The M2 selective antagonist AF-DX 116 shows high affinity for mnscarine receptors in bovine tracheal membranes

Summary We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using ³H-dexetimide/pirenzepine and ³H-dexetimide/AF-DX 116 competition studies. Pirenzepinc exhibited low (M₂) affinity binding to both preparations; K _d was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M₂) affinity binding to left ventricle (K _d = 95.6 nM); in tracheal membranes it bound with high (M₂) affinity (K _d = 40.7 nM) to 74% of the receptors and with low (M₃) affinity (K _d = 2.26 M) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M₂ (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M₃ (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors. Send offprint requests to A. F. Roffel at the above address     

Characterization of the muscarine receptor type on paracrine cells activated by McN-A-343 in the mouse isolated stomach

Summary An attempt was made to characterize the muscarine receptor type(s) involved in acid secretion in the mouse isolated stomach when stimulated by the muscarinic agonist McN-A-343. A series of 8 muscarinic antagonists was used with preference for Mr receptors (telenzepine and pirenzepine), M₁ and M₂ receptors (secoverine), M₂ receptors (AF-DX 116 and himbacine) and M₁ and M₃ receptors (p-F-HHSiD and HHSiD). BTM-1086 was used as a high affinity antagonist at the M₁ receptor however with little selectivity.Receptor type preferences were determined in binding experiments with [³H]telenzepine in cortical membranes (M₁) and [³H]N-methylscopolamine in atrial (M₂) or salivary gland (M₃) membranes, derived from guinea pigs. No antagonist with M₃ preference could be identified in the binding studies.A fixed antagonist concentration of 1 mol/l was used to antagonize acid secretion stimulated by 10 mo1/l McN-A-343. By plotting the percentage inhibition obtained in the functional test against the Ki values determined in binding experiments for each antagonist at M₁, M₂ and M₃ binding sites, an affinity-inhibition curve could only be constructed when based on the antagonist affinities to the Mr receptor. No statistically significant fit was found using antagonist affinities to the M₂ or M₃ receptor.Thus, in accordance with the presumed Mr selectivity of the agonist McN-A-343, the rank order of potencies of different antagonists point to the M₁ nature of the muscarine receptor which stimulates acid secretion in the mouse isolated stomach upon activation by McN-A-343. Though M₂ receptors were completely ruled out, M₃ receptors may still contribute to some extent to the acid stimulating effect of McN-A-343 in this tissue. Send offprint request to W. Kromer at the above address     

Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes

Summary The non-selective labelled antagonist [³H]N-methyl-scopolamine ([³H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a K_D-value of 0.2–0.3 nmol/l and a receptor concentration (B_max) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M₁ (rat cerebral cortex), M₂ (rat heart), M₃ (rat submandibular gland) and M₄ (data from Dörje et al. 1991) receptors. The M₂-selective agent AF-DX 116, the group of M₂/M₄-selective compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the M3-selective HHSiD showed affinities corresponding to M₂ and/or M₄ sites. The intermediate affinity of 4-DAMP favours a mixed M₂/M₄ receptor population mainly containing M₂ receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [³⁵S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level. Send offprint requests to C. Liebmann at the above address     

Prejunctional muscarine receptors in the rabbit ear artery differ from M1, M2 and M3 muscarine receptors

Summary The ability of several selective muscarine receptor antagonists to inhibit the effect of carbachol on prejunctional muscarine receptors on sympathetic nerve endings in the rabbit isolated ear artery was investigated to characterise the receptor subtype involved.Carbachol did not reduce responses to exogenous noradrenaline and the inhibitory effect of carbachol on responses to nerve stimulation was unaffected by hexamethonium (10 M) indicating that the effect of the muscarine agonist was exerted prejunctionally and was not modulated by nicotine receptor stimulation.The dissociation constants or apparent dissociation constants obtained using (±)-benzhexol (pK_B; 6.63), (R)-benzhexol methiodide (8.11), dicyclomine (5.86), (±)-telenzepine (7.34), AF-DX 116 (6.95), himbacine (7.60), (±)-hexahydrosiladiphenidol (5.39) and a bisquaternary ammonium compound, heptane-1,7-bis(dimethyl-3-phthalimidopropyl ammonium bromide) (5.84), indicate that the muscarine receptor subtype involved is not of the M₁, M₂ or M₃ subtype. Send offprint requests to F. Mitchelson at the above address     

Characterization of porcine coronary muscarinic receptors

Summary To determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [³H]N-methyl-scopolamine ([³H]NMS) binding to membrane preparations from porcine coronary arteries. [³H]NMS binds to a single population of muscarinic binding sites with a K_D of 135 pM and a B_max of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((–((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N-bis (6-((2-methoxybenzyl) amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladi-fenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M₂ and M₃ subtype.Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pK_i values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA₂ values.It is concluded that a mixed population of the M₂ and M₃ muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M₂ subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M₃ receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m₃, m₄ and m₅ receptors the involvement of muscarinic receptors different from M₁, M₂ and M₃ cannot be excluded. Send offprint requests to M. Entzeroth at the above address     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

The relative potencies of cholinomimetics and muscarinic antagonists on the rat iris in vivo: effects of pH on potency of pirenzepine and telenzepine

Summary The effects of cholinomimetics and muscarinic antagonists were compared following topical administration to the eyes of anaesthetized rats. For tests with cholinomimetics, clonidine (0.3 mg/kg) was used to induce mydriasis via central inhibition of parasympathetic tone. Full, dose-dependent miosis was induced by acetylcholinesterase inhibitors [physostigmine > neostigmine > tetrahydroaminoacridine (THA)] and by membrane channel blockers (4-aminopyridine > 3,4-diaminopyridine). Oxotremorine was the most potent direct agonist tested [oxotremorine > arecaidine propargylester (APE) > arecolne > carbachol > ethoxyethyltrimethyl-ammonium iodide (EOE) > RS 86]. Some putative M1 selective agonists were weakly active or behaved as partial agonists (pilocarpine > AH6405 > Mc-A-343 > isoarecoline). Of the antagonists, compared in non-clonidine treated rats, scopolamine hydrochloride was the most potent. Of the receptor selective antagonists the M₂ (ileal) selective compounds hexahydrosiladifenidol and 4-DAMP were more potent than either M₁ selective (pirenzepine, telenzepine) or M₂ (atrial) selective (AF DX 116) drugs. These data tentatively suggest the involvement of an M₂ (ileal) type muscarinic receptor. Potency was lower for quaternary structures, probably due to impaired corneal penetration. The potency of pirenzepine and telenzepine was increased 60-fold at low pH following topical administration. Acid induced corneal damage does not appear to account for this potency shift as the effects of scopolamine and several agonists (oxotremorine, pilocarpine and McN-A-343) were not substantially altered by acid media. For pirenzepine the potency shift appears to be related to protonation of the second amino group (N1) in the piperazine tail (pK _a = 2.05). Intraocular injections suggest that diprotonation facilitates penetration through the cornea. This anomalous behaviour of pirenzepine may contribute to its potency in gastric acid inhibition where the acid environment of the stomach would favour the diprotonated state and therefore penetration through the epithelium. Send offprint requests to J. J. Hagan at the above address     

AF-DX 116 differentiates between prejunctional muscarine receptors located on noradrenergic and cholinergic nerves

Summary Prejunctional affinity constants of the cardioselective muscarine receptor antagonist AF-DX 116 (11-[(2[(diethyl-amino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro6 H-pyrido [2,3-b] [1,4] benzodiazepine-6-one) were determined for muscarine autoreceptors on cholinergic nerves of the guinea-pig ileum and for heteroreceptors on noradrenergic nerves of the rat heart and guinea-pig iris. AF-DX 116 antagonized with low affinity the muscarinic inhibition induced by arecaidine propargyl ester of the stimulation-evoked [³H]acetylcholine overflow (pA₂ 6.74) from the guinea-pig ileum. In contrast, AF-DX 116 was more potent in antagonizing the methacholine-induced inhibition of the stimulation-evoked [³H]noradrenaline overflow from rat heart (pA₂ 7.29) or guinea-pig iris (pA₂ 7.57). The data confirm previously reported differences between prejunctional muscarine heteroreceptors in the rat heart which belong to the cardiac subtype (M₂ or M₂) and autoreceptors in the guinea-pig ileum that cannot be distinguished from the ileal subtype (M₂) or (M₃). Send offprint requests to H. Fuder at the above address     

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart

Summary The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [³H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA₂ values for M₁-, M₂- and M₃-receptors revealed that the presynaptic autoreceptor meets the criteria of an M₁-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by leakage release (non-exocytotic), as it was independent of extracellular Ca²⁺ . Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10^–6M tacrine or pretreatment with 10^–4M DFP, the ACh overflow was partially Ca²⁺-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (±)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [³H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis. The dramatic enhancement of ACh release by the ChE inhibition in combination with a block of presynaptic muscarinic autoinhibition was not inhibited by (+)-tubocurarine but by atropine (10^–9 to 10^–7 M) or 10^–6 M telenzepine. It is concluded that basal release of ACh in the heart was due to non-exocytotic leakage release. Inhibition of ChE led to a marked stimulation of excitatory muscarinic receptors of the intrinsic parasympathetic neuron with a consecutive postganglionic release of ACh. The strong postganglionic excitation was obvious when the inhibitory muscarinic autoreceptors were selectively blocked. Of the two described muscarinic receptors found in the parasympathetic postganglionic neuron of the chicken heart only the inhibitory was classified as being M₁, whereas the subtype of the excitatory one is unlike M₁ and remains to be identified.Preliminary results have been presented at the Spring meeting of the German Pharmacological Society in 1992 (Brehm and Lindmar 1992) Correspondence to R. Lindmar at the above address     

Muscarinic inhibition of endogenous noradrenaline release from rabbit isolated trachea: receptor subtype and receptor reserve

The aim of the present study was to characterize putative muscarine receptors on sympathetic nerve terminals in the rabbit trachea. Release of endogenous noradrenaline from in vitro incubated rabbit tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) and quantified by high performance liquid chromatography with electrochemical detection.The muscarine receptor agonist oxotremorine inhibited the evoked release of noradrenaline completely at 1 ol/l (EC₅₀: 64 nmol/l). The concentration response curve was very steep (Hill coefficient of 2.3). Scopolamine shifted the concentration response curve of oxotremorine to the right (–log K_B 8.48) demonstrating specific, inhibitory muscarine receptors. Several subtype-preferring muscarine receptor antagonists also shifted the concentration response curve of oxotremorine to the right. The rank order of potency was (–log K_B or pA^* ₂): scopolamine (8.48) > AF DX 384 (7.88^*; slope of Schild plot 1.1) > (R)-trihexyphenidyl (7.87) > 4-DAMP (7.85) > AQ-RA 741 (7.77) methoctramine 6.18 > pirenzepine (6.0) >p-fluoro-hexahydrosiladifenidol (p-FHHSiD, 5.68). When these affinity constants were plotted against reported –log K_i values determined in binding studies on human cloned muscarine receptor subtypes (m₁-m₅), the best correlation was obtained for m₂. Indomethacin (3 mol/l), which on its own increased the evoked noradrenaline release by about 45%, affected neither the inhibitory effect of oxotremorine nor the antagonistic potency of methoctramine or p-FHHSiD. After preincubation for 48 min with 300 mol/l phenoxybenzamine, which has been shown to inactivate muscarine receptors irreversibly, the concentration response curve of oxotremorine was shifted 5.2 fold to the right and the maximal inhibition was reduced by 50%, whereas the slope remained steep (Hill coefficient 2.6). These experiments indicated that a fraction of about 22% of the muscarine receptors has to be occupied by oxotremorine to produce half-maximum inhibition of noradrenaline release; the dissociation constant of oxotremorine at the prejunctional muscarine receptors was 0.33 mol/l.In conclusion, the sympathetic nerve terminals in the rabbit trachea are endowed with inhibitory M₂-like muscarine receptors for which methoctramine displayed a low affinity. Since a large receptor reserve could be excluded, the steep concentration response curve of oxotremorine suggests that activation of muscarine receptors has to reach a threshold level before the onset of an inhibitory effect. Correspondence to: K. Racké at the above address     

Binding of muscarine receptor antagonists to pig coronary smooth muscle

Summary In order to characterize the muscarinic binding site on coronary smooth muscle, we investigated the binding properties of (³H)quinuclidinyl benzilate (QNB) in membrane preparations of pig coronary arteries and atria. Scatchard analysis and Hill plot showed that (³H)QNB binds to a single population of sites in both tissues. The binding profiles of the muscarine receptor antagonists atropine, 11-((2-((dimethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one (AF-DX 116), pirenzepine, and 4-diphenylacetoxy-Nmethylpiperidine methiobromide (4-DAMP) in both tissues were compared with binding data from other tissues, representative for different muscarinic binding site subtypes. It is concluded that the pig coronary smooth muscle muscarinic binding site is different from M₁ and M₂ binding sites investigated so far. Send offprint requests to I. Rinner     

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.

     

Characterization of 3H-GABA receptor binding to rat brain synaptosomal membranes: Effect of non GABAergic compounds

Characteristics of ³H-GABA binding to rat brain synaptic membranes in vitro have been investigated. The specific binding of ³H-GABA displays saturation kinetics. Only one single population of receptor sites was found (K _m=31.3 nM) with a concentration of 2.09 pmol/mg protein. Only GABA agonists show inhibitory effect on the binding, whereas GABA antagonists, GABA-uptake inhibitors, and inhibitors of GAD and GABA-T are without effect. The order of potencies for GABA agonists are: Muscimol > GABA 4,5-dihydromuscimol > 3-aminopropane sulphonic acid > isoguvacine > THIP > 3-hydroxy-GABA > imidazol-4-acetic acid. Agonists and antagonists from other neurone systems as well as neuroleptics and benzodiazepines had no or only a very slight potency in the binding test.     

Dose-response curves of pirenzepine in man in relation to M1- and M2-cholinoceptor occupancy

Summary The aim of the present study was to investigate the M-cholinoceptor subtype selectivity of pirenzepine in man. In parallel with effects on the heart rate and salivary flow, M-cholinoceptor subtype occupancy by antagonist present in plasma samples was detected in radioreceptor assays. Bovine cerebral cortex membranes labelled with ³H-pirenzepine (M₁) and rat salivary gland membranes labelled with ³H-N-methylscopolamine (M₂) were used in these in vitro assays. A half-maximal occupancy of M₁-cholinoceptors in the in vitro assay of plasma samples was detected after 0.25 mg of pirenzepine i.v. The respective half-maximal M₂-cholinoceptor occupancy was observed after 10 mg. Doses < 3 mg decreased the heart rate by maximally 10.7 beats/min with an ED₅₀ of about 0.1 mg. An increase in heart rate (relative to control values) was observed at doses > 10 mg. This bivalent dose-response relationship was also observed after -blockade. Salivary flow tended to increase at doses < 1 mg and was half-maximally inhibited after 10 mg. Combining the in vitro and in vivo results, the typical antimuscarinic effects (tachycardia and inhibition of salivary flow) can be attributed to the blockade of M₂-cholinoceptors, whereas the reduction of heart rate coincides with the blockade of the M₁-subtype. With respect to the typical antimuscarinic effects, pirenzepine was 70-fold less potent than atropine; in contrast, with respect to the reduction of heart rate, pirenzepine was equipotent with atropine. It is concluded that pirenzepine does not discriminate between cardiac and salivary gland M₂-cholinoceptors but shows pronounced selectivity for the M₁-cholinoceptors through which it mediates the decrease in heart rate. The latter effect may be explained by inhibitory M₁-auto-receptors. Send offprint requests to H. F. Pitschner at the above addressParts of the results were reported at the spring meeting 1987 of the Deutsche Gesellschaft für Pharmakologie und Toxikologie (Wellstein et al. 1987)     

The moderate affinity of clozapine at H3 receptors is not shared by its two major metabolites and by structurally related and unrelated atypical neuroleptics

We determined the affinity and/or potency of two metabolites of clozapine (clozapine-N-oxide and N-desmethylclozapine) and of five atypical neuroleptics, chemically related (olanzapine) or unrelated to clozapine (remoxipride, risperidone, thioridazine, zotepine), at H₃ receptors.The specific binding of ³H-N-methylhistamine to rat brain cortex homogenates was inhibited by the seven compounds; the pK_i values were: N-desmethyl-clozapine (5.33); clozapine-N-oxide (4.18); olanzapine (5.45); thioridazine (4.91); zotepine (4.75); remoxipride (4.51) and risperidone (4.43). Three compounds were examined in a functional H₃ receptor model as well. The electrically evoked tritium overflow from superfused mouse brain cortex slices, which represents quasi-physiological noradrenaline release, was not affected by N-desmethylclozapine (3.2 and 10 M), clozapine-N-oxide (3.2–100 M) and olanzapine (3.2–32 M). On the other hand, the three compounds shifted to the right the concentration-response curve of histamine for its inhibitory effect on the evoked overflow; the apparent pA₂ values were 5.84, 4.21 and 5.80, respectively.The present study shows that five atypical neuroleptics of different chemical classes and the two major metabolites of clozapine possess a lower affinity and/or antagonistic potency at H3 receptors than clozapine itself (pK_i 6.15, pA₂ 6.33; Kathmann M, Schlicker E, Göthert M (1994). Psychopharmacology 116: 464–468).     

Pharmacological comparison of the sigma recognition site labelled by [3H]haloperidol in human and rat cerebellum

Summary The radioligand binding characteristics of [³H]haloperidol (in the presence of spiperone, 25 nmolL^–1) were investigated in rat and human cerebellar membranes.In both rat and human cerebellar membrane preparations saturation studies with [³H]haloperidol (non-specific binding defined by pentazocine, 10 molL^–1) demonstrated high affinity saturable specific binding to a homogenous population of binding sites (rat, Bmax 6693 ± 1242 fmol mg^–1 protein, pK_D 8.33 ± 0.08; human, Bmax 2550 ± 437 fmol mg^–1 protein, pK_D 8.59 ± 0.11; mean ± SEM, n = 3–6). Competition studies employing a wide range of structurally diverse competing compounds displayed that the [³H]haloperidol binding site was pharmacologically similar in both preparations and comparable to sigma recognition sites previously identified in various tissues originating from different species. In addition, with reference to the potential subtypes of sigma recognition sites, the labelling of these sites by low nanomolar concentrations of [³H]haloperidol provides evidence that they belong to the sigma-1 recognition site subtype.The present findings suggest that the pharmacology of the rat and human cerebellar sigma recognition site are directly comparable and provides further supporting evidence towards the use of [³H]haloperidol radioligand binding studies in the rat to detect sigma receptor ligands with potential therapeutic activity. Send offprint requests to: N.M. Barnes at the above address     

Sialogogic activities of SNI-2011 compared with those of pilocarpine and McN-A-343 in rat salivary glands: Identification of a potential therapeutic agent for treatment of Sjörgen's syndrome

• 1.1. We examined the sialogogic activities in rat major salivary glands of SNI-2011, in comparison with those of pilocarpine and McN-A-343, and we characterized the subtypes of muscarine receptors that are involved in the sialogogic responses to SNI-2011 and McN-A-343.
• 2.2. SNI-2011 at doses ranging from 1 to 10 mg/kg (i.v.) increased the secretion of saliva in a dose-dependent manner. The dose-response curves for SNI-2011 were approximately parallel to curves for pilocarpine but the potency of SNI-2011 was about 25-fold lower than that of pilocarpine.
• 3.3. The total volume of saliva secreted in response to McN-A-343 was very much less than that secreted in response to SNI-2011.
• 4.4. The salivation induced by SNI-2011 and by McN-A-343 was inhibited by various antagonists with the following rank order of potency: 4-DAMP⪢pirenzepine⪢AF-DX 116.
• 5.5. Our results suggest that the sialogogic effects of SNI-2011 and McN-A-343 are mediated by direct stimulation of M₃ receptors in salivary glands and that SNI-2011 may prove useful in the management of xerostomia in patients with Sjögren's syndrome.

     

The agonist activities of the putative antipsychotic agents,L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor

1. Dopamine D₄ receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced.
2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D_4.4 receptor (HEK293/D₄ cells). [³H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response.
3. [³H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D₄ cell homogenates (B_max 505±90 fmol mg⁻¹ protein, pK_D 9.5±0.1, n=3). Inhibition of specific [³H]-spiperone binding was observed with spiperone (pK_i 9.6±0.1, n=3), clozapine (pK_i 7.4±0.1, n=4), L-745,870 (pK_i 8.5±0.1, n=3) and U-101958 (pK_i 8.9±0.1, n=3). By contrast, raclopride was very weak (pK_i<5, n=3).
4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent fashion (E_max 71±2% inhibition of forskolin-stimulated levels, pEC₅₀ 8.7±0.1, n=10). This effect was mimicked by the dopamine D₂-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).
5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC₅₀) values of L-745,870 and U-101958 were 9.0±0.2 (n=4) and 8.7±0.3 (n=3), consistent with pK_i values determined in radioligand binding studies.
6. Dopamine, L-745,870 and U-101958 (up to 1 μM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells.
7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D₄ cells could be antagonized by spiperone (pK_B 8.2–8.8) and clozapine (pK_B 7.1), but not by raclopride (pK_B<5). None of these antagonists had any significant agonist activity at concentrations up to 10 μM.
8. These results show that the putative dopamine D₄ receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D_4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D₄ receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

     

Different muscarine receptors mediate the prejunctional inhibition of [3H]-noradrenaline release in rat or guinea-pig iris and the contraction of the rabbit iris sphincter muscle

Summary To investigate the muscarine receptor type mediating inhibition of [³H]-noradrenaline release from the isolated rat and guinea-pig iris we have determined the potency of antimuscarinic drugs to antagonize the methacholine-induced inhibition of [³H]-noradrenaline overflow evoked by field stimulation (3 Hz, 2 min). The prejunctional apparent affinities were compared with those obtained for postjunctional muscarine receptors mediating the methacholine-induced contraction of the isolated rabbit iris sphincter muscle.Prejunctional apparent affinity constants of pirenzepine (6.67), himbacine (8.51), methoctramine (7.92), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.00), hexahydro-difenidol enantiomers (6.92, (R); 5.77, (S)) in the rat iris and methoctramine (7.58) in the guinea-pig iris indicate the presence of M₂ receptors. Although the post-junctional affinity constants in the rabbit iris sphincter of methoctramine (5.93), gallamine (3.92), and 4-DAMP (9.07) confirm our previous suggestions of the presence of M₃-like receptors, the results obtained with the hexahydro-difenidol enantiomers do not agree with that concept. The post-junctional affinity constants of the hexahydro-difenidol enantiomers were not different from the prejunctional values (6.86, (R); 5.55, (S)), indicating a similar and low degree of stereoselectivity for these stereoisomers at both receptor sites (14 and 17, (R)/(S)-ratios, respectively). Hence, the postjunctional muscarine receptor in the rabbit iris sphincter fails to exhibit the high degree of stereo selectivity observed for hexahydro-difenidol enantiomers at M₃ receptors on other smooth muscles.This study was supported by the Deusche Forschungsgemeinschaft (Fu 163/2) Send offprint requests to H. Fuder at the above address     

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M₁ receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M₁(m₁)-M₄(m₅) receptors in functional studies and binding experiments.The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A₁ and P₂ purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC₅₀): arecaidine propargyl ester (0.062 M), 4-Cl-McN-A-343 (0.32 M), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN⁺; 0.48 M) and methacholine (0.86 M). The affinity constants of most of the muscarinic antagonists [atropine: pK_B = 9.47; (R)-trihexyphenidyl: pK_B = 9.18; pirenzepine: pA₂ = 7.68; methoctramine: pK_B = 6.90] are consistent with estimates of these antagonists at M₁(m₁) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN⁺ argue for the involvement of M₁, and against that of M₂ and M₃ receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pK_B of 8.30 for himbacine is not in accordance with an M₁ receptor; by contrast, it would be compatible with the presence of M₂ or M₄ receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M₁ receptors inhibiting neurogenic twitch contractions, and are not of the M₂, M₃ or m₅ subtype. Correspondence to: U. Grimm at the above address