Natriuretic Receptor Research Articles

Exploring the molecular cardioprotection mechanism of the natriuretic-like peptide lebetin 2: An in silico study

Lebetin 2 (L2), a natriuretic-like peptide, exerts potent cardioprotective effects in experimental myocardial ischemia (MI), as well as additional cardiac actions compared to human B-type natriuretic peptide (BNP). Here, we determined by a computational approach the dynamic interaction of L2 with natriuretic receptors (NPRs), to understand its molecular mechanism of action and to develop therapeutics for the treatment of coronary disease. We used homology modeling, protein-peptide docking and molecular dynamics (MD) simulation to assess interaction parameters between L2 and NPRs. L2 binding affinity was studied for different NPR species (human, rat, mice and chicken), and L2 dynamic interaction was assessed for human NPRs. Our data show that L2 has a higher binding affinity and a stronger interaction for the three human NPRs (NPR-A, -B and -C) compared to BNP. L2 binds NP receptors with an affinity preference to NPR-A>NPR-C>NPR-B. L2 also binds to human NPR-A and NPR-C with a higher affinity than in other species. NPR-C binding to L2 was found to be higher than that of BNP in all species. Likewise, the L2/NPR-C complex has been shown to have the most favorable interaction compared to other NP receptors. Using an in silico approach, we determined that, compared to BNP, L2 has a higher binding affinity and a stronger interaction to human NPRs, especially to types A and C receptors. Our results suggest that the discrepancies previously reported between L2 and BNP actions during MI could be attributed to differences in their affinities to natriuretic receptors. More specifically, the high affinity of L2 to NPR-C could provide further explanation on how L2 acts during cardiac ischemia. These features make L2 a promising candidate for drug design towards new compounds with high activity, affinity, stability, and fewer adverse effects, and pave the way for the future design of small molecule therapeutics.

Exploring the molecular cardioprotection mechanism of the natriuretic-like peptide lebetin 2: An in silico study

Lebetin 2 (L2), a natriuretic-like peptide, exerts potent cardioprotective effects in experimental myocardial ischemia (MI), as well as additional cardiac actions compared to human B-type natriuretic peptide (BNP). Here, we determined by a computational approach the dynamic interaction of L2 with natriuretic receptors (NPRs), to understand its molecular mechanism of action and to develop therapeutics for the treatment of coronary disease. We used homology modeling, protein-peptide docking and molecular dynamics (MD) simulation to assess interaction parameters between L2 and NPRs. L2 binding affinity was studied for different NPR species (human, rat, mice and chicken), and L2 dynamic interaction was assessed for human NPRs. Our data show that L2 has a higher binding affinity and a stronger interaction for the three human NPRs (NPR-A, -B and -C) compared to BNP. L2 binds NP receptors with an affinity preference to NPR-A>NPR-C>NPR-B. L2 also binds to human NPR-A and NPR-C with a higher affinity than in other species. NPR-C binding to L2 was found to be higher than that of BNP in all species. Likewise, the L2/NPR-C complex has been shown to have the most favorable interaction compared to other NP receptors. Using an in silico approach, we determined that, compared to BNP, L2 has a higher binding affinity and a stronger interaction to human NPRs, especially to types A and C receptors. Our results suggest that the discrepancies previously reported between L2 and BNP actions during MI could be attributed to differences in their affinities to natriuretic receptors. More specifically, the high affinity of L2 to NPR-C could provide further explanation on how L2 acts during cardiac ischemia. These features make L2 a promising candidate for drug design towards new compounds with high activity, affinity, stability, and fewer adverse effects, and pave the way for the future design of small molecule therapeutics.

Natriuretic peptides appeared after their receptors in vertebrates

BackgroundIn mammals, the natriuretic system contains three natriuretic peptides, NPPA, NPPB and NPPC, that bind to three transmembrane receptors, NPR1, NPR2 and NPR3. The natriuretic peptides are known only in vertebrates. In contrast, the receptors have orthologs in all the animal taxa and in plants. However, in non-vertebrates, these receptors do not have natriuretic properties, and most of their ligands are unknown. How was the interaction of the NP receptors and the NP established in vertebrates? Do natriuretic peptides have orthologs in non-vertebrates? If so, what was the function of the interaction? How did that function change? If not, are the NP homologous to ancestral NPR ligands? Or did the receptor’s binding pocket completely change during evolution?MethodsIn the present study, we tried to determine if the pairs of natriuretic receptors and their ligands come from an ancestral pair, or if the interaction only appeared in vertebrates. Alignments, modeling, docking, research of positive selection, and motif research were performed in order to answer this question.ResultsWe discovered that the binding pocket of the natriuretic peptide receptors was completely remodeled in mammals. We found several peptides in non vertebrates that could be related to human natriuretic peptides, but a set of clues, as well as modeling and docking analysis, suggest that the natriuretic peptides undoubtedly appeared later than their receptors during animal evolution. We suggest here that natriuretic peptide receptors in non vertebrates bind to other ligands.ConclusionsThe present study further support that vertebrate natriuretic peptides appeared after their receptors in the tree of life. We suggest the existence of peptides that resemble natriuretic peptides in non-vertebrate species, that might be the result of convergent evolution.

Abstract P2047: Human Renal Proximal Tubule Cells Cultured Under High Salt Conditions Increase Angiotensin Type 2 Receptor Abundance In Secreted Exosomes

The dopamine type 1 (D 1 R) and angiotensin type 2 (AT 2 R) are both natriuretic receptors that regulate renal sodium homeostasis. Aberrant D 1 R and AT 2 R mechanisms are implicated in hypertension, salt-sensitive hypertension, and in normotensive salt sensitives. Increased AT 2 R recruitment to the plasma membrane has been shown to occur when the D 1 R is stimulated by dopamine and increases with increased extracellular and intracellular sodium. RPTCs secrete exosomes which are plasma membrane vesicles between 50 and 90 nm in diameter and which encapsulate proteins and microRNA from the cell. We hypothesized that exosomal AT 2 R may reflect plasma membrane abundance of the RPTCs responding to the exposure to increased sodium. This study examined the abundance of the AT 2 R found on the surface of these exosomes in response to changing salt concentrations. Immortalized hRPTC were treated in serum free media cultured overnight with either an additional 50 mM sodium chloride or choline chloride as an osmotic equivalent, and exosomes were purified and concentrated from cell culture supernatant. The exosomes were then captured with magnetic lectin affinity beads and immunostained with fluorescent AT 2 R that increased in high salt conditions compared to vehicle control treated cells (VEH 71,884±7099 RFU, n=17, 190 mM Sodium, 94,180±5487 RFU, n=16, p= 0.018, t-test). The increase levels of AT 2 R are appropriate for their natriuretic activity. Future work will examine this response as it relates to salt sensitivity and inverse salt sensitivity of blood pressure as we previously reported using miRNA.

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.

Abstract P320: Cooperative Signaling and Membrane Recruitment of the Dopamine-1 (D 1 R) and Angiotensin Type 2 (AT 2 R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

The D 1 R and AT 2 R are interdependent natriuretic receptors that are critical for the regulation of renal sodium balance and are implicated in both hypertension and salt-sensitivity. D 1 R and AT 2 R stimulation with the D 1 R agonist fenoldopam (FEN) and Angiotensin III (AngIII), selectively increases cAMP and cGMP, respectively, leading to D 1 R and AT 2 R recruitment to the cell surface (measured by fluorescent extracellular receptor epitope specific antibodies). The interdependence of the D 1 R and AT 2 R recruitment was investigated following AT 2 R stimulation with AngIII (10 nmol/L 1 hour) which leads to D 1 R cell surface recruitment using a normally D 1 R/Gs coupled cell line (i22) (VEH 8209±863, AngIII 19485±3425 RFU, n=6, p<0.05) but not in a D 1 R uncoupled from Gs cell line (i19). These data were verified by increasing intracellular sodium with monensin (100 μM, 1 hr) and measuring cell surface binding with a fluorescent labelled D 1 R agonist SKF83566 (10 nmol/L, 1 hr, i22 1.67±0.7 fold, n=5, p<0.0001, i19, NS). The importance of dopamine in regulating these receptor actions was shown by stimulating the AT 2 R with AngIII and measuring D 1 R recruitment followed by blockade of amino acid decarboxylase using either carbidopa or benzeraside (100 μM each, 1 hour). Additionally, FEN stimulated D 1 R surface recruitment is blocked by the AT 2 R inhibitor PD123319 (1 μM, 1 hour) and the coupling defect is fully rescued by 8Br-cGMP (1 mmol/L, 1 hour), a cell permeable second messenger normally thought to signal specifically through the AT 2 R. We then show that FEN stimulation leads to increased production of AngIII only in the D 1 R/Gs coupled i22 cell line (5.82±0.43 fold, n=6, p<0.05) but not in i19 the D 1 R/Gs uncoupled cell line and this production was blocked with an aminopeptidase A inhibitor, EC-33 (10 μmol/L, 1 hour). Addition of 8Br-cAMP (1mmol/L 1 hour) not only leads to an increase in AngIII production, but also cGMP production (4.33±0.36 fold, n=6, p<0.05) that is blocked by PD123319 (potent, selective, non-peptide angiotensin AT 2 R antagonist, 1 μM, 1 hour). In summary we not only show that dopaminergic stimulation leads to increased AngIII production in a D 1 R coupled manner, but also that AngIII stimulation of AT 2 R leads to dopaminergic activation in a D 1 R coupled manner.

Acromesomelic dysplasia (Marotaeux type) associated with craniovertebral junction anomaly: A report of a rare case and review of literature

Acromesomelic dysplasia Maroteaux type (AMDM) is a rare autosomal recessive osteochondrodysplasia. The responsible gene, AMDM gene, in human beings, has been mapped on 9p13-q12 chromosome by homozygous mapping and pathogenic mutation was later identified in natriuretic receptor B (NPR-B) which has been implicated in the regulation of skeletal growth. Till now, around 40 to 50 cases of AMDM have been described in the world literature. Association of the congenital craniovertebral (CV) junction anomaly has not been reported. Here we are presenting a case of AMDM, with CV junction anomaly. A 10-year boy presented with short stature (122 cm) with short distal limbs, symptomatic for thoracic kyphoscoliosis with back pain. On examination there were no neurological deficits. On radiological investigation, he was found to have short and broad phalanges and toes, thoracic kyphoscoliosis, abnormal pelvic ring, mild ventriculomegaly, cervical syringomyelia and tonsillar descent below foramen magnum, hydrocephalus, os odontoideum with Klippel-Feil anomaly. This was diagnosed as AMDM with congenital os odontoideum, Klippel-Feil anomaly with Arnold-Chiari malformation (ACM) type-1. The patient underwent posterior fossa decompression by removal of foramen magnum ring along with C1 arch for ACM type-1. Kyphosis was left for conservative treatment till further observation and required orthopedic correction in his further age. To the best of our knowledge this is a very rare entity of AMDM with congenital CV junction anomaly.

ANP system activity predicts variability of fat mass reduction and insulin sensitivity during weight loss

IntroductionIn weight loss trials, a considerable inter-individual variability in reduction of fat mass and changes of insulin resistance is observed, even under standardized study conditions. The underlying mechanisms are not well understood. Given the metabolic properties of the atrial natriuretic peptide (ANP) system, we hypothesized that ANP signaling might be involved in this phenomenon by changes of ANP secretion or receptor balance. Therefore, we investigated the impact of systemic, adipose and myocellular ANP system on metabolic and anthropometric improvements during weight loss. MethodsWe comprehensively investigated 143 subjects (31 male, 112 female) before and after a 3 month-standardized weight loss program. The time course of BMI, fat mass, insulin sensitivity, circulating mid-regional proANP (MR-proANP) levels as well as adipose and myocellular natriuretic receptor A (NPR-A) and C (NPR-C) mRNA expression were investigated. ResultsBMI decreased by −12.6±3.7%. This was accompanied by a remarkable decrease of adipose NPR-C expression (1005.0±488.4 vs. 556.7±465.6; p<0.001) as well as a tendency towards increased adipose NPR-A expression (4644.7±946.8 vs. 4877.6±869.8; p=0.051). Weight loss induced changes in NPR-C (ΔNPR-C) was linked to relative reduction of total fat mass (ΔFM) (r=0.281; p<0.05), reduction of BMI (r=0.277; p<0.01), and increase of free fatty acids (ΔFFA) (r=−0.258; p<0.05). Basal NPR-C expression and weight loss induced ΔNPR-C independently explained 22.7% of ΔFM. In addition, ΔMR-proANP was independently associated with improvement of insulin sensitivity (standardized ß=0.246, p<0.01). DiscussionANP receptor expression predicted the degree of weight loss induced fat mass reduction. Our comprehensive human data support that peripheral ANP signalling is involved in control of adipose tissue plasticity and function during weight loss. (Funded by the Deutsche Forschungsgemeinschaft (KFO281/2), the Berlin Institute of Health (BIH) and the German Centre for Cardiovascular Research (DZHK/BMBF); ClinicalTrials.gov number: NCT00850629).

0309 : Cardioprotective effect of a novel snake venom derived natriuretic peptide during myocardial ischemia reperfusion injury

In this study, we aimed at testing the therapeutic potential of a novel Natriuretic Peptide (NPs), identified from Tunisian snake venom, when administered during reperfusion. Langendorff perfused male Wistar rat hearts were subjected to 30min regional coronary artery occlusion followed by 90min reperfusion. Hearts were treated either with brain natriuretic peptide BNP (10nM) or NPs (200nM). In another set of experiments, hearts were pretreated with either isatin (100μM), a natriuretic peptide receptors blocker or 5HD (10μM), a mitochondrial KATP channels blocker. Post ischemic cardiac haemodynamic parameters, using Labt Chart (ADInstruments) and infarct size (IS), using planimetry, were evaluated. Western blotting experiments for studying cGMP and Reperfusion Injury Salvage Kinases pathways were performed. Calcium Retention Capacity (CRC) to evaluate the mitochondrial function was also assessed by oxygraphy. BNP and NPs significantly decreased the IS by 60% and 62% respectively compared to control non treated hearts (p<0.001). Regarding hemodynamic parameters, both BNP and NPs improved the developed pressure (DP) by 135% and 152% respectively (p<0.05). These beneficial effects are abolished after pretreatment by isatin or 5HD. NPs significantly increased the expression of pAKT, pGSK3ß and PKCεwhen BNP increased pERK1/2 compared to control group. Both BNP and NPs increased the CRC by 124% and 86% respectively compared to control group. Our results demonstrate that NPs and BNP have cardioprotective effects during acute myocardial ischemia. These effects are mediated, for both drugs, by natriuretic receptors and by the activation of mitochondrial KATP channels. The cardioprotection involves the two PI3K/Akt/ERK and cGMP-PKC signaling pathways which converge to the mitochondria. Administration of NPs may provide a novel therapeutic strategy in acute myocardial isch

Dipyridamole-induced C-type natriuretic peptide mRNA overexpression in a minipig model of pacing-induced left ventricular dysfunction

Dipyridamole (DP) restores ischemic tissue blood flow stimulating angiogenesis in eNOS-dependent pathways. C-type natriuretic peptide (CNP) is expected to mimic the migration-stimulatory effect of NO via a cGMP-dependent mechanism. Aim of this study was to assess the role of concomitant treatment with DP on CNP levels in blood and myocardial tissue of minipigs with left ventricular dysfunction (LVD) induced by pacing at 200bpm in the right ventricular apex. Minipigs with DP therapy (DP+, n=4) or placebo (DP−, n=4) and controls (C-SHAM, n=4) underwent 2D-EchoDoppler examination and blood collection before and after 4 weeks of pacing, when cardiac tissue was collected. Histological/immunohistochemical analyses were performed. CNP levels were determined by radioimmunoassay; cardiac CNP, BNP, natriuretic receptors expression by Real-Time PCR. After pacing, cardiac parameters resulted less impaired in DP+ compared to DP−. Histological sections presented normal morphology while the arteriolar density resulted: C-SHAM: 9.0±1.2; DP−: 4.9±0.3; DP+: 6.5±0.6number/mm2; C-SHAM vs DP− and DP+ p=0.004, p=0.04, respectively. CNP mRNA resulted lower in DP− compared to C-SHAM and DP+ as well as NPR-B (p=0.011, DP− vs DP+). Both NPR-A/NPR-C mRNA expressions were significantly (p<0.001) lower both in DP− and DP+ compared to C-SHAM. BNP mRNA was higher in LVD. CNP plasma levels showed a similar trend with respect to gene expression (C-SHAM: 30.5±15; DP−: 18.6±5.5; DP+: 21.2±4.7pg/ml). These data suggest that DP may serve as a preconditioning agent to increase the protective CNP-mediated endocrine response in LVD. This response, mediated by its specific receptor NPR-B, may offer new insights into molecular targets for treatment of LVD.

Epidermal growth factor-network signaling mediates luteinizing hormone regulation of BNP and CNP and their receptor NPR2 during porcine oocyte meiotic resumption.

The epidermal growth factor (EGF) network, induced by luteinizing hormone (LH), plays an essential role during the regulation of oocyte maturation, cumulus expansion, and ovulation. Binding of brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) to natriuretic receptor 2 (NPR2) generates cyclic guanosine monophosphate (cGMP), a key inhibitor that sustains porcine oocyte meiotic arrest. This correlation suggests that LH interacts with natriuretic-peptide signaling, possibly via the EGF network, to promote porcine meiotic resumption. In testing this hypothesis, we found that the majority of porcine oocytes remain arrested in the germinal-vesicle stage after 44 hr of co-culturing cumulus-oocyte complexes with 10(7) granulosa cells, which secreted active BNP and CNP; these natriuretic peptides associate with NPR2 on cumulus cells, thereby inhibiting porcine oocyte maturation. This inhibitory effect of BNP and CNP was relieved by EGF-like growth factors, whose expression naturally increases in granulosa cells 18 hr after human chorionic gonadotropin injection. LH and the EGF-like peptide amphiregulin (AREG) decreased BNP and CNP production in granulosa cells and down-regulated NPR2 expression in cumulus cells, which together decreased oocyte cGMP to levels that permit meiotic resumption. The effects of AREG on the gene expression of natriuretic-peptide signaling components and on oocyte maturation were completely blocked by the EGF receptor kinase inhibitor AG1478; the effect of LH, however, was only partially reversed by AG1478. Based on these results, LH regulates natriuretic-peptide signaling, although other pathways also cooperate with the EGF network to induce porcine oocyte maturation.

New elements in the C-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption.

C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.

Brain natriuretic peptide is a potent vasodilator in aged human microcirculation and shows a blunted response in heart failure patients.

BackgroundBrain natriuretic peptide (BNP) is normally present in low levels in the circulation, but it is elevated in parallel with the degree of congestion in heart failure subjects (CHF). BNP has natriuretic effects and is a potent vasodilator. It is suggested that BNP could be a therapeutic alternative in CHF. However, we postulated that the high levels of circulating BNP in CHF may downregulate the response of microvascular natriuretic receptors. This was tested by comparing 15 CHF patients (BNP > 3000 ng/L) with 10 matched, healthy controls.MethodsCutaneous microvascular blood flow in the forearm was measured by laser Doppler Flowmetry. Local heating (+44°C, 10 min) was used to evoke a maximum local dilator response.ResultsNon-invasive iontophoretic administration of either BNP or acetylcholine (ACh), a known endothelium-dependent dilator, elicited an increase in local flow. The nitric oxide synthase inhibitor, l-N-Arginine- methyl-ester (L-NAME), blocked the BNP response (in controls). Interestingly, responses to BNP in CHF patients were reduced to about one third of those seen in healthy controls (increase in flow: 251% in CHF vs. 908% in controls; P < 0.001). In contrast, the vasodilator responses to ACh and to local heating were only somewhat attenuated in CHF patients. Thus, dilator capacity and nitric oxide signalling were not affected to the same extent as BNP-mediated dilation, indicating a specific downregulation of the latter response.ConclusionsThe findings show for the first time that microvascular responses to BNP are markedly reduced in CHF patients. This is consistent with the hypothesis of BNP receptor function is downregulated in CHF.

CGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

Abstract 463: Transregulation and Plasma Membrane Recruitment of the Dopamine-1 (D1R) and Angiotensin Type 2 (AT2R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

Both the D1R and AT2R are key natriuretic receptors in that stimulation by dopamine agonists or AT2R agonist angiotensin III (AngIII) increases cAMP and cGMP, respectively, causing inhibition of sodium reabsorption. The interdependence of D1R and AT2R stimulation on receptor translocation has not been studied. We utilized normally coupled RPTC (nRPTC) and RPTC with defective D1R coupling to adenylyl cyclase (uRPTC). A 1 hr addition of the cell permeable cyclic nucleotide analogs 8Br-cGMP (1 mM) or 8Br-cAMP (1 mM) caused a similar recruitment of both D1R and AT2R, as measured by cell surface biotinylation and western blotting (see table below). Flow cytometry confirmed these results using fluorescently labeled extracellular epitope-specific D1R and AT2R antibodies. When 8Br-cAMP and 8Br-cGMP were added together, no added effects were seen. AngIII-alone increased plasma membrane recruitment of D1R (1.23±0.04 fold, n=6, p&lt;0.05, VEH=0.63) and AT2R (1.65±0.10 fold, n=6, p&lt;0.05, VEH=0.69) in nRPTC but not in uRPTC. The nRPTC response was completely blocked by the AT2R specific antagonist PD123319 or the D1R antagonist LE300. Inhibition of either PKA with adenosine-3’, 5’-cyclic monophosphorothioate Rp-Isomer (100 μM) or PKG with 8-(4-chlorophenylthio) guanosine-3’, 5’ -cyclic monophosphorothioate Rp-isomer (10 μM) completely inhibited the cell surface recruitment of both D1R and AT2R when stimulated with 8Br-cAMP or 8Br-cGMP, respectively. In conclusion, we found a novel AngIII-interdependent AT2R and D1R cell surface recruitment in human nRPTC cells. Furthermore, defects in this pathway in uRPTC can be bypassed by incubating the cells with either 8Br-cAMP or 8Br-cGMP.

Chronic natriuretic peptide protein therapeutics

The natriuretic peptides (NP) consist of a group of genetically distinct peptides with a 17 amino acid disulfide ring structure that exert important paracrine and endocrine actions in cardiorenal homeostasis. ANP and BNP bind to the natriuretic peptide-A receptor (NPR-A), which via 3',5'-cyclic guanosine monophosphate (cGMP) mediates natriuresis, vasodilatation, renin-aldosterone inhibition, anti-mitogenesis, and positive lusitropism. CNP lacks natriuretic actions, but possesses vasodilating and important growth- and fibrotic-inhibiting actions via the guanylyl cyclase-linked natriuretic peptide-B receptor (NPR-B). CD-NP now represents the first designer NP which, unlike ANP, BNP or CNP, co-activates both NPR-A and NPR-B. In both experimental and human heart failure (HF), investigations have supported the hypothesis that the capacity of the cardiomyocytes to produce and/or release the NP may be overwhelmed in HF due to the increased demands of the system, thus leading to a state of relative deficiency of biologically active endogenous NP. Hence, giving the favorable cardiorenal and humoral proprieties of the NP, their chronic exogenous delivery is now viewed as favorable therapeutic opportunity for the treatment of cardiovascular diseases. We have previously reported that subcutaneous (SQ) administration of BNP in experimental HF resulted in improved cardiac output with reduced systemic vascular resistance and cardiac filling pressures. Importantly, we translated these studies to humans and completed a pilot study to establish safety and efficacy of 8 weeks of chronic SQ BNP administration in human symptomatic systolic HF. More importantly, we have recently completed 2 proof of concept studies to assess the biological effects of 12 weeks of SQ BNP administration in patients with preclinical systolic dysfunction and patients with preclinical diastolic dysfunction (Stage B HF). These studies demonstrated that chronic SQ BNP induced a sustained generation of cGMP, which, importantly, was associated with persistent enhanced renal natriuretic response to volume overload. Going beyond BNP, CD-NP (Cenderitide) is in clinical development as an outpatient therapy during the post-acute HF period, to be administered continuously for up to 90 days after hospital discharge via subcutaneous pump with the goals of reducing rehospitalization. Recently, a phase 1 study in HF patients was completed which demonstrated that SQ CD-NP infusion achieved desirable pharmacokinetic levels and was well tolerated. Pre-clinically, we have recently designed an in situ polymer precipitation delivery system suitable for the chronic and sustained release of CD-NP. With the appropriate polymer-gel formulation, CD-NP release could be sustained over 3 weeks, thus inducing prolonged cGMP activation and favourable biological actions. Preclinical and early phase clinical studies have demonstrated the potential beneficial actions of chronic NP therapeutics. Further studies are warranted to determine if these functional responses can be translated into improved clinical outcomes and contribute to delaying the progression of HF.

Physiology and clinical significance of natriuretic hormones

The natriuretic system consists of the atrial natriuretic peptide (ANP) and four other similar peptides including the wrongly named brain natriuretic peptide (BNP). Chemically they are small peptide hormones predominantly secreted by the cardiac myocytes in response to stretching forces. The peptide hormones have multiple renal, hemodynamic, and antiproliferative effects through three different kinds of natriuretic receptors. Clinical interest in these peptide hormones was initially stimulated by the use of these peptides as markers to differentiate cardiac versus noncardiac causes of breathlessness. Subsequently work has been done on using these peptides to prognosticate patients with acute and chronic heart failure and those with acute myocardial infraction. Synthetic forms of both atrial- and brain-natriuretic peptides have been studied and approved for use in acute heart failure with mixed results. This review focuses on the biochemistry and physiology of this fascinating hormone system and the clinical application of these hormones.

Chimeric natriuretic peptide ACNP stimulates both natriuretic peptide receptors, the NPRA and NPRB

Here, we investigated the receptor profile of the newly designed natriuretic peptide (NP) ACNP consisting of the N- and C-terminus of human ANP and the ring structure of CNP, its potency/efficacy in stimulating cGMP generation in primary cells, and its stability towards peptidase activity. ACNP stimulated both human natriuretic peptide receptors (NPRs), NPRA and NPRB, as potent as their native ligands in receptor transfected cells. Consequently, ACNP was more efficient in generating cGMP compared to ANP, BNP, and CNP, in primary cells expressing both NPRs. All NPs have been similarly degraded by neprilysin, except the neprilysin-resistant BNP. However, ACNP was fastest degraded in serum, while CNP was most stable. Congruently, CNP but not ACNP reduced blood pressure most significantly after acute peptide infusion in normotensive mice. Our data identify ACNP as the first compound being able to stimulate both natriuretic receptors with similar potency and efficacy as their respective ligands.

Abstract 495: A Novel Test for Low Salt Sensitivity: Angiotensin type-II Receptor Recruitment After Dopamine-1 Receptor Stimulation in Urine-Derived Renal Proximal Tubule Cells

Salt-sensitivity of blood pressure is an inappropriate increase in blood pressure following high salt intake. Subjects in our clinical study were typed according to their salt-sensitivity status into 3 categories: High-Salt-Sensitive (HSS; ≥ 7 mmHg increase in mean arterial pressure (MAP) on a high salt diet of 300 mEq of sodium, 17% prevalence), Low-Salt-Sensitive (LSS:, who paradoxically showed a ≥ 7 mmHg increase in MAP on a low salt diet of 10 mEq of sodium, 11% prevalence), and Salt-Resistant (SR, individuals who showed no significant increase in blood pressure on either diet, 72% prevalence). We previously demonstrated that LSS subjects show increased recruitment of the natriuretic dopamine-1 receptor (D1R) to the plasma membrane following a salt stimulation as compared to HSS subjects. Stimulation of the D1R in RPTC with fenoldopam (dopaminergic agonist) results in recruitment of the natriuretic angiotensin type-2 receptor (AT2R) to the cell surface. We hypothesized that LSS individuals may also demonstrate an enhanced AT2R RPTC membrane recruitment compared to HSS individuals when challenged with fenoldopam. In order to gain access to fresh RPTC from each subject, we isolated exfoliated RPTC from randomly voided urine from SR, LSS, and HSS subjects from our clinical study. We measured three subjects from each category with a minimum of three voids for each subject. We counted individual cells as independent events using both the confocal microscope (n=245) and the flow cytometer (n=5344). We found an inverse correlation between AT2R recruitment and the degree of salt-sensitivity of blood pressure. Fenoldopam stimulated AT2R recruitment as measured by confocal microscopy (y = -0.0047x + 0.4966, R2 = 0.2488, P&lt;0.0001) and flow cytometry (y =-0.057x + 1.5645, R2=0.2912, P=0.0185). Flow cytometry provided a more sensitive diagnostic for LSS than HSS subjects. AT2R recruitment was more predictive of LSS than HSS. AT2R recruitment may be used as a rapid method to test for LSS individuals who need to be identified and encouraged to increase their sodium intake in order to avoid paradoxical hypertension.

Mechanisms of Dopamine D 1 and Angiotensin Type 2 Receptor Interaction in Natriuresis

Renal dopamine D(1)-like receptors (D(1)Rs) and angiotensin type 2 receptors (AT(2)Rs) are important natriuretic receptors counterbalancing angiotensin type 1 receptor-mediated tubular sodium reabsorption. Here we explore the mechanisms of D(1)R and AT(2)R interactions in natriuresis. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D(1)R agonist fenoldopam induced a natriuretic response that was abolished by the AT(2)R-specific antagonist PD-123319 or by microtubule polymerization inhibitor nocodazole but not by actin polymerization inhibitor cytochalasin D. By confocal microscopy and immunoelectron microscopy, fenoldopam translocated AT(2)Rs from intracellular sites to the apical plasma membranes of renal proximal tubule cells, and this translocation was abolished by nocodazole. Because D(1)R activation induces natriuresis via an adenylyl cyclase/cAMP signaling pathway, we explored whether this pathway is responsible for AT(2)R recruitment and AT(2)R-mediated natriuresis. Renal interstitial coinfusion of the adenylyl cyclase activator forskolin and 3-isobutly-1-methylxanthine induced natriuresis that was abolished either by PD-123319 or nocodazole but was unaffected by specific the D(1)R antagonist SCH-23390. Coadministration of forskolin and 3-isobutly-1-methylxanthine also translocated AT(2)Rs to the apical plasma membranes of renal proximal tubule cells; this translocation was abolished by nocodazole but was unaffected by SCH-23390. The results demonstrate that D(1)R-induced natriuresis requires AT(2)R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D(1)Rs and AT(2)Rs act in concert to promote sodium excretion in vivo.