Angiotensin-(1-5), an active mediator of renin-angiotensin system,stimulates ANP secretion via Mas receptor

Angiotensin-(1-5) [Ang-(1-5)], which is a metabolite of Angiotensin-(1-7) [Ang-(1-7)] catalyzed by angiotensin-converting enzyme (ACE), is a pentapeptide of the renin-angiotensin system (RAS). It has been reported that Ang-(1-7) and Ang-(1-9) stimulate the secretion of atrial natriuretic peptide (ANP) via Mas receptor (Mas R) and Ang II type 2 receptor (AT₂R), respectively. However, it still remains unknown whether Ang-(1-5) has a similar function to Ang-(1-7). We investigated the effect of Ang-(1-5) on ANP secretion and to define its signaling pathway using isolated perfused beating rat atria. Ang-(1-5) (0.3, 3, 10 μM) stimulated high pacing frequency-induced ANP secretion in a dose-dependent manner. Ang-(1-5)-induced ANP secretion (3 μM) was attenuated by the pretreatment with an antagonist of Mas R (A-779) but not by an antagonist of AT₁R (losartan) or AT₂R (PD123,319). An inhibitor for phosphatidylinositol 3-kinase (PI3K; wortmannin), protein kinase B (Akt; API-2), or nitric oxide synthase (NOS; L-NAME) also attenuated the augmentation of ANP secretion induced by Ang-(1-5). Ang-(1-5)-induced ANP secretion was markedly attenuated in isoproterenol-treated hypertrophied atria. The secretagogue effect of Ang-(1-5) on ANP secretion was similar to those induced by Ang-(1-9) and Ang-(1-7). These results suggest that Ang-(1-5) is an active mediator of renin-angiotensin system to stimulate ANP secretion via Mas R and PI3K-Akt-NOS pathway.     

Ventricular expression of natriuretic peptides in Npr1(-/-) mice with cardiac hypertrophy and fibrosis

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that regulate blood pressure and volume, and exert their biological actions via the natriuretic peptide receptor-A gene (Npr1). Mice lacking Npr1 (Npr(-/-)) have marked cardiac hypertrophy and fibrosis disproportionate to their increased blood pressure. This study examined the relationships between ANP and BNP gene expression, immunoreactivity and fibrosis in cardiac tissue, circulating ANP levels, and ANP and BNP mRNA during embryogenesis in Npr1(-/-) mice. Disruption of the Npr1 signaling pathway resulted in augmented ANP and BNP gene and ANP protein expression in the cardiac ventricles, most pronounced for ANP mRNA in females [414 +/- 57 in Npr1(-/-) ng/mg and 124 +/- 25 ng/mg in wild-type (WT) by Taqman assay, P < 0.001]. This increased expression was highly correlated to the degree of cardiac hypertrophy and was localized to the left ventricle (LV) inner free wall and to areas of ventricular fibrosis. In contrast, plasma ANP was significantly greater than WT in male but not female Npr1(-/-) mice. Increased ANP and BNP gene expression was observed in Npr1(-/-) embryos from 16 days of gestation. Our study suggests that cardiac ventricular expression of ANP and BNP is more closely associated with local hypertrophy and fibrosis than either systemic blood pressure or circulating ANP levels.     

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor

Aims

Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion.

Main methods

We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria.

Key findings

Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 μM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT₂R) antagonist but not by AT₁R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 μM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT₂R antagonist but not by Mas receptor antagonist.

Significance

These results suggest that Ang-(1-9) stimulates ANP secretion via the AT₂R-PI3K-Akt-NO-cGMP pathway.     

Infusion of iso-rANP(1-45) or (17-45) increases plasma immunoreactive ANP and lowers plasma renin activity and aldosterone

We have reported that a second rat atrial natriuretic peptide, iso-rANP (1-45), as well as the putative ANP homologue, iso-rANP (17-45), elicited circulatory and renal responses in the rat similar to those found after administration of ANP. Iso-rANP also interacted with ANP to potentiate the observed biological activity in the rat. In the present studies in awake dogs, intravenous infusion of low doses (6.3-50 pmol.kg-1.min-1) of iso-rANP(1-45) and iso-rANP(17-45) increased plasma immunoreactive ANP and suppressed plasma renin activity (PRA) and aldosterone. Iso-rANP, like ring-deleted analogues of ANP, may have displaced ANP from ANP clearance receptors to increase plasma ANP concentration, since factors influencing myocardial ANP release were not changed. The effect of iso-rANP (1-45) and (17-45) in lowering PRA and plasma aldosterone may therefore have been indirect, via ANP stimulation of active guanylate cyclase-linked ANP receptors. However, an additional direct effect of iso-rANP on an active receptor cannot be excluded.     

Microbial polysaccharide, HS-142-1, competitively and selectively inhibits ANP binding to its guanylyl cyclase-containing receptor.

During the search for ANP receptor ligands of microbial origin, we isolated a novel polysaccharide, HS-142-1, from culture broth of Aureobasidium sp. HS-142-1 inhibited [125I]-rANP binding to ANP receptor in rabbit kidney cortex membranes with an IC50 of 0.3 mu g/ml, but gave no effects on specific binding of [125I]-Endothelin nor [125I]-Angiotensin II to their respective receptors in bovine lung membranes. HS-142-1 competitively and selectively inhibited ANP binding to its guanylyl cyclase-containing receptor purified from solubilized bovine adrenocortical membranes and blocked cGMP production elicited by ANP. HS-142-1 is the first non-peptide antagonist selective for ANP functional receptor and will be a powerful tool to elucidate the physiological functions of ANP.     

HS-142-1, a novel polysaccharide of microbial origin, specifically recognizes guanylyl cyclase-linked ANP receptor in rat glomeruli.

HS-142-1, a novel polysaccharide, of microbial origin had been characterized as a specific antagonist of guanylyl cyclase-linked atrial natriuretic peptide (ANP) receptors (ANP-GC receptor) in bovine adrenal cortex. The effect of HS-142-1 on ANP receptors of rat glomeruli were examined. HS-142-1 blocked rat ANP (r-ANP)-stimulated cGMP production in a concentration-dependent manner, although it caused only slight inhibition in the specific binding of [125I]-rANP to the glomeruli where only a small portion of the binding sites are coupled to guanylyl cyclase. HS-142-1 recognized the 135K ANP receptor which is thought to be ANP-GC receptors but did not recognized 60K receptor, guanylyl cyclase-free type from affinity cross-linking studies with glomerular membranes. These results indicate that HS-142-1 is a specific antagonist for the ANP-GC receptor in rat glomeruli, and that it will be a powerful tool for understanding the physiological roles of ANP in renal responses.     

The 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides: application to the synthesis of Alzheimer's disease-related amyloid beta peptide (Abeta) 1-42.

An efficient 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides was applied successfully to the synthesis of amyloid beta peptide (Abeta) 1-42 via a water-soluble O-acyl isopeptide of Abeta1-42, i.e. '26-O-acyl isoAbeta1-42' (6). This paper describes the detailed synthesis of Abeta1-42 focusing on the importance of resin selection and the analysis of side reactions in the O-acyl isopeptide method. Protected '26-O-acyl isoAbeta1-42' peptide resin was synthesized using 2-chlorotrityl chloride resin with minimum side reactions in comparison with other resins and deprotected crude 26-O-acyl isoAbeta1-42 was easily purified by HPLC due to its relatively good purity and narrow elution with reasonable water solubility. This suggests that only one insertion of the isopeptide structure into the sequence of the 42-residue peptide can suppress the unfavourable nature of its difficult sequence. The migration of O-acyl isopeptide to intact Abeta1-42 under physiological conditions (pH 7.4) via O--N intramolecular acyl migration reaction was very rapid and no other by-product formation was observed while 6 was stable under storage conditions. These results concluded that our strategy not only overcomes the solubility problem in the synthesis of Abeta1-42 and can provide intact Abeta1-42 efficiently, but is also applicable in the synthesis of large difficult sequence-containing peptides at least up to 50 amino acids. This synthesis method would provide a biological evaluation system in Alzheimer's disease research, in which 26-O-acyl isoAbeta1-42 can be stored in a solubilized form before use and then rapidly produces intact Abeta1-42 in situ during biological experiments.     

Increased central AT(1)-receptor activation, not systemic vasopressin, sustains hypertension in ANP knockout mice

We tested the hypothesis that hypertension in atrial natriuretic peptide (ANP) knockout mice is caused in part by disinhibition of angiotensin II-mediated vasopressin release. Inactin-anesthetized F(2) homozygous ANP gene-disrupted mice (-/-) and wild-type (+/+) littermates were surgically prepared for carotid arterial blood pressure measurement (ABP) and background intravenous injection of physiological saline or vasopressin V(1)-receptor antagonist (Manning compound, 10 ng/g body wt) and subsequent intracerebroventricular (left lateral ventricle) injection of saline (5 microl) or ANP (0.5 microg) or angiotensin II AT(1)-receptor antagonist losartan (10 microg). Only (-/-) showed significant decrease in ABP after intracerebroventricular ANP or losartan. Both showed significant hypotension after intravenous V(1) antagonist, but there was no difference between their responses. We conclude that 1) vasopressin contributes equally to ABP maintenance in ANP-disrupted mice and wild-type controls; 2) permanently elevated ABP in ANP knockouts is associated with increased central nervous angiotensin II AT(1)-receptor activation; 3) disinhibition of central nervous angiotensin II AT(1) receptors in ANP-deficient animals does not lead to a significant increase in the importance of vasopressin as a mechanism for blood pressure maintenance.     

Microbial hydroxylation of (+/-)- and (-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into (+/-)- and (-)-xanthoxin acid by Cunninghamella echinulata

Microbial hydroxylation of (+/-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid (3a) with Cercospora cruenta, a fungus producing (+)-abscisic acid, gave a four-stereoisomeric mixture consisting of (+)- and (-)-xanthoxin acid (4a), and (+)- and (-)-epi-xanthoxin acid (5a) by an HPLC analysis with a chiral column. Screening of the microorganisms capable of oxidizing (+/-)-3a showed that Cunninghamella echinulata stereoselectively oxidized (+/-)-3a to xanthoxin acid (4a) with the some degree of enantioselectivity as (-)-3a to (-)-4a.     

G(i-1)/G(i-2)-dependent signaling by single-transmembrane natriuretic peptide clearance receptor

Single-transmembrane natriuretic peptide clearance receptor (NPR-C), which is devoid of a cytoplasmic guanylyl cyclase domain, interacts with pertussis toxin (PTx)-sensitive G proteins to activate endothelial nitric oxide synthase (eNOS) expressed in gastrointestinal smooth muscle cells. We examined the ability of NPR-C to activate other effector enzymes in eNOS-deficient tenia coli smooth muscle cells; these cells expressed NPR-C and NPR-B but not NPR-A. Atrial natriuretic peptide (ANP), the selective NPR-C ligand cANP-(4-23), and vasoactive intestinal peptide (VIP) inhibited (125)I-ANP and (125)I-VIP binding to muscle membranes in a pattern indicating high-affinity binding to NPR-C. Interaction of VIP with NPR-C was confirmed by its ability to inhibit (125)I-ANP binding to membranes of NPR-C-transfected COS-1 cells. In tenia muscle cells, all ligands selectively activated G(i-1) and G(i-2); VIP also activated G(s) via VIP(2) receptors. All ligands stimulated phosphoinositide hydrolysis, which was inhibited by ANP-(1-11), PTx, and antibodies to phospholipase C-beta3 (PLC-beta3) and Gbeta. cANP-(4-23) contracted tenia muscle cells; contraction was blocked by U-73122 and PTx and by antibodies to PLC-beta3 and Gbeta in intact and permeabilized muscle cells, respectively. VIP and ANP contracted muscle cells only after inhibition of cAMP- and cGMP-dependent protein kinases. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP in a PTx-sensitive fashion. We conclude that NPR-C is coupled to activation of PLC-beta3 via betagamma-subunits of G(i-1) and G(i-2) and to inhibition of adenylyl cyclase via alpha-subunits.     

D-galactosyl-beta1-1'-sphingosine and D-glucosyl-beta1-1'-sphingosine induce human natural killer cell apoptosis

Natural killer (NK) cells perform multiple biological functions including tumor cell lysis and eradicating virally infected cells. Here, we report for the first time that D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1- 1' sphingosine damage human NK cells. We show that these cells express T-cell-associated gene-8, the receptor for glycosphingolipids. D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine induce the in vitro chemotaxis of human NK cells. Both D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine inhibit the cytotoxicity and IFN-gamma secretion by these cells. Further analysis shows that the glycosphingolipids D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine but not any other lipid examined, which include D-lactosyl-beta1-1' sphingosine, sphingosine 1-phosphate, sphingosine, lysophosphatidic acid, and phosphatidic acid, induce the apoptosis, globoid-like formation, and multinucleation in human NK cells. These results may have important implications on diseases where glycosphingolipids accumulate.     

Synthesis of (1R,2S)-1-(3'-chloro-4' -methoxyphenyl)-1,2-propanediol (trametol) and (1R,2S)-1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol,chlorinated fungal metabolites in the natural environment

(1R,2S)-1-(3'-Chloro-4'-methoxyphenyl)-1,2propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.     

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1'-(acridin-3,6-diyl)-3',3'-dialkyldiureas

The synthesis of novel 1',1'-(acridin-3,6-diyl)-3',3'-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1'-(Acridin-3,6-diyl)-3',3'-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.     

The concentration of atrial natriuretic peptide (ANP) is decreased in plasma but not in atria in hypophysectomised rats

To study the role of the pituitary gland in the release of Atrial Natriuretic Peptide (ANP) plasma and atrial concentrations were measured both in intact and in hypophysectomized rats. The plasma concentration of ANP (pg/ml) was significantly (p less than 0.01) decreased from 143 +/- 35 to 82 +/- 29 (mean +/- SD, n) while the tissue concentration (ng/wet tissue mg) remained unchanged, 192 +/- 46 and 194 +/- 39, respectively. The total atrial amount of ANP (ug) was, however, significantly (p less than 0.01) decreased from 29.7 +/- 7.8 to 17.0 +/- 3.3 after hypophysectomy. In intact animals, a volume load (1.1ml/100 body weight g 0.9% NaCl) resulted in 2-fold (p less than 0.001) increase in the plasma ANP levels whereas similar load had no effects on plasma ANP levels in hypophysectomized animals. In both groups, the right atrial pressure was increased from about 2 to about 6 mmHg. We conclude that in the absence of pituitary gland the right atrial pressure and the atrial ANP concentration do not change but plasma ANP levels and the response to volume stimulus are attenuated.     

Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells

A single class of saturable, specific binding sites for the circulating form of atrial natriuretic peptides, ANP(99-126), was identified in rat thymus and spleen and in isolated thymocytes and spleen cells using quantitative autoradiographic techniques. In the thymus, the relative potency of ANP analogs to inhibit [125I] ANP(99-126) binding was ANP(99-126) = ANP(103-126) greater than ANP(111-126) greater than ANP(103-125). ANP(103-123) could not displace [125I]ANP(99-126) binding. Addition of ANP(99-126) stimulated the formation of cyclic GMP in isolated thymocytes and spleen cells in a dose-dependent manner. Our results indicate that immune cells have specific ANP receptors which could be coupled to guanylate cyclase activation and may play a role in the regulation of the immune response.     

Stable expression of natriuretic peptide receptors: effects of HS-142-1, a non-peptide ANP antagonist.

We established clonal cell lines stably expressing each of two subtypes of membrane bound guanylate cyclases (GC-A and GC-B), which are known as natriuretic peptide receptors. Using these cell lines, we showed that GC-A is an ANP/BNP receptor, whereas GC-B is a specific receptor for CNP. Effects of HS-142-1, a novel non-peptide ANP antagonist, on GC-A and GC-B were examined by using these cells. In cells expressing either GC-A or GC-B, HS-142-1 inhibited cGMP production elicited by ANP or CNP with IC50 values of 1.8 micrograms/ml and 1.5 micrograms/ml, respectively, and also competitively blocked specific binding of the natriuretic peptides with IC50 values of 2.2 micrograms/ml and 3.3 micrograms/ml, respectively. These results indicate that HS-142-1 is a potent antagonist of CNP as well as ANP. We also showed that CNP suppressed the growth of cells expressing GC-B by 22% and that HS-142-1 blocked the antiproliferative action of CNP.     

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.