Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.     

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.     

Functional expression of components of the natriuretic peptide system in human ocular nonpigmented ciliary epithelial cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP > ANP >/= BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65-75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4-23 elicited an inhibitory effect (30-37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75-85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.     

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.     

Activation of protein kinase C stimulates the dephosphorylation of natriuretic peptide receptor-B at a single serine residue: a possible mechanism of heterologous desensitization

The binding of atrial natriuretic peptide and C-type natriuretic peptide (CNP) to the guanylyl cyclase-linked natriuretic peptide receptors A and B (NPR-A and -B), respectively, stimulates increases in intracellular cGMP concentrations. The vasoactive peptides vasopressin, angiotensin II, and endothelin inhibit natriuretic peptide-dependent cGMP elevations by activating protein kinase C (PKC). Recently, we identified six in vivo phosphorylation sites for NPR-A and five sites for NPR-B and demonstrated that the phosphorylation of these sites is required for ligand-dependent receptor activation. Here, we show that phorbol 12-myristate 13-acetate, a direct activator of PKC, causes the dephosphorylation and desensitization of NPR-B. In contrast to the CNP-dependent desensitization process, which results in coordinate dephosphorylation of all five sites in the receptor, phorbol 12-myristate 13-acetate treatment causes the dephosphorylation of only one site, which we have identified as Ser(523). The conversion of this residue to alanine or glutamate did not reduce the amount of mature receptor protein as indicated by detergent-dependent guanylyl cyclase activities or Western blot analysis but completely blocked the ability of PKC to induce the dephosphorylation and desensitization of NPR-B. Thus, in contrast to previous reports suggesting that PKC directly phosphorylates and inhibits guanylyl cyclase-linked natriuretic peptide receptors, we show that PKC-dependent dephosphorylation of NPR-B at Ser(523) provides a possible molecular explanation for how pressor hormones inhibit CNP signaling.     

A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signal was weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as but more specific than the (32)PO(4) labeling method. The two approaches were highly correlated (R(2) = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levels are best detected by Western blot analysis, not SYPRO Ruby staining.     

The natriuretic peptide system in eels: a key endocrine system for euryhalinity?

The natriuretic peptide system of a euryhaline teleost, the Japanese eel (Anguilla japonica), consists of three types of hormones [atrial natriuretic peptide (ANP), ventricular natriuretic peptide (VNP), and C-type natriuretic peptide (CNP)] and four types of receptors [natriuretic peptide receptors (NPR)-A, -B, -C, and -D]. Although ANP is recognized as a volume-regulating hormone that extrudes both Na(+) and water in mammals, ANP more specifically extrudes Na(+) in eels. Accumulating evidence shows that ANP is secreted in response to hypernatremia and acts to inhibit the uptake and to stimulate the excretion of Na(+) but not water, thereby promoting seawater (SW) adaptation. In fact, ANP is secreted immediately after transfer of eels to SW and ameliorates sudden increases in plasma Na(+) concentration through inhibition of drinking and intestinal absorption of NaCl. ANP also stimulates the secretion of cortisol, a long-acting hormone for SW adaptation, whereas ANP itself disappears quickly from the circulation. Thus ANP is a primary hormone responsible for the initial phase of SW adaptation. By contrast, CNP appears to be a hormone involved in freshwater (FW) adaptation. Recent data show that the gene expression of CNP and its specific receptor, NPR-B, is much enhanced in FW eels. In fact, CNP infusion increases (22)Na uptake from the environment in FW eels. These results show that ANP and CNP, despite high sequence identity, have opposite effects on salinity adaptation in eels. This difference apparently originates from the difference in their specific receptors, ANP for NPR-A and CNP for NPR-B. VNP may compensate the effects of ANP and CNP for adaptation to respective media, because it has high affinity to both receptors. On the basis of these data, the authors suggest that the natriuretic peptide system is a key endocrine system that allows this euryhaline fish to adapt to diverse osmotic environments, particularly in the initial phase of adaptation.     

ATP-independent activation of natriuretic peptide receptors

Natriuretic peptide receptor A (NPR-A) is an essential cardiovascular regulator that is stimulated by atrial natriuretic peptide and B-type natriuretic peptide, whereas natriuretic peptide receptor B (NPR-B) stimulates long bone growth in a C-type natriuretic peptide-dependent manner. Many reports indicate that ATP is essential for NPR-A and NPR-B activation. Current models suggest that natriuretic peptide binding to receptor extracellular domains causes ATP binding to intracellular kinase homology domains, which derepresses adjacent catalytic domains. Here, we report 100-fold activations of natriuretic peptide receptors in the absence of ATP. The addition of a nonhydrolyzable ATP analog had no effect at early time periods (measured in seconds) but increased cGMP production about 2-fold after longer incubations (measured in minutes), consistent with a stabilization, not activation, mechanism. These data indicate that ATP does not activate natriuretic peptide receptors as has been repeatedly reported. Instead, ATP increases activity primarily by maintaining proper receptor phosphorylation status but also serves a previously unappreciated enzyme stabilizing function.     

Ligand-independent oligomerization of natriuretic peptide receptors. Identification of heteromeric receptors and a dominant negative mutant.

Activation of many single-transmembrane receptors requires ligand-induced receptor oligomerization. We have examined the oligomerization of the atrial natriuretic peptide receptor, NPR-A, using epitope-tagged receptor in a co-immunoprecipitation assay. Unlike other single-transmembrane receptors, NPR-A oligomerized in a ligand-independent fashion. Extracellular receptor sequences were both necessary and sufficient for oligomer formation. NPR-A was also able to oligomerize with the related natriuretic peptide receptor, NPR-B. A truncated NPR-A lacking most of the cytoplasmic domain blocked activation of the full-length receptor, presumably through formation of an inactive heteromer. These results indicate that oligomerization of this single-transmembrane receptor is important for the transduction of a conformational change across the plasma membrane but are not consistent with models in which natriuretic peptide receptor oligomerization serves merely to bring intracellular domains together.     

Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptors

Natriuretic peptides are a family of hormones/paracrine factors that regulate blood pressure, cardiovascular homeostasis and bone growth. The mammalian family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). A family of three cell surface receptors mediates their physiologic effects. Two are receptor guanylyl cyclases known as NPR-A/GC-A and NPR-B/GC-B. Peptide binding to these enzymes stimulates the synthesis of the intracellular second messenger, cGMP, whereas a third receptor, NPR-C, lacks enzymatic activity and functions primarily as a clearance receptor. Here, we provide a brief review of how various desensitizing agents and/or conditions inhibit NPR-A and NPR-B by decreasing their phosphorylation state.     

Involvement of the atrial natriuretic peptide in the reduction of arterial pressure induced by swimming but not by running training in hypertensive rats

The aim of this study was to compare, under resting conditions, the influence of chronic training in swimming or running on mean arterial pressure (MAP) and the involvement of the natriuretic peptide system in this response. Two-month-old male spontaneously hypertensive rats (SHR) were divided into three groups—sedentary (SD), swimming (SW) and running (RN)—and were trained for eight weeks under regimens of similar intensities. Atria tissue and plasma atrial natriuretic peptide (ANP) concentrations were measured by radioimmunoassay. ANP mRNA levels in the right and left atria as well as the natriuretic peptide receptors (NPR), NPR-A and NPR-C, mRNA levels in the kidney were determined by real-time PCR. Autoradiography was used to quantify NPR-A and NPR-C in mesenteric adipose tissue. Both training modalities, swimming and running, reduced the mean arterial pressure (MAP) of SHR. Swimming, but not running, training increased plasma levels of ANP compared to the sedentary group (P < 0.05). Expression of ANP mRNA in the left atrium was reduced in the RN compared to the SD group (P < 0.05). Expression of NPR-A and NPR-C in the kidneys of the SW group decreased significantly (P < 0.05) compared to the SD group. Although swimming increased ¹²⁵I-ANP binding to mesenteric adipose tissue, displacement by c-ANF was reduced, indicating a reduction of NPR-C. These results suggest that the MAP reduction induced by exercise in SHR differs in its mechanisms between the training modalities, as evidenced by the finding that increased levels of ANP were only observed after the swimming regimen.     

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.     

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.     

Comparison of infarct-derived and control ovine cardiac myofibroblasts in culture: response to cytokines and natriuretic peptide receptor expression profiles

This study investigated whether gene expression profiles of myofibroblasts derived from infarcted myocardium differ from normal cardiac fibroblasts. We compared the expression of cytoskeletal proteins in cultured ovine cardiac fibroblasts derived from infarcted (ID) and noninfarcted ovine myocardium (NID) and the levels of expression of the natriuretic peptide receptors (NPR)-A and NPR-B in response to treatment with transforming growth factor (TGF)-beta1 and/or platelet-derived growth factor (PDGF). Transformation of cultured cardiac fibroblasts to myofibroblasts, as indicated by alpha-smooth muscle actin and vimentin expression, was independent of the presence of TGF-beta1, PDGF, or cell origin. ID fibroblasts had higher basal levels than NID fibroblasts of NPR-A (ID: 58.0 +/- 32.2 arbitrary density units, NID: undetectable), NPR-B (ID: 780 +/- 155, NID: 330 +/- 38 arbitrary density units) and collagen I (ID: 17.2 +/- 0.5, NID: 10.5 +/- 1.7 pg mRNA/mug total RNA, P < 0.05) but lower levels of alpha-SMa expression (ID: 50.2 +/- 7.9, NID: 76.9 +/- 3.2 fluorescence units, P < 0.05). NPR-A mRNA in ID fibroblasts showed a rapid fourfold increase in response to TGF-beta1 and/or PDGF at 4 and 2 h, respectively, followed by a profound decline; in NID cells, NPR-A mRNA was undetectable. In ID fibroblasts, cytokines reduced NPR-B mRNA below control levels; in NID fibroblasts, TGF-beta1 and PDGF elicited prompt increments in expression: a fourfold increase with TGF-beta1 at 8 h and a twofold increase with PDGF at 4 h (P < 0.05). In summary, transformation of cardiac fibroblasts to myofibroblasts in culture is independent of cytokine treatment. Moreover, whether the cultured cardiac fibroblasts are from infarct tissue is a major determinant of NPR expression levels and cytokine responses, even after four to five passages.     

Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response

Natriuretic peptides (NPs) are cyclic vasoactive peptide hormones with high therapeutic potential. Three distinct NPs (ANP, BNP, and CNP) can selectively activate natriuretic peptide receptors, NPR-A and NPR-B, raising the cyclic GMP (cGMP) levels. Insulin-degrading enzyme (IDE) was found to rapidly cleave ANP, but the functional consequences of such cleavages in the cellular environment and the molecular mechanism of recognition and cleavage remain unknown. Here, we show that reducing expression levels of IDE profoundly alters the response of NPR-A and NPR-B to the stimulation of ANP, BNP, and CNP in cultured cells. IDE rapidly cleaves ANP and CNP, thus inactivating their ability to raise intracellular cGMP. Conversely, reduced IDE expression enhances the stimulation of NPR-A and NPR-B by ANP and CNP, respectively. Instead of proteolytic inactivation, IDE cleavage can lead to hyperactivation of BNP toward NPR-A. Conversely, decreasing IDE expression reduces BNP-mediated signaling. Additionally, the cleavages of ANP and BNP by IDE render them active with NPR-B and a reduction of IDE expression diminishes the ability of ANP and BNP to stimulate NPR-B. Our kinetic and crystallographic analyses offer the molecular basis for the selective degradation of NPs and their variants by IDE. Furthermore, our studies reveal how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to biased stochastic, non-sequential cleavages and the ability of IDE to switch its substrate selectivity. Thus, the evolutionarily conserved IDE may play a key role in modulating and reshaping the strength and duration of NP-mediated signaling.     

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.     

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.