Rat ANP Research Articles

Abstract P095: Arginine Vasopressin Infusion Is Sufficient To Cause Pregnancy-specific Cardiac Hypertrophy

Preeclampsia is a prevalent gestational disorder characterized by hypertension, proteinuria, and renal damage. Mothers with a history of preeclampsia (PE) are at increased risk of future cardiovascular disease (CVD). Yet, the mechanism by which preeclampsia predisposes patients to future CVD is unknown. Previously published data demonstrated that arginine vasopressin (AVP) infusion in mice recapitulates clinical features of PE including pregnancy-specific hypertension. We hypothesized that chronic AVP infusion in pregnant mice would cause cardiac hypertrophy. C57Bl6/J mice were implanted with an osmotic minipump to deliver 24ng/hr of AVP throughout gestation until necropsy on gestational day 18. Blood pressures were monitored by tail cuff plethysmography and showed no increase in mean arterial pressure (MAP) in nonpregnant mice. However in pregnant mice, an increase of 18.8 ± 7.4 mmHg (82.1 vs 100.8, p=0.05) in MAP was observed in AVP-infused mice compared to saline-infused pregnant mice. Similarly, there was pregnancy-specific 13.4 ± 0.9 mg (93.6 vs 106.9, p=0.0001) increase in the heart weight of AVP-infused mice. Plasma levels of NT-ProBNP were measured by ELISA and showed a significant 163.6 ± 61.00 pg/ml (426.0 vs 589.6, p=0.01) increase in pregnant AVP-infused mice. Human 3rd trimester maternal plasma samples were obtained from the Iowa Maternal Fetal Tissue Bank (IRB# 200910784) for a case control study. In plasma samples from preeclamptic patients there was a significant 264.6 ± 79.72 pg/ml (185.1 vs 449.8, p=0.001) and a 27.9 ± 11.5 pg/ml (64.6, n=32 vs 92.5, n=66, p=0.02) increase in NT-ProBNP and MR-proANP respectively compared to normal pregnancy plasma. H9C2 rat cardiomyocytes treated with pooled plasma from preeclamptic patients showed a 6.6 and 1.8 fold increase in rat ANP and BNP expression respectively. These data suggest that AVP infusion in mice specifically during pregnancy is sufficient to cause cardiac hypertrophy beyond typical hypertrophy seen in a normal pregnancy and that unknown circulating factors in preeclamptics may contribute to the development of hypertrophy. Importantly, these data support using AVP-infusion as a novel mouse model of pregnancy-specific cardiac hypertrophy to study postpartum cardiomyopathies.

Angiotensin II As A Natural Suppressor On Cardiac Natriuretic Peptide System: Molecular Insights And Novel Therapeutic Design

<h3>Introduction</h3> Therapeutic strategies harnessing both renin-angiotensin system (RAS) and natriuretic peptide system (NPS) mediated effects, such as Sacubitril/Valsartan, has been proven to be promising in treating human heart failure (HF). However, molecular understanding of the interaction between RAS and NPS remains to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the only functional receptor for both atrial and b-type natriuretic peptide (ANP and BNP), has not been investigated. <h3>Hypothesis</h3> We hypothesize that ANGII naturally suppresses pGC-A and the inhibition of this crosstalk would be a novel therapeutic strategy to combat cardiovascular disease (CVD) of which RAS hyperactivation is a hallmark feature. <h3>Methods</h3> Plasma ANP, BNP, cGMP, and ANGII were measured in 128 healthy humans to define the balance between ANGII and NPS. cGMP responses to acute infusion of ANP alone and in the presence of ANGII was characterized in normal rats. The molecular mechanisms including involvement of ANGII type I receptor (AT1R) and its downstream target protein kinase C (PKC) were investigated in HEK293 cell overexpressing pGC-A and together with AT1R. CRRL-58, an innovative dual-functional peptide was designed to activate pGC-A and inhibit PKC, and tested for cGMP generation in HEK293 cells. <h3>Results</h3> In healthy human, plasma cGMP was positively associated with ANP or BNP only in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels (see Figure). Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII (50 pmol/kg/min). Notably, the suppressive effect of ANGII on pGC-A was recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. Meanwhile, we found PKC, a well-established downstream target of AT1R, was a key mediator for this observed crosstalk, as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Notably, CRRL-58 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in vitro. <h3>Conclusions</h3> Our human and experimental data demonstrate that ANGII is a natural suppressor of pGC-A activation via AT1R and PKC signaling, but not via pGC-A per se. Moreover, we report for the first time that CRRL-58, an innovative bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Together, our study provides new insights into the interaction between RAS and NPS, and highlights a novel and mechanism-based therapeutic avenue for treating HF and other CVDs.

Abstract 10303: CRRL290: A Novel and Mechanism-Based Therapeutic Peptide Targeting the Crosstalk Between Angiotensin II and the Natriuretic Peptide System for Cardiorenal Disease

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (&gt; 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

The Protective Role of the Long Pentraxin PTX3 in Spontaneously Hypertensive Rats with Heart Failure.

Pentraxin 3 (PTX3) is synthesized locally and released into the circulation, reflecting local inflammation in the cardiovascular system. Therefore, we conducted a study to explore the effect of PTX3 in spontaneously hypertensive heart failure (SHHF) rats. Sprague Dawley (SD) and SHHF rats were treated with recombinant PTX3 protein, and the blood pressure (BP) and echocardiographic parameters were collected. Radioimmunoassay, enzyme immunoassay and enzyme-linked immunosorbent assay (ELISA) were applied to detect plasma levels of atrial/B-type natriuretic peptide (ANP/BNP) and PTX3. The pathological changes in the myocardial tissues were observed by hematoxylin and eosin (HE) and Masson stainings. The mRNA and protein expressions were detected by quantitative real-time reverse-transcription polymerase chain reaction (qPCR) and western blotting. Cardiomyocyte apoptosis was evaluated by TUNEL staining and DNA fragmentation test. Increased plasma concentrations of PTX3 were found in SHHF rats compared with SD rats, which was further enhanced by recombinant PTX3 protein. After injection with recombinant PTX3 protein, the heart function was improved in SHHF rats with the decreased systolic and diastolic BP, and the reduced plasma levels of ANP and BNP. Moreover, PTX3 improved the myocardial damage and interstitial fibrosis in SHHF rats with reduced cardiomyocyte apoptosis and decreased mRNA expressions of pro-inflammatory factors in myocardial tissues. PTX3 could decrease the BP and plasma levels of ANP and BNP in SHHF rats, as well as improve the inflammation, cardiomyocyte apoptosis, and pathological changes of myocardial tissues, suggesting it may be a useful intervention in the treatment of SHHF.

The chemical profiling of aqueous soluble fraction from Lagopsis supina and its diuretic effects via suppression of AQP and RAAS pathways in saline-loaded rats

Ethnopharmacological relevanceLagopsis supina (Steph.) Ik. -Gal. ex Knorr. has been widely used as a remedy treatment for diuresis and edema in China over 2500 years. Our previous results showed that the aqueous soluble fraction from L. supina (LSB) possessed acute diuretic effect. Aim of the studyThe aim of this study was to appraise the acute (6 h) and prolonged (7 d) diuretic effects, underlying mechanisms, and chemical profiling of LSB. Materials and methodsThe chemical profiling of LSB was performed by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS). Then, oral administration of LSB (40, 80, 160 and 320 mg/kg) and furosemide (10 mg/kg) once daily for 7 consecutive days to evaluate the diuretic effects in saline-loaded rats. The body weight, food consumption, and water intake were recorded once daily. The urinary volume, pH and electrolyte concentrations (Na+, K+, Cl−, and Ca2+) were measured after administration drugs for acute and prolonged diuretic effects. In addition, the serum levels of Na+-K+-ATPase, angiotensin II (Ang II), anti-diuretic hormone (ADH), aldosterone (ALD), atriopeptin (ANP), aquaporins (AQPs)-1, 2 and 3 were determined by ELISA kits. The mRNA expressions and protein levels of AQPs-1, 2 and 3 were analyzed by real-time quantitative PCR and Western blot assays, respectively. Results30 compounds were identified in LSB based on accurate mass and MS/MS fragmentation compared to literature, among which phenylpropanoids and flavonoids could be partly responsible for the major diuretic effect. Daily administration of LSB (160 or 320 mg/kg) prominently increased urinary excretion volume after the 2 h at the first day of treatment, remaining until the 7th day. LSB did not cause Na+ and K+ electrolyte abnormalities, and has minor effect on Cl− and Ca2+ concentrations at 320 mg/kg. Furthermore, LSB observably suppressed renin-angiotensin-aldosterone system (RAAS) activation, including decreased serum levels of Ang II, ADH, and ALD, and prominently increased serum level of ANP in rats. LSB treatment significantly down-regulated the serum levels, mRNA expressions and protein levels of AQP1, AQP2, and AQP3. ConclusionLSB has a prominent acute and prolonged diuretic effects via suppression of AQP and RAAS pathways in saline-loaded rats, and support the traditional folk use of this plant. Taken together, LSB might be a potential diuretic agent.

Effects of Oryeongsan in 2K1C rats through regulation of renin‐angiotensin‐aldosterone system and ANP system

Oryeongsan (ORS) which was first recorded in SangHanLoon, the classic of traditional Chinese medicine describing treatments of disordered body fluid balance. Renal hypertension is caused by renovascular dysfunction through an activation of renin‐angiotensin‐aldosterone system (RAS). The RAS is one of the most important regulators in maintenance of body fluid homeostasis and in long‐term regulation of blood pressure. The purpose of this study is to identify effects of ORS on regulation of renal function, change of blood pressure and RAS in the two‐kidney one‐clip (2K1C) renal hypertensive rats. Experiments were performed in four experimental groups: sham rats, 100 mg/kg/day ORS in sham rats, 2K1C rats and ORS in 2K1C rats, respectively. After 4 weeks of operation, sham and 2K1C rats were administered with vehicle or ORS for 3 weeks. Urinary volume, urinary excretion of Na+, K+, Cl−, osmolality and creatinine were measured. The 2K1C rats showed significantly higher systolic blood pressure (SBP) compared with sham rats. ORS treatment for 3 weeks markedly decreased the SBP in 2K1C rats. ORS significantly increased urinary volume, urinary excretion of electrolytes, and clearance of creatinine (CCr) in 2K1C rats. ORS significantly suppressed plasma level of PRA, Angiotensin II and aldosterone in 2K1C rats. Also, ORS suppressed the upregulated RAS components in the kidney from 2K1C rats. However, ORS increased plasma level of ANP in 2K1C rats. These findings suggest that ORS may elicit renal and vascular effects via two ways: directly and indirectly through activation of the ANP system and inhibition of the RAS.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of glutamine on the effect of resistance exercise on cardiac ANP in rats

Various nutritional supplements (herbs, vitamins, and micronutrients) improve responses and adaptations to resistance exercise. ANP is a heart hormone that contributes to fluid, electrolyte and blood pressure homeostasis through its natriuretic and vasodilative actions. In the present study, the adaptation of ANP in response to resistance exercise was investigated in rats supplemented with glutamine for five weeks. The results showed that supplementation with glutamine did not influence the number of ANP granules per atrial cardiocyte in sedentary animals. In exercised-trained rats, the number and diameter of the granules was significantly higher in comparison with the control group and in exercised animals supplemented with glutamine there was significant increase in the number and diameter of ANP granules compared with controls. Altogether, these data indicated that in resistance exercise rats, glutamine significantly enhances cardiac ANP thus implicating the beneficial effects of glutamine supplementation to the ANP system.

The Involvement of SWI/SNF chromatin remodeling enzymes in Transcriptional Regulation of Pathological Cardiac Hypertrophy

Pathological cardiac hypertrophy is characterized by transcriptional reprogramming of gene expression which involves certain chromatin modulators. Little is known about the chromatin modifications involved in hypertrophy. SWI/SNF chromatin remodeling enzymes are epigenetic modulators playing pertinent roles in gene regulation. To understand the involvement of various subunits in the SWI/SNF complex and to study the chromatin modifications occurring in hypertension induced cardiac hypertrophy, we performed expression analysis as well as chromatin immunoprecipitaions (CHIPs) on Dahl salt sensitive and resistant rat hearts. The relative protein expression revealed a significant increase of Brg1, Baf 60c and Baf 180 in sensitive rat hearts. Moreover CHIPs demonstrated an increased enrichment of these subunits on promoters of ANP and BNP (hypertrophic markers) in sensitive models. Also there was an increased relative enrichment of acetylated H4, H3K4 trimethylation (active transcription marks) and decreased enrichment of H3k9 trimethylation, H3k27 trimethylation (negative transcription marks) on ANP and BNP promoters in sensitive rats. Our studies for the first time revealed the importance of SWI/SNF complex in promoting cardiac hypertrophy in Dahl salt sensitive and resistant models and suggest what we believe to be a novel mechanism for reprogramming of gene expression involved in hypertrophy.

Hagfish natriuretic peptide changes urine flow rates and vascular tensions in a hagfish

Since the first report of their natriuretic effect on mammalian kidneys the relative influences of natriuretic peptides (NPs) on volume and salt regulation in vertebrates have been debated. As marine osmoconformers, with plasma ionic concentrations similar to seawater, the actions of NPs on hagfishes may provide information on their primordial role. A synthetic natriuretic peptide derived from Eptatretus burgeri (hNP) increased urine production rates in E. cirrhatus at 3 × 10 − 8 M. It also contracted afferent branchial and segmental arteries at low concentrations (1 × 10 − 10 M) and relaxed them at 3 × 10 − 8 M. Thus, hNP has a renal effect and at higher concentrations causes vascular relaxation suggesting a role in volume regulation and the prevention of cardiac overloading. Rat ANP (rANP) stimulated sodium efflux from both isolated, perfused gill pouches and the whole animal. rANP also reduced subcutaneous sinus haematocrit relative to that in the ventral aorta, which is consistent with a vasodilatory role.

Opposite regulation of ANP and renin in hyperleptinemic rats

The kidney expresses the leptin receptor, and various renal effects of leptin have been reported. This study is aimed to investigate the effects of leptin on regulation of body fluid. Sprague‐Dawley rats were received adenovirus containing the rat leptin cDNA (AdCMV‐leptin) or b‐galactosidase gene (AdCMV‐bGal) and their metabolism was observed for 2–4weeks. Food intake and body weight were markedly decreased in hyperleptinemic rats. However, no significant differences in water balance and urinary excretions of electrolytes were observed between hyperleptinemic rats and pair‐feeding rats. Plasma renin activity was increased whereas plasma ANP level was decreased. No significant change in ANP binding density in kidney was observed. The ratio between plasma cAMP and cGMP levels was decreased. In isolated perfused beating atria from hyperleptinemic rats, no differences in basal ANP release, contractility and ECF translocation were observed. The response of isoproterenol‐induced decrease of ANP release was similar. These results suggest that leptin may stimulate renin secretion rate from kidney, which was followed by water retention and suppression of ANP release. Supported by KRF‐2005‐204‐E0007 and the KOSEF (R01‐2006‐000‐10554‐0).

Expression and genomic organization of a medaka fish novel membrane form of guanylyl cyclase/orphan receptor.

A novel membrane guanylyl cyclase (membrane GC), OlGC8, was identified in the medaka fish Oryzias latipes by the isolation of full-length cDNA (4958 bp) and genomic DNA (14.3 kbp) clones. Phylogenetic analysis indicated that OlGC8 does not belong in any known vertebrate membrane GC subfamily. OlGC8 consists of an extracellular domain (214 residues), a transmembrane segment (19 residues), and an intracellular protein kinase-like domain (284 residues) and a cyclase catalytic domain (228 residues), although the extracellular domain is about half the length (around 450 residues) of other known vertebrate membrane GCs. OlGC8 transiently expressed in COS-7 cells exhibited only basal guanylyl cyclase activity. None of the known ligands (rat ANP, BNP, CNP, and C-ANF) and various medaka fish tissue extracts, which activated OlGC1, OlGC2, and OlGC7 differentially, stimulated basal activity, suggesting that OlGC8 is an orphan receptor. The OlGC8 gene consists of 24 exons and exists as a single copy on the medaka fish genome. Northern blot hybridization showed that a 5 kb-OlGC8 mRNA was expressed in the kidney and the testis at a high level and a 3.3 kb-OlGC8 mRNA was expressed only in the brain. The RNase protection, RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE), and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that the 3.3 kb-OlGC8 mRNA detected in the brain is transcribed from the second transcription initiation site, and contains an intron at the position prior to the catalytic domain, the translation product of which appears to be a protein lacking the cyclase catalytic domain.

Effect of a regulatory mutation on the rat atrial natriuretic peptide gene transcription

To investigate the functional relevance of a regulatory mutation affecting the enhancer element PEA2 of the rat ANP gene we transfected rat cardiomyocytes and aortic endothelial cells with either the mutant or the wild-type ANP promoter construct (-683 +54) and performed CAT assays both at baseline and in response to Phenylephrine and Angiotensin II. In the myocardial cells we also determined the DNA/nuclear protein interaction through electrophoretic mobility shift assay. These studies showed a significantly lower degree of ANP transcription in the presence of the mutant PEA2 site, thus demonstrating its functional significance and the biological relevance of ANP gene structural alterations.

Functional Analysis of Natriuretic Peptide Receptors in the Bladder of the Toad, Bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific 125I-rat atrial natriuretic peptide (125I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with 125I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to 125I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH2, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 μmol l−1in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 μmol l−1: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH2 in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 μmol l−1was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat.

Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Natriuretic peptide receptors in the central vasculature of the toad, Bufomarinus

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus, were characterized using autoradiographical, molecular, and physiological techniques. Specific 125I-rat ANP binding sites were present in the carotid and pulmonary arteries, the lateral aorta, the pre- and post-cava, and the jugular vein, and generally occurred in each layer of the blood vessel. The 125I-rat ANP binding was partially displaced by the specific natriuretic peptide receptor C ligand, C-ANF, which indicates the presence of two types of natriuretic peptide receptors in the blood vessels. This was confirmed by a RT-PCR study, which demonstrated that guanylyl cyclase receptor (NPR-GC) and NPR-C mRNAs are expressed in arteries and veins. An in vitro guanylyl cyclase assay showed that frog ANP stimulated the production of cGMP in arterial membrane fractions. Physiological recordings from isolated segments of the carotid and pulmonary arteries and the lateral aorta, which had been pre-constricted with arginine vasotocin, showed that rat ANP, frog ANP and porcine CNP relaxed the vascular smooth muscle with relatively similar potency. Together, the data show that the central vasculature contains two types of natriuretic peptide receptors (NPR-C and NPR-GC) and that the vasculature is a target for ANP and CNP.

Evidence of a guanylyl cyclase natriuretic peptide receptor in the gills of the new zealand hagfish Eptatretus cirrhatus (Class Agnatha).

Natriuretic peptide binding sites were examined in the gills of the hagfish Eptatretus cirrhatus (Class Agnatha, subfamily Eptatretinae) using radio-ligand binding techniques, molecular cloning and guanylyl cyclase assays. Iodinated rat atrial natriuretic peptide ((125)I-rANP) and iodinated porcine C-type natriuretic peptide ((125)I-pCNP) bound specifically to the lamellar folds and cavernous tissue of E. cirrhatus gills, and 0.3 nmol l(-1) rat ANP competed for 50 % of specific (125)I-rANP binding sites. Affinity cross-linking of (125)I-rANP to gill membranes followed by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a single binding site of 150 kDa. In the presence of Mn(2+), 0.1 nmol l(-1) rANP inhibited cGMP production, whereas 1 micromol l(-1) rANP stimulated cGMP production rates. At 1 micromol l(-1), pCNP also stimulated cGMP production. The production of cGMP was also measured in the presence and absence of ATP with either Mn(2+) or Mg(2+). Reverse transcriptase polymerase chain reaction (RT-PCR) of hagfish gill RNA, followed by cloning and sequencing of PCR products, produced a partial cDNA sequence of a natriuretic peptide guanylyl cyclase receptor. The deduced amino acid sequence indicated 87-91 % homology with other natriuretic peptide guanylyl cyclase receptors. This study indicates the presence of a natriuretic peptide guanylyl cyclase receptor in the gills of E. cirrhatus that is similar to the natriuretic peptide guanylyl cyclase receptors in higher vertebrates. These observations demonstrate that the coupling of natriuretic peptide receptors with guanylyl cyclase has a long evolutionary history.

Antiproliferative effects of NO and ANP in cultured human airway smooth muscle.

Airway smooth muscle (ASM) hypertrophy and hyperplasia are important determinants of bronchial responsiveness in asthma, and agents that interfere with these processes may prevent airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC) proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP; 10(-6) to 10(-4) M) and sodium nitroprusside (10(-5) to 10(-3) M) and human atrial natriuretic peptide [ANP-(1-28); 10(-8) to 10(-6) M], which activate soluble and particulate guanylyl cyclases, respectively, inhibited serum- and thrombin-induced proliferation of cultured HASMCs. The antimitogenic effect of SNAP was reversed by hemoglobin (10(-5) M), an NO scavenger, suggesting that NO donation was involved. The antiproliferative effects of SNAP and ANP-(1-28) were potentiated by the cGMP-specific phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP (10(-6) to 10(-3) M), suggesting that cGMP-dependent mechanisms were involved. However, first, ANP-(1-28) produced a smaller antiproliferative effect than SNAP in contrast to their abilities to elevate cGMP, and second, rat ANP-(104-126), which binds selectively to ANP clearance receptors without elevating cGMP, had a small antiproliferative effect, suggesting that cGMP-independent mechanisms were also involved. These results provide evidence for a novel antiproliferative effect of NO and ANP in HASMCs mediated through cGMP-dependent and cGMP-independent mechanisms.

Distribution and Characterization of Natriuretic Peptide Receptors in the Kidney of the Toad, Bufo marinus

The location and characteristics of atrial natriuretic peptide binding sites in the kidney of the toad, Bufo marinus, were determined. Specific 125I-rANP binding sites were observed on glomeruli and blood vessels, but little if any binding was observed over regions corresponding to the renal tubules. 125I-rANP binding in tissue sections and/or isolated membranes was completely displaced in the presence of 1 μM rat ANP, frog ANP, and porcine C-type natriuretic peptide (membranes only); however, residual binding remained after incubation with 1 μM of the NPR-C ligand, C-ANF, indicating the presence of two distinct binding sites. Electrophoresis of kidney membranes cross-linked to 125I-rANP identified specific bands at approximately 70 and 140 kDa which correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP production rates in membrane preparations, while C-ANF had no stimulatory effect. Two partial cDNA clones generated using primers based on conserved regions of vertebrate natriuretic peptide receptors showed high homology to an NPR-C and the natriuretic peptide guanylate cyclase receptors (NPR-GC), respectively. This study provides evidence that the kidney of B. marinus contains both NPR-C and NPR-GC and that the glomerulus is potentially the principal site of ANP regulation in the kidneys.

Natriuretic peptides and the control of catecholamine release in two freshwater teleost and a marine elasmobranch fish

Experiments were carried out in situ and in vivo to investigate the relationship between natriuretic peptides (NPs) and humoral catecholamine secretion in the American eel (Anguilla rostrata), rainbow trout (Oncorhynchus mykiss) and spiny dogfish ( Squalus acanthias). In situ perfusion of the chromaffin tissue of A. rostrata with homologous atrial NP (ANP; 10 9 mol l 1 ) or ventricular NP (VNP; 10 9 mol l 1 ), or O. mykiss with either rat ANP (10 9 mol l 1 ), eel VNP (10 9 mol l 1 ), or trout VNP (10 9 mol l 1 ), did not significantly affect basal or carbacholelicited (10 5 mol kg 1 ) catecholamine release. Bolus injections of homologous ANP (10 9 mol kg 1 ) or VNP (10 9 mol kg 1 )i nA. rostrata in vivo elicited a rapid and prolonged reduction in arterial blood pressure and an increase in heart rate (fH ); circulating plasma catecholamine levels were unaffected. In O. mykiss, bolus injections of rat ANP (10 9 mol kg 1 ) or trout VNP (10 9 mol kg 1 ) elicited a significant bi-phasic pressor-depressor response and a marked increase in fH . Neither the acute pressor or the longer-term depressor effects of NPs in O. mykiss were associated with any change in circulating plasma catecholamine levels. In S. acanthias, bolus injections of homologous C-type natriuretic peptide (CNP; 10 9 mol kg 1 ) elicited a bi-phasic pressor-depressor response, an increase in systemic resistance, a decrease in cardiac output and stroke volume, but no change in fH . Plasma noradrenaline levels were elevated 15-fold after CNP injection while circulating adrenaline levels remained unchanged. These results show that NPs of systemic origin do not directly or indirectly affect basal or cholinergically-mediated catecholamine release in A. rostrata and O. mykiss and that the initial pressor response to NP’s in trout cannot be attributed to an elevation of circulating catecholamines. Conversely, CNP appears to be a potent secretagogue (direct or indirect) of noradrenaline release in S. acanthias and thus there is likely to be a significant humoral adrenergic component to the cardiovascular effects of NPs in this species.