Effect of bay K 8644, a calcium channel agonist,on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [³H]QNB binding with K _i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [³H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [³H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K _i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K _i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [³H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

新抗胆碱药[3H]三环哌酯对人脑M受体的作用

用放射配体受体结合试验法,研究了新化合物三环哌酯与人大脑皮质M受体的结合特性,并与QNB作了比较。饱和实验结果显示,[³H]三环哌酯的结合参数与[³H]QNB相近,两种配体的作用均符合单位点模型。竞争性抑制实验结果表明二者作用强度相当。[³H]三环哌酯的结合和解离速率常数均较[³H]QNB大,且其与皮质M受体的解离受季铵酚的变构调节,结果提示,两种配体与M受体有一些不同的结合特性,在M受体研究中,[³H]三环哌酯可以作为[³H]QNB的补充工具。     

Interaction of class III antiarrhythmic drugs with muscarinic M2 and M3 receptors: radioligand binding and functional studies

We have recently reported that class III antiarrhythmic drugs inhibit the muscarinic acetylcholine (ACh) receptor-operated K⁺ current (I _K, ACh) in guinea-pig atrial cells by different molecular mechanisms. The data obtained from the patch-clamp study suggest that d,l-sotalol inhibits I _{K, ACh} by blocking the muscarinic receptors, whereas MS-551 inhibits the K⁺ current by blocking the muscarinic receptors and depressing the function of the K⁺ channel itself and/or the guanine nucleotide-binding protein (G protein). This study was undertaken to determine whether the class III antiarrhythmic drugs d,l-sotalol and MS-551 interact with the muscarinic receptors of cardiac and peripheral tissues. Both drugs inhibited concentration dependently the specific [³H]N-methylscopolamine ([³H]-NMS) binding to membrane preparations obtained from guinea-pig atria and submandibular glands. The competition curves of these drugs for [³H]-NMS binding to glandular membranes were monophasic, suggesting competition with [³H]-NMS at a single site. Although the competition curve of d,l-sotalol for [³H]-NMS binding to atrial membranes was monophasic, that of MS-551 was biphasic and showed high- and low-affinity states of binding. d,l-Sotalol showed slightly, but significantly, higher affinity for cardiac-type muscarinic receptors (M₂) than for glandular-type muscarinic receptors (M₃). The inhibition constant (K _i) for MS-551 in glandular membranes was also slightly greater than the high-affinity K _i value for the drug in atrial membranes. In guinea-pig left atria and ilea, d,l-sotalol shifted the concentration-response curves for the negative inotropic effect and the contracting effect of carbachol in a parallel manner. The slopes of Schild plot were not significantly different from unity, suggesting competitive antagonism, and the pA₂ for d,l-sotalol in left atria was slightly greater than that in ilea. MS-551 also shifted the concentration response curve for the negative inotropic effect of carbachol in atrial preparations to a greater extent than that for the contracting effect in ileal preparations, although MS-551 failed to show a pure competitive antagonism. These results suggest that both d,l-sotalol and MS-551 interact with cardiac M₂ and peripheral M₃ receptors, and that at high concentrations they exert anticholinergic activity in cardiac and peripheral tissues.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

Adenosine receptor agonists: Binding and adenylate cyclase stimulation in rat liver plasma membranes

Summary N⁶-Cyclohexyl[³H]adenosine([³H]CHA),[³H]adenosine, and 5N-ethylcarboxamide[³H]adenosine ([³H]NECA), potent agonists in adenosine-responsive cellular systems, have been used to identify adenosine binding sites in rat liver plasma membranes. Endogenous ligands were removed by prior dialysis of the membranes. Specific binding of the ligands tested was characterized by rapid forward and reverse kinetics and heterogeneity as indicated by curvilinear Scatchard plots. The K _D in the high affinity range was 80 nM for [³H]adenosine, 84 nM for [³H]NECA, and 168 nM for [³H]CHA; the respective binding capacities of 1.19, 1.03, and 1.05 pmol/mg protein were of virtually the same magnitude, suggesting labeling of identical sites. However, all ligands also displayed binding to large numbers of low affinity sites. This high level of apparently non-receptor binding markedly influenced the adenosine structure-activity profile of [³H]CHA displacement, which differs with pharmacological findings. — NECA and CHA stimulated hepatic adenylate cyclase with an apparent ED₅₀ of 60 and 580 nM, respectively; adenosine was stimulatory at a concentration range from 0.1–2.0 M, but inhibitory at higher concentrations. Hence, estimation of the true ED₅₀ was not possible. Because the K _D of high affinity binding and the ED₅₀ of the biological effect of NECA and CHA are in the same range, it may be reasonable to assume that the high affinity sites represent adenosine receptors, recently classified as R_a-site receptors.Preliminary reports of this study have been presented at the 22nd Spring Meeting of the Deutsche Pharmakologische Gesellschaft (Naunyn-Schmiedeberg's Arch Pharmacol 316, R10, 1981) and at the 8th International Congress of Pharmacology [Tokyo 1981 (P 1459)]     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max values of [³H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K⁺-dependency of the [³H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [³H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [³H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [³H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

Circadian rhythm of the B max of [3H]-imipramine binding in rabbit platelets

Summary [³H]-imipramine binding was measured in rabbit blood platelet membranes on a 24 h cycle. Animals were kept on a 14 h light (L) 10 h dark (D) schedule, and blood samples were collected at L + 2, L + 8, D + 2, D + 8 and L – 2 h on a following cycle. Significant differences were found for B_max values of [³H]-imipramine binding, with highest values during the dark phase and lowest during the light phase. No significant differences were found in K _d values. These results suggest the existence of a circadian rhythm for the B_max of [³H]-imipramine binding in blood platelets. Send offprint requests to S. Z. Langer     

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     

Hawthorn (<Emphasis Type="Italic">Crataegus monogyna</Emphasis> Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, l-[benzylic-4,4′-³H] ([³H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [³H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.     

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     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

[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.     

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     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

The influence of guanyl-5′-yl imidodiphosphate and sodium chloride on the binding of the muscarinic agonist, [3H] cis methyldioxolane

[³H]Quinuclydinyl benzylate([³H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with K_D 0.25 nM, Hill number 0.89 and B_max 114 pmol/g protein. The local anesthetics procaine, tetracaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 μM, inhibited [³H]QNB binding with K_i varying between 9 μM for procaine and 80 μM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [³H]QNB binding sites. Neither 1 mM Ca²⁺ nor Mg²⁺ affected [³H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [³H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action of local anesthetics on synaptic transmission is discussed.     

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     

Cannabinoid CB1 receptor-mediated inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain

Guinea-pig hippocampal slices preincubated with [³H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [³H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB₁ receptors was determined in rat brain cortex membranes using [³H]SR 141716. In guinea-pig hippocampal slices preincubated with [³H]noradrenaline, tritium overflow stimulated by NMDA 100 μM and 1000 μM and by kainate 1000 μM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB₁ receptor antagonist SR 141716 increased the NMDA (1000 μM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 μM or kainate 1000 μM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 μM)- and kainate (1000 μM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [³H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 μM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [ ³ H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pK _i 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pK _i < 5). In conclusion, activation of CB₁ receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB₁ receptors or that these receptors are activated by endogenous cannabinoids. Received: 25 February 1999 / Accepted: 12 April 1999