C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C)

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.     

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Concentration of mRNA for the natriuretic peptide receptor-C in hypertrophic chondrocytes of the fetal mouse tibia

The roles of natriuretic peptides in cardiovascular homeostasis have been well characterized. A recent study revealed that mice lacking natriuretic peptide receptor-C (NPR-C) exhibit skeletal-overgrowth. We therefore, performed in situ hybridization with riboprobes to determine the localization of mRNAs for receptors for natriuretic peptides in the growth plate of the fetal mouse tibia The amount of mRNA for NPR-A was below the detectable level in the growth plate. The mRNA for NPR-B was detected predominantly in proliferating chondrocytes. By contrast, high levels of mRNA for NPR-C were found in hypertrophic chondrocytes. In other regions of the growth plate, the levels of mRNA for NPR-C were very low. The patterns of expression of mRNAs for NPR-B and NPR-C, namely, subtype switching during differentiation from proliferating chondrocytes to hypertrophic chondrocytes, suggest that these receptors might be involved in the growth and differentiation of the growth plate during fetal development in the mouse.     

Diabetes-induced loss of gastric ICC accompanied by up-regulation of natriuretic peptide signaling pathways in STZ-induced diabetic mice

Our previous study demonstrated that natriuretic peptides (NPs) play an inhibitory role in regulation of gastric smooth muscle motility. However, it is not clear whether NPs are involved in diabetics-induced loss of gastric interstitial cell of Cajal (ICC). The present study was designed to investigate the relationship between diabetics-induced loss of gastric ICC and natriuretic peptide signaling pathway in streptozotocin (STZ)-induced diabetic mice. The results showed that the protein expression levels of c-Kit and membrane-bound stem cell factor (mSCF) in gastric smooth muscle layers were decreased in STZ-induced diabetic mice. However, both mRNA and protein expression levels of natriuretic peptide receptor (NPR)-A, B and C were increased in the same place of the diabetic mice. The amplitude of spontaneous contraction in gastric antral smooth muscles was inhibited by C-type natriuretic peptide (CNP) dose-dependently and the inhibitory effect was potentiated in diabetic mice. Pretreatment of the cultured gastric smooth muscle cells (GSMCs) with different concentration of CNP can significantly decrease the mSCF expression level. 8-Bromoguanosine-3′,5′-cyclomo-nophosphate (8-Br-cGMP), a membrane permeable cGMP analog, mimicked the effect of CNP but not cANF (a specific NPR-C agonist). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay showed that high concentration of cANF (10⁻⁶ mol/L) inhibited cell proliferation in cultured GSMCs. These findings suggest that up-regulation of NPs/NPR-A, B/cGMP and NPs/NPR-C signaling pathways may be involved in diabetes-induced loss of gastric ICC.     

Characterization of the phosphorylation state of natriuretic peptide receptor-C

Many internalized receptors are known to be phosphorylated within their cytoplasmic domain. Natriuretic peptide receptor-C (NPR-C) is a covalent homodimer primarily involved in the internalization of bound ligand resulting in tissue uptake and degradation of natriuretic peptides. In this report, we have investigated the phosphorylation state of NPR-C receptors present at high level in rat aortic smooth muscle cells (RASM).³² P labeled cells, NPR-C purification and phosphoamino acid analysis clearly demonstrate that NPR-C exists as a phosphoprotein in RASM cells and that phosphorylation occurs exclusively on serine residues. Transient expression of bovine NPR-C in Cos-P cells of kidney origin confirmed that phosphorylation occurs within the cytoplasmic domain of the receptor. These results provide the first evidence for NPR-C phosphorylation as well as a model for future studies of its role in altering receptor function.     

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

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

NPs/NPRs Signaling Pathways May Be Involved in Depression-Induced Loss of Gastric ICC by Decreasing the Production of mSCF

It is well known that natriuretic peptides (NPs) are involved in the regulation of gastrointestinal motility. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal motility and gastrointestinal dyskinesia is one of the important digestive tract symptoms of depression. However, it is unclear whether they are involved in depression-induced loss of ICC. The aim of the present study was to investigate the relationship between the natriuretic peptide signaling pathway and depression-induced loss of gastric ICC in depressed rats. These results showed that the expression of c-kit and stem cell factor (SCF) in smooth muscle layers of stomach were down-regulated in depressed rats at the mRNA and protein levels. The expression of natriuretic peptide receptor (NPR)-A, B and C were up-regulated in the stomach of depressed rats at the mRNA and protein levels. NPR-A, B and C can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells (GSMCs) obtained from normal rats with different concentrations of C-type natriuretic peptide (CNP). Pretreatment of cultured GSMCs with 8-Brom-cGMP (8-Br-cGMP, a membrane permeable cGMP analog), cANF (a specific NPR-C agonist) and CNP (10⁻⁶ mol/L) demonstrated that 8-Br-cGMP had a similar effect as CNP, but treatment with cANF did not. The results of the methyl thiazolyl tetrazolium bromide (MTT) assay indicated that high concentrations of cANF (10⁻⁶ mol/L) restrained the proliferation of cultured GSMCs. Taken together, these results indicate that the up-regulation of the NPs/NPR-C and NPs/NPR-A, B/cGMP signaling pathways may be involved in depression-induced loss of gastric ICC.     

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.     

Novel bifunctional natriuretic peptides as potential therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.     

Natriuretic peptides stimulate steroidogenesis in the fetal rat testis

To study the regulation of fetal testicular steroidogenesis in the rat, we examined effects of members of the natriuretic peptide family, that is, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), on testosterone production of dispersed Leydig cells of rat fetuses at Embryonic Day (E) 18.5. All three peptides stimulated testosterone production, with significant effect at concentrations > or =1 x 10(-8) mol/L of ANP, > or =1 x 10(-9) mol/L of BNP, and > or =1 x 10(-6) mol/L of CNP. Likewise, receptors for all three peptides (i.e., NPR-A, NPR-B, and NPR-C) were expressed in the fetal testis as early as E15.5. The natriuretic peptides had no effect on cAMP production by fetal Leydig cells. When tested in combination with two other peptides previously shown to stimulate fetal testicular steroidogenesis, vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27), the combined effects did not differ significantly from the maximum effect with any one of the peptides alone. In conclusion, our present findings provide both functional and molecular evidences for NPR-A, NPR-B, and NPR-C in the fetal testis. Because ANP has previously been detected in fetal plasma and we now demonstrate the expression of BNP and CNP in fetal testes, these findings indicate involvement of the natriuretic peptides in endocrine and paracrine regulation during the early phase of fetal testicular steroidogenesis at E15.5--19.5 (i.e., before the onset of pituitary LH secretion).     

Natriuretic peptides inhibit adenylyl cyclase activity in dispersed eel gill cells

The effect of natriuretic peptides on forskolin-evoked adenylyl cyclase activity was investigated in dispersed gill cells from the Australian short-finned eel (Anguilla australis). Molecular cloning techniques were employed to identify the putative G-protein-activating motif within the intracellular domain of the eel natriuretic peptide C receptor. Eel ANP, eel CNP and the NPR-C-specific C-ANF inhibited the forskolin-stimulated production of cyclic AMP. This effect was abolished by pretreatment of cells with pertussis toxin. Eel VNP was without effect on adenylyl cyclase activity. PCR and molecular cloning indicated that the intracellular domain of A. australis NPR-C has the same amino acid sequence as Anguilla japonica. Alignment of these sequences with Rattus norvegicus NPR-C indicated conservation of the putative G-protein-activating motif BB...BBXXB (B=basic, X=nonbasic residues). These data suggest that branchially-expressed NPR-C may play a physiological role additional to that of ligand clearance.Abbreviations ANP atrial natriuretic peptide - CNP C-type natriuretic peptide - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - eANP-NH₂ amidated form of eel ANP - GC guanylyl cyclase - G_i inhibitory G-protein - IBMX isobutylmethylxanthine - NP natriuretic peptide - NPR natriuretic peptide receptor - PCR polymerase chain reaction - PTX pertussis toxin - VNP ventricular natriuretic peptideCommunicated by I.D. Hume     

NPR-C receptors are involved in C-type natriuretic peptide response on bile secretion

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family. Previous studies reported the presence of natriuretic peptide receptors and mRNA CNP in the liver. In the present work, we sought to establish the role of CNP in the regulation of bile secretion in the rat and the possible pathways involved.CNP diminished basal as well as bile salt-evoked bile flow and bile acid output in a dose-dependent manner. It also reduced the excretion of sodium, chloride, and potassium but did not modify bile pH or the excretion of phospholipids, total proteins, and glutathione. Neither parasympathetic nor sympathetic blockade abolished CNP inhibitory response on bile secretion. The selective NPR-C agonist, C-ANP-(4-23) amide, diminished bile flow and the co-administration of both peptides did not further decrease it. CNP did not alter mean arterial pressure or portal venous pressure at any given doses.CNP decreased bile acid-dependent flow without affecting bile acid-independent flow. The inhibitory effect of CNP did not involve the participation of the autonomic nervous system or hemodynamic changes. The participation of NPR-C receptors in CNP response is strongly supported by present findings. The present study shows that CNP modulates bile secretion in the rat, suggesting that CNP may be part of the large family of peptides involved in the regulation of gastrointestinal physiology.     

Natriuretic peptides--their receptors and role in cardiovascular system

Natriuretic peptides belong to a family of small proteins that play a major role in modulation of natriuresis, diuresis and vasodilatation. They counteract the activity of renin-angiotensin-aldosterone system. They are also involved in the regulation of homeostasis, fat metabolism and long bone growth. Natriuretic peptides family in mammals consists of three main members: atrial natriuretic peptide (ANP) - secreted by the atrial myocardium; brain natriuretic peptide (BNP)--secreted mainly by the ventricular myocardium, and C-type natriuretic peptide (CNP)--produced and released by endothelial cells. Secretion of these peptides is stimulated by atrial and ventricular distension, increased blood pressure, hypoxia or renal dysfunction. Natriuretic peptides play their roles via interactions with NPR-A and NPR-B receptors which are transmembrane guanylyl cyclases. Their local concentrations, regulated by internalization and degradation, are mediated by the NPR-C receptor and by neutral endopeptidase. The paper presents the current knowledge of structure and biological function of natriuretic peptides.     

Atrial natriuretic factor negatively modulates secretin intracellular signaling in the exocrine pancreas

We previously reported that atrial natriuretic factor (ANF) stimulates pancreatic secretion through NPR-C receptors coupled to PLC and potentiates secretin response without affecting cAMP levels. In the present study we sought to establish the intracellular signaling mechanism underlying the interaction between both peptides. In isolated pancreatic acini 100 nM ANF abolished cAMP accumulation evoked by any dose of secretin. Lower doses of ANF (1 fM, 1 pM, 1 and 10 nM) dose dependently reduced EC50 secretin-evoked cAMP. Although ANF failed to affect cAMP stimulated by amthamine (selective H2 agonist) or isoproterenol (beta-adrenergic agonist), it abolished VIP-induced cAMP formation. ANF inhibitory effect was prevented by U-73122 (PLC inhibitor) and GF-109203X (PKC inhibitor) but unaltered by PKG and nitric oxide synthase inhibition, supporting that the PLC/PKC pathway mediated the effect. ANF response was mimicked by cANP (4-23 amide) and abolished by pertussis toxin, strongly supporting NPR-C receptor activation. In vivo studies showed that ANF at 0.5 microg x kg(-1) x h(-1) enhanced secretion stimulated by 1 U x kg(-1) x h(-1) secretin but at 1 and 2 microg x kg(-1) x h(-1) it abolished secretin response. However, ANF at such doses failed to modify the secretion evoked by carbachol or CCK. Present results show that ANF negatively modulated secretin secretory response and intracellular signaling through the activation of NPR-C receptors coupled to the PLC/PKC pathway. Furthermore, the finding that ANF also inhibited VIP-evoked cAMP supports a selective modulation of class II G-protein coupled receptors by ANF. Present findings suggest that ANF may play a protective role by reducing secretin response to avoid overstimulation.     

BNP-induced activation of cGMP in human cardiac fibroblasts: interactions with fibronectin and natriuretic peptide receptors

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin (FN). FN contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the natriuretic peptide receptor A (NPR-A) sequence contains both RGD and DDX sequences. The goal of the current investigation was to determine potential interactions between FN and NPR-A on BNP induction of cGMP in cultured human cardiac fibroblasts (CFs). Further, we sought to determine whether a Mayo designed NPR-A specific RGD peptide could modify this interaction. Here we reconfirm the presence of all three natriuretic peptide receptors (NPR) in CFs. CFs plated on FN demonstrated a pronounced increase in cGMP production to BNP compared to non-coated plates. This production was also enhanced by the NPR-A specific RGD peptide, which further augmented FN associated cGMP production. Addition of HS-142-1, a NPR-A/B antagonist, abrogated the responses of BNP to both FN and the NPR-A specific RGD peptide. Finally, we defined a possible role for the NPR-C through non-cGMP mechanisms in mediating the anti-proliferative actions of BNP in CFs where the NPR-C antagonist cANF 4-28 but not HS-142-1 blocked BNP-mediated inhibition of proliferation of CFs. We conclude that NPR-A interacts with components of the ECM such as FN to enhance BNP activation of cGMP and that a small NPR-A specific RGD peptide augments this action of BNP with possible therapeutic implications. Lastly, the NPR-C may also have a role in mediating anti-proliferative actions of BNP in CFs.     

The receptor attributable to C-type natriuretic peptide-induced differentiation of osteoblasts is switched from type B- to type C-natriuretic peptide receptor with aging

C-type natriuretic peptide (CNP) stimulates the differentiation and inhibits the proliferation of osteoblastic lineage cells. In this study, we examined whether the effects of CNP on osteoblastic functions change with aging using calvarial osteoblast-like cells from 25-week-old (young) and 120-week-old (aged) rats. CNP inhibited DNA synthesis and stimulated collagen synthesis and mineralized bone nodule formation. These effects were less pronounced in aged rat cells, suggesting the age-related attenuation of CNP-induced signaling. They were also blocked by the treatment of young rat cells with KT5823, a protein kinase G (PKG) inhibitor, but not by the treatment of aged rat cells with KT5823. CNP stimulated cGMP production in young rat cells, but not in aged rat cells. Natriuretic peptide receptor (NPR)-B, which has a guanylyl cyclase activity domain, and NPR-C, which has no enzyme activity domain, were predominantly expressed in young and aged rat cells, respectively. C-ANF, an NPR-C agonist, mimicked the effects of CNP on the proliferation and differentiation of aged rat cells; these effects were inhibited by the treatment with pertussis toxin (PTX), a Gi protein inhibitor. CNP and C-ANF evoked intracellular levels of inositol-1,4,5-triphosphate and Ca(2+), which are markers for phospholiase C (PLC) activation, in aged rat cells, and the effects of these two peptides were also blocked by the treatment with PTX. From these results, we concluded that CNP acts as a positive regulator of bone formation by osteoblasts and that the signaling pathway for CNP is switched from NPR-B/cGMP/PKG to NPR-C/G(i) protein/PLC with aging.     

Immunolocalization of natriuretic peptides and their receptors in goat (Capra hircus) heart

Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors.