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.

     

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.

     

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.

     

[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.

     

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.

     

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

Summary To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [³H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue.During the incubation of the tracheae with [³H]choline a significant synthesis of [³H]acetylcholine (35,000 dpm/preparation) and [³H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [³H]phosphorylcholine was enhanced, whereas the content of [³H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [³H]phosphorylcholine (900 dpm/3 min) and [³H]choline (800 dpm/3 min); [³H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [³H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [³H]phosphorylcholine (1,900 dpm) without affecting the outflow of [³H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [³H]acetylcholine (2,400 dpm), but only a small outflow of [³H]phosphorylcholine. Chemical stimulation (30 mol/1 veratridine) also caused a large release of [³H]acetylcholine (1,700 dpm) without affecting the outflow of [³H]phosphorylcholine or [³H]choline. Indomethacin (3 mol/1) enhanced the electrically-evoked release of [³H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [³H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release. Send offprint requests to I. Wessler at the above address     

Tachykinin receptors in the guinea-pig isolated oesophagus: a complex system

1. The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea-pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar⁹,Met(O₂)¹¹]SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB, selective for NK₁, NK₂ and NK₃, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μM), thiorphan (1 μM) and amastatin (20 μM), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.
2. All agonists produced concentration-dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration-response curves of SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB significantly leftwards and the concentration-response curve of NKB was shifted significantly rightwards. However, the EC₅₀ values were significantly different only for [Nle¹⁰]NKA(4–10) and NKB.
3. In the presence of the peptidase inhibitors, the EC₅₀ values of the selective agonists, [MePhe⁷]NKB (0.6 nM) and [Nle¹⁰]NKA(4–10) (66 nM) indicated the presence of both tachykinin NK₃ and NK₂ receptors. [MePhe⁷]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar⁹,Met(O₂)¹¹]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK₁ receptors were also present.
4. Assuming competitive antagonism, the NK₂-selective antagonist SR 48,968 (30 nM) gave apparent pK_B values of 8.13 and 8.65 for [Nle¹⁰]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK₂ receptors.
5. The NK₃-selective antagonist SR 142,801 (0.1 μM), suppressed responses to low (0.1–10 nM) concentrations of [MePhe⁷]NKB. These contractile responses to [MePhe⁷]NKB were also abolished by atropine (0.6 μM) suggesting that this response was mediated via cholinergic nerves.
6. It is concluded that the guinea-pig oesophagus is a complex system which has both NK₂ and NK₃ receptors and possibly some NK₁ receptors as well.

     

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.

     

Prostanoid receptors of the EP3 subtype mediate the inhibitory effect of prostaglandin E2 on noradrenaline release in the mouse brain cortex

Mouse or rat brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the effects of prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂) and related drugs on the electrically evoked (50 mA, 2 ms, 0.3 Hz) tritium overflow.PGE₂ inhibited the electrically evoked tritium overflow from mouse brain cortex slices; the maximum effect of PGE₂ (79010) was attenuated by the ₂-adrenoceptor agonist talipexole (to 52010) and enhanced by the ₂-adrenoceptor antagonist rauwolscine (to 92%). Rauwolscine was added to the superfusion medium in all subsequent experiments. The effect of PGE₂ was readily reversible upon withdrawal from the medium and remained constant upon prolonged exposure of the tissue to the prostanoid. Studies with EP receptor agonists, mimicking the inhibitory effect of PGE₂, showed the following potencies (pIC₅₀): sulprostone (8.22); misoprostol (8.00); PGE₂ (7.74); PGEZ (7.61); iloprost (5.86). The concentration-response curve of PGE₂ was marginally shifted to the right by the EP₁ receptor antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; apparent pA₂ 3.97) and by the TP receptor antagonist vapiprost (4.50). AH 6809, by itself, did not affect the evoked overflow whereas vapiprost increased it. PGD2 inhibited the evoked overflow at high concentrations (pIC₅₀ 4.90); this effect was not altered by the DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2hydroxyethylamino)hydantoin), which, by itself, did not affect the evoked overflow. Indometacin slightly increased the evoked overflow and tended to increase the inhibitory effect of PGE₂. PGE₂ inhibited the electrically evoked tritium overflow also in rat brain cortex slices. The maximum effect (obtained in the presence of rauwolscine) was 61%; the pIC₃₀ value was 7.67.The present study suggests that PGE₂ inhibits noradrenaline release from mouse brain cortex via EP₃ receptors and that its maximum effect is more marked in the mouse than in the rat. The inhibitory effect of PGD₂ (in the mouse brain) does not involve DP receptors and may also be related to EP₃ receptors. The EP₃ receptors interact with a2-adrenoceptors and may be activated by endogenous prostanoids.     

Characterization of the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes

1. SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [³H]-SB-204269 in rat forebrain membranes.
2. Specific [³H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (K_D) of 32±1 nM and a maximum binding capacity (B_max) of 253±18 fmol mg⁻¹ protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na⁺, K⁺, Ca²⁺ or Mg²⁺ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues.
3. Specific [³H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action.
4. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [³H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na⁺ or K⁺ channels or various other receptors did not demonstrate any affinity for the binding site.
5. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.

     

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Summary Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [³H]choline to measure the synthesis and the release of [³H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used.The radiochromatogram following the injection of a [³H]choline-standard and a [¹⁴C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [³H]choline caused the synthesis of [³H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [³H]acetylcholine, whereas the overflow of [³H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mol/l) suppressed the release of [³H]acetylcholine by 60%. Scopolamine (0.1 mol/l) prevented this inhibition and, given alone, enhanced the release of [³H]acetylcholine by 43%. The release of [³H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies.The present experiments show the synthesis and the calcium-dependent release of [³H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats. However, some differences (frequency-dependency of [³H]acetylcholine release, spontaneous neuronal activity) are evident between both species. Reverse phase HPLC is a useful method to separate radioactive choline and acetylcholine with a high recovery rate.Send offprint requests to I. Wessler at the above address     

Differences between proximal and distal portions of the male rabbit posterior urethra in the physiological role of muscarinic cholinergic receptors

1. The aim of the present study was to elucidate functional differences between embryologically different portions of the posterior urethra of male rabbits in response to muscarinic acetylcholine receptor (mAChR) stimulation using in vitro isometric tension experiments and radioligand binding studies.
2. In the in vitro isometric tension experiments, carbachol, produced a dose-dependent contraction of the proximal portion under the resting state, but did not change the basal tone of the distal portion. Contraction of the proximal portion by 10⁻⁵ M noradrenaline (NA) was dose-dependently enhanced by carbachol either in the presence or absence of N^G-nitro-L-arginine (NOARG). In contrast, carbachol induced relaxation of the distal portion contracted by 10⁻⁵ M NA, which was reversed to dose-dependent contraction in the presence of NOARG.
3. Both portions of the urethra had a similar number of [³H]-quinuclidinyl benzilate ([³H]-QNB) binding sites (195.3±74.1 fmols mg⁻¹ protein for the proximal portion and 146.5±8.5 fmols mg⁻¹ protein for the distal portion) with similar affinities (115.0±45.4 pM for the proximal portion and 79.9± 2.9 pM for the distal portion).
4. The concentration-response curves to carbachol in both portions were shifted to the right in a parallel manner in the presence of pirenzepine (an M₁ antagonist), 11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5, 11-dihydro-6H-pyrido-2,3-b)-(1,4)-benzodiazepin-6-one (AFDX-116, an M₂ antgonist) and 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP, an M₁/M₃ antagonist). The pA₂ values for pirenzepine, AFDX-116 and 4-DAMP were 7.5±0.1, 7.2±0.02 and 9.3±0.1 respectively for the contraction of the proximal portion, and 7.2±0.1, 7.1±0.2 and 9.1±0.2, respectively for the relaxation of the distal portion.
5. In conclusion mAChR subtypes distribute in a similar fashion throughout the length of the male rabbit posterior urethra with the discrepant responses to carbachol attributable to the differential involvement of the NO pathway in mAChR-generated reactions.

     

Release of [3H]acetylcholine from the isolated rat or guinea-pig trachea evoked by preganglionic nerve stimulation; a comparison with transmural stimulation

Summary Basal and stimulated outflow of radioactive acetylcholine, phosphorylcholine and choline from rat and guinea-pig isolated tracheae were measured by reverse phase HPLC followed by liquid-scintillation-spectrometry. Tracheae were stimulated either by an electrical field (transmural stimulation) or by a local stimulation of the innervating parasympathetic nerves (preganglionic stimulation). Epithelium was removed in most experiments, as the epithelium inhibits acetylcholine release.The basal tritium efflux (1,600 dpm/3min) from rat isolated tracheae incubated with [³H]choline consisted of 56% [³H]phosphorylcholine and 38% [³H]choline. Preganglionic stimulation (15 Hz, 1,200 pulses) caused a 2-fold increase in tritium outflow that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. The stimulated outflow of tritium induced by preganglionic nerve stimulation was caused by an exclusive release of [³H]acetylcholine, whereas the efflux of [³H]phosphorylcholine and [³H]choline remained unaffected by this stimulation mode. Transmural stimulation of the rat or guinea-pig trachea, however, caused, in addition to the release of [³H]acetylcholine, the outflow of [³H]phosphorylcholine. Hexamethonium (300 mol/l) or tubocurarine (100 mol/l) inhibited (80%) the increase in tritium outflow evoked by preganglionic stimulation, but did not affect tritium outflow evoked by transmural stimulation. Oxotremorine reduced [³H]acetylcholine release evoked by both stimulation modes, but oxotremorine was less potent with transmural stimulation. Scopolamine (0.3 mol/l) enhanced (120%) the release of [³H]acetylcholine evoked by preganglionic nerve stimulation indicating the blockade of an endogenous negative muscarinic feedback mechanism. Epithelium-dependent inhibition of [³H]acetylcholine release was evident with both preganglionic and transmural stimulation.The present experiments demonstrate the release of [³H]acetylcholine evoked from the isolated trachea by stimulation of the preganglionic trunk of the parasympathetic cholinergic nerves. Qualitative and quantitative differences were observed in comparison to transmural stimulation. Preganglionic nerve stimulation allows a selective excitation of pulmonary, parasympathetic nerve fibres, mimics the physiological excitation of intramural neurones and is not followed by the liberation of phosphorylcholine from non-neuronal cells. Send offprint requests to I. Wessler 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.

     

Glycine-evoked release of [3H]acetylcholine from rat striatal slices is independent of the NMDA receptor

Summary KCl-, NMDA-, and glycine-evoked release of [³H]acetylcholine was studied in superfused rat striatal slices. KCl-evoked release of [³H]acetylcholine was inhibited by 1.2 mM MgC12 and 100 M lidocaine. Similarly, NMDA-evoked release was inhibited by MgCl₂ and lidocaine as well as 10 M CGS 19755, a competitive antagonist at NMDA receptors, and 10 nM MK-801, a noncompetitive antagonist of NMDA-induced responses. Glycine-evoked release was calcium-dependent and was inhibited by 0.1 M strychnine whereas KCl- and NMDA-evoked release were resistant to strychnine. In addition, lidocaine inhibited the glycine-induced response. Cross-tachyphylaxis was not observed between NMDA- and glycine-evoked release. These results indicate that the strychnine-sensitive, glycine-evoked release of [³H]acetylcholine is independent of the NMDA receptor.     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

Modulation of electrically evoked [3H]-noradrenaline release from cultured chick sympathetic neurons

In the present study we attempted a comprehensive characterization of modulation of noradrenaline release from chick sympathetic neurons. To this purpose sympathetic neurons derived from chick lumbosacral paravertebral ganglia and kept in culture for 7 days were loaded with 0.05 mol/l [³H]-noradrenaline and subjected to electrical field stimulation (36 pulses/3 Hz). Since the released transmitter was partially recaptured, superfusion was usually performed in the presence of (+)-oxaprotiline, an inhibitor of noradrenaline re-uptake. [³H]-Noradrenaline was released in a manner which was dependent on extracellular Ca²⁺ and sensitive to tetrodotoxin (TTX). -Conotoxin (-CTX; 100 nmol/l) abolished [³H]-noradrenaline release indicating that influx through -CTX-sensitive Ca²⁺-channels was essential for transmitter release. 1,4-dihydro-2,6-dimethyl-5-nitro4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester ((±)Bay K 8644) and 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester ((±)-202-791), agonists at L-type voltage sensitive Ca²⁺-channels (VSCCs), increased noradrenaline release and induced, in addition, an overflow of tritium which was Ca²⁺-dependent and prevented by the presence of TTX. The L-type VSCC antagonists (–)-202-791 and (+)-4-(4-benzofurazanyl)-1,4-dihydro2,6-dimethyl-3,5-pyridinedicar boxylic acid methyl, isopropyl ester ((+)-PN 200–110) diminished [³H]-noradrenaline release. These data suggest that L-type VSCCs, probably located on the cell body of the neuron, play an additional role in modulation of release. The full ₂-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline ( UK-14,304) and noradrenaline significantly inhibited noradrenaline release, whereas clonidine, a partial a2-agonist, produced only a slight inhibition even at 10 mol/l. The facilitation of noradrenaline release observed in the presence of the ₂-adrenoceptor antagonist rauwolscine was very low in comparison to that obtained with brain slices and isolated smooth muscle tissues. These results corroborate the observation that noradrenaline release from chick sympathetic neurons is regulated by an ₂-adrenoceptor which needs further subtype characterization. The experiments were mostly performed at 25°C, since a rise in temperature to 37°C increased the resting outflow, but not the evoked overflow of tritium, approximately 4-fold. In the presence of pargyline to block monoamine oxidase, however, the temperature-dependent enhancement was diminshed and the release showed properties comparable to those observed at 25°C (with respect to TTX-sensitivity, Ca²⁺ dependence and modulation via ₂-adrenoceptors). In addition to the ₂-adrenoceptors, we detected inhibitory -adrenoceptors, opioid and receptors, and P₂ purinoceptors as well as facilitatory prostaglandin (PG) E receptors. No indication was found for a functional relevance of 5-hydroxytryptamine (5-HT), opioid , PGD, adenosine A₁ or glutamate receptors. In conclusion, electrically evoked noradrenaline release from cultured chick sympathetic neurons shows the properties of action-potential-induced transmitter release and is bidirectionally regulated by various substances. Therefore, sympathetic neurons in culture offer the possibility to investigate directly the mechanisms bringing about receptor-coupled modulation of transmitter release.Abbreviations ATP adenosine 5-triphosphate - Bay K 8644 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester - DAGO (d-Ala²,N-methyl-Phe⁴,Gly-ol⁵)-enkephalin - DPDPE (d-Pen ^2,5)-enkephalin - 5-HT 5-hydroxytryptamine - -CTX -conotoxin - KRBB modified Krebs-Ringer bicarbonate buffer - NMDA N-methyl-d-aspartic acid - PG prostaglandin - PN 200-110 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxy lic acid methyl, isopropyl ester - R-PIA R(–)-N⁶-(2-phenyl-isopropyl)-adenosine - TTX tetrodotoxin - U-50,488H trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide - UK-14,304 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline - VSCC voltage sensitive Ca²⁺-channel - 202-791 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester Correspondence to: C. Allgaier at the above address