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.

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.

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.

Effects of androgen on alpha- and beta-adrenergic receptors in membranes from the rat seminal vesicle.

The effects of castration and androgen-replacement on adrenergic receptors in membranes from the rat seminal vesicle were studied. Membranes from seminal vesicles showed saturable and high-affinity binding sites for the beta-adrenergic receptor antagonist, [3H]dihydroalprenolol ([3H]DHA), and the alpha 1-adrenergic receptor antagonist, [3H]prazosin. Castration markedly reduced beta-adrenergic receptors with decreasing the effect of GTP modulating the receptor-ligand affinity, suggesting defects in both the receptor per se and the guanine-nucleotides-regulating mechanism after castration. In contrast, castration increased alpha 1-adrenergic receptors and androgen-replacement reversed this change. The effects of GTP decreasing the alpha 1-receptor binding affinity to the radioligand were observed to a similar extent in the castrated and control membranes. These results demonstrate an inverse regulation by androgen on beta- and alpha 1-adrenergic receptors in membranes of the rat seminal vesicle.

Alpha-adrenoceptors.

Major advances have been made in our understanding of the molecular structure and function of the alpha-adrenoceptors. Many new subtypes of the alpha-adrenoceptor have been identified recently through biochemical and pharmacological techniques and several of these receptors have been cloned and expressed in a variety of vector systems. Currently, at least seven subtypes of the alpha-adrenoceptor have been identified and the molecular structure and biochemical functions of these subtypes are beginning to be understood. The alpha-adrenoceptors belong to the super family of receptors that are coupled to guanine nucleotide regulatory proteins (G-proteins). A variety of G-proteins are involved in the coupling of the various alpha-adrenoceptor subtypes to intracellular second messenger systems, which ultimately produce the end-organ response. The mechanisms by which the alpha-adrenoceptor subtypes recognize different G-proteins, as well as the molecular interactions between receptors and G-proteins, are the topics of current research. Furthermore, the physiological and pathophysiological role that alpha-adrenoceptors play in homeostasis and in a variety of disease states is also being elucidated. These major advances made in alpha-adrenoceptor classification, molecular structure, physiologic function, second messenger systems and therapeutic relevance are the subject of this review.

Alpha-1 adrenergic receptor number and receptor density in isolated hepatocytes from foetal, juvenile and adult rats.

Alpha-1 adrenergic receptor number was defined by [3H]-prazosin binding in crude membrane preparations of hepatocytes and in intact hepatocytes isolated from foetal (day 22 of gestation), juvenile (12 days old), adult female and adult male (90-150 days old) rats and compared with the alpha-1 adrenergic response (measured by epinephrine stimulated glucose liberation in presence of the beta-antagonist propranolol). The alpha-1 receptor number (expressed as fmol bound [3H]-prazosin/mg membrane protein or as receptor number/cell) increases in an age-dependent fashion reaching the highest values in hepatocytes of adult female and male rats. Statistically significant differences could be found between foetal, juvenile and adult rat hepatocytes. No differences in [3H]-prazosin binding were observed between hepatocytes of adult female and adult male rats. The receptor density (expressed as receptor number/microns 2 cell surface), however, was found to be equal in juvenile and adult rats. There are no differences of alpha-1 adrenergic response in juvenile, adult female and adult male rat hepatocytes, whereas the values in foetal hepatocytes were significantly lower. So the biological response is closely correlated with the receptor density and not with the receptor number per cell.

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.

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.

Regional and laminar distributions of alpha 1-adrenoceptors and their subtypes in human and rat hippocampus.

The distributions of the alpha 1-adrenoceptor and its subtypes (alpha 1A and alpha 1B) in human and rat hippocampus are analysed by quantitative receptor autoradiography. alpha 1-Adrenoceptors are labelled by [3H]prazosin. The alpha 1A subtype is visualized by [3H]prazosin after irreversible blockade of alpha 1B adrenoceptors with chloroethylclonidine or directly by [3H]5-methyl-urapidil. The alpha 1B subtype is investigated by [3H]prazosin binding in the presence of the alpha 1A antagonist 5-methyl-urapidil. Considerable differences in the regional and laminar patterns of alpha 1-adrenoceptors are found between rat and human hippocampi. The rat hippocampus is characterized by a low overall density and a rather homogeneous regional and laminar distribution. This is in contrast to the human pattern, which shows a much higher overall level of alpha 1 receptor density and a restriction of alpha 1 receptors to the CA3 region of Ammon's horn and the dentate gyrus. Moreover, alpha 1A and alpha 1B receptors of the human hippocampus are differentially distributed with the alpha 1A subtype concentrated in the hilus and lucidum layer of CA3, and the alpha 1B subtype concentrated in the molecular layer of the dentate gyrus. Additionally, the distribution of alpha 1 receptors is compared with the distribution of 5-hydroxytryptamine 1A receptors. The subtype specific pattern is correlated with the distribution of glutamatergic systems in the human (but not in the rat) hippocampus. alpha 1A Receptor localization coincides with the target area of the mossy fibre system, and alpha 1B receptors are preferentially localized in the target area of the hippocampal associational fibres and partly of the perforant pathway. This result points to possible interactions between noradrenaline- and glutamate-mediated neurotransmission differentiated by topographically segregated alpha 1-adrenoceptor subtypes.

Heterologous expression of the cloned guinea pig alpha 2A, alpha 2B, and alpha 2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene.

Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.

The alpha 1-adrenoceptor is inactivated by alterations in membrane phospholipids.

The influence of the membrane environment on the alpha 1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the alpha 1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic alpha 1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the alpha 1-adrenoceptor.

Estimation of the biophase concentration of noradrenaline at presynaptic alpha 2-adrenoceptors in brain slices.

The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic alpha 2-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the alpha 2-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [3H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC50 value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the alpha 2-adrenoceptor antagonist yohimbine (1 or 10 mumol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of an alpha 2-adrenoceptor in human coronary arteries by radioligand binding assay.

A single high affinity binding site for an alpha 2-adrenoceptor in human coronary arteries was identified by radioligand binding assay. Human coronary arteries were obtained at autopsy within 6 hours of death. A crude membrane solution was incubated with (3H)-rauwolscine at 25 degrees C for 30 min. The binding of (3H)-rauwolscine was rapidly saturable and reversible. Kd was 1.2 +/- 0.2 (SE) nM and Bmax 22 +/- 3 fmol/mg protein. This is the first study which has shown the presence of an alpha 2-adrenoceptor in human coronary arteries using a radioligand binding assay method.

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.