn/cn humeri, femurs, and tibias were a mean of 0.65 (±0.03, n = 34, 17 ratios) compared to cn/+bones. The main physeal abnormality was a markedly shortened hypertrophic zone with the ratio of hypertrophic zone to entire physis 0.17 (±0.063) in the cn/cn and 0.30 (±0.052) in the cn/+mice. Ratio assessments were similar comparing humeral, femoral, and tibial growth plates as were ratios from each of the 3 time periods. Ultrastructural assessments from the resting zone to the lower hypertrophic zone-metaphyseal junction showed no specific individual cell abnormalities in cn/cn compared to cn/+physes.
The disorder causes a shortened physeal hypertrophic zone but normal ultrastructure of cn/cn chondrocytes points to abnormality primarily affecting the hypertrophic zone rather than a structural cell or matrix synthesis problem.
We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.
The second messenger cyclic GMP affects synaptic transmission and modulates synaptic plasticity and certain types of learning and memory processes. The impact of the natriuretic peptide receptor B (NPR-B) and its ligand C-type natriuretic peptide (CNP), one of several cGMP producing signaling systems, on hippocampal synaptic plasticity and learning is, however, less well understood. We have previously shown that the NPR-B ligand CNP increases the magnitude of long-term depression (LTD) in hippocampal area CA1, while reducing the induction of long-term potentiation (LTP). We have extended this line of research to show that bidirectional plasticity is affected in the opposite way in rats expressing a dominant-negative mutant of NPR-B (NSE-NPR-BΔKC) lacking the intracellular guanylyl cyclase domain under control of a promoter for neuron-specific enolase. The brain cells of these transgenic rats express functional dimers of the NPR-B receptor containing the dominant-negative NPR-BΔKC mutant, and therefore show decreased CNP-stimulated cGMP-production in brain membranes. The NPR-B transgenic rats display enhanced LTP but reduced LTD in hippocampal slices. When the frequency-dependence of synaptic modification to afferent stimulation in the range of 1–100 Hz was assessed in transgenic rats, the threshold for both, LTP and LTD induction, was shifted to lower frequencies. In parallel, NPR-BΔKC rats exhibited an enhancement in exploratory and learning behavior. These results indicate that bidirectional plasticity and learning and memory mechanism are affected in transgenic rats expressing a dominant-negative mutant of NPR-B. Our data substantiate the hypothesis that NPR-B-dependent cGMP signaling has a modulatory role for synaptic information storage and learning.
cGMP; exploratory; hippocampus; LTP; LTD; metaplasticity; NPR-B; memory
Acromesomelic dysplasia Maroteaux type (AMDM) is an autosomal recessive disorder belonging to the group of acromesomelic dysplasias. AMDM is characterised by severe dwarfism with shortening of the middle and distal segments of the limbs. An AMDM gene has recently been mapped to human chromosome 9p13-q12 by homozygosity mapping in four consanguineous families. Here, we show linkage of the disease gene to chromosome 9p13-q12 in four of five consanguineous AMDM families and its exclusion in a fifth family with two children affected with a mild form of the disease. This study suggests that genetic heterogeneity accounts for the variable clinical and radiological severity of AMDM.
Keywords: acromesomelic dysplasia Maroteaux type; acromesomelic dysplasias; homozygosity mapping; chromosome 9
Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.
Millions of people suffer from debilitating hearing defects, ranging from a complete inability to detect sound to more subtle changes in how sounds are encoded by the nervous system. Many forms of deafness are due to mutations in genes that impair the development or function of hair cells, which are responsible for changing sound into electrical signals that can be processed by the brain. Both mice and humans carrying these mutations fail standard hearing tests. In contrast, very little is known about the genetic basis of central auditory processing disorders, which are poorly defined and difficult to diagnose, since these patients can still detect sounds. By finding genes that are required for the normal wiring of central auditory circuits in mice, we can investigate how changes at the circuit level affect circuit function and therefore improve our understanding of central auditory processing disorders. Here, we show that the natriuretic peptide receptor Npr2 is required to establish frequency maps in the mouse central auditory system. Surprisingly, despite a dramatic change in circuit organization, Npr2 mutant mice are still able to respond to sounds with normal sensitivity and timing, underscoring the need for better hearing diagnostic methods in mice as in humans.
Atrial natriuretic peptide (ANP) binds to natriuretic peptide receptor-A (NPR-A), a membrane guanylyl cyclase, and to natriuretic peptide receptor-C (NPR-C), which plays a role in peptide clearance. Rat ANP (rANP) mutants that bind rat NPR-A selectively over rat NPR-C were isolated from randomized libraries of rANP-display phage by differential panning. One variant was identified with reduced NPR-C binding; rANP (G16R, A17E, Q18A) [rANP(REA18)]. Synthetic rANP(REA18) was equipotent with rANP in stimulating cGMP production from cloned rat NPR-A (ED50 = 1.8 nM) and was reduced in NPR-C binding by approximately 200-fold. When infused into conscious rats at 0.325 microg/min for 30 min rANP elicited an identical decrease in blood pressure compared with 0.25 microg/min of rANP(REA18), however the natriuretic (P < 0.05) and diuretic (P = 0.07) responses to rANP(REA18) were greater. These data are consistent with a role for NPR-C as a local decoy receptor attenuating NPR-A effects in the kidney, where these receptors are coexpressed. Improved NPR-A specificity could provide more effective natriuretic peptides for treatment of acute renal failure or heart failure.
Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA, and subsequent human chorionic gonadotropin (hCG) treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with equine chorionic gonadotropin to further define the mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by estradiol (E2), with augmentation by follicle-stimulating hormone (FSH), but FSH or luteinizing hormone (LH) alone had no effect. Thus, estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. Human chorionic gonadotropin increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.
Expression of natriuretic peptide type C (NPPC) in mouse granulosa cells is regulated by estrogen, gonadotropins, and EGF, but ovarian NPPC peptide levels are unaffected by mutation of the NPR3 clearance receptor.
epidermal growth factor; estradiol/estradiol receptor; estrogens; follicle-stimulating hormone; gonadotropins; granulosa cells; luteinizing hormone; natriuretic peptide receptor 3; natriuretic peptide type C
Understanding of the regulatory mechanisms of gene expression in the control of blood pressure and fluid volume is a key issue in cardiovascular medicine. Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signalling antagonizes the physiological and pathophysiological effects mediated by the renin–angiotensin–aldosterone system (RAAS) in the regulation of cardiovascular homeostasis.The targeted-disruption of the Npr1 gene (coding for GC-A/PRA) leads to activation of the cardiac RAAS involved in the hypertrophic remodelling process, which influences cardiac size, expression of pro-inflammatory cytokine genes and the behaviour of various hypertrophy marker genes. The Npr1 gene-knockout (Npr1−/−) mice exhibit 35–40 mmHg higher systolic blood pressure and a significantly greater heart weight to bodyweight ratio than wild-type (Npr1+/+) mice.The expression of both angiotensin-converting enzyme (ACE) and angiotensin II AT1a receptors are significantly increased in hearts from Npr1−/− mice compared with hearts from Npr1+/+ mice. In parallel, the expression of interleukin-6 and tumour necrosis factor-α is also markedly increased in hearts from Npr1−/− mice.These findings indicate that disruption of NPRA/cGMP signalling leads to augmented expression of the cardiac RAAS in conjunction with pro-inflammatory cytokines in Npr1-null mutant mice, which promotes the development of cardiac hypertrophy and remodelling.
angiotensin-converting enzyme; AT1 receptor; atrial natriuretic peptide; cardiac hypertrophy; fibrosis; gene expression; gene knockout; guanylyl cyclase/natriuretic peptide receptor-A; pro-inflammatory cytokines
Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is the principal receptor for the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) and an important effector molecule in controlling of extracellular fluid volume and blood pressure homeostasis. We have utilized RNA interference to silence the expression of GC-A/NPRA gene (Npr1), providing a novel system to study the internalization and trafficking of NPRA in intact cells. MicroRNA (miRNA)-mediated small interfering RNA (siRNA) elicited functional gene-knockdown of NPRA in stably transfected human embryonic kidney 293 (HEK-293) cells expressing a high density of recombinant NPRA. We artificially expressed three RNA polymerase II-driven miRNAs that specifically targeted the Npr1 gene, but shared no significant sequence homology with any other known mouse genes. Reverse transcription-PCR (RT-PCR) and Northern blot analyses identified two highly efficient Npr1 miRNA sequences to knockdown the expression of NPRA. The Npr1 miRNA in chains or clusters decreased NPRA expression more than 90% as compared with control cells. ANP-dependent stimulation of intracellular accumulation of cGMP and guanylyl cyclase activity of NPRA were significantly reduced in Npr1 miRNA-expressing cells by 90-95% as compared with control cells. Treatment with Npr1 miRNA caused a drastic reduction in the receptor density subsequently a deceased internalization of radiolabeled 125I-ANP-NPRA ligand-receptor complexes. Only 12%-15% of receptor population was localized in the intracellular compartments of microRNA silenced cells as compared to 70%-80% in control cells.
Atrial natriuretic peptide; guanylyl cyclase/natriuretic peptide receptor-A; gene-knockdown; receptor internalization; human embryonic kidney cells; microRNA
Atrial natriuretic peptide (ANP) exerts an inhibitory effect on juxtaglomerular (JG) renin synthesis and release by activating guanylyl cyclase/ natriuretic peptide receptor-A (GC-A/NPRA). Renin has also been localized in connecting tubule cells; however, the effect of ANP/NPRA signaling on tubular renin has not been determined. In the present study, we determined the role of NPRA in regulating both JG and tubular renin using Npr1 (coding for NPRA) gene-disrupted mice, which exhibit a hypertensive phenotype. Renin-positive immunoreactivity in Npr1-/- homozygous null mutant mice was significantly reduced compared with Npr1+/+ wild-type mice (23% vs 69% renin-positive glomeruli). However, after chronic diuretic treatment, Npr1-/- mice showed an increment of JG renin immunoreactivity compared with Npr1+/+ mice (70% vs 81% renin-positive glomeruli). There were no significant differences in the distal tubule renin between Npr1+/+ and Npr1-/- mice. However, after diuretic treatment, Npr1-/- mice showed a significant decrease in renin immunoreactivity in principal cells of cortical collecting ducts (p<0.05). The increased JG renin immunoreactivity after reduction in blood pressure in diuretic-treated Npr1-/- mice, demonstrates an inhibitory action of ANP/NPRA system on JG renin; however, a decreased expression of distal tubular renin suggests a differential effect of ANP/NPRA signaling on JG and distal tubular renin.
Distal tubular and juxtaglomerular renin; immunohistochemistry; atrial natriuretic peptide receptor-A; gene-disrupted mice; hypertension
The objective of the present study was to gain insight into the cooperative role of Ets-1 and p300 in transcriptional regulation and expression of Npr1 gene (coding for guanylyl cyclase-A/natriuretic peptide receptor-A; GC-A/NPRA). Overexpression of Ets-1 and p300 in mouse mesangial cells (MMCs) increased Npr1 promoter activity by 12-fold, NPRA mRNA levels by 5-fold, and ANP-dependent intracellular accumulation of cGMP by 26-fold. Knockdown of Ets-1 and p300 expression by small interfering RNA (siRNA) inhibited Npr1 gene transcription by 90%. Sequential chromatin immunoprecipitation assay demonstrated a direct physical association between p300 and Ets-1 upon binding to Npr1 promoter, suggesting that a physical interaction between Ets-1 and p300 is important to enhance Npr1 gene transcription. Mutant p300 lacking histone acetyltransferase (HAT) activity did not show functional effect with Ets-1, suggesting that HAT activity of p300 is required for the cooperative interaction in modulating Npr1 gene transcription. Overexpression of wild-type adenovirus E1A significantly decreased the Npr1 promoter activity by 40%, whereas mutant E1A, which is incapable of binding to p300, did not show any effect. The results indicate that Npr1 gene transcription is critically controlled by histone acetyltransferase p300 and Ets-1. The present findings should yield important insights into the molecular signaling governing Npr1 gene transcription, an important regulator in the control of hypertension and cardiovascular events.
Atrial natriuretic peptide; guanylyl cyclase-A/natriuretic peptide receptor-A; gene transcription; gene expression; histone acetyltransferase
Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias) – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components.
Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX.
We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor).
Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors.
C-type natriuretic peptide (CNP) signaling through its receptor natriuretic peptide receptor B (NPR-B) is a key molecule for mammalian reproduction, and known to play important roles in female fertility. However, the function of these peptides in mouse male reproduction remains largely unknown. To determine the role of CNP/NPR-B signaling in male reproduction we investigated phenotype of Npr2-deficient short-limbed-dwarfism (Npr2
) mice, which have been shown to have gastrointestinal (GI) abnormalities.
In homozygous Npr2
mice, spermatogenesis is developmentally delayed at both 2 and 4 weeks of age, with vacuolation and degenerating apoptotic germ cells being observed at 3 weeks age. However, the adult Npr2
mice exhibited apparently normal spermatogenesis, albeit with some aberrant spermatids, suggesting that developmental delay was overcome. In addition, the adult Npr2
mice showed abnormal penile morphology (paraphimosis).
The potential role of CNP signaling via the NPR-B receptor in male fertility appears to be mediated not through germ-cell development, but may be through maintenance of normal penile function.
CNP; NPR-B; cGMP; SLW mouse; Smooth muscle; Paraphimosis; Infertility; Spermatogenesis
Background: The receptors for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPR-A), have been reported to be expressed in lung cancer, prostate cancer, ovarian cancer. NPR-A expression and signaling is important for tumor growth, its deficiency protect C57BL/6 mice from lung, skin, and ovarian cancers, and these result suggest that NPR-A is a new target for cancer therapy. Recently, NPR-A has been demonstrated to be expressed in pre-implantation embryos and in ES cells, it has a novel role in the maintenance of self-renewal and pluripotency of ES cells. However, the direct role of NPR-A signaling in gastric cancer remains unclear. Method: NPR-A expression was downregulated by transfection of shRNA. The proliferation of gastric cancer cells was measured by Hoechst 33342 stain. Cell proliferation and invasion were determined via BrdU and transwell assays, respectively. Results: Down-regulation of NPR-A expression by shNPR-A induced apoptosis, inhibited proliferation and invasion in AGS cells. The mechanism of shNPR-A-induced anti-AGS effects was linked to NPR-A-induced expression of KCNQ1, a gene to be overexpressed in AGS and significantly reduced by shNPR-A. Conclusion: Collectively, these results suggest that NPR-A promotes gastric cancer development in part by regulating KCNQ1. Our findings also suggest that NPR-A is a target for gastric cancer therapy.
NPR-A; gastric cancer; KCNQ1
The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-dNle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH2), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (Kis of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC50s of 2, 3 and 14 nm). PL-3994 has a Ki of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 µm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC50s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm–100 µm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1–1000 µg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.
PL-3994; Natriuretic peptide receptors; Atrial natriuretic peptide; Brain natriuretic peptide; Neutral endopeptidase sensitivity; Bronchodilator
Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain largely unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gαi-βγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is co-expressed in TRPV1-expressing mouse DRG neurons. NPR-C can be co-immunoprecipitated with Gαi, and CNP treatment induced translocation of PKCε to the plasma membrane of these neurons, which was inhibited by pertussis toxin pre-treatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons, and increased neuronal firing frequency, an effect that was absent in DRG neurons from TRPV1−/− mice. CNP-induced sensitization of TRPV1 activity was attenuated by pre-treatment of DRG neurons with the specific inhibitors of Gβγ, PLCβ or PKC, but not of PKA, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Further, CNP injection into mouse hind paw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγ or TRPV1, and was also absent in TRPV1−/− mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity.
Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents the precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C, Nppc, mRNA while cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC elevated cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.
Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1−/−) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1+/+ ) mice. A significant up-regulation of cytokine genes such as TNF-α (5- fold), IL-6 (3-fold) and TGF-β1 (4-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-κB binding activity was almost 5-fold greater in the nuclear extract of Npr1−/− mutant mice hearts as compared with wild-type Npr1+/+ mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10-fold and 6-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-κB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.
Natriuretic peptide receptor-A; GC activity; cGMP signaling; and cytokines
The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.
Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF4-23. Nitric oxide (NO) production, prostaglandin E2 (PGE2) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.
The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE2 release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF4-23 had the opposite effect and reduced NO and PGE2 release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE2 release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE2 release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.
Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.
The heart expresses the three natriuretic peptide receptors (NPR), namely NPR-A, NPR-B, and NPR-C. We have examined the temporal relationship between the expression of mRNA transcripts for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their receptors in the heart during the development of cardiac hypertrophy in the aortovenocaval fistula rat. Messenger RNAs were measured by cDNA amplification. Progressive cardiac hypertrophy was accompanied by increased ANP mRNA prevalence throughout the heart and increased BNP mRNA in the left atrium. The most striking observation was the gradual disappearance of NPR-C transcripts (the putative "clearance" receptor) in all chambers; this was in marked contrast to the increase in mRNA levels for NPR-A and NPR-B (the guanylyl cyclase-linked receptors). Our observations have important therapeutic implications if the transcript changes are mirrored at the receptor protein level because (a) the apparent down-regulation of NPR-C may enhance the local action of natriuretic peptides on the heart, and (b) the loss of NPR-C, particularly if it is widespread, may reduce the rate of elimination of the natriuretic peptides, restricting the therapeutic potential of specific NPR-C ligands designed to reduce peptide clearance.
To demonstrate the presence of natriuretic peptide receptors (NPRs) in primary human corneal epithelial cells (p-CEPI), SV40-immortalized CEPI cells (CEPI-17-CL4) and in human corneal epithelium, and to define the pharmacology of natriuretic peptide (NP)-induced cGMP accumulation.
NPR presence was shown by RT–PCR, western blot analysis, and indirect immunofluoresence. cGMP accumulation was determined using an enzyme immunoassay.
p-CEPI and CEPI-17-CL4 cells expressed mRNAs for NPR-A and NPR-B. Proteins for both NPRs were present in these cells and in human corneal epithelium. C-type NP (CNP), atrial NP (ANP) and brain NP (BNP) stimulated the accumulation of cGMP in a concentration-dependent manner in p-CEPI cells (potency; EC50s): CNP (1–53 amino acids) EC50=24±5 nM; CNP fragment (32–53 amino acids) EC50=51±8 nM; ANP (1–28 amino acids) EC50=>10 µM; BNP (32 amino acids) EC50>10 µM (all n=3–4). While the NPs were generally more potent in the CEPI-17-CL4 cells than in p-CEPI cells (n=4–9; p<0.01), the rank order of potency of the peptides was essentially the same in both cell types. Effects of CNP fragment in p-CEPI and CEPI-17-CL4 cells were potently blocked by HS-142–1, an NPR-B receptor subtype-selective antagonist (Ki=0.25±0.05 µM in CEPI-CL4–17; Ki=0.44±0.09 µM in p-CEPIs; n=6–7) but less so by an NPR-A receptor antagonist, isatin (Ki=5.3–7.8 µM, n=3–7).
Our studies showed the presence of NPR-A and NPR-B (mRNAs and protein) in p-CEPI and CEPI-17-CL4 cells and in human corneal epithelial tissue. However, detailed pharmacological studies revealed NPR-B to be the predominant functionally active receptor in both cell-types whose activation leads to the generation of cGMP. While the physiologic role(s) of the NP system in corneal function remains to be delineated, our multidisciplinary findings pave the way for such future investigations.
The heart adapts to an increased workload through the activation of a hypertrophic response within the cardiac ventricles. This response is characterized by both an increase in the size of the individual cardiomyocytes and an induction of a panel of genes normally expressed in the embryonic and neonatal ventricle, such as atrial natriuretic peptide (ANP). ANP and brain natriuretic peptide (BNP) exert their biological actions through activation of the natriuretic peptide receptor-1 (Npr1). The current study examined mice lacking Npr1 (Npr1−/−) activity and investigated the effects of the absence of Npr1 signaling during cardiac development on embryo viability, cardiac structure and gene and protein expression. Npr1−/−embryos were collected at embryonic day (ED) 12.5, 15.5 and neonatal day 1 (ND 1). Npr1−/−embryos occurred at the expected Mendelian frequency at ED 12.5, but knockout numbers were significantly decreased at ED 15.5 and ND 1. There was no indication of cardiac structural abnormalities in surviving embryos. However, Npr1−/−embryos exhibited cardiac enlargement (without fibrosis) from ED 15.5 as well as significantly increased ANP mRNA and protein expression compared to wild-type (WT) mice, but no concomitant increase in expression of the hypertrophy-related transcription factors, Mef2A, Mef2C, GATA-4, GATA-6 or serum response factor (SRF). However, there was a significant decrease in Connexin-43 (Cx43) gene and protein expression at mid-gestation in Npr1−/−embryos. Our findings suggest that the mechanism by which natriuretic peptide signaling influences cardiac development in Npr1−/− mice is distinct from that seen during the development of pathological cardiac hypertrophy and fibrosis. The decreased viability of Npr1−/−embryos may result from a combination of cardiomegaly and dysregulated Cx43 protein affecting cardiac contractility.
Akt1; Atrial natriuretic peptide; Calcineurin A; Cardiac hypertrophy; Connexin 43; Gene expression; Heart development; Npr1; Transcription factor
Natriuretic peptide signaling is important in the regulation of blood pressure as well as in the growth of multiple cell types. To examine the role of natriuretic peptide signaling in atherosclerosis, we crossbred mice that lack natriuretic peptide receptor A (NPRA; Npr1−/−) with atherosclerosis-prone mice that lack apolipoprotein E (apoE; Apoe−/−).
Methods and Results
Doubly deficient Npr1−/− Apoe−/− mice have increased blood pressure relative to Npr1+/+ Apoe−/− mice (118±4 mm Hg compared with 108±2 mm Hg, P<0.05) that is coincident with a 64% greater atherosclerotic lesion size (P<0.005) and more advanced plaque morphology. Additionally, aortic medial thickness is increased by 52% in Npr1−/− Apoe−/− mice relative to Npr1+/+ Apoe−/− mice (P<0.0001). Npr1−/− Apoe−/− mice also have significantly greater cardiac mass (9.0±0.3 mg/g body weight) than either Npr1+/+ Apoe−/− mice (5.8±0.2 mg/g) or Npr1−/− Apoe+/+ mice (7.1±0.2 mg/g), suggesting that the lack of both NPRA and apoE synergistically enhances cardiac hypertrophy.
These data provide evidence that NPR1 is an atherosclerosis susceptibility locus and represents a potential link between atherosclerosis and cardiac hypertrophy. Our results also suggest roles for Npr1 as well as Apoe in regulation of hypertrophic cell growth.
natriuretic peptide receptor A; atherosclerosis; cardiac hypertrophy; vascular smooth muscle cells
The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.
We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.
Methods and Results
Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.
These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.