Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.     

Inhibitory effects of quinolone antibacterial agents on gamma-aminobutyric acid binding to receptor sites in rat brain membranes.

The specific binding of 3H-labeled gamma-aminobutyric acid ([3H]GABA) to synaptic plasma membranes from rat brains was inhibited by various quinolonecarboxylic acid derivatives (quinolones), and these inhibitions were concentration dependent. The binding of [3H]muscimol to GABAA sites was also inhibited. These inhibitory potencies differed widely among the quinolones examined. The Dixon plots showed that a newly developed difluorinated quinolone, NY-198 [1-ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3- quinolinecarboxylic acid hydrochloride], competitively inhibits the receptor bindings of [3H]GABA and [3H]muscimol. In conclusion, our findings suggest that the inhibition of GABA binding to receptors (including uptake sites) in the brain may be involved in the induction of epileptogenic neurotoxicities by quinolones.     

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. II. Evidence for N-methyl-D-aspartate receptor antagonist properties

The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidineethanol] have been analyzed in a number of models indicative of N-methyl-D-aspartate (NMDA) antagonistic potential in vitro and in vivo. Ifenprodil and SL 82.0715 potently and noncompetitively antagonize the stimulatory effects of NMDA on cyclic GMP production in immature rat cerebellar slices (IC50 values, 0.4 and 10 microM, respectively), as well as the NMDA-evoked [3H]acetylcholine release in adult rat striatal slices (IC50 values, 1.6 and 6.6 microM, respectively). Ifenprodil is 10 times more potent than (+/-)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) but less active than the reference noncompetitive NMDA channel blockers [MK 801, ((+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine ], phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP)] in these models. Ifenprodil and SL 82.0715 partially displace (maximal displacement 40-50% at 10 microM) the NMDA receptor ligand [3H]CPP from its binding site to rat brain membranes (IC50 values, 0.1 and 0.3 microM, respectively) in a noncompetitive manner; in the micromolar range the two agents also partially displace the NMDA channel ligand [3H]TCP from its binding site to rat brain membranes, and noncompetitively antagonize the L-glutamate-induced increase in [3H]TCP binding. Ifenprodil (0.01-1 microM) partially antagonizes the depolarizing effects of NMDA on the immature rat hemisected spinal cord in vitro. In mouse cultured spinal cord neurons, ifenprodil dose-dependently antagonizes the depolarizing effects of micropressure applied NMDA. Inhibition of the effects of NMDA in this model by ifenprodil and SL 82.0715 is noncompetitive. In vivo and after systemic i.p. administration, ifenprodil and SL 82.0715 antagonize the stimulatory effects of intrastriatally dialyzed NMDA on striatal dopamine release in rats (ID50 values, 0.9 and 0.3 mg/kg, respectively), and block the harmaline-evoked increase in cerebellar cyclic GMP production in mice (ID50 values, 3 and 4 mg/kg, respectively). These results indicate that ifenprodil is a noncompetitive NMDA antagonist which has a mechanism of action distinct from either the reference competitive NMDA receptor antagonists (CPP and 2-amino-5-phosphonovalerate) or the noncompetitive NMDA channel blockers (phencyclidine, TCP and MK 801). The potent NMDA antagonistic effects of the ifenprodil class of compounds are likely to be related to the demonstrated anti-ischemic potential of these compounds.     

SL651498: an anxioselective compound with functional selectivity for alpha2- and alpha3-containing gamma-aminobutyric acid(A) (GABA(A)) receptors.

SL651498 [6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one] is a novel pyridoindole derivative that displays high affinity for rat native GABA(A) receptors containing alpha(1) (K(i) = 6.8 nM) and alpha2 (K(i) = 12.3 nM) subunits, and weaker affinity for alpha5-containing GABA(A) receptors (K(i) = 117 nM). Studies on recombinant rat GABA(A) receptors confirm these data (K(i), alpha1beta2gamma2 = 17, alpha2beta2gamma2 = 73, alpha5beta3gamma2 = 215 nM) and indicate intermediate affinity for the alpha3beta2gamma2 subtype (K(i) = 80 nM). SL651498 behaves as a full agonist at recombinant rat GABA(A) receptors containing alpha2 and alpha3 subunits and as a partial agonist at recombinant GABA(A) receptors expressing alpha1 and alpha5 subunits. SL651498 elicited anxiolytic-like activity similar to that of diazepam [minimal effective dose (MED): 1-10 mg/kg, i.p.] in three conflict models, in the elevated plus-maze, the light/dark test, and the defense test battery in rats and mice. Results from activity tests and electroencephalogram analysis indicated that SL651498 induced muscle weakness, ataxia, or sedation at doses much higher than those producing anxiolytic-like activity (MED > or = 30 mg/kg, i.p.). Repeated treatment for 10 days with SL651498 (30 mg/kg, i.p., b.i.d.) in mice was not associated with the development of tolerance to its anticonvulsant effects or physical dependence. Furthermore, SL651498 was much less active than diazepam in potentiating the depressant effects of ethanol in mice. The "anxioselective" profile of SL651498 points to a major role for GABA(A) alpha2 subtype in regulating anxiety and suggests that selectively targeting GABA(A) receptor subtypes can lead to drugs with increased clinical specificity.     

Acute pentylenetetrazol injection reduces rat GABAA receptor mRNA levels and GABA stimulation of benzodiazepine binding with No effect on benzodiazepine binding site density.

The effects of a single convulsive dose of pentylenetetrazol (PTZ, 45 mg/kg i.p.) on rat brain gamma-aminobutyric acid type A (GABAA) receptors were studied. Selected GABAA receptor subunit mRNAs were measured by Northern blot analysis (with beta-actin mRNA as a standard). Four hours after PTZ, the GABAA receptor gamma2-mRNA was decreased in hippocampus, cerebral cortex, and cerebellum; alpha1-mRNA was decreased in cerebellum; and beta2 subunit mRNA was decreased in cortex and cerebellum. The alpha5 subunit mRNA level was not altered. Those mRNAs that had been reduced were increased in some brain regions at the 24-h time point, and these changes reverted to control levels by 48 h. PTZ effect on GABAA receptors was also studied by autoradiographic binding assay with the benzodiazepine agonist [3H]flunitrazepam (FNP), the GABAA agonist [3H]muscimol, and the benzodiazepine antagonist [3H]flumazenil. There was an overall decrease in [3H]FNP binding 12 but not 24 h after PTZ treatment. In contrast, [3H]muscimol binding was minimally affected, and [3H]flumazenil binding was unchanged after PTZ treatment. Additional binding studies were performed with well-washed cerebral cortical homogenates to minimize the amount of endogenous GABA. There was no PTZ effect on specific [3H]FNP binding. However, there was a significant reduction in the stimulation of [3H]FNP binding by GABA. The results showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels without altering receptor number but affected the coupling mechanism between the GABA and benzodiazepine sites of the GABAA receptor.     

gamma-Aminobutyric acidA receptor heterogeneity in rat central nervous system: studies with clonazepam and other benzodiazepine ligands.

The properties of [3H]clonazepam, [3H]diazepam and [3H]zolpidem (N,N,6[trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitratrate) binding to synaptic membranes of cerebellum, cortex, olfactory bulb, striatum and spinal cord of rat were compared to the binding properties of [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. In the cerebellar, cortical and olfactory bulb membranes, the density of high-affinity binding sites of all these tritiated benzodiazepine (BZ) ligands is almost identical. In contrast, in the striatum, the density of [3H]clonazepam and [3H]zolpidem binding sites is approximately 60 and 30%, respectively, of the density of [3H]diazepam, [3H]flunitrazepam or [3H]flumazenil sites. In spinal cord membranes, the number of high-affinity binding sites of [3H]clonazepam and [3H]zolpidem is less than 20% of the number of binding sites for [3H]diazepam, [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. Moreover, the displacement of [3H]flunitrazepam from spinal cord membranes by clonazepam and zolpidem was characterized by high IC50 values and Hill slopes significantly less than 1. Because [3H]BZ ligand binding in the spinal cord is enhanced by gamma-aminobutyric acid (GABA), these data suggest that different regions of the rat central nervous system may contain different GABA-BZ receptor subtypes. The different pharmacological properties of clonazepam, diazepam and zolpidem (i.e., regarding their ability to enhance bicuculline seizure threshold, to decrease locomotor activity, to induce ataxia or to elicit anticonflict action) further support the concept that in the rat central nervous system preferential occupancy of heterogeneous GABAA receptors by these drugs can be related to their effects on behavior.     

Presence of a gamma-aminobutyric acid (GABA) uptake system on cholinergic terminals of rat hippocampus: evidence for neuronal coexistence of acetylcholine and GABA?

The effect of gamma-aminobutyric acid (GABA) on the basal release of [3H]acetylcholine ([3H]ACh) was investigated using synaptosomes prepared from rat hippocampus and superfused after prelabeling with [3H]choline. Exogenous GABA added to the superfusion medium caused a long-lasting and concentration-dependent enhancement of the basal efflux of [3H]ACh. The effect of GABA was not antagonized by bicuculline or picrotoxin. Muscimol increased slightly but not significantly the release of [3H]ACh, whereas (+/-)-baclofen or (-)-baclofen were ineffective. The effect of GABA was counteracted by SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], SK&F 100330A [N-(4,4-diphenyl-3-butenyl)-guvacine] and SK&F 100561 [N-(4,4-diphenyl-3-butenyl)-homo-beta-proline], three novel inhibitors of GABA uptake, but was unaffected by hemicholinium-3 or by beta-alanine. Nipecotic acid, a substrate-inhibitor of the GABA transporter, mimicked GABA and enhanced [3H]ACh release. The results indicate that a GABA transport system is present on cholinergic terminals.     

Characterization of COR627 and COR628, two novel positive allosteric modulators of the GABA(B) receptor

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.     

SL 84.0418: a novel, potent and selective alpha-2 adrenoceptor antagonist: in vitro pharmacological profile.

One novel, potent and selective alpha-2 adrenoceptor antagonist is 2-(4,5-dihydro-1H-imidazol-2-yl)-1,2,4,5-tetrahydro-2- propylpyrrolo[3,2,1-hi]-indole hydrochloride (SL 84.0418). It inhibits with high affinity the radioligand binding to rat cortical alpha-2 adrenoceptors, as well as to human platelet alpha-2 adrenoceptors labeled with [3H]idazoxan (Ki = 7 nM). SL 84.0418 has low affinity for alpha-1 adrenoceptors labeled with [3H]prazosin (Ki = 3.3 microM). In vitro, SL 84.0418 has no alpha agonist properties, whereas it is a potent alpha-2 adrenoceptor antagonist at both pre- and postsynaptic alpha-2 adrenoceptors. In contrast, it possesses low potency as an antagonist at postsynaptic alpha-1 adrenoceptors demonstrating a more than 1000-fold selectivity toward alpha-2 compared with alpha-1 adrenoceptors. In the same tests, the alpha-2 adrenoceptor antagonist idazoxan had a selectivity ratio of 200. SL 84.0418 is the racemic mixture of two enantiomers, SL 86.0715 [(+) enantiomer] and SL 86.0714 [(-) enantiomer]. The alpha-2 adrenoceptor blocking activities reside with SL 86.0715. Similar to idazoxan, SL 84.0418 increases in a concentration-dependent manner the electrically evoked release of [3H]norepinephrine from rat hypothalamic slices through the blockade of the presynaptic inhibitory alpha-2 adrenoceptors. In isolated hamster adipocytes, SL 84.0418 potently antagonizes the inhibition of lipolysis induced by UK 14,304. In addition, SL 84.0418 inhibits epinephrine-induced aggregation of rabbit platelets, effects mediated by postsynaptic alpha-2 adrenoceptors. SL 84.0418 does not inhibit (IC50 > 1,000 nM) radioligand binding to other receptors or recognition sites, nor does it inhibit calcium, sodium or potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ifenprodil and SL 82.0715 as cerebral antiischemic agents. III. Evidence for antagonistic effects at the polyamine modulatory site within the N-methyl-D-aspartate receptor complex

Ifenprodil and SL 82.0715 are noncompetitive N-methyl-D-aspartate (NMDA) antagonists whose inhibitory actions are not explained by antagonistic effects at any of the three commonly recognized sites within the NMDA receptor complex (recognition, channel and modulatory glycine sites). We presently show that ifenprodil and SL 82.0715 antagonize the effects of NMDA via a selective action at the recently described polyamine modulatory site. Spermine and spermidine (0.5-100 microM) increase the binding of [3H]1-[1-(2-thienyl)cyclohexyl] piperidine to washed rat forebrain membranes in the presence of glutamate (10 microM). This effect is antagonized by ifenprodil and SL 82.0715 (0.1-10 microM) at concentrations which do not displace [3H]1-(2-thienyl)cyclohexyl] piperidine in the absence of added polyamine. Spermine and spermidine (up to 100 microM) do not significantly alter the binding of [3H]glycine but increase the binding of the NMDA recognition site ligand [3H](+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. Ifenprodil and SL 82.0715 (0.1-10 microM) antagonize this effect; ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine maleate) or 7-chlorokynurenate (100 microM) are ineffective. In immature rat cerebellar slices, spermine and spermidine (10-1000 microM) potentiate the maximal effects of NMDA (80-160 microM) on cyclic GMP production. Spermine (100-1000 microM) reverses the antagonistic effects of ifenprodil (0.15-50 microM) but not of ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine acid or kynurenate on the NMDA receptor-mediated increase in cyclic GMP levels. Ifenprodil (0.01-1 microM) potently but only partially antagonizes the depolarizing effects of NMDA (10 microM) on the immature rat spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid anesthetics and naturally occurring analogs modulate the gamma-aminobutyric acid receptor complex at a site distinct from barbiturates

The steroid anesthetic alphaxalone and a series of naturally occurring analogs were compared in potency and efficacy with each other and the hypnotic barbiturate pentobarbital for interaction with gamma-aminobutyric acid (GABA) receptors:binding sites in rat brain membranes and functional activity in 36Cl- flux measurements with rat hippocampal slices. The steroids enhanced [3H]muscimol binding to GABA receptor sites, enhanced [3H] flunitrazepam binding to benzodiazepine receptors and inhibited [35S]t-butyl bicyclophosphorothionate binding to picrotoxin/convulsant binding sites on the GABA receptor-chloride channel complex. The same steroids that were active in modulating the binding of ligands to the various receptor sites on the GABA receptor complex at micromolar concentrations enhanced muscimol-stimulated 36Cl- flux in rat hippocampal slices. The steroids, like the barbiturates, increased the maximal response to muscimol but produced little or no potentiation of basal 36Cl- flux in the absence of GABA agonist. Although the effects of steroids and barbiturates were similar, separate sites of action were demonstrated conclusively by the observation that the two classes of compounds, when included together, gave additive or synergistic effects on binding, as well as on 36Cl- flux in the absence of GABA agonist. Structure-activity studies showed that the synthetic steroid anesthetic alphaxalone was the most potent compound tested, followed by the naturally occurring steroids tetrahydro-deoxycorticosterone, allo-tetrahydrocorticosterone, cis-androsterone and 5 alpha-androstan-17 beta-ol-3-one. The ability of several naturally occurring steroids to enhance GABA-mediated inhibition in the brain suggests the possibility of an endogenous steroid modulator of neuronal function.     

Release of gamma-[3H]aminobutyric acid (GABA) from electrically stimulated rat cortical slices and its modulation by GABAB autoreceptors

Slices of rat temporo-parietal cortex were prelabeled with gamma-[3H]aminobutyric acid ([3H]GABA), in the presence of the glial GABA uptake inhibitor beta-alanine. The slices were then superfused with a medium containing the GABA transaminase inhibitor aminooxyacetic acid and stimulated electrically (5 min, 2 msec, 36 mA at 5 or 10 Hz), in the presence of the neuronal GABA reuptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid] and of beta-alanine. Representative experiments showed that the tritium released could be accounted for almost entirely by authentic [3H]GABA. The electrically evoked overflow of [3H]GABA was tetrodotoxin sensitive and largely calcium-dependent. Exogenous GABA, added to the superfusion medium at 3 to 30 microM, reduced in a concentration-dependent manner the electrically evoked (5 Hz) release of [3H]GABA. The GABAB receptor agonist (-)-baclofen, but not the GABAA receptor agonist muscimol, mimicked GABA and produced a concentration-inhibition curve almost superimposable to that of the natural transmitter. The effects of GABA and of (-)-baclofen were much more pronounced at 5 than at 10 Hz. The GABA-induced inhibition of [3H]GABA release was sensitive to the novel GABAB receptor antagonist beta-(p-chlorophenyl)-3-amino propyl phosphonic acid which, by itself, increased the [3H]GABA overflow. The inhibitory effect of GABA was not counteracted by the GABAA receptor antagonists bicuculline or SR 95531 [2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide]. The results are compatible with the presence in the rat cerebral cortex of autoreceptors mediating inhibition of GABA release and belonging to the GABAB type. These autoreceptors may be activated tonically under physiological conditions.     

Benzodiazepines and barbiturate potentiate the pre- and postsynaptic gamma-aminobutyric acid (GABA)A receptor-mediated response in the enteric nervous system of guinea pig small intestine

To determine whether or not presynaptic gamma-aminobutyric acid (GABA) receptors regulate the release of GABA, we examined properties of the presynaptic GABA receptor and compared the findings within the case of the postsynaptic GABA receptor, using the longitudinal muscle with myenteric plexus (L-M) preparation of guinea pig small intestine. Muscimol, but not baclofen, reduced the Ca++-dependent release of [3H]GABA evoked by high K+ in the presence of tetrodotoxin from L-M preparation of the small intestine preloaded with [3H]GABA. Bicuculline, picrotoxin and furosemide antagonized the effect of muscimol. Diazepam, clonazepam and pentobarbital potentiated the muscimol-induced inhibition of high K+-evoked release of [3H]GABA. The potentiating effect of clonazepam was antagonized by Ro 15-1788. Muscimol induced a Ca++-dependent and tetrodotoxin-sensitive release of [3H]acetylcholine from L-M preparation preloaded with [3H]choline. The effect of muscimol was antagonized by bicuculline, picrotoxin and furosemide. Diazepam, clonazepam and pentobarbital potentiated the muscimol-evoked release of [3H]ACh. The potentiation of muscimol effect by clonazepam was inhibited by Ro 15-1788. These results indicate that both the GABA autoreceptor and postsynaptic receptor may possess the same property which is related to benzodiazepine and barbiturate binding sites in the enteric nervous system of the guinea pig small intestine. The benzodiazepine binding site seems to be of central type.     

Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benzodiazepine receptors are coupled to a subpopulation of gamma-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study

The colocalization and interaction of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors in the rat brain were characterized using standardized, quantitative, light microscopic autoradiographic methods. In serial sections, striking differences were observed in the distribution of high affinity GABA and BZ receptors in areas such as the cerebral cortex, globus pallidus, thalamus, hypothalamus and cerebellar cortex. However, in a semiquantitative visual examination of more than 200 brain regions, added exogenous GABA increased BZ binding in all regions. In a quantitative analysis of 19 regions, exogenous GABA uniformly stimulated [3H]flunitrazepam binding, the effect being proportional to the regional density of BZ receptors. No relationship was seen between the magnitude of the stimulation and the distribution of high affinity GABA receptors. In the mounted tissue sections, BZ binding appeared influenced by endogenous GABA since it was reduced by preincubation or by the addition of bicuculline. Taken together, these data suggest that most or all BZ receptors can be influenced by GABA and are coupled to a type of GABA receptor. However, the BZ-linked GABA receptor could represent either a subpopulation of GABA binding sites or a distinct receptor not labeled under the conditions used in these and other experiments.     

Fengabine, a novel antidepressant GABAergic agent. II. Effect on cerebral noradrenergic, serotonergic and GABAergic transmission in the rat

The effects of fengabine (a novel benzylidene derivative possessing clinically demonstrated antidepressant action) on neurochemical parameters related to norepinephrine, serotonin and gamma-aminobutyric acid (GABA) neurons have been investigated in the rat and mouse brain. When given acutely, fengabine (50-1000 mg/kg i.p.) does not alter norepinephrine uptake but accelerates the turnover rate of norepinephrine in the rat brain as demonstrated by the enhancement of: the alpha-methyl-p-tyrosine-induced disappearance of norepinephrine in the hypothalamus; 3,4-dihydroxyphenylacetic acid levels in noradrenergic cell body areas; the pargyline-induced accumulation of normetanephrine in the hypothalamus; and 3,4-dihydroxyphenylethyleneglycol levels in the hypothalamus, septum and spinal cord. No tolerance to the effect of fengabine on the latter biochemical parameter was observed after repeated treatment for 2 weeks at doses of 100 or 200 mg/kg i.p., b.i.d. Fengabine (100 or 200 mg/kg i.p., b.i.d.), given for 14 days, causes a desensitization of isoprenaline-stimulated adenylate cyclase in septal and cortical slices of the rat but fails to modify cortical beta, alpha-1 or alpha-2 adrenoceptor binding sites. Fengabine (up to 400 mg/kg i.p.) has no effect on rat cerebral serotonin uptake, synthesis or metabolism. Moreover, when given subacutely (100 or 200 mg/kg i.p., b.i.d. for 2 weeks), it fails to alter rat cortical serotonine receptors or [3H]imipramine binding sites. Fengabine (up to 50-100 microM) is also inactive in vitro on [3H] GABA binding to GABAA or GABAB receptors in the rat brain or on GABA transaminase activity in the mouse brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preclinical evaluation of McN-5707 as a potential antidepressant

Based on its activity in a variety of tests in vivo and in vitro McN-5707 [trans-6-(2-chlorophenyl)-1,2,3,5,6,10b-hexahydropyrrolo- (2,1-a)isoquinoline] is a novel potential antidepressant. McN-5707 blocked tetrabenazine-induced sedation and ptosis in mice and rats, and potently inhibited the uptake of norepinephrine by synaptosomes from rat hypothalamus (Ki approximately 2 nM), and the uptake of serotonin by synaptosomes from rat cerebral cortex (Ki approximately 10 nM). McN-5707 also inhibited the uptake of dopamine by synaptosomes from rat striatum (Ki approximately 40 nM); however, the stereotypic behavior often caused by dopamine uptake inhibitors was not evident in rats at doses of 300 mg/kg (p.o.) or less. In receptor binding assays, McN-5707 potently inhibited ketanserin binding to serotonin 5-HT2 receptors in synaptic membranes from rat cerebral cortex (apparent Ki approximately 8 nM). In mice, McN-5707 antagonized 5-hydroxytryptophan-induced head twitches. Spiperone binding to dopamine D2 receptors in synaptic membranes from rat striatum was weakly inhibited by McN-5707 (apparent Ki approximately 400 nM), as was the binding of WB4101 to alpha-1 adrenergic receptors (apparent Ki approximately 150 nM). McN-5707 was essentially inactive as an inhibitor of [3H]clonidine binding to alpha-2 adrenergic receptors and of [3H]quinuclidinyl benzilate binding to muscarinic receptors. In experiments with guinea pig ileum, McN-5707 weakly antagonized histamine-induced contractions and exhibited virtually no cholinergic or anticholinergic activity. Our observations indicate McN-5707 possesses attributes of both tricyclic and newer atypical antidepressants because it inhibits the uptake of both norepinephrine and serotonin, and blocks 5-HT2 receptors, but lacks some of the anticholinergic and behavioral properties often associated with them.     

FK 224, a novel cyclopeptide substance P antagonist with NK1 and NK2 receptor selectivity.

We have discovered a novel cyclopeptide substance P (SP) antagonist, FK 224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2-pentylphenyl )- propionyl]-L-threonyl]tyrosyl-L-leucynyl]-D-phenylalanyl]-L-allo- threonyl]-L-asparaginyl]-L-serine-nu-lactone), which inhibited [3H]SP binding to guinea pig lung membranes in a dose-dependent manner. According to Rosenthal analysis, the inhibitory effect of FK 224 on [3H]SP binding appears to be competitive. In order to clarify the receptor subtype selectivity of FK 224, we have studied the interaction of FK 224 with three tachykinin receptors (NK1, NK2 and NK3) by using receptor binding techniques and in vitro bioassays, and have also compared FK 224 with the novel nonpeptide antagonist, (+/-)-CP-96,345. In binding experiments, FK 224 dose-dependently inhibited [3H]SP binding to rat cerebral cortical membranes (NK1) and [3H]neurokinin (NK) A (NKA) binding to rat duodenum smooth muscle membranes (NK2), but did not affect [3H]eledoisin binding to rat cerebral cortical membranes (NK3). In bioassay experiments, FK 224 inhibited SP-induced contraction of guinea pig ileum (NK1) and NKA-induced contraction of rat vas deferens (NK2) in a dose-dependent manner, but did not affect NKB-induced contraction of rat portal vein (NK3). In contrast, (+/-)-CP-96,345 inhibited SP-induced contraction of guinea pig ileum, but not NKA-induced contraction of rat vas deferens or NKB-induced contraction of rat portal vein. In the presence of FK 224, SP dose-response curves and NKA dose-response curves were shifted to the right in parallel with no depression of the maximal contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfiram and diethyldithiocarbamate are competitive inhibitors at the peripheral benzodiazepine receptor.

In the present study in vitro interactions of disulfiram (an agent used to induce ethanol intolerance in alcoholics), diethyldithiocarbamate (DDC), and metronidazole with central benzodiazepine receptors (CBR) and peripheral benzodiazepine (BZ) receptors (PBR) were investigated in rat tissues. Disulfiram displaced specific binding of [3H]PK 11195 from PBR in the cerebral cortex with an IC50 value of 5 x 10(-7) M. The binding of [3H]PK 11195 and [3H]Ro 5-4864 to PBR in the kidney was displaced by disulfiram with IC50 values of 7 x 10(-7) and 2 x 10(-7) M, respectively. DDC displaced [3H]PK 11195 binding to kidney membranes with an IC50 value of 5 x 10(-5) M. Binding of [3H] flunitrazepam to CBR in the cerebral cortex was not affected by either disulfiram or DDC. Metronidazole (up to 10(-4) M), a disulfiram congener, did not affect [3H]flunitrazepam (FNZ) and [3H] PK 11195 binding to CBR and PBR, respectively. Scatchard analysis of [3H]PK 11195 binding to kidney membranes, performed in the absence or presence of 7 x 10(-7) M disulfiram, decreased ligand affinity without influencing the maximal number of binding sites, suggesting a competitive inhibition. Beta-Mercaptoethanol (2 x 10(-2) M), which blocks the inhibitory activity of disulfiram and DDC at the acetaldehyde dehydrogenase, did not affect the inhibitory potency of disulfiram at the kidney PBR. Removal of disulfiram from kidney by repeated washing with Tris-HCl buffer resulted in the restoration of binding properties to control values, suggesting reversibility of disulfiram binding to PBR.     

Effect of a protracted antidepressant treatment on signal transduction and [3H](-)-baclofen binding at GABAB receptors

In membranes prepared from frontal cortex of rats receiving desmethylimipramine (10 mg/kg i.p. twice daily) or imipramine (7.5 mg/kg i.p. twice daily) for 3 weeks, the density of high-affinity gamma-aminobutyric acid (GABA)B recognition sites is increased when measured by [3H]GABA binding in the presence of an excess of bicuculline, but it is unchanged when measured by [3H](-)-baclofen binding. When the atypical antidepressant maprotiline was administered (10 mg/kg i.p. twice daily for 3 weeks), no change in the density of GABAB recognition sites was observed using either [3H]GABA or [3H](-)-baclofen as ligands. In addition, a protracted treatment with imipramine, desmethylimipramine and maprotiline failed to change GABAB receptor-coupled signal transduction as monitored by the ability of (-)-baclofen to inhibit the forskolin-stimulated adenylate cyclase activity in membranes prepared from frontal cortex and hippocampus or the cyclic AMP formation in slices from frontal cortex. Hence, after protracted antidepressant treatment, the increase of [3H]GABA binding may not reflect changes in the characteristics of the recognition sites of the GABAB receptors subclass coupled to the adenylate cyclase through a guanine nucleotide binding protein inhibitory (Ni).