New series of N-substituted phenyl ketone oxime ethers: synthesis and bovine beta3-adrenergic agonistic activities.

A series of ten novel phenyl ketone oxime ethers substituted on the terminal nitrogen by either 1,3 benzodioxole, alkyl, aralkyl or aryl moiety were synthesized and tested for their activity at bovine beta3-adrenoceptors. The best compound, which was the benzodioxole dicarboxylate derivative, showed potent beta3-adrenergic agonistic activities in Chinese hamster ovary cells expressing the bovine beta3-adrenoceptors with Kact and Ki values better than compound CL 316,243 used as reference (14 +/- 6 nM and 203 +/- 71 nM, respectively). In this series three compounds showed an antagonistic activity. Structure-activity relationships in these ketone oxime ethers are discussed.

Synthesis and bovine beta 3-adrenergic agonistic activities of a novel series of aryloxypropanolamines.

We synthesized a novel series of 21 aryloxypropanolamine compounds characterized by N-alkyl, aralkyl, and aryl substituents. The compounds showed potent beta 3-adrenergic agonistic activities in Chinese hamster ovary cells expressing the bovine beta 3-adrenoceptors with Kact and Ki values of 4.2 +/- 3.0 nM and 459 +/- 169 nM respectively, for the ligand with the best compromise between potency and affinity. Structure-activity relationships are discussed.

Site-directed mutagenesis of the human beta3-adrenoceptor--transmembrane residues involved in ligand binding and signal transduction.

All three subtypes of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-binding protein. Nevertheless, the beta3 adrenoceptor (beta3-AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent beta1-AR and beta2-AR antagonists. Furthermore, the human beta3-AR is quite different from the animal beta3-AR. Molecular modelling studies followed by site-directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the beta3-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first transmembrane domain at positions 48-50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no 'rodent-like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human beta3-AR, has been suggested to participate in beta2-/beta3-AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta2-AR, left the beta3-AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the beta3-AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal-transduction pathway.

Genomic cloning and species-specific properties of the recombinant canine beta3-adrenoceptor.

A molecular clone encoding a beta3-adrenoceptor was isolated from a canine genomic library. The cloned receptor exhibited a pharmacological profile similar to that of other species: in particular, high efficiency of the two selective beta3-adrenoceptor agonists, CL 316,243 (disodium(R,R)-5[2[[2-(chlorophenyl)-2hydroxyethyl]-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate) and ICI 201651 ((R)4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl)phe noxy acetic acid) and a low affinity for the radioligand (-)-[3-(125)I]-iodocyanopindolol. Interestingly, CGP 12177A ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one), which is described as a partial agonist for the human receptor, was a full agonist for the canine receptor. After expression and stimulation of the canine beta3-adrenoceptor in stably transfected Chinese hamster ovary cells there was a very low accumulation of cAMP, suggesting weak coupling to Gs-protein and adenylyl cyclase. However, the response was much better in human embryonal kidney cells transfected with the canine beta3-adrenoceptor gene. The cloning of the canine beta3-adrenoceptor and the insights gained from its pharmacological characterization may allow the development of selective compounds for use in the treatment of obese dogs.

The biochemical effect of the naturally occurring Trp64-->Arg mutation on human beta3-adrenoceptor activity.

A Trp-->Arg mutation at amino acid position 64 in the human beta3-adrenoceptor is reportedly associated with morbid obesity; carriers suffer from increased gain in mass, early-onset diabetes, insulin resistance, and an increased waist-to-hip ratio [Clément, K., Vaisse, C., Manning, B. S., Basdevant, A., Guy-Grand, B., Ruiz, J., Silver, K. D., Shuldiner, A. R., Froguel, P. & Strosberg, A. D. (1995) N. Engl. J. Med. 333, 352-354]. Here, we report the stable expression of the genes encoding the wild-type or the [Arg64]beta3-adrenoceptor in two different cell types: hamster CHO-K1 and human HEK293. The mutated receptor displayed unchanged pharmacological values compared to the wild type for the binding inhibition (Ki) and adenylyl cyclase activation constants (K(act)) in two independent clones of both cell lines. However, maximal cAMP accumulation was significantly reduced in response to various beta3-adrenergic agonists, including endogenous catecholamines, (-)-epinephrine and (-)-norepinephrine, the non-selective agonist (-)-isoproterenol, and the beta3-adrenergic selective agonist CGP 12177A. Treatment with Pertussis toxin did not restore the adenylyl cyclase activity to that of the wild type, suggesting that the reduction in cAMP accumulation observed in cells expressing [Arg64]beta3-adrenoceptor is not due to enhanced interaction of the beta3-adrenoceptor with the inhibitory Gi protein.

Human immortalized brown adipocytes express functional beta3-adrenoceptor coupled to lipolysis.

Human brown pre-adipocytes were immortalized by microinjection of the genes encoding simian virus 40 T and t antigens under the control of the human vimentin promotor. The transfected pre-adipocytes were cultured for several months with no loss of their morphological characteristics. These cells accumulate lipids and differentiate into adipocytes when treated with insulin, triiodothyronine and dexamethazone. The mRNA of various adipocyte markers was detected by reverse transcriptase-polymerase chain reaction analysis, including hormone-sensitive lipase, lipoprotein lipase, adipsin, glucose transporters 1 and 4, the uncoupling protein (specific of brown adipocytes), and leptin, the product of the ob gene. Pharmacological analyses indicated that the beta3-adrenoceptor is the predominant beta-adrenoceptor subtype in PAZ6 cells and that this receptor subtype is functionally coupled to adenylate cyclase and lipolysis. The immortalization of human adipocytes will permit pharmacological analysis of the human beta3-adrenoceptor function in adipose cells and will allow detailed studies of human adipocyte differentiation.

Function and regulation of the beta 3-adrenoceptor.

The cloning, sequencing and expression in model systems of the previously unidentified beta 3-adrenoceptor recently led to an extensive functional characterization. Ligand binding and adenylate cyclase activation studies helped define a specific profile that is quite distinct from that of the beta 1- and beta 2-adrenoceptors, but strongly reminiscent of most of the 'atypical' beta-adrenoceptor-mediated responses reported in earlier pharmacological studies. More recently, a naturally occurring variation in the human beta 3-adrenoceptor has been correlated with hereditary obesity and with increased dynamic capacity to add on weight and develop non-insulin dependent diabetes in Western obese patients. Donny Strosberg and France Pietri-Rouxel describe how results now provide a consistent picture of an important role for the human beta 3-adrenoceptor in the regulation of lipid metabolism and as an obvious target for drugs to treat some forms of obesity and diabetes.

Molecular cloning and pharmacological characterization of the bovine beta 3-adrenergic receptor.

A full-length clone encoding a beta-adrenergic receptor was isolated from a bovine brown adipose tissue cDNA library. By comparative sequence analysis, and pharmacological characterization of a Chinese hamster ovary cell line expressing the full-length cDNA, it was shown that the product of the cloned gene is the bovine equivalent of the atypical beta 3-adrenergic receptor previously described in human, mouse, and rat [Strosberg, A. D. (1993) Prot. Sci. 2, 1198-1209]. The cloned receptor exhibits a pharmacological profile very similar to those from other species. In particular, the receptor has high affinity for BRL 37344 [(RR,SS)-(+/-)-4-(2'-[2-hydroxy-2-(3- chlorophenyl)ethylamino]propyl)phenoxyacetate sodium salt sesquihydrate], and low affinity for the iodinated ligand(-)-[3-125I]-iodocyanopindolol. The bovine beta 3-adrenergic receptor has high affinity for beta 1-adrenergic receptor and beta 2-adrenergic receptor antagonists including ICI 201651 [(R)-4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2- methoxyethyl)phenoxy acetic acid], carazolol, and CGP 12177A [(+/-)-4-(3-t-butylamino-2- hydroxypropoxy)benzimidazol-2-one]. In contrast to the murine beta 3-adrenergic receptor, both bupranolol and (-)-propranolol were partial agonists of the bovine receptor. The isolation of the bovine beta 3-adrenergic receptor, and information obtained from detailed pharmacological profiling may allow for the development of selective compounds for producing beef cattle with a low-body-mass index, and also aid the ongoing search for more selective agonists for the human receptor.

Long term phorbol ester treatment down-regulates the beta 3-adrenergic receptor in 3T3-F442A adipocytes.

The role of protein kinase C (PKC) in the regulation of the beta 3-adrenergic receptor (beta 3-AR) gene was examined in murine 3T3-F442A adipocytes, which express this receptor subtype at a high level. We also investigated the involvement of this kinase in the modulation of beta 3-AR gene expression by insulin. Long term exposure of 3T3-F442A adipocytes to phorbol 12-myristate 13-acetate (PMA) decreased beta 3-AR mRNA content in a time- and concentration-dependent manner, with maximal changes observed at 6 h (6.5-fold decrease) and at 100 nM PMA. This inhibition was selective for beta 3-AR transcripts, since beta 1- and beta 2-AR mRNA content remained unchanged. Also, (-)-[125I]cyanopindolol saturation and competition binding experiments on adipocyte membranes indicated that PMA induced an approximately 2-fold decrease in beta 3-AR expression, while that of the two other subtypes was not affected. This correlated with a lower efficacy of beta 3-AR agonists to stimulate adenylyl cyclase. Conversely, long term exposure to PMA did not alter adenylyl cyclase activity in response to guanosine 5'-O-(3-thiotriphosphate) or forskolin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not repress beta 3-AR mRNA levels. Inhibition of beta 3-AR mRNA by PMA was suppressed by the PKC-selective inhibitor bisindolylmaleimide, and was not observed in PKC-depleted cells, indicating that PKC was involved in this response. mRNA turnover experiments showed that the half-life of beta 3-AR transcripts was not affected by long term PMA exposure. When 3T3-F442A adipocytes were pretreated with PMA for 24 h to down-regulate PKC, or with bisindolylmaleimide, the insulin-induced inhibition of beta 3-AR mRNA levels was reduced by 44-67%. These findings demonstrate that sustained PKC activation exerts a specific control of beta 3-AR gene expression and is involved, at least in part, in the modulation by insulin of this adrenergic receptor subtype.

Pharmacological characteristics and species-related variations of beta 3-adrenergic receptors.

Beta-adrenergic receptors (beta-AR) belong to the large multigenic family of receptors coupled to GTP-binding proteins. Three subtypes have been identified: beta 1-, beta 2- and beta 3-AR. Much of the work delineating the precise pharmacological comparison of the three beta-ARs has come from investigations with stably transfected Chinese hamster ovary cells (CHO cells). This review discusses the structure and function of beta 3-AR in various species and presents new findings on a number of beta 3-AR ligands including carazolol, tertatolol and CL 316,243 which were found to be selective and potent beta 3-AR agonists and ZD 2079 and salmeterol which appear to display full but non-subtype selective agonistic activity. Species-related variations of the beta 3-AR pharmacology have been shown for propranolol and bupranolol. With the ongoing characterization of the beta 3-AR at the molecular and cellular level, and with the advent of computer-assisted molecular modelling to aid in the determination of the three-dimensional structure of the receptor, it is thought that novel beta 3-AR compounds will become available with improved selectivity and potency.

The inositol 1,4,5-trisphosphate receptor: kinetic properties and regulation.

Inositol 1,4,5-triphosphate (InsP3) is a second messenger responsible for the mobilization of intracellular Ca2+ after receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. InsP3 binds to a specific receptor located on the membrane of an intracellular compartment and opens a Ca2+ channel causing the cytosolic Ca2+ concentration to increase. Measurement of radiolabelled InsP3 binding and InsP3-induced Ca2+ release in parallel experiments indicated that the liver InsP3 receptor exists in two main states: an active state (A) and an inactive one (I). The "I" form of the receptor is found in the presence of high Ca2+ concentrations (above 1 microM). The binding properties of the "A" and the "I" states of the receptor have been characterized by analysing a membrane fraction enriched in InsP3 receptors. The inactive "I" state displays a high affinity (Kd = 2 nM) and slow rates of association and dissociation. The active state "A" of the receptor displays complex kinetic properties. The rate of association and the rate of dissociation of labelled InsP3 are rapid phenomena probably involving several components. The apparent Kd for the InsP3 binding is about 40 nM in a low Ca2+ medium. The affinity of the "A" state of the receptor is increased by Ca2+ (at concentrations lower than 0.5 microM) and by thiol reagents. The increase of the affinity of the receptor is due to a decrease of the dissociation rate constants. This lowers the threshold such that Ca2+ is released at lower concentrations of InsP3. These data indicate that the binding of InsP3 to its receptor is a complex phenomenon involving the transition among several states.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor.

Thiol reagents have been shown to increase cytosolic Ca2+ in several cell types. In non-muscle cells, these agents induce Ca2+ spikes by increasing the sensitivity of the intracellular Ca2+ stores to D-myo-inositol-1,4,5-trisphosphate (InsP3). We have investigated the effects of thimerosal and oxidized glutathione on the binding properties of the InsP3 receptor in permeabilized hepatocytes and liver and cerebellar membranes. Thimerosal, at the maximal concentration of 100 microM, decreased the KD for the InsP3 binding to permeabilized hepatocytes and cerebellar membranes from 16 to 3 nM and from 25 to 8 nM, respectively, without affecting the maximal binding capacities. On liver membranes, both thimerosal and high Ca2+ concentrations increased the affinity for InsP3 binding. The Ca2+ and the thimerosal effects were differentiated by kinetic experiments. In low Ca2+ media, two kinetic components were identified and thimerosal decreased the rate of dissociation from both these components without affecting the rate of association. In the high Ca2+ medium, a single kinetic component was found with a very slow rate of dissociation. These data suggest that the InsP3 receptor exists in different states. The high-affinity inactive state induced by high Ca2+ concentrations displays slow rates of association and dissociation. The binding properties of the receptor in its active state can be regulated by thiol reagents which increase the affinity by decreasing the dissociation rate constants. At a resting concentration of 100-200 nM, Ca2+ has two effects: it increases the affinity of the active state of the receptor as thiol reagents do and transforms part of the receptors into the inactive high-affinity state.