Effects of nitric oxide and arginine vasopressin on sodium intake induced by central angiotensin II. Part 2

We study the effects of angiotensin receptors antagonists, arginine vasopressin receptor antagonist, L-arginine and L-NAME, injected into supraoptic nucleus of the hypothalamus (SON) on sodium intake induced by the injection of angiotensin II (ANGII). Holtzman rats weighing 200-250 g with canulae implanted into the SON were used. The drugs were injected in 0.5 μL over 30-60 sec. Sodium intake after injection of saline SAL+SAL 0.15 M NaCl was 0.10±00.1 mL 2 h -1; SAL+ANGII injected into SON increased sodium intake. Losartan injected prior to ANGII into SON decreased sodium intake induced by ANGII. PD123319 injected prior to ANGII produced no changes in sodium intake induced by ANGII. AVPA receptor V 1 antagonist injected prior to ANGII reduced sodium intake with a less intensity than losartan. L-arginine injected prior to ANGII decreases sodium intake at a same intensity than losartan. L-NAME injected prior to ANGII potentiated sodium intake induced by ANGII. Losartan injected simultaneously with L-arginine prior to ANGII blocked the natriorexigenic effect of ANGII. These results confirm the importance of SON in the control of sodium intake. Also suggest that both AT 1 and arginine vasopressin V 1 receptors interact with nitrergic pathways within the SON influencing the sodium metabolism by changing sodium appetite induced by ANGII. © 2007 Asian Network for Scientific Information.

Role of central α1- and α2-adrenoceptors on the dipsogenic and cardiovascular effect of angiotensin II

We investigated the effects of previous central treatment with prazosin (an α1-adrenoceptor antagonist) or clonidine (an α2-adrenoceptor agonist) on the dipsogenic, pressor and tachycardic responses produced by intracerebroventricular (ICV) injection of angiotensin II (AII) in conscious rats. Holtzman rats with a chronic cannula implanted in the lateral ventricle were tested for dipsogenic and cardiovascular (arterial pressure and heart rate) responses in separate experiments. Previous ICV treatment with clonidine (20, 40, 80 and 120 nmol) abolished the pressor, tachycardic and dipsogenic effects of ICV AII. After all doses of prazosin (40, 80 and 120 nmol), AII induced bradycardic responses, but only the 80 and 120 nmol doses of prazosin reduced the pressor responses to AII. Prazosin produced no alteration in the dipsogenic effect of AII. The results show that the periventricular α1-adrenoceptors are involved only in the cardiovascular responses produced by central AII, whereas clonidine acting through α2-adrenergic and/or imidazole receptors can modulate all actions of AII. © 1990.

Genetic transfer of a nonpeptide antagonist binding site to a previously unresponsive angiotensin receptor.

Mutational analysis based on the pharmacological differences between mammalian and amphibian angiotensin II receptors (AT receptors) previously identified 7 aa residues located in transmembrane domains (TMs) III (Val-108), IV (Ala-163), V (Pro-192, Thr-198), VI (Ser-252), and VII (Leu-300, Phe-301) of the rat AT receptor type 1b (rAT1b receptor) that significantly influenced binding of the nonpeptide antagonist Losartan. Further studies have shown that an additional 6 residues in the rAT1b receptor TMs II (Ala-73), III (Ser-109, Ala-114, Ser-115), VI (Phe-248), and VII (Asn-295) are important in Losartan binding. The 13 residues required for Losartan binding in the mammalian receptor were exchanged for the corresponding amino acids in the Xenopus AT receptor type a (xATa receptor) to generate a mutant amphibian receptor that bound Losartan with the same affinity as the rAT1b receptor (Losartan IC50 values: rAT1b, 2.2 +/- 0.2 nM: xATa, > 50 microM; mutant, 2.0 +/- 0.1 nM). To our knowledge, this is the first report of a gain-of-function mutant in which the residues crucial to formation of a ligand binding site in a mammalian peptide hormone receptor were transferred to a previously unresponsive receptor by site-directed mutagenesis. Ala substitutions and comparison of mammalian and amphibian combinatorial mutants indicated that TM III in the rAT1b receptor plays a key role in Losartan binding. Identification of residues involved in nonpeptide ligand binding will facilitate studies aimed at elucidating the chemical basis for ligand recognition in the AT receptor and peptide hormone receptors in general.

Induction of multiple reinstatements of ethanol- and sucrose-seeking behavior in Long–Evans rats by the α-2 adrenoreceptor antagonist yohimbine

Rationale Developing models to efficiently explore the mechanisms by which stress can mediate reinstatement of drug-seeking behavior is crucial to the development of new pharmacotherapies for alcohol use disorders. Objectives We examined the effects of multiple reinstatement sessions using the pharmacological stressor, yohimbine, in ethanol- and sucrose-seeking rats in order to develop a more efficient model of stress-induced reinstatement. Methods Long–Evans rats were trained to self-administer 10% ethanol with a sucrose-fading procedure, 20% ethanol without a sucrose-fading procedure, or 5% sucrose in 30-min operant self-administration sessions, followed by extinction training. After reaching extinction criteria, the animals were tested once per week with yohimbine vehicle and yohimbine (2 mg/kg), respectively, 30 min prior to the reinstatement sessions or blood collection. Levels of reinstatement and plasma corticosterone (CORT) were determined each week for four consecutive weeks. Results Yohimbine induced reinstatement of ethanol- and sucrose-seeking in each of the 4 weeks. Interestingly, the magnitude of the reinstatement decreased for the 10% ethanol group after the first reinstatement session but remained stable for the 20% ethanol group trained without sucrose. Plasma CORT levels in response to injection of both vehicle and yohimbine were significantly higher in the ethanol-trained animals compared to sucrose controls. Conclusions The stable reinstatement in the 20% ethanol group supports the use of this training procedure in studies using within-subject designs with multiple yohimbine reinstatement test sessions. Additionally, these results indicate that the hormonal response to stressors can be altered following extinction from self-administration of relatively modest amounts of ethanol.

Inhibition of form-deprivation myopia by a GABAAOr receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA), in guinea pigs

Purpose To investigate the effects of the relatively selective GABAAOr receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on form-deprivation myopia (FDM) in guinea pigs. Methods A diffuser was applied monocularly to 30 guinea pigs from day 10 to 21. The animals were randomized to one of five treatment groups. The deprived eye received daily sub-conjunctival injections of 100 μl TPMPA at a concentration of (i) 0.03 %, ( ii) 0.3 %, or (iii) 1 %, a fourth group (iv) received saline injections, and another (v) no injections. The fellow eye was left untreated. An additional group received no treatment to either eye. Prior to and at the end of the treatment period, refraction and ocular biometry were performed. Results Visual deprivation produced relative myopia in all groups (treated versus untreated eyes, P < 0.05). The amount of myopia was significantly affected by the drug treatment (one-way ANOVA, P < 0.0001); myopia was less in deprived eyes receiving either 0.3 % or 1 % TPMPA (saline = −4.38 ± 0.57D, 0.3 % TPMPA = −3.00 ± 0.48D, P < 0.01; 1 % TPMPA = −0.88 ± 0.51D, P < 0.001). The degree of axial elongation was correspondingly less (saline = 0.13 ± 0.02 mm, 0.3 % TPMPA = 0.09 ± 0.01 mm, P < 0.01, 1 % TPMPA = 0.02 ± 0.01 mm, P < 0.001) as was the VC elongation (saline = 0.08 ± 0.01 mm, 0.3 % TPMPA = 0.05 ± 0.01 mm, P < 0.01, 1 % TPMPA = 0.01 ± 0.01 mm; P < 0.001). ACD and LT were not affected (one-way ANOVA, P > 0.05). One percent TPMPA was more effective at inhibiting myopia than 0.3 % (P < 0.01), and 0.03 % did not appreciably inhibit the myopia (0.03 % TPMPA versus saline, P > 0.05). Conclusions Sub-conjunctival injections of TPMPA inhibit FDM in guinea pig models in a dose-dependent manner.

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO2 group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.

Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

Effect of angiotensin-related antihypertensives on brain neurotransmitter levels in rats.

The extensive clinical experience of angiotensin converting enzyme inhibitors and angiotensin AT(1) receptor antagonists as antihypertensive agents provide numerous examples of anecdotal evidence of improvements in cognition and mood. This study aimed to determine the effect of chronic treatment with the angiotensin converting enzyme inhibitor, perindopril, and the angiotensin AT(1) receptor antagonist, candesartan, on central neurotransmitter levels in the rat. Perindopril (1.0mg/kg/day) or candesartan (10mg/kg/day) was administered via the drinking water at for 1 week, while controls received water alone. At the end of treatment rats were sacrificed, brains removed and discrete regions dissected and analysed for noradrenaline, dopamine and its major metabolites, and serotonin content. As shown previously we found an increase in striatal dopamine levels after perindopril treatment, though this did not extend to the mesolimbic system with neurotransmitter levels unchanged in the hippocampus, nucleus accumbens and frontal cortex. Conversely, candesartan administration produced no change in dopamine, but significant decreases in both DOPAC and HVA in the striatum. In addition chronic candesartan infusion produced a significant increase in the levels of hippocampal noradrenaline and serotonin; and frontal cortex serotonin content. These results demonstrate that while angiotensin converting enzyme inhibitors and angiotensin AT(1) receptor antagonists act as antihypertensives by affecting the renin-angiotensin system, they have divergent actions on brain neurochemistry.

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro(3))GIP receptor antagonist in type 2 diabetes

In this study, we tested the biological activity of a novel acylated form of (Pro(3))glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys(16) to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro(3))GIP, (Pro(3))GIPLys(16)PAL was completely stable to the actions of DPP-IV and significantly (p <0.01 to p <0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro(3))GIPLys(16)PAL also significantly (p <0.05 to p <0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro(3))GIPLys(16)PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro(3))GIPLys(16)PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p <0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p <0.05) and pancreatic insulin was significantly reduced (p <0.05) in the (Pro(3))GIPLys(16)PAL-treated mice. These data demonstrate that acylation of Lys(16) with palmitic acid in (Pro(3))GIP does not improve its biological effectiveness as a GIP receptor antagonist.

Comparison of Dynamics of Extracellular Accesses to the β(1) and β(2) Adrenoceptors Binding Sites Uncovers the Potential of Kinetic Basis of Antagonist Selectivity.

From the molecular mechanism of antagonist unbinding in the ß(1) and ß(2) adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of ß(1) -selective Esmolol and ß(2) -selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the ß(1) adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the ß(2) adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the ß(1) adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the ß(2) adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. © 2012 John Wiley & Sons A/S.

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Near 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signalling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting ß-arrestin2 (CCK2Rß) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150,013X), acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rß. Several structural elements on both GV150,013X and in CCK2R binding cavity, which hinder binding of GV150,013X only to the CCK2Rß were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulphur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rß state. These data establish structural evidences for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.