Design, synthesis and cardiovascular evaluation of some aminoisopropanoloxy derivatives of xanthone.

A series of aminoisopropanoloxy derivatives of xanthone has been synthesized and their pharmacological properties regarding the cardiovascular system has been evaluated. Radioligand binding and functional studies in isolated organs revealed that title compounds present high affinity and antagonistic potency for α1-(compound 2 and 8), ß-(compounds 1, 3, 4, 7), α1/ß-(compounds 5 and 6) adrenoceptors. Furthermore, compound 7, the structural analogue of verapamil, possesses calcium entry blocking activity. The title compounds showed hypotensive and antiarrhythmic properties due to their adrenoceptor blocking effect. Moreover, they did not affect QRS and QT intervals, and they did not have proarrhythmic potential at tested doses. In addition they exerted anti-aggregation effect. The results of this study suggest that new compounds with multidirectional activity in cardiovascular system might be found in the group of xanthone derivatives.

Sustained adrenergic stimulation is required for the nuclear retention of TORC1 in male rat pinealocytes.

The process involved in relocation of the coactivator, transducer of regulated cAMP-regulated element-binding protein (TORC) to the cytoplasm, unlike its activation, is not well understood. Using cultured pineal cells prepared from male rats, we found that although both α- and ß-adrenergic stimulation could cause TORC1 dephosphorylation, only α-adrenergic stimulation was effective in the norepinephrine (NE)-mediated translocation of TORC1 into the nucleus. In contrast, blockade of either the α- or the ß-adrenergic receptor after NE stimulation was effective in causing the rephosphorylation and rapid relocation of TORC1 into the cytoplasm. Studies with phosphoprotein phosphatase (PP) inhibitors indicated that although both PP2A and PP2B could dephosphorylate TORC1, only PP2B could cause translocation into the nucleus. However, after NE stimulation, treatment with either PP2A or PP2B inhibitors could cause the rephosphorylation and cytoplasmic relocation of TORC1. These results indicate a requirement of continuous activation of both α- and ß-adrenergic receptors as well as PP2A and PP2B activities for the nuclear retention of TORC1 during NE stimulation. Knockdown of salt-inducible kinase 1 (SIK1) had no effect on the phosphorylation or localization of TORC1. Although overexpressing SIK1 could induce TORC1 phosphorylation in the nucleus, it did not reduce TORC1 level in the nucleus, indicating that SIK1-mediated TORC1 phosphorylation may not be sufficient for its relocation into the cytoplasm. Together, these results demonstrate that, in the rat pineal gland, different mechanisms are involved in regulating the nuclear entry and exit of TORC1 and that the SIK1-mediated phosphorylation of TORC1 may not lead to its nuclear exit.

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions.

UNLABELLED: Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. CONCLUSIONS: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.

Antiadrenergic and hemodynamic effects of ranolazine in conscious dogs.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 µg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 µg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 µM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 µM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and ß) effects.

Synthesis and blocking activities of isoindolinone- and isobenzofuranone-containing phenoxylalkylamines as potent α(1)-adrenoceptor antagonists.

This paper describes the synthesis and blocking activities of twelve new isoindolinone- and isobenzofuranone-containing phenoxylalkylamines as potent α(1)-Adrenoceptor antagonists. These compounds were synthesized in moderate to good yields starting from 3,4-dimethylphenol, and characterized with (1)H-NMR, MS, IR and elemental analysis. Their blocking activities toward α(1)-Adrenoceptors were evaluated on isolated rat anococcygeus muscles. The results indicated that these compounds were very strong in blocking α(1)-Adrenoceptors, and most of them exhibited activities that were comparable to that of known potent α(1)-Adrenoceptor antagonist 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (DDPH).

Cell surface delivery and structural re-organization by pharmacological chaperones of an oligomerization-defective alpha(1b)-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers.

Many G-protein-coupled receptors, including the alpha(1b)-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the alpha(1b)-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this alpha(1b)-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of alpha(1b)-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the alpha(1b)-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant alpha(1b)-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between alpha(1b)-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the alpha(1b)-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the alpha(1b)-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

Potential relevance of alpha(1)-adrenergic receptor autoantibodies in refractory hypertension.

BACKGROUND: Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target. METHODOLOGY/PRINCIPAL FINDINGS: To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca(2+) in cardiomyocytes and induce mesentery artery segment contraction. CONCLUSIONS/SIGNIFICANCE: Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor.

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.

The in silico insights of alpha-adrenergic receptors over the last decade: methodological approaches and structural features of the 3D models.

Homology modeling has been widely used in the latest years in order to overcome the lack of adequate structural information. This technique has also been successfully applied in the very difficult but challenging field of G-protein coupled receptors where the need of three-dimensional insight is significantly more essential. Here we will review the latest advancements in this topic taking as case studies alpha-adrenergic receptors theoretical models and their structural features.

Biological chiral recognition: the substrate's perspective.

A novel stereocenter-recognition (SR) model has been proposed recently for describing the stereoselectivity of biological and other macromolecules towards substrates that have multiple stereocenters, based on the topology of substrate stereocenters (Sundaresan and Abrol, Prot Sci 11:1330-1339, 2002). The SR model provides the minimum number of substrate locations interacting with receptor sites that need to be considered for understanding stereoselectivity characteristics. Interactions between substrate locations and receptor sites may be binding, nonbinding or repulsive in nature and may occur in a many-to-one or one-to-many fashion, but for a receptor to be stereoselective, its interactions with substrate stereoisomers have to involve a minimum number of locations, in the correct geometry. The SR model is topologically rigorous, explains several previous experimental observations, and is predictive in nature. It predicts that stereoselectivity towards a substrate with N stereocenters in a linear structure involves a minimum of N + 2 substrate locations, distributed over all stereocenters in the substrate, such that effectively at least three locations per stereocenter interact with one or more receptor sites. This article uses the SR model to provide an insight into the chiral recognition process from a substrate's perspective that is intuitive and simple, furnishing a rigorous stereochemical basis for explaining stereoselectivity characteristics of many biological systems.

Estimation of the lipophilicity of antiarrhythmic and antihypertensive active 1-substituted pyrrolidin-2-one and pyrrolidine derivatives.

The lipophilicity of some antiarrhythmic and antihypertensive active 1-[2-hydroxy- or 1-[2-acetoxy-3-(4-aryl-1-piperazinyl)propyl]pyrrolidin-2-one derivatives (1-12) has been investigated. Their lipophilicity (R(MO) and log k') was determined by reversed-phase thin-layer chromatography and reversed-phase high-performance liquid chromatography with mixtures of acetonitrile and Tris buffer as mobile phases. The partition coefficients of compounds 1-12 (log P(ScilogP)) were also calculated with the ScilogP program. Comparison of R(MO), log k' and calculated log D(7.0 ScilogP) values enabled calculation of clog D(7.0 TLC) and clog D(7.0 HPLC) values. Preliminary quantitative structure-activity relationship studies indicated that for active compounds there is a dependence between affinity for alpha(2)-adrenoceptors and their clog D(7.0 HPLC) values.

Myocyte adrenoceptor signaling pathways.

Adrenoceptors (ARs), members of the G protein-coupled receptor superfamily, form the interface between the sympathetic nervous system and the cardiovascular system, with integral roles in the rapid regulation of myocardial function. However, in heart failure, chronic catecholamine stimulation of adrenoceptors has been linked to pathologic cardiac remodeling, including myocyte apoptosis and hypertrophy. In cardiac myocytes, activation of AR subtypes results in coupling to different G proteins and induction of specific signaling pathways, which is partly regulated by the subtype-specific distribution of receptors in plasma membrane compartments containing distinct complexes of signaling molecules. The Connections Maps of the Adrenergic and Myocyte Adrenergic Signaling Pathways bring into focus the specific signaling pathways of individual AR subtypes and their relevant functions in vivo.

Towards a general model for protein-substrate stereoselectivity.

Protein-substrate interactions in enzymatic, neurological, and immunological systems are typically characterized by a high degree of stereoselectivity towards complex substrates. We propose a novel stereocenter-recognition (SR) model for stereoselectivity of proteins (or receptors in general) towards substrates that have multiple stereocenters, based on the topology of substrate stereocenters. The model provides the minimum number of substrate locations that need to enter into binding, nonbinding, or repulsive interactions with receptor sites, for stereoselectivity to occur. According to this model, a substrate location may interact with multiple receptor sites, or multiple substrate locations may interact with a single receptor site, but a stereoselective receptor has to offer, in the correct geometry, at least as many interactions as the required minimum number of substrate locations. The SR model predicts that stereoselectivity towards an acyclic substrate with N stereocenters distributed along a single chain requires interactions involving a minimum of N + 2 substrate locations, distributed over all stereocenters in the substrate, such that effectively three locations exist per stereocenter. Thus, enantioselective recognition of molecules with one chiral center requires a protein to interact with a minimum of three substrate locations, while stereoselectivity towards substrates with two or three stereocenters requires interactions with a minimum of four or five substrate locations, respectively, and so on. We demonstrate the general applicability of this model to protein-substrate interactions by interpreting several previous experimental observations.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Cis- and trans-N-benzyl-octahydrobenzo[g]quinolines. Adrenergic and dopaminergic activity studies.

In vitro assays on a series of cis- and trans-octahydrobenzo[g]quinolines indicated an unusual trend of affinities at the dopaminergic receptors and alpha adrenoceptors. The trans N-benzyl analogues exhibited affinity at the alpha2 as well as the D1-like receptors whereas their N-unsubstituted congeners showed a distinct preference for the alpha2 adrenoceptor. Enhanced activity for the alpha2 receptors was also exhibited by the cis N-benzylated isomers. These observations are interpreted by theoretical calculations.

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis.

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

Identification of a three-amino acid deletion in the alpha2B-adrenergic receptor that is associated with reduced basal metabolic rate in obese subjects.

The alpha2-adrenergic receptors mediate part of the actions of the catecholamines noradrenaline and adrenaline on the regulation of energy balance. As part of an ongoing study on the genetics of obesity, the entire coding sequence of the alpha2B-adrenoceptor gene was screened in 58 obese, nondiabetic Finns by PCR-single stranded conformational analysis (PCR-SSCA). A polymorphism that leads to a deletion of 3 glutamic acids from a glutamic acid repeat element (Glu x 12, amino acids 297-309) present in the third intracellular loop of the receptor protein was identified. This repeat element has previously been shown to be important for agonist-dependent receptor desensitization. Of 166 genotyped subjects, 47 (28%) had 2 normal (long) alleles (Glu12/Glu12), 90 (54%) were heterozygous (Glu12/Glu9), and 29 (17%) were homozygous for the short (Glu9/Glu9) form. The basal metabolic rate, determined by indirect calorimetry and adjusted for fat-free body mass, fat mass, sex, and age, was 94 Cal/day (5.6%) lower (95% confidence interval for difference, 32, 156) in subjects homozygous for the short allele than in subjects with two long alleles (F = 4.84; P = 0.009, by ANOVA). Thus, a genetic polymorphism of the alpha2B-adrenoceptor subtype can partly explain the variation in basal metabolic rate in an obese population and may therefore contribute to the pathogenesis of obesity.

Phenylalanine in the second membrane-spanning domain of alpha 1A-adrenergic receptor determines subtype selectivity of dihydropyridine antagonists.

The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization.

We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.