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1.  Novel mutations in natriuretic peptide receptor-2 gene underlie acromesomelic dysplasia, type maroteaux 
BMC Medical Genetics  2012;13:44.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2350-13-44
PMCID: PMC3458994  PMID: 22691581
Acromesomelic dysplasia-type Maroteaux; gene NPR2; missence mutation (T907M); splice site mutation c.2986 + 2 T > G
2.  Exclusion of chromosome 9 helps to identify mild variants of acromesomelic dysplasia Maroteaux type 
Journal of Medical Genetics  2000;37(1):52-54.
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
doi:10.1136/jmg.37.1.52
PMCID: PMC1734441  PMID: 10633136
3.  Anesthesia for a patient of acromesomelic dysplasia with associated hydrocephalus, Arnold Chiari malformation and syringomyelia 
Acromesomelic dysplasias are autosomal recessive osteochondrodysplasias. Acromesomelic dysplasia Maroteaux-type (AMDM), also known as St Helena dysplasia, is of two types: The classical and the mild variety. About 50 cases of AMDM have been reported till date, most of them being the classical variety. There is scarcity of literature on anesthesia for such patients. We are reporting a case of general anesthetic management of AMDM, associated with hydrocephalus, Arnold Chiari malformation type-1 and syringomyelia. The patient was a 10-year-old short-statured boy who presented with symptomatic thoracic kyphoscoliosis, gibbus deformity and back pain. On examination, there was no neurological deficit. Radiology revealed thoracic kyphoscoliosis, mild ventriculomegaly and upper cervical syringomyelia. The patient underwent posterior fossa decompression in the prone position under general anesthesia. We will discuss the anesthetic considerations for such patients and review the pertinent literature.
doi:10.4103/0970-9185.119153
PMCID: PMC3819856  PMID: 24249999
Acromesomelic dysplasia; arnold chiari malformation; osteochondrodysplasia; short stature; syringomyelia
4.  An Overgrowth Disorder Associated with Excessive Production of cGMP Due to a Gain-of-Function Mutation of the Natriuretic Peptide Receptor 2 Gene 
PLoS ONE  2012;7(8):e42180.
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.
doi:10.1371/journal.pone.0042180
PMCID: PMC3411678  PMID: 22870295
5.  Granulosa Cell Ligand NPPC and Its Receptor NPR2 Maintain Meiotic Arrest in Mouse Oocytes 
Science (New York, N.Y.)  2010;330(6002):366-369.
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.
doi:10.1126/science.1193573
PMCID: PMC3056542  PMID: 20947764
6.  Disruption of Npr1 gene differentially regulates the juxtaglomerular and distal tubular renin levels in null mutant mice 
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.
PMCID: PMC3466495  PMID: 23071870
Distal tubular and juxtaglomerular renin; immunohistochemistry; atrial natriuretic peptide receptor-A; gene-disrupted mice; hypertension
7.  Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes 
Developmental Biology  2012;366(2):308-316.
In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 minutes, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2 hours, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.
doi:10.1016/j.ydbio.2012.04.019
PMCID: PMC3358460  PMID: 22546688
oocyte; meiosis; mouse ovarian follicle; luteinizing hormone; guanylyl cyclase; cyclic GMP
8.  Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases. 
Molecular and Cellular Biology  1992;12(6):2581-2590.
The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.
Images
PMCID: PMC364451  PMID: 1350322
9.  Resistance to endotoxic shock in mice lacking natriuretic peptide receptor-A 
British Journal of Pharmacology  2010;160(8):2045-2054.
Background and purpose:
Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.
Experimental approach:
Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.
Key results:
LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NOx] and cyclic GMP).
Conclusions and implications:
These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition.
doi:10.1111/j.1476-5381.2010.00830.x
PMCID: PMC2913103  PMID: 20649600
atrial natriuretic peptide; inducible nitric oxide synthase; cyclic GMP; endotoxaemia; hypotension; vascular smooth muscle
10.  Resistance to endotoxic shock in mice lacking natriuretic peptide receptor-A 
British Journal of Pharmacology  2010;160(8):2045-2054.
Background and purpose:
Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.
Experimental approach:
Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.
Key results:
LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NOx] and cyclic GMP).
Conclusions and implications:
These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition.
doi:10.1111/j.1476-5381.2010.00830.x
PMCID: PMC2913103  PMID: 20649600
atrial natriuretic peptide; inducible nitric oxide synthase; cyclic GMP; endotoxaemia; hypotension; vascular smooth muscle
11.  ENHANCED ACTIVATION OF PRO-INFLAMMATORY CYTOKINES IN MICE LACKING NATRIURETIC PEPTIDE RECEPTOR-A 
Peptides  2006;28(4):893-899.
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.
doi:10.1016/j.peptides.2006.12.009
PMCID: PMC2743377  PMID: 17267074
Natriuretic peptide receptor-A; GC activity; cGMP signaling; and cytokines
12.  Influence of natriuretic peptide receptor-1 on survival and cardiac hypertrophy during development 
Biochimica et biophysica acta  2009;1792(12):1175-1184.
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.
doi:10.1016/j.bbadis.2009.09.009
PMCID: PMC2894466  PMID: 19782130
Akt1; Atrial natriuretic peptide; Calcineurin A; Cardiac hypertrophy; Connexin 43; Gene expression; Heart development; Npr1; Transcription factor
13.  Characterization and functional expression of the natriuretic peptide system in human lens epithelial cells 
Molecular Vision  2010;16:630-638.
Purpose
The family of natriuretic peptides (NPs); atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three associated receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C) has never been documented in human lens epithelial cells. The study described herein was designed to demonstrate both expression and functionality of components of the natriuretic peptides and natriuretic peptide receptors in the human lens epithelial cell line, HLE-B3 and in normal human lens epithelial cell cultures (nHLE).
Methods
Reverse transcriptase-polymerase chain reaction (RT-PCR) along with confirmation by DNA sequencing and real-time quantitative RT–PCR was used to identify and demonstrate expression of mRNA for the natriuretic peptide family. Authentication of protein expression of the natriuretic peptide receptors was determined by using formaldehyde-fixed, Saponin-permeabilized cells (HLE-B3) or methanol:acetone-fixed and permeabilized cells (nHLE) using conventional immunofluorescence techniques. Enzyme-linked immunosorbent assay was used to determine cyclic GMP (cGMP) activity as stimulated by exogenous addition of natriuretic peptides.
Results
Using RT–PCR with confirmation by DNA sequencing and real-time quantitative RT–PCR, HLE-B3 cells were shown to express mRNA for ANP, BNP, and CNP along with their associated receptors. Conventional immunofluorescence on the permeabilized cells confirmed positive diffuse staining indicating the presence of the three natriuretic peptide receptors in both HLE-B3 and nHLE cells. All three natriuretic peptides educe a cGMP response in the rank order CNP>>ANP≈BNP indicating that the natriuretic peptide family is functional in HLE-B3 cells.
Conclusions
The data indicates that ANP, BNP, and CNP and natriuretic peptide receptor transcripts are expressed and are functional in human lens epithelial cells. The cellular expression of NPs and NPRs, as well as the demonstration that all three NPs activate guanylyl cyclase suggests a potential role in maintaining lens epithelial cell homeostasis.
PMCID: PMC2852620  PMID: 20700369
14.  The C-type Natriuretic Peptide Induces Thermal Hyperalgesia Through a Non-canonical Gβγ-dependent Modulation of TRPV1 Channel 
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.
doi:10.1523/JNEUROSCI.1330-12.2012
PMCID: PMC3461320  PMID: 22933780
15.  Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor 
Molecular and Cellular Biology  1998;18(4):2164-2172.
Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (∼20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.
PMCID: PMC121455  PMID: 9528788
16.  A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization 
Molecular Biology of the Cell  1999;10(6):1811-1820.
Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.
PMCID: PMC25375  PMID: 10359598
17.  NPR-B natriuretic peptide receptors in human corneal epithelium: mRNA, immunohistochemistochemical, protein, and biochemical pharmacology studies 
Molecular Vision  2010;16:1241-1252.
Purpose
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.
Methods
NPR presence was shown by RT–PCR, western blot analysis, and indirect immunofluoresence. cGMP accumulation was determined using an enzyme immunoassay.
Results
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).
Conclusions
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.
PMCID: PMC2903464  PMID: 20664698
18.  Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide 
British Journal of Pharmacology  2011;164(2b):584-597.
BACKGROUND AND PURPOSE
C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the Gi-coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis.
EXPERIMENTAL APPROACH
Proliferation of human umbilical vein endothelial cells (HUVEC), rat aortic smooth muscle cells (RAoSMC) and endothelial and vascular smooth muscle cells from NPR3 knockout (KO) mice was investigated in vitro.
KEY RESULTS
CNP (1 pM–1 µM) facilitated HUVEC proliferation and inhibited RAoSMC growth concentration-dependently. The pro- and anti-mitogenic effects of CNP were blocked by the NPR3 antagonist M372049 (10 µM) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (30 µM) and were absent in cells from NPR3 KO mice. Activation of ERK 1/2 by CNP was inhibited by Pertussis toxin (100 ng·mL−1) and M372049 (10 µM). In HUVEC, ERK 1/2 activation enhanced expression of the cell cycle promoter, cyclin D1, whereas in RAoSMC, ERK 1/2 activation increased expression of the cell cycle inhibitors p21waf1/cip1 and p27kip1.
CONCLUSIONS AND IMPLICATIONS
A facet of the vasoprotective profile of CNP is mediated via NPR3-dependent ERK 1/2 phosphorylation, resulting in augmented endothelial cell proliferation and inhibition of vascular smooth muscle growth. This pathway may offer an innovative approach to reversing the endothelial damage and vascular smooth muscle hyperplasia that characterize many vascular disorders.
doi:10.1111/j.1476-5381.2011.01400.x
PMCID: PMC3178781  PMID: 21457229
natriuretic peptide receptor-C; C-type natriuretic peptide; endothelial cell; vascular smooth muscle cell; atherosclerosis; restenosis; extracellular signal-regulated kinase; knockout mice
19.  Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide 
British Journal of Pharmacology  2011;164(2b):584-597.
BACKGROUND AND PURPOSE
C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the Gi-coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis.
EXPERIMENTAL APPROACH
Proliferation of human umbilical vein endothelial cells (HUVEC), rat aortic smooth muscle cells (RAoSMC) and endothelial and vascular smooth muscle cells from NPR3 knockout (KO) mice was investigated in vitro.
KEY RESULTS
CNP (1 pM–1 µM) facilitated HUVEC proliferation and inhibited RAoSMC growth concentration-dependently. The pro- and anti-mitogenic effects of CNP were blocked by the NPR3 antagonist M372049 (10 µM) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (30 µM) and were absent in cells from NPR3 KO mice. Activation of ERK 1/2 by CNP was inhibited by Pertussis toxin (100 ng·mL−1) and M372049 (10 µM). In HUVEC, ERK 1/2 activation enhanced expression of the cell cycle promoter, cyclin D1, whereas in RAoSMC, ERK 1/2 activation increased expression of the cell cycle inhibitors p21waf1/cip1 and p27kip1.
CONCLUSIONS AND IMPLICATIONS
A facet of the vasoprotective profile of CNP is mediated via NPR3-dependent ERK 1/2 phosphorylation, resulting in augmented endothelial cell proliferation and inhibition of vascular smooth muscle growth. This pathway may offer an innovative approach to reversing the endothelial damage and vascular smooth muscle hyperplasia that characterize many vascular disorders.
doi:10.1111/j.1476-5381.2011.01400.x
PMCID: PMC3178781  PMID: 21457229
natriuretic peptide receptor-C; C-type natriuretic peptide; endothelial cell; vascular smooth muscle cell; atherosclerosis; restenosis; extracellular signal-regulated kinase; knockout mice
20.  Differential gene expression of the three natriuretic peptides and natriuretic peptide receptor subtypes in human liver. 
Gut  1997;40(1):145-150.
BACKGROUND: Various effects of atrial natriuretic peptide (ANP) on the liver have been observed. However, there is limited information about the types of receptors for natriuretic peptides expressed by the human liver. AIM: To investigate gene expression of the three NP receptor types (NPR) as well as of the NP in human liver. METHODS: Presence of mRNA coding for all three NPR and for ANP, brain and C-type natriuretic peptide (BNP, CNP) was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Human liver tissues and hepatocellular carcinoma tissues were examined. RESULTS: Specific PCR products for all three NPR, namely NPR-A, B, and C, could be detected. Moreover, ANP and CNP, but not BNP mRNA was detectable. The concentration of ANP transcripts was up to fivefold higher in hepatocellular carcinoma compared with non-tumorous liver tissue of the same subjects. No difference in the expression of NP receptors relative to GAPDH mRNA of tumorous and non-tumorous tissue was observed except of slightly increased NPR-A transcripts. CONCLUSION: These data show that NPR transcripts are coexpressed with ANP and CNP mRNA in the human liver. This provides evidence for a local NP system in the human liver.
Images
PMCID: PMC1027025  PMID: 9155593
21.  Differential regulation of natriuretic peptide receptor messenger RNAs during the development of cardiac hypertrophy in the rat. 
Journal of Clinical Investigation  1993;92(6):2702-2712.
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.
Images
PMCID: PMC288468  PMID: 7902846
22.  Atrial natriuretic peptide signal pathway upregulated in stomach of streptozotocin-induced diabetic mice 
AIM: To investigate atrial natriuretic peptide (ANP) secretion from gastric mucosa and the relationship between the ANP/natriuretic peptide receptor type A (NPR-A) pathway and diabetic gastroparesis.
METHODS: Male imprinting control region (ICR) mice (4 wk old) were divided into two groups: control mice, and streptozotocin-induced diabetic mice. Eight weeks after injection, spontaneous gastric contraction was recorded by using physiography in control and streptozotocin-induced diabetic mice. The ANP-positive cells in gastric mucosa and among dispersed gastric epithelial cells were detected by using immunohistochemistry and flow cytometry, respectively. ANP and natriuretic peptide receptor type A (NPR-A) gene expression in gastric tissue was observed by using the reverse transcriptase polymerase chain reaction.
RESULTS: The frequency of spontaneous gastric contraction was reduced from 12.9 ± 0.8 cycles/min in the control group to 8.4 ± 0.6 cycles/min in the diabetic mice (n = 8, P < 0.05). However, the amplitude of contraction was not significantly affected in the diabetic group. The depletion of interstitial cells of Cajal in the gastric muscle layer was observed in the diabetic mice. ANP-positive cells were distributed in the gastric mucosal layer and the density index of ANP-positive cells was increased from 20.9 ± 2.2 cells/field in control mice to 51.8 ± 2.9 cells/field in diabetic mice (n = 8, P < 0.05). The percentage of ANP-positive cells among the dispersed gastric epithelial cells was increased from 10.0% ± 0.9% in the control mice to 41.2% ± 1.0% in the diabetic mice (n = 3, P < 0.05). ANP and NPR-A genes were both expressed in mouse stomach, and the expression was significantly increased in the diabetic mice.
CONCLUSION: These results suggest that the ANP/NPR-A signaling pathway is upregulated in streptozotocin-induced diabetic mice, and contributes to the development of diabetic gastroparesis.
doi:10.3748/wjg.v16.i1.48
PMCID: PMC2799916  PMID: 20039448
Diabetes mellitus; Atrial natriuretic peptide; Gastric mucosa; Gastroparesis
23.  Novel analog of atrial natriuretic peptide selective for receptor-A produces increased diuresis and natriuresis in rats. 
Journal of Clinical Investigation  1996;98(4):969-976.
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.
PMCID: PMC507512  PMID: 8770869
24.  Natriuretic Peptide Receptor-3 Gene (NPR3) Nonsynonymous Polymorphism Results in Significant Reduction in Protein Expression Because of Accelerated Degradation 
Background
The primary role of natriuretic peptide receptor-3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups.
Methods and Results
DNA samples from 96 European American, 96 African American, and 96 Han Chinese American healthy subjects were used to resequence NPR3 exons, splice junctions, and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous single-nucleotide polymorphisms, 7 were novel. Expression constructs were created for the nonsynonymous single-nucleotide polymorphisms. HEK293 cells were transfected with constructs for wild type and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of wild-type protein, primarily because of autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with wild-type conformation and could result in protein misfolding or instability.
Conclusions
Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity because of degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.
doi:10.1161/CIRCGENETICS.112.964742
PMCID: PMC3685298  PMID: 23493048
natriuretic peptide receptor-3; natriuretic peptides; pharmacogenetics; polymorphism
25.  Characterization of Two ENU-Induced Mutations Affecting Mouse Skeletal Morphology 
G3: Genes|Genomes|Genetics  2013;3(10):1753-1758.
Using the N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we have identified two skeletal morphology mutants, Skm1 and Skm2. Positional cloning and candidate gene sequencing localized the causative point mutations within the genes coding for natriuretic peptide receptor C (NPR-C) and filamin b (FLNB), respectively. Mice that carry a mutation in Npr3 exhibit a skeletal overgrowth phenotype, resulting in an elongated body and kyphosis. Skm2 mice, carrying a mutation in Flnb, present with scoliosis and lordosis. These mutant mice will serve as useful models for the study of vertebral malformations.
doi:10.1534/g3.113.007310
PMCID: PMC3789799  PMID: 23979929

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