Guanylate cyclase-B Research Articles

The role of the cartilage guanylyl cyclase-B receptor in craniofacial skeletal development

ObjectiveC-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent endochondral bone growth factor that exerts its biological effects via the guanylate cyclase B (GC-B) receptor. We previously demonstrated that CNP knockout (KO) mice exhibited midfacial hypoplasia along the sagittal plane; however, the effects of GC-B (the receptor for CNP) on endochondral ossification in the maxillofacial region remain unclear, and the mechanism of the CNP/GC-B system has not been elucidated. MethodsWe investigated the physiological significance of GC-B in the cartilage of the craniofacial region through analysis of cartilage-specific GC-B KO mice. Morphological assessments were performed at 12 weeks old, with histological analyses performed at 2 weeks old. ResultsGC-B-KO mice exhibited sagittal midfacial hypoplasia, foramen magnum stenosis, and spinal canal stenosis. Histological examination revealed reduced thickness in the spheno-occipital synchondrosis (SOS), a critical growth center in cranio-maxillofacial skeletal development. The hypertrophic zone of the SOS exhibited reduced thickness, accompanied by a reduction in cell count in this area. ConclusionsThis study highlights the essential role of GC-B receptors in craniofacial morphology contributing to our understanding of the mechanisms underlying facial morphological abnormalities, foramen magnum stenosis, and spinal canal stenosis.

C-type natriuretic peptide (CNP)/guanylate cyclase B (GC-B) system and endothelin-1(ET-1)/ET receptor A and B system in human vasculature.

To assess the physiological and clinical implications of the C-type natriuretic peptide (CNP)/guanylyl cyclase B (GC-B) system in the human vasculature, we have examined gene expressions of CNP and its receptor, GC-B, in human vascular endothelial cells (ECs) and smooth muscle cells (SMCs) and have also compared the endothelin-1(ET-1)/endothelin receptor-A (ETR-A) and endothelin receptor-B (ETR-B) system in human aortic ECs (HAECs) and vascular SMCs (HSMCs) in vitro. We also examined these gene expressions in human embryonic stem (ES)/induced pluripotent stem cell (iPS)-derived ECs and mural cells (MCs). A little but significant amount of mRNA encoding CNP was detected in both human ES-derived ECs and HAECs. A substantial amount of GC-B was expressed in both ECs (iPS-derived ECs and HAECs) and SMCs (iPS-derived MCs and HSMCs). ET-1 was expressed solely in ECs. ETR-A was expressed in SMCs, while ETR-B was expressed in ECs. These results indicate the existence of a vascular CNP/GC-B system in the human vascular wall, indicating the evidence for clinical implication of the CNP/GC-B system in concert with the ET-1/ETR-A and ETR-B system in the human vasculature.

Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation

In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

Natriuretic peptides in brain physiology

Abstract Natriuretic peptides (NPs) regulate salt and water homeostasis by inducing natriuresis and diuresis in the kidney. These actions in addition to those via the heart and vascular system play important roles in the regulation of blood pressure. In the central nervous system NPs play a significant role in neuronal development, synaptic transmission and neuroprotection. Currently, six different human NPs have been described: atrial natriuretic peptide (ANP), urodilatin (URO, renal natriuretic peptide), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as guanylin and uroguanylin. ANP, URO and BNP activate the natriuretic peptide receptor A (NPR-A or guanylate cyclase A (GC-A)) while CNP activates natriuretic peptide receptor B (NPR-B or guanylate cyclase B (GC-B)). Guanylin and uroguanylin are known to activate guanylate cyclase C (GC-C). The receptors GC-A, GC-B, and GC-C are widely expressed in the human body. Currently, GC-B and CNP seems to have the highest expression in central nervous system compared to other NPs and their receptors. All known NPs generate intracellular cyclic GMP (cGMP) by activating their specific guanylate cyclase receptors. Subsequently, cGMP is able to activate protein kinase I or II (PKG I or II) and/or directly regulate transmembrane proteins such as ion channels, transporters and pumps. NPs also bind to the natriuretic peptide receptor C (also called clearance receptor NPR-C) which is a major pathway for the degradation of NPs and has no guanylate cyclase activity. In this review we will focus on new insights regarding the physiological effects of NPs in the brain, especially specific areas of their signaling pathways in neurons and glial cells.

Recent advances in cartilage metabolism research

The past few years have seen an enormous increase in the knowledge of the signaling pathways and the transcription factors that control endochondral bone development. Recent studies of rare chondrodysplasias that exhibit disproportionate dwarfism due to the impairment of endochondral ossification have identified many responsible genes and have clarified pathogenic mechanisms of these diseases by using molecular genetics and biology. The details of human diseases related to collagen type II alpha1 (COL2A1), collagen type X alpha1 (COL10A1), parathyroid hormone (PTH) /parathyroid hormone-related protein (PTHrP) receptor, guanylate cyclase B (GC-B), sry-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), which play important roles in the process of endochondral ossification, will be outlined.

Enzyme-ultracytochemical study of adenylate and guanylate cyclases in normal and pathologic human nasal mucosa.

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.

Characterization of VIP and PACAP receptors in cultured rat penis corpus cavernosum smooth muscle cells and their interaction with guanylate cyclase-B receptors.

Penile corpus cavernosum smooth muscle relaxation can be induced by both cyclic AMP and cyclic GMP-elevating agents, but possible interactions between these two signalling pathways are still poorly understood. Using in vitro cultured rat penile corpus cavernosum smooth muscle (CCSM) cells, we have characterized the local expression and functional activities of receptors for the cAMP-elevating peptides, PACAP and VIP, and for the cGMP-elevating peptides, CNP and ANP. Stimulation of the cells with various concentrations of PACAP(-27/-38) or VIP resulted in rapid and dose-dependent increases in cyclic AMP levels. RT-PCR analyses revealed gene expression of PAC(1) and VPAC(2) but not of VPAC(1) receptors in the cells. The natriuretic peptide, CNP, and the nitric oxide donor, sodium nitroprusside, were capable of enhancing cyclic GMP formation, indicating the presence of membrane-associated in addition to soluble guanylate cyclase (sGC) activities in these cells. Findings that cyclic GMP formation was preferentially activated by CNP but not by the related peptide, ANP, were consistent with RT-PCR analyses, demonstrating gene expression of the CNP receptor, GC-B, but not of the ANP receptor, GC-A, in these cells. Prior exposure of the cells to 10(-8) M PACAP resulted in a marked down-regulation of GC-B activity, whereas sGC was not affected. These findings provide functional and molecular evidence for the presence of three receptors, PAC(1), VPAC(2) and GC-B, involved in cyclic nucleotide signalling in penile CCSM cells. The observed cross-talk of the PACAP/VIP receptors with GC-B but not with sGC may have implications for the therapy of erectile dysfunction.

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population.

We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart.

C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization.

We recently reported that C-type natriuretic peptide (CNP) occurs in vascular endothelial cells and acts as a vascular-type natriuretic peptide. In the present study, we stimulated the cGMP cascade in proliferating smooth muscle cells (SMCs), in which particulate guanylate cyclase-B, the specific receptor for CNP, is predominantly expressed, by use of an adenovirus encoding rat CNP cDNA (Ad.CNP). In the Ad.CNP-treated cultured SMCs, CNP caused the growth inhibition of SMCs at G(1) phase with an early increase of p21(CIP1/WAF1) expression and subsequent upregulation of p16(INK4a). The expression of smooth muscle myosin heavy chain-2, which is the molecular marker of highly differentiated SMCs, was reinduced in the Ad.CNP-treated SMCs. The Ad.CNP-treated SMCs also reexpressed particulate guanylate cyclase-A, which shows high affinity to atrial and brain natriuretic peptide and is exclusively expressed in well-differentiated SMCs. CNP, which was overexpressed in rabbit femoral arteries in vivo at the time of balloon injury, significantly suppressed neointimal formation. Furthermore, an enhancement of the expression of smooth muscle myosin heavy chain-2 occurred in the residual neointima. In addition, early regeneration of endothelial cells was observed in the Ad.CNP-infected group. Thus, stimulation of cGMP cascade in proliferating dedifferentiated SMCs can induce growth inhibition and redifferentiation of SMCs with accelerated reendothelialization.

Pergnancy increases C-type natriuretic peptide (CNP) and its receptor particulate guanylate cyclase-B levels in ovine uterine but not systemic arteries

Pergnancy increases C-type natriuretic peptide (CNP) and its receptor particulate guanylate cyclase-B levels in ovine uterine but not systemic arteries

Pregnancy increases C-type Natriuretic Peptide (CMP) and its receptor participate guanylate cyclase-B levels in ovine uterine but not systemic arteries

Pregnancy increases C-type Natriuretic Peptide (CMP) and its receptor participate guanylate cyclase-B levels in ovine uterine but not systemic arteries

C-Type Natriuretic Peptide as an Autocrine/Paracrine Regulator of Osteoblast: Evidence for Possible Presence of Bone Natriuretic Peptide System

C-type natriuretic peptide (CNP) is a local regulator in the brain and vascular wall. We present data to demonstrate the production and action of CNP in the osteoblast. CNP increased cGMP production, far more potently than atrial natriuretic peptide (ANP) in an osteoblastic cell line, MC3T3-E1. Since ANP and CNP are the ligands for two particulate guanylate cyclases, guanylate cyclase-A (GC-A) and guanylate cyclase-B (GC-B), respectively, these results reveal the expression of GC-B in MC3T3-E1. In addition, CNP mRNA and CNP-like immunoreactivity were detected in cell extracts from MC3T3-E1 and its culture medium, respectively. Both CNP and 8-bromo cGMP dose-dependently decreased [3H]thymidine uptake, without affecting alkaline phosphatase activity. These results indicate that CNP is a novel autocrine/paracrine regulator of osteoblast and suggest the presence of “bone natriuretic peptide system.”

Altered gene expression of natriuretic peptide receptor subtypes in the kidney of stroke-prone spontaneously hypertensive rats.

1. To elucidate the physiological and pathophysiological role of the natriuretic peptide system in the progression of hypertensive renal disease, we examined the gene expression of natriuretic peptide receptor subtypes, guanylate cyclase-A (GC-A), guanylate cyclase-B (GC-B) and clearance receptor (C receptor), in the kidney of stroke-prone spontaneously hypertensive rats (SHRSP) at 8 and 20 weeks of age, and compared them with their gene expression in age-matched Wistar-Kyoto (WKY) rats. 2. Northern blot analyses revealed that messages for three natriuretic peptide receptor subtypes were expressed in the kidney, and their expressions were higher in the glomeruli than in the whole kidney in each strain. 3. In 20 week old rats with established hypertension, the glomerular concentration of GC-A mRNA was significantly higher in SHRSP than in WKY. The concentrations of GC-B and C receptor mRNA in the glomeruli tended to increase and decrease, respectively, but they were not statistically significant in SHRSP. 4. In 8 week old rats, the glomerular concentrations of GC-A, GC-B and C receptor mRNA were not significantly different between SHRSP and WKY. 5. This study demonstrates that in the progression of hypertension, the expression of GC-A, which mediates biological actions of natriuretic peptides, is enhanced in the kidney of SHRSP compared to that of WKY. Together with the augmented secretion of the ligands previously revealed, altered expression of natriuretic peptide receptor subtypes in SHRSP may have a deterrent role in the development of hypertension and its renal complications.

Selective downregulation of ANP-clearance-receptor gene expression in lung of rats adapted to hypoxia

To test the hypothesis that expression of atrial natriuretic peptide (ANP)-receptor genes is modified to provide a compensatory mechanism against hypoxic pulmonary hypertension, steady state mRNA levels for the ANP-A receptor (or guanylate cyclase-A; ANPAR), ANP-B receptor (or guanylate cyclase-B; ANPBR), and ANP-clearance receptor (ANPCR) were quantitated by Northern blot and slot-blot analysis in lung, kidney, spleen, and liver of hypoxia-adapted rats and air controls. Exposure of rats to short-term (48 h) and chronic (4 wk) hypoxia (10% O2, 1 atm) did not affect lung ANPAR-mRNA levels. Lung ANPBR-mRNA levels were unchanged by short-term hypoxia but selectively increased (approximately twofold) by chronic hypoxia. ANPCR-mRNA levels were selectively and significantly downregulated by 48-h and 4-wk hypoxia in lung but were unchanged or upregulated in other tissues. Lung ANPCR gene transcription, assessed by nuclear-runoff analysis, was decreased by hypoxia. These data support the conclusion that altered pulmonary ANP-receptor gene expression modulates the development of hypoxic pulmonary hypertension.

Capillary and interstitial responses to inflammation and burn injury

C-type natriuretic peptide (CNP) is a local regulator in the brain and vascular wall. We present data to demonstrate the production and action of CNP in the osteoblast. CNP increased cGMP production, far more potently than atrial natriuretic peptide (ANP) in an osteoblastic cell line, MC3T3-E1. Since ANP and CNP are the ligands for two particulate guanylate cyclases, guanylate cyclase-A (GC-A) and guanylate cyclase-B (GC-B), respectively, these results reveal the expression of GC-B in MC3T3-E1. In addition, CNP mRNA and CNP-like immunoreactivity were detected in cell extracts from MC3T3-E1 and its culture medium, respectively. Both CNP and 8-bromo cGMP dose-dependently decreased [3H]thymidine uptake, without affecting alkaline phosphatase activity. These results indicate that CNP is a novel autocrine/paracrine regulator of osteoblast and suggest the presence of “bone natriuretic peptide system.”

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)

Effects of a high-salt diet on hepatic atrial natriuretic peptide receptor expression in Dahl salt-resistant and salt-sensitive rats.

To investigate the effects of a salt diet and of salt-induced hypertension on hepatic atrial natriuretic peptide (ANP) receptors in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats. DS and DR rats were maintained for 5 weeks on either normal- (0.8% w:w NaCl) or high- (8% w:w NaCl) salt diets. Blood pressures were recorded by a tail-cuff analyser and plasma ANP concentrations were determined by radioimmunoassay. ANP binding and guanylate cyclase activities in purified liver plasma membrane fractions were determined by conventional radioreceptor and enzymatic techniques. DS rats exhibited higher blood pressure than DR rats on the equivalent diet and in both groups the high-salt diet significantly increased systolic blood pressures. The high-salt diet significantly reduced plasma ANP concentrations in DR rats but not DS rats. Membrane fractions from DS rats exhibited increased ANP receptor densities compared to membranes isolated from DR rats on the equivalent diet. The high-salt diet induced a significant increase in receptor density in the DS but not the DR group. The fractional displacement of [125I]-ANP binding by the truncated, ring-deleted analogue des[QSGLG]-4,23-ANP-NH2 was reduced in membrane fractions isolated from DS rats maintained on the high-salt diet. There was no change in ANP receptor affinity. Increases in receptor density in DS rats were accompanied by increases in both basal and ANP-stimulated guanylate cyclase activities. These results indicate that plasma membrane isolated from the liver of DS rats exhibit increased expression of the guanylate cyclase-linked ANP-B (guanylate cyclase-A and/or guanylate cyclase-B) receptors over similar preparations isolated from DR rats. ANP B receptor density is further increased when DS rats are maintained on a high-salt diet.

Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclase-A (GC-A), the ANP-B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured cells with the aid of the binding assay, Northern blot technique, and the cGMP production method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP greater than CNP greater than BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP greater than or equal to BNP much greater than CNP, but that via the ANP-B receptor (GC-B) was CNP greater than ANP greater than or equal to BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system.

Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide

To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclase-A (GC-A), the ANP-B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured cells with the aid of the binding assay, Northern blot technique, and the cGMP production method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP greater than CNP greater than BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP greater than or equal to BNP much greater than CNP, but that via the ANP-B receptor (GC-B) was CNP greater than ANP greater than or equal to BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system.