CRRL269.

RATIONALE: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. OBJECTIVE: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. METHODS AND RESULTS: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. CONCLUSIONS: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Embryonal brain tumor with unknown primary lesion and massive cerebrospinal fluid dissemination: A case report.

The 2007 World Health Organization Classification of Tumors of the Central Nervous System (CNS) categorized embryonal tumors of the CNS into three classes: medulloblastoma, CNS primitive neuroectodermal tumor, and atypical teratoid/rhabdoid tumor. Due to the lack of specific histological features, it was sometimes difficult to accurately differentiate CNS embryonal tumors pathologically. Here, we report a case of a young man, who presented with headache. Gadolinium-enhanced magnetic resonance imaging demonstrated massive lesions in the cerebrospinal fluid space, which strongly suggested leptomeningeal dissemination of a brain tumor. The histology showed the tumor comprised densely packed, small cells with scant cytoplasm. Immunoreactivities were positive for synaptophysin and chromogranin A, and negative for glial fibrillary acidic protein, S-100, EMA, and CD20. Because the tumors were located in multiple sites and most of them were within the cerebrospinal fluid space, the primary lesion could not be determined. We diagnosed this case as 'CNS primitive neuroectodermal tumor' by the patient age and predominantly supratentorial distribution of the lesions. After the induction therapy, WHO published its updated classification in 2016. Considering the possibility that the diagnosis is medulloblastoma, we performed additional immunohistochemical analyses, and diagnosed Group 3 medulloblastoma because of the expression of natriuretic peptide receptor 3.

Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

OBJECTIVE: Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. STUDY DESIGN: Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. RESULTS: Corin concentrations were significantly higher in mild (2.78 ±â€¯0.67 ng/ml, p < .05) and severe (2.53 ±â€¯0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ±â€¯0.08 ng/ml) and CHT (1.55 ±â€¯0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ±â€¯0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ±â€¯0.24 ng/ml, p < .01) than in normotensive (0.48 ±â€¯0.06 ng/ml) and mild preeclampsia (0.52 ±â€¯0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. CONCLUSIONS: ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.

Adipose tissue natriuretic peptide receptor expression is related to insulin sensitivity in obesity and diabetes.

OBJECTIVE: Cardiac natriuretic peptides (NPs) bind to two receptors (NPRA-mediator of signaling; NPRC-clearance receptor) whose ratio, NPRR (NPRA/NPRC), determines the NP bioactivity. This study investigated the relationship of NP receptor gene expression in adipose tissue and muscle with obesity and glucose intolerance. Prospectively, the study also assessed whether changes in NP receptor expression and thermogenic gene markers accompanied improvements of insulin sensitivity. METHODS: A cross-sectional study of subjects with a wide range of BMI and glucose tolerance (n = 50) was conducted, as well as a randomized 12-week trial of subjects with type 2 diabetes mellitus (T2DM) treated with pioglitazone (n = 9) or placebo (n = 10). RESULTS: NPRR mRNA was significantly lower in adipose tissue of subjects with obesity when compared with lean subjects (P ≤ 0.001). NPRR decreased with progression from normal glucose tolerance to T2DM (P < 0.01) independently of obesity. Treatment of subjects with T2DM with pioglitazone increased NPRR in adipose tissue (P ≤ 0.01) in conjunction with improvements in insulin sensitivity and increases of the thermogenic markers PPARγ coactivator-1α and uncoupling protein 1 (P ≤ 0.01). CONCLUSIONS: Decreased adipose tissue NPRR was associated with obesity, glucose intolerance, and insulin resistance. This relationship was not observed for skeletal muscle NPRR. Pharmacological improvement of insulin sensitivity in subjects with T2DM was tied to improvement in NPRR and increased expression of genes involved in thermogenic processes.

Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP.

Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP-NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP.

A patient with medulloblastoma in its early developmental stage.

Medulloblastoma is the most frequent malignant brain tumor of the posterior fossa in children and is considered an embryonal tumor. It has been suggested that medulloblastomas be categorized into 4 distinct molecular subgroups- WNT (DKK1), SHH (SFRP1), Group 3 (NPR3), or Group 4 (KCNA1)-since each subgroup is distinct and there is no overlap. The authors report on a 13-year-old boy with medulloblastoma. He presented with sudden-onset nausea and vomiting due to intratumoral hemorrhage. The medulloblastoma was thought to be in an early developmental stage because the tumor volume was extremely small. Immunohistochemical analysis showed that the tumor was mainly composed of DKK1- and NPR3-positive areas. The individual areas of the tumor stained only for DKK1 or NPR3, with no overlap-that is, DKK1 and NPR3 expression were mutually exclusive. Samples obtained by laser microdissection of individual areas and subjected to mass spectrometry confirmed that the expression patterns of proteins were different. Fluorescence in situ hybridization for chromosome 6 showed there were 2 distinct types of cells that exhibited monosomy or disomy of chromosome 6. These results demonstrated that distinct subtypes of medulloblastoma may be present within a single tumor, an observation that has not been previously reported. Our findings in this case indicate that early-stage medulloblastoma may include more than 1 distinct subtype and hint at factors involved in the origin and development of medulloblastomas.

Nanoparticle PET/CT imaging of natriuretic peptide clearance receptor in prostate cancer.

Atrial natriuretic peptide has been recently discovered to have anticancer effects via interaction with cell surface natriuretic peptide receptor A (NPRA) and natriuretic peptide clearance receptor (NPRC). In a preclinical model, NPRA expression has been identified during tumor angiogenesis and may serve as a potential prognostic marker and target for prostate cancer (PCa) therapy. However, the presence of NPRC receptor in the PCa model has not yet been assessed. Furthermore, there is still no report using nanoparticle for PCa positron emission tomography (PET) imaging. Herein, an amphiphilic comb-like nanoparticle was synthesized with controlled properties through modular construction containing C-atrial natriuretic factor (CANF) for NPRC receptor targeting and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator for high specific activity Cu-64 radiolabeling. The pharmacokinetics of (64)Cu-CANF-Comb exhibited tuned biodistribution and optimized in vivo profile in contrast to the nontargeted (64)Cu-Comb nanoparticle. PET imaging with (64)Cu-CANF-Comb in CWR22 PCa tumor model showed high blood pool retention, low renal clearance, enhanced tumor uptake, and decreased hepatic burden relative to the nontargeted (64)Cu-Comb. Immunohistochemistry staining confirmed the presence of NPRC receptor in tumor tissue. Competitive PET receptor blocking study demonstrated the targeting specificity of (64)Cu-CANF-Comb to NPRC receptor in vivo. These results establish a new nanoagent for prostate cancer PET imaging.

C-type natriuretic peptide and its receptors in atherosclerotic plaques of the carotid artery of clinically asymptomatic patients.

OBJECTIVES: C-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative and anti-migratory properties. No data exist on the presence of CNP in human atherosclerotic plaques of the carotid artery. Therefore, this study aimed to analyse qualitatively the distribution pattern and characteristics of CNP and its receptors in both, early and advanced human carotid plaques, as well as in stable and unstable lesions. In addition, the aim of this study was to evaluate CNP and its receptors as possible biomarkers to predict plaque stability in advanced lesions. METHODS: Advanced carotid artery plaques of 40 asymptomatic patients (20 histologically stable and 20 histologically unstable) and early arteriosclerotic lesions of three patients were analysed. RESULTS: Serum level of CNP was similar in patients with stable and unstable plaques (196 ± 19 pg ml(-1) vs. 198 ± 25 pg ml(-1), p = 0.948). Expression level of natriuretic peptide receptor 3 (NPR3) was significantly higher in unstable plaques compared to stable plaques (5.6 ± 1.8% vs. 1.7 ± 0.5%, p = 0.045). Expression levels of CNP and NPR2 were higher in unstable plaques but the differences were not statistically significant. The distribution pattern of CNP, NPR2 and NPR3 varied qualitatively between early and advanced carotid plaques. No relevant histological differences were observed with respect to plaque stability. CONCLUSIONS: This study shows the presence of CNP and its receptors in atherosclerotic plaques of human carotid artery, with increased expression of NPR3 in histologically unstable plaques. In this study, serum CNP was not associated with histological plaque stability. In future, larger studies are required to further evaluate whether proteins of the CNP axis would be useful as biomarkers.

Atrial natriuretic peptide ameliorates peritoneal fibrosis in rat peritonitis model.

BACKGROUND: Atrial natriuretic peptide (ANP) was recently reported to ameliorate fibrosis in the heart and experimental renal diseases and vascular thickening after balloon injury. Peritoneal fibrosis is an important complication of long-term peritoneal dialysis, and peritonitis is a factor in its onset. In the present study, we investigated the effects of ANP in a rat peritonitis-induced peritoneal fibrosis model. METHODS: As pretreatment, an osmotic pump containing vehicle (saline) or ANP (0.15 or 0.3 µg/min) was inserted through the carotid vein in male Sprague-Dawley rats. ANP or saline was continuously infused using the osmotic pump. Three days after administration of ANP or saline, rats underwent peritoneal scraping in a blind manner and were sacrificed on Day 14. The effects of ANP were evaluated based on peritoneal thickness, immunohistochemistry and real-time polymerase chain reaction. In each experiment, we evaluated messenger RNA (mRNA) expression of the ANP receptor natriuretic peptide receptor A (NPR-A) in the peritoneum after scraping. The effects of ANP were also studied in cultured peritoneal fibroblasts and mesothelial cells. RESULTS: We observed a significant increase in NPR-A mRNA in the peritoneum. Peritoneal thickness increased with time and peaked on Day 14, but ANP significantly reduced peritoneal thickness. Parameters such as number of macrophages and CD-31-positive vessels and expression of type III collagen/transforming growth factor-ß/plasminogen activator inhibitor-1 (PAI-1)/connective tissue growth factor (CTGF) were significantly suppressed by ANP. In cultured peritoneal fibroblasts and mesothelial cells, ANP suppressed angiotensin II-induced upregulation of CTGF and PAI-1. CONCLUSIONS: Our results suggest that ANP is useful in preventing inflammation-induced peritoneal fibrosis.

Knockdown of natriuretic peptide receptor-A enhances receptor C expression and signalling in vascular smooth muscle cells.

AIMS: Natriuretic peptide receptor-A (NPR-A) knockout mice exhibited an increased blood pressure that may also be attributed to the up-regulation of NPR-C and associated signalling; however, the interaction between the two receptors has not been investigated. In the present study, we investigated the effect of knockdown of NPR-A using NPR-A antisense (AS) on the expression of NPR-C and adenylyl cyclase (AC) signalling in A10 vascular smooth muscle cells (VSMC). METHODS AND RESULTS: The receptor and G protein expression was determined by western blotting, and AC activity was determined by measuring [(32)P]cAMP formation from [α-(32)P]ATP. Treatment of A10 VSMC with NPR-A AS decreased NPR-A and enhanced NPR-C expression without altering the levels of angiotensin II AT1 and muscarinic M2 receptors. In addition, siRNA-NPR-A also resulted in the up-regulation of NPR-C. The re-expression of NPR-A in AS-treated cells reversed the enhanced expression of NPR-C to control levels. In addition, NPR-C-, AT1, and M2 receptor-mediated inhibition of AC and Giα protein expression was enhanced in AS-treated cells, whereas NPR-A-mediated cyclic GMP (cGMP) formation and Gsα-mediated stimulation of AC were significantly reduced. Pertussis toxin treatment attenuated the AS-induced enhanced inhibition of AC to control levels. Furthermore, the enhanced levels of NPR-C and Giα proteins were reversed to control levels by 8-bromo-cGMP (8Br-cGMP) and PD98059, an MEK inhibitor. In addition, 8Br-cGMP also attenuated AS-induced enhanced ERK1/2 phosphorylation to control levels. CONCLUSION: These results demonstrate that knockdown of NPR-A up-regulates the expression of NPR-C, Giα proteins, and NPR-C-linked AC signalling and suggests a cross-talk between NPR-A and NPR-C.

B-type natriuretic peptide receptors in hypertrophied adult rat cardiomyocytes.

Brain natriuretic peptide (BNP) binds to three types of natriuretic peptide receptors, NPR-A, -B and -C (NPRs). The expression shape of BNP and NPRs seems to be an important modulator factor in the pathogenesis of cardiac hypertrophy. The aim of this study was to evaluate the expression of NPRs in an animal model of pressure overload hypertrophy. Left ventricular hypertrophy was induced by chronic abdominal aortic banding in adult male Wistar rats. After six weeks, NPRs gene expression was evaluated with RT-PCR, BNP plasma concentration and BNP positive myocytes were measured with ELISA and immunohistochemistry techniques respectively. NPR-A and NPR-C mRNA expression was significantly increased in left ventricular hypertrophied cardiomyocytes by 1.6-fold and 2.1-fold respectively (P<0.01). Abdominal aortic banding increased significantly BNP plasma concentration (630+/-8pg/ml vs 106+/-4pg/ml; P<0.01). The percentage of BNP positive cells in normal myocardial tissue were 40% while in the hypertrophied one it raised to 80%. The data suggest that in our left ventricular hypertrophy model, the NPR-A and NPR-C receptors were increased in association to the increased BNP level. This relationship may amplify beneficial paracrine/autocrine effects of BNP on cardiac remodelling in response to hemodynamic overload.

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, alphaT3-1 and LbetaT2 gonadotroph cells.

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Cardiac and kidney hormones cure up to 86% of human small-cell lung cancers in mice.

BACKGROUND: Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, long acting natriuretic peptide and kaliuretic peptide, and the kidney hormone urodilatin have anticancer effects in vitro. MATERIALS AND METHODS: These cardiac hormones and urodilatin were infused subcutaneously for 28 days with weekly fresh hormones since they lose biological effects at body temperature for more than a week at 0.3 nm kg(-1) body weight in athymic mice bearing human small-cell lung carcinomas. RESULTS: Long acting natriuretic peptide, vessel dilator, kaliuretic peptide, atrial natriuretic peptide and urodilatin eliminated 86%, 71%, 57%, 43% (P < 0.001 for the cardiac hormones) and 25% (P < 0.05; urodilatin) of the human small-cell lung carcinomas. The treated small-cell lung carcinomas that were not cured grew rapidly, similar to the untreated controls, whose volume was 7 fold larger in 1 week, 18-fold increased in 2 weeks, 39-fold increased in 3 weeks, 63-fold increased in 1 month and 97-fold increased in volume in 6 weeks. One vessel dilator treated small-cell lung carcinoma animal developed a large tumour (8428 mm3 volume) on treatment and this tumour was eliminated with utilizing atrial natriuretic peptide and then long acting natriuretic peptide sequentially. CONCLUSIONS: Four cardiac hormones eliminate up to 86% of human small-cell lung carcinomas in athymic mice. Urodilatin can also eliminate small-cell lung carcinomas but at a lower cure rate of 25%. Unresponsive lesions can be eliminated by utilizing different hormones synthesized by the atrial natriuretic peptide gene in a sequential manner.

Changes in A-type natriuretic peptide and its receptors induced by a neutral endopeptidase inhibitor in a rat model of sepsis.

PURPOSE: Elevated plasma A-type natriuretic peptide (ANP) levels in sepsis cause fluid transfer into extravascular spaces. We investigated the changes in ANP concentrations and natriuretic peptide receptor (NPR) expression induced by thiorphan, a neutral endopeptidase (NEP) inhibitor, in a rat model of sepsis. METHODS: Fifteen male rats were divided into three groups: a control group (n = 5), a lipopolysaccharide (LPS) group (n = 5), and an LPS-thiorphan group (n = 5). We measured ANP concentrations in the plasma and lung, and NPR mRNA expression in the lung 4 h after administering LPS, and compared the values with those in the control group. RESULTS: Plasma and lung ANP levels in the LPS group were significantly higher than those in the control group (P < 0.05), but were significantly decreased by thiorphan administration (P < 0.05). NPR-A mRNA levels did not differ significantly among the groups. NPR-C mRNA levels in the LPS-thiorphan group were significantly higher than those in the other groups (P < 0.05). CONCLUSIONS: Elevated ANP levels were decreased by thiorphan administration, which increased NPR-C mRNA levels in the lung. Thus, thiorphan might be effective for reducing elevated ANP levels in sepsis.

Expression of natriuretic peptide-activated guanylate cyclases by cholinergic and dopaminergic amacrine cells of the rat retina.

Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPR-A and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina.

Inhibitory effect of dendroaspis natriuretic peptide on spontaneous contraction in gastric antral circular smooth muscles of guinea pigs.

AIM: To determine whether the natriuretic peptide receptor (NPR) is present in the stomach of guinea pigs and to investigate the effect of dendroaspis natriuretic peptide (DNP) on the gastric motility of guinea pigs and its mechanism. METHODS: The distribution of the NPR was analyzed by autoradioimmunography. The spontaneous contraction of gastric antral circular muscles of guinea pigs was recorded by a 4-channel physiograph. The whole cell patch-clamp technique was introduced to record calcium-activated potassium currents in the gastric myocytes isolated by collagenase. RESULTS: The NPR existed in the gastric fundus, gastric body, and gastric antrum of guinea pigs, and its density was largest in the gastric antrum. DNP inhibited spontaneous contraction and exhibited a dose-dependent manner. The DNP-induced inhibition was diminished by LY83583 (a guanylate cyclase inhibitor) and was potentiated by zaprinast (a cGMP-sensitive phosphoesterase inhibitor). The inhibitory effect of DNP on spontaneous contraction was also inhibited by tetraethylammonium (a non-selective potassium channel blocker); 10 nmol/L DNP increased the calcium-activated potassium currents in the gastric circular myocytes of guinea pigs. CONCLUSION: The NPR is most common in the gastric antrum of guinea pigs. DNP significantly inhibits gastric motility in the gastric antrum of guinea pigs. The inhibitory effect occurs via a cGMP-dependent pathway, and a calcium-activated potassium channel may be also involved in the relaxation induced by DNP in gastric antral circular smooth muscles.

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout.

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.

Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation.

BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.