Helmut Bittiger

CGP 35348 : a centrally active blocker of GABAB receptors

The biochemical, electrophysiological and pharmacological properties of the new GABAB receptor blocker CGP 35348 are described. In a variety of receptor binding assays CGP 35348 showed affinity for the GABAB receptor only. CGP 35348 had an IC50 of 34 microM at the GABAB receptor. The compound antagonized (100, 300, 1000 microM) the potentiating effect of L-baclofen on noradrenaline-induced stimulation of adenylate cyclase in rat cortex slices. In electrophysiological studies CGP 35348 (10, 100 microM) antagonized the effect of L-baclofen in the isolated rat spinal cord. In the hippocampal slice preparation CGP 35348 (10, 30, 100 microM) blocked the membrane hyperpolarization induced by D/L-baclofen (10 microM) and the late inhibitory postsynaptic potential. CGP 35348 appeared to be 10-30 times more potent than the GABAB receptor blocker phaclofen. Ionophoretic and behavioural experiments showed that GABAB receptors in the brain were blocked after i.p. administration of CGP 35348. This compound may be of considerable value in elucidating the roles of brain GABAB receptors.

CGP 20712 A: a useful tool for quantitating β1- and β2-adrenoceptors

Abstract CGP 20712 A (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxyl]-2-propanol methanesulfonate), a specific β 1 -adrenoceptor antagonist, was tested for resolution of β 1 - and β 2 -adrenoceptors in an in vitro [ 3 H]dihydroalprenolol ([ 3 H]DHA) binding assay. Competition experiments, using rat neocortical and cerebellar membranes, yielded two dissimilar concentration-effect curves. A distinct biphasic curve was evident for neocortex, with a plateau at 100 nM CGP 20712 A (60% [ 3 H]DHA displacement). This plateau indicated a differentiation between β 1 - and β 2 -adrenoceptors; the ratio of IC 50 -β 2 to IC 50 -β 1 was ∼ 10 000. In contrast, only a monophasic curve was obtained for cerebellum. CGP 20712 A is a useful tool for estimating percentages of β 1 - and β 2 -adrenoceptors in a given tissue.

MUTAGENESIS AND MODELING OF THE GABAB RECEPTOR EXTRACELLULAR DOMAIN SUPPORT A VENUS FLYTRAP MECHANISM FOR LIGAND BINDING

The γ-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity.

1 The pharmacological properties of CGP 37849 (dl‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentenoic acid; 4‐methyl‐APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N‐methyl‐d‐aspartate (NMDA) receptor antagonist, 2‐amino‐5‐phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2 Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nm) and competitively inhibited NMDA‐sensitive l‐[3H]‐glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]‐(±)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonate (CPP), with a Ki of 35 nm, and was 4, 5 and 7 fold more potent than the antagonists ((±)‐cis‐4‐phosphonomethylpiperidine‐2‐carboxylic acid) (CGS 19755), CPP and d‐AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the d‐stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3 CGP 37849 and CGP 39551 were inactive as inhibitors of l‐[3H]‐glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4 In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA‐evoked increases in CA1 pyramidal cell firing rate. In slices bathed in medium containing low Mg2+ levels, concentrations of CGP 37849 up to 10 μm suppressed burst firing evoked in CA1 neurones by stimulation of Schaffer collateral‐commissural fibres without affecting the magnitude of the initial population spike; CGP 39551 exerted the same effect but was weaker. In vivo, oral administration to rats of either CGP 37849 or CGP 39551 selectively blocked firing in hippocampal neurones induced by ionophoretically‐applied NMDA, without affecting the responses to quisqualate or kainate. 5 CGP 37849 and CGP 39551 suppressed maximal electroshock‐induced seizures in mice with ED50s of 21 and 4 mg kg−1 p.o., respectively. 6 CGP 37849 and CGP 39551 are potent and competitive NMDA receptor antagonists which show significant central effects following oral administration to animals. As such, they may find value as tools to elucidate the roles of NMDA receptors in brain function, and potentially as therapeutic agents for the treatment of neurological disorders such as epilepsy and ischaemic brain damage in man.

Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin

A peripheral injection of DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] produced a marked, selective, and lasting depletion of norepinephrine in certain regions of the rat central nervous system. This depletion at 10 days after injection was associated with regional alterations in some, but not all, adrenergic binding sites (receptors) as determined by in vitro [3H]prazosin (alpha 1), [3H]p-aminoclonidine (alpha 2), and [3H]dihydroalprenolol (beta) binding. The neocortical alpha 1-receptor was not changed. The alpha 2-receptor in several regions was altered as indicated by an increase in ligand affinity; additionally, the density of this receptor was slightly decreased in some regions. Depending on the region, the beta-receptor either increased in density or was unchanged. The increased density of this receptor in neocortex corresponded to an increased activity of isoproterenol-sensitive adenylate cyclase. These two changes were not affected by subchronic treatment with desipramine, a norepinephrine uptake inhibitor. The changes were, however, partially or completely reversed by subchronic administration of clenbuterol, a centrally-acting beta-receptor agonist. The dopaminergic receptor in various regions was unaltered as assessed by in vivo and/or in vitro binding of [3H]spiperone. The in vivo binding of this ligand also indicated that the serotoninergic receptor in frontal neocortex was unchanged. Assessment of adrenergic receptors in neocortex at 50 days after injection indicated only the above affinity change of the (presumably postsynaptic) alpha 2-receptor. The alpha 1-receptor remained unaltered. The density of the beta-receptor had normalized, as had the activity of isoproterenol-sensitive adenylate cyclase. Implicit in these findings is the following rank order of receptor sensitivity to chronic norepinephrine depletion: alpha 2 greater than beta greater than alpha 1. The use of DSP-4 has clear advantages over other methods of depleting central norepinephrine. This neurotoxin can be administered by intraperitoneal injection, the depletion of norepinephrine can be readily checked by absence of the post-decapitation reflex, and the changes in other neurotransmitter concentrations are relatively minor or nonexistent. The alteration of alpha 2- and beta-receptors, as a consequence of DSP-4 treatment, may form the basis of a new animal model of adrenergic receptor supersensitivity. Such a model may clarify the importance of these central receptors to physiological and behavioral processes.

Central actions of somatostatin

Somatostatin (SRIF) was applied microiontophoretically to neurons in the frontal and parietal neocortex, the hippocampus and the striatum of rats anaesthetized with either urethane or chloral hydrate. Qualitatively identical results were obtained under both anaesthetic conditions. In urethane-treated rats SRIF elicited a dose-dependent increase of the firing rate of 74% of the neurons studied in the frontal cortex and of 46% of the neurons studied in the parietal cortex. All cortical cells identified as pyramidal cells were excited. In the hippocampus SRIF provoked excitatory responses in two thirds of all neurons. Six out of the nine cells identified as pyramidal cells were excited by SRIF. In the striatum 80% of all neurons were excited. Following repeated exposure of central neurons to SRIF, the magnitude of the excitatory response gradually diminished, indicating desensitisation. SRIF in concentrations ranging from 10(-8) to 10(-4) M did not interfere with the binding of (3H)-muscimol to GABA receptor sites. The release of GABA from synapses preloaded with (3H-GABA) was not influenced by SRIF in the concentration range from 10(-6) to 10(-4) M. These results indicated that SRIF does not evoke the excitatory responses through attenuation of GABA-mediated inhibition. In conclusion, the findings support the hypothesis that somatostatin may function as a neurotransmitter in the central nervous system.

GABA and glutamate release affected by GABAB receptor antagonists with similar potency : no evidence for pharmacologically different presynaptic receptors

1 The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared. 2 The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50s) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348). 3 Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofens effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4 Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5 In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

High affinity beta-2-adrenergic receptors in mononuclear leucocytes: Similar density in young and old normal subjects

Abstract In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (−)- 3 H-dihydroalprenolol ( 3 H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K D ) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced 3 H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (−) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of 3 H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.

Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain

Recently, two N-terminal splice variants of the metabotropic receptor for GABA (gamma-amino-butyric acid) were cloned. Here, we describe an antiserum that recognizes the two receptor variants. We demonstrate that these proteins are identical with GABAB receptors that are photoaffinity labeled with [125I]CGP71872 in rat brain. The C-terminal epitopes recognized by the antiserum are conserved in several vertebrate species but not in chicken. No hints for the existence of additional closely related receptor subtypes or variants are found in double-labeling experiments with antibody and photoaffinity ligand. Western blot analysis reveals widespread expression of the GABABR1 receptor proteins in rat brain with the highest level of expression at early postnatal stages. The binding affinity of the GABAB receptor agonist L-baclofen at native R1a and R1b variants is similar. In early postnatal development the affinity at R1a and R1b is 10-fold lower than in adult brain and gradually increases with aging.

In vivo [3H]spiperone binding to the rat hippocampal formation: involvement of dopamine receptors.

The existence of DA receptors in the rat hippocampus was demonstrated with an in vivo [3H]spiperone radioreceptor assay. Kinetic studies revealed that maximum binding of [3H]spiperone in hippocampus was much smaller than in striatum and frontal cortex but much higher than in cerebellum. In inhibition studies of [3H]spiperone binding, all neuroleptics tested were active in hippocampus as well as in striatum. In contrast, 5HT antagonists were definitely less potent in these two brain regions than in frontal cortex. Finally, even when 5HT receptors were blocked, dipropyl-ATN and haloperidol remained fully effective in hippocampus, striatum, but also in frontal cortex although to a lesser degree. From these results it was concluded that [3H]spiperone binds mainly to DA receptors in hippocampus as well as in striatum, whereas both 5HT and DA receptors are present in frontal cortex.