Profile of SB-204269, a mechanistically novel anticonvulsant drug,in rat models of focal and generalized epileptic seizures

1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test.
2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays.
3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1–30 mg kg⁻¹) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MES)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg⁻¹, p.o.
4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K⁺ rat hippocampal slice model at concentrations (0.1–10 μM) that had no effect on normal synaptic activity and neuronal excitability.
5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy.
6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series.
7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg⁻¹, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED₅₀ in the MES test) for SB-204269 of >31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively.
8. At concentrations (⩾10 μM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 μM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na⁺ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS.
9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.

     

Presynaptic imidazoline receptors and non-adrenoceptor[3H]-idazoxan binding sites in human cardiovascular tissues

1. In segments of human right atrial appendages and pulmonary arteries preincubated with [³H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [³H]-noradrenaline release was investigated.
2. In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [³H]-noradrenaline release. The inhibition was not affected by blockade of α₂-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA₂ against aganodine and DTG: 5.55 and 5.21, respectively).
3. In the presence of 1 μM rauwolscine, [³H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine.
4. In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [³H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta.
5. When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α₂-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [³H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [³H]-noradrenaline release.
6. In human atrial appendages, non-adrenoceptor [³H]-idazoxan binding sites were identified and characterized. The binding of [³H]-idazoxan was specific, reversible, saturable and of high affinity (K_D: 25.5 nM). The specific binding of [³H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model.
7. The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I₂-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue.
8. It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I₁ and I₂ imidazoline binding sites.

     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

Effects of [3H]-BIDN,a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [³H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([³H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC₅₀, 35±3 nM) and dieldrin (IC₅₀, 93±7 nM), displaced [³H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [³H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [³H]-BIDN from rootworm membranes by a competitive mechanism.
3. Rat brain membranes were also shown to possess a population of saturable, specific [³H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [³H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA_B receptors were unaffected.
5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl⁻ channels to which a number of insecticidally-active molecules bind.

     

Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes

1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol.
2. [³H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [³H]-5-HT uptake with apparent K_is of 0.27±0.04 and 0.76±0.04 μM, respectively. Morphine essentially failed to inhibit [³H]-5-HT uptake (K_i 0.50±0.30 M).
3. Methadone, morphine and tramadol were active in the hot plate test with ED₅₀s of 3.5, 4.3 and 31 mg kg⁻¹, respectively. At the highest tested dose (80 mg kg⁻¹) tramadol produced only 77±5.3% of the maximal possible effect.
4. When [³H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg⁻¹, s.c., equal to the ED₅₀ in the hot plate test) and methadone (35 mg kg⁻¹, s.c., equal to the ED₉₀ in the hot plate test) decreased uptake.
5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg⁻¹ and 15 to 120 mg kg⁻¹, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg⁻¹, s.c.) or tramadol (31 mg kg⁻¹, s.c.) was administered. [³H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%.
6. Rats chronically-treated with methadone showed a significant increase in [³H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [³H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals.
7. These results further support the hypothesis that [³H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [³H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi

1. The release of neuronal [³H]acetylcholine (ACh) from isolated human bronchi after labelling with [³H]choline was measured to investigate the effects of prostanoids.
2. A first period of electrical field stimulation (S₁) caused a [³H]ACh release of 320±70 and 200±40 Becquerel (Bq) g⁻¹ in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S_n, n=2, 3, and 4) released less [³H]ACh, i.e. decreasing S_n/S₁ values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).
3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E₂, nocloprost, and sulprostone (EP₁ and EP₃ selective) inhibited evoked [³H]ACh release in a concentration dependent manner with IC₅₀ values between 4–14 nM and maximal inhibition of about 70%.
4. The inhibition of evoked [³H]ACh release by prostaglandin E₂, nocloprost and sulprostone was not affected by the DP-, EP₁- and EP₂-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA₂ values) at the respective receptors.
5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP₁-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [³H]ACh release.
6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [³H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [³H]ACh release.
7. In conclusion, applied prostanoids appear to inhibit [³H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP₃ subtype.

     

Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [³H]-L-2-amino-4-phosphonobutyrate ([³H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors.
2. Specific [³H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [³H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum.
3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [³H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [³H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [³H]-L-AP4 uptake into cerebellar membranes, [³H]-L-AP4 binding to brain sections was sensitive to guanosine-5′-O-(3-thio)trisphosphate-γ-S.
4. In the molecular layer of the cerebellar cortex, [³H]-L-AP4 binding showed a maximal binding density (B_max) of 0.52±0.06 pmol mg⁻¹ tissue and an affinity (K_d) of 346 nM. The rank order of affinity for displacement of [³H]-L-AP4 binding to rat brain sections was: L-AP4>L-serine-O-phosphate>glutamate>(L)-2-aminomethyl-4-phosphonobutanoate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.

     

Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719

1. The functional profile of the long form of the human cloned 5-HT₇ receptor (designated h5-HT_7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT_7(a) receptor.
2. The receptor binding profile, determined by competition with [³H]-5-CT, was consistent with that previously reported for the h5-HT_7(a) receptor. The selective 5-HT₇ receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzene sulfonamide) displayed high affinity (pK_i 7.5) for the receptor.
3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT>5-HT>8-OH-DPAT) was the same in functional and binding studies.
4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA₂ of 7.2±0.2 and slope not significantly different from 1, consistent with competitive antagonism.
5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT₇ receptor-mediated and to reflect inverse agonism.
6. It is concluded that in this expression system, the h5-HT_7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.

     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Inhibition of [3H]resiniferatoxin binding to rat dorsal root ganglion membranes as a novel approach in evaluating compounds with capsaicin-like activity

Summary We have recently reported the specific binding of [³H]resiniferatoxin to sensory ganglion membranes; this binding appears to represent the postulated vanilloid (capsaicin) receptor. In the present report, we compare the structure/activity relations for binding to rat dorsal root ganglion membranes and for biological responses in the rat, using a series of vanilloids of the capsaicin (homovanilloyl-decylamide, homovanilloyl-dodecylamide, homovanilloyl-cyclododecylamide, homovanilloyl-hexadecylamide, homovanilloyl-piperidine and nonenoyl-homoveratrylamide) and resiniferatoxin (tinyatoxin, 12-deoxyphorbol 13-phenylacetate 20-homovanillate) classes. We find that all the tested biologically active vanilloids, but not the inactive structure analogs, compete for the [³H]resiniferatoxin binding sites in rat dorsal root ganglion membranes, and we conclude that the [³H]resiniferatoxin binding assay may provide an efficient approach for evaluating such compounds. We also provide evidence that the [3H]resiniferatoxin receptor is likely to recognize vanilloids which are inserted into the membranes; and that the apparent activity of capsaicinoids may be significantly influenced by factors other than equilibrium binding affinities. Send offprint requests to P. M. Blumberg at the above address     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Inhibition of [3H]-U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 ((5a,7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benzeneacetamide), a specific κ-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine.
2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ-receptor stimulation by a specific κ-receptor agonist, U50,488H (trans-(±-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective κ-receptor antagonist, nor-BNI (nor-binaltorphimine,17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-binorphinan-3,4′,14, 14′-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED₅₀ values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca²⁺ influx in the cardiac muscle.
3. Measurement of [Ca²⁺]_i in the absence of free extracellular Ca²⁺ by a spectrofluorometric method, with fura-2 as Ca²⁺ indicator, showed that U50,488H 5×10⁻⁵ M slowly increased [Ca²⁺]_i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 μM), or ryanodine (5 μM). Verapamil 1 and 10 μM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca²⁺ transient.
4. The observations suggest that CCBs may inhibit the actions of κ-receptor stimulation at the level of the κ-receptor.

     

Light-dark differences in [3H]-paroxetine binding to rabbit platelet membranes

Summary [³H]-Paroxetine binding to rabbit blood platelet membranes from samples obtained under light and dark conditions was examined. Animals were kept on a 14 h light (L) — 10 h dark (D) schedule and blood samples were collected at L + 7 and D + 5 h. Significant differences were found for B _max values of [³H]-paroxetine binding, with low B _max values during the light period and high B _max values during the dark period. The K _d values were not significantly different. These results confirm previous observations on light-dark differences of [³H]-imipramine binding in rabbit blood platelets suggesting the existence of a circadian rhythm for the 5-HT transporter complex.Send offprint requests to S. Z. Langer at the above address     

Attenuation by electroshock treatment of the haloperidol-induced rise in the binding of 3H-imipramine to rat brain membranes

Studies were conducted in rats to investigate whether chronic haloperidol treatment, electroshock treatment (EST), or a combination of both affects the high-affinity binding of ³H-imipramine to cerebral membranes. Chronic haloperidol (2 mg/kg SC daily for 28 days) resulted in a significant decrease in the density of binding sites in animals killed 1 h after the last treatment, but binding was markedly enhanced after a washout period of 5 days. In animals subjected to EST (20–30 mA, 1.5 s, 60 Hz; three times a week for 4 weeks) and killed 1 h after the last treatment, there were no apparent changes, but when animals were allowed a recovery period of 5 days there appeared to be a slight increase in binding. The marked increase in binding seen after haloperidol withdrawal was no longer apparent in animals treated with both haloperidol and EST, indicating that EST acts to attenuate the increase in binding seen after withdrawal of chronic haloperidol. It is suggested that chronic haloperidol may influence presynaptic recognition sites that regulate serotonin uptake and that EST may interact with haloperidol to stabilize these presynaptic sites.     

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
2. In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA₂ value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D₁ receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [³H]-N-methylscopolamine ([³H]-NMS) bound to striatal M₄ receptors with a monophasic inhibitory curve and a pK_i value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC₅₀ of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC₅₀=50 nM) and stimulating at higher concentrations (EC₅₀=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pK_i value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
5. As in rat striatum, in CHO cell membranes the displacement of [³H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
6. Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [³⁵S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [³⁵S]-GTPγS binding with a pA₂ value of 7.48.
7. These results show that at the striatal M₄ receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

     

Use of the radioligand [(125)I]-SB-217644 in the characterisation and solubilisation of the novel binding site for the anticonvulsant SB-204269 in rat brain membranes and cell lines

SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [(3)H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [(125)I]-labelled form of SB-217644 (trans-6-acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [(125)I]-SB-217644 shows a similar binding profile to that of [(3)H]-SB-204269, with a maximum specific binding capacity (B(max)) of 286+/-12 fmol/mg protein, but has twenty-fold higher affinity (K(d) value 1.7+/-0.1 nM). The high affinity and high specific activity of [(125)I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [(125)I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K(d) value of 2.7+/-0.3 nM and a B(max) value of 55+/-11 fmol/mg protein, with a 7.3+/-0.3% yield of solubilised binding sites. [(125)I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [(125)I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [(125)I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch