Quinuclidinyl Benzilate Research Articles

Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies.

In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [(3)H]quinuclidinyl benzilate ([(3)H]QNB) demonstrated binding sites of high affinity (K(d)=0.45 nM) and low capacity ( approximately 8000 sites/cell). Pharmacological characterisation of muscarinic receptors was assessed by evaluating the effects of the M(3)>M(1)>>M(2) antagonist 4-DAMP (4-diphenylacetyl-N-methylpiperidine) and the M(1)=M(4)> M(3)>>M(2) antagonist pirenzepine on the binding of [(3)H]QNB and carbachol-induced cortisol production. For both parameters, the potency of 4-DAMP was about two orders of magnitude higher than that of pirenzepine. Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M(2), M(3) and M(4) receptors revealed the expression of only M(3) mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M(3) receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells.

Autoradiographic demonstration of quinuclidinyl benzilate binding sites in the vestibular organs of the gerbil

Gerbil vestibular tissues were isolated by microdissection and incubated in vitro with 3 H -quinuclidinyl benzilate ( 3 H -QNB). Control tissues were incubated in medium containing unlabeled atropine to differentiate non-specific from specific binding. Autoradiographic grain densities were determined by morphometric techniques and evaluated by two-tailed t-test. The label densities of sensory epithelia from experimental preparations of ampulla, utricle and saccule were found to be significantly higher than those in the adjacent endolymphatic compartment and also higher than those of adjacent stromal tissue comprising connective tissue, nerve fibers and capillaries. In contrast, no tissue region in atropine controls showed label density significantly above that of the endolymphatic compartment. Label density of ampullar sensory epithelium incubated with 3 H -QNB alone was significantly higher than that of sensory epithelium from utricle or saccule. Grain density was greater in the peripheral regions of the ampullar crista compared to the vertex. Appreciable label was also present in nerve bundles beneath the sensory epithelium of the ampulla. The current study demonstrates the existence of putative muscarinic neurotransmitter/neuromodulator receptor sites in mammalian vestibular sense organs at locations corresponding to efferent innervation, with particularly significant concentrations in the ampulla.

Depolarization Affects the Binding Properties of Muscarinic Acetylcholine Receptors and Their Interaction with Proteins of the Exocytic Apparatus

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca(2+) channels open, level of intracellular Ca(2+) increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K(d) of approximately 20 nM) and they shift to a low affinity state (K(d) of tens of microM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca(2+) influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca(2+) influx, enables release of ACh to commence.

Site-directed mutagenesis of the putative human muscarinic M 2 receptor binding site

Experimental probing of the model of the muscarinic M 2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8–15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [ 3 H ] N-methylscopolamine and [ 3 H ] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [ 3 H ] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [ 3 H ] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (<10-fold) to the binding site of the M 2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M 2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M 2 receptor, as predicted by the proposed model.

Changes in binding to muscarinic and nicotinic cholinergic receptors in the chick telencephalon, following passive avoidance learning

Changes in nicotinic and muscarinic cholinergic receptors 30 min after one-trial passive avoidance training were studied in day-old chicks ( Gallus domesticus), by quantitative receptor autoradiography. [ 3H]-α-bungarotoxin (α-BgT) and [ 3H]-quinuclidinyl benzilate (QNB) were used to monitor changes in 15 forebrain regions for nicotinic and muscarinic receptors, respectively. A significant increase occurred bilaterally in the quantity of bound α-BgT in the lobus parolfactorius. while the amount of bound QNB decreased significantly, and bilaterally, in the hippocampus, hyperstriatum ventrale, lobus parolfactorius and posterolateral telencephalon, pars dorsalis. The data support an involvement of cholinergic receptor types in the neural mechanisms underlying passive avoidance learning.

Comparative Neurochemical Effects of Repeated Methyl Parathion or Chlorpyrifos Exposures in Neonatal and Adult Rats

Several studies have reported higher sensitivity based on lethality in young animals compared to adults following acute exposure to organophosphorus insecticides (OPs). We propose that age-related differences in sensitivity to OPs may differ qualitatively and quantitatively with different OPs and varying exposure conditions (e.g., high vs. low dose, acute vs. repeated). To test this hypothesis, we treated neonatal (7 days of age) and adult (90 days of age) rats with either methyl parathion (MPS) or chlorpyrifos (CPF) daily for 14 days and measured neurochemical endpoints {cholinesterase (ChE) inhibition, total muscarinic receptor ([3H]quinuclidinyl benzilate, QNB) and muscarinic M2 subtype-preferential ([3H]AF-DX 384) binding} in frontal cortex and striatum at timepoints both during (1 day after the 7th and 14th dose) and after (8 days after the 14th dose) exposures. Repeated CPF exposures were associated with relatively similar degrees of ChE inhibition between the age groups during dosing but more extensive inhibition was noted in adults after termination of exposures. Relatively similar changes in muscarinic receptor binding were also noted between age groups following CPF exposures. Moreover, the degree of muscarinic receptor binding reduction relative to ChE inhibition appeared similar in both age groups following CPF exposures. In contrast, ChE activity and muscarinic receptor binding were generally more reduced in neonatal relative to adult brain regions following repeated MPS exposures. Furthermore, the relationship between the degree of ChE inhibition and the reduction in cortical muscarinic receptor binding appeared different between the age groups, i.e., more extensive reduction was noted in neonates compared to adults with a given level of ChE inhibition. We conclude that OP-selective differences in in vivo ChE sensitivity, differential rates of enzyme recovery following inhibition, and age-dependent differences in muscarinic receptor adaptations can all influence the nature of age-related susceptibility to OPs.

Amnesic effects of the anticholinergic drugs, trihexyphenidyl and biperiden: differences in binding properties to the brain muscarinic receptor

An amnesic effect of anticholinergic drugs was previously described from several behavioral studies. We examined this effect induced by trihexyphenidyl and biperiden, clinically used in the parkinsonism and schizophrenic patients, by using passive avoidance tasks. Both of these drugs (0.1–10 mg/kg, s.c.) showed dose-dependent amnesic effects in the acquisition and retrieval phases. However, the effect induced by trihexyphenidyl was transient, whereas that of biperiden was long-lasting. To clarify the reason for the different duration of the amnesic activity, binding to the muscarinic receptor was examined. In the Scatchard analysis, trihexyphenidyl competed with [ 3 H ]quinuclidinyl benzilate ([ 3 H ]QNB) on the muscarinic receptor (showed increased K d and unchanged B max value), while biperiden decreased [ 3 H ]QNB binding ( B max value) significantly. Furthermore, in an exchange assay for receptor inactivation, trihexyphenidyl binding to muscarinic receptors was exchanged by [ 3 H ]QNB completely, but biperiden decreased the exchangeable binding of [ 3 H ]QNB in a dose dependent manner (0.1–100 nM). These results suggested that the binding of trihexyphenidyl and biperiden to muscarinic receptor might be completely reversible and partially irreversible, respectively, whereas the K i values of these two drugs were similar. In conclusion, this difference in binding property may explain the difference in the time-course of the amnesic effect induced by trihexyphenidyl and biperiden.

Expression of muscarinic m2 receptor mRNA in dorsal root ganglia of neonatal rat

The expression of mRNA coding for m2 subtype of muscarinic cholinergic receptors was assessed in dorsal root ganglia (DRG) of 15-day post-natal rats. Northern blot analysis on total RNA using a mixture of two different oligonucleotide probes, indicated the presence of a single prominent band of approximately 6.5 kb in rat DRG; a band of the same size was observed both in brainstem and cortex taken as positive controls. Analysis by RT-PCR of the mRNA coding for a region of the third cytoplasmic loop of m2 receptor showed a single signal both in rat DRG and hippocampus. In situ hybridization was then used to identify the neuronal subpopulations expressing the mRNA for M 2. The transcripts were preferentially localized in medium–small neurons of the ganglion as well as in satellite cells surrounding the neuron cell body. Large neurons were usually negative. Finally, competition binding experiments, performed in the presence of [ 3 H ]-quinuclidinyl benzilate (QNB) and methoctramine (a selective competitor for M 2 receptors), demonstrated the presence of M 2 receptor protein ( K i=100 nM), as previously observed in chick DRG. The preferential localization of M 2 in medium–small neurons of the ganglion suggests the involvement of this receptor subtype in the transduction of nociceptive stimuli.

Muscarinic M 1 and M 3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) and [ 3H]diphenyl-acetoxy-N-methyl-piperidine ([ 3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [ 3H]QNB and [ 3H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M 1, M 3 subtypes selective antagonist) > atropine (general) > pirenzepine (M 1). Methoctramine (M 2) didn’t antagonise the [ 3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca 2+ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M 1 and M 3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.

Characterization of the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([ 3 H ]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (−logEC 50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-( m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with p A 2 values in parentheses): atropine (8.95), 4-diphenylacetoxy- N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6 H-pyrido[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (−logIC 50) was 4-DAMP (8.35)>tropicamide (6.72)>himbacine (6.54)>pirenzepine (6.31)>AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M 2 and/or M 4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [ 3 H ]quinuclidinyl benzilate binding site ( K d=0.92 nM; B max=126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with p K i value and Hill coefficient in parentheses): atropine (8.22 and 0.93)>4-DAMP (8.18 and 0.94)>tropicamide (6.78 and 0.93)>pirenzepine (5.46 and 0.92)>AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M 3 receptors present on smooth muscle cells.

Long-term changes in brain following continuous phencyclidine administration: an autoradiographic study using flunitrazepam, ketanserin, mazindol, quinuclidinyl benzilate, piperidyl-3,4-3H(N)-TCP, and AMPA receptor ligands.

Phencyclidine induces a model psychosis which can persist for prolonged periods and presents a strong drug model of schizophrenia. When given continuously for several days to rats, phencyclidine and other N-methyl-D-aspartate (NMDA) antagonists induce neural degeneration in a variety of limbic structures, including retrosplenial cortex, hippocampus, septohippocampal projections, and piriform cortex. In an attempt to further clarify the mechanisms underlying these degeneration patterns, autoradiographic studies using a variety of receptor ligands were conducted in animals 21 days after an identical dosage of the continuous phencyclidine administration employed in the previous degeneration studies. The results indicated enduring alterations in a number of receptors: these included decreased piperidyl-3,4-3H(N)-TCP (TCP), flunitrazepam, and mazindol binding in many of the limbic regions in which degeneration has been reported previously. Quinuclidinyl benzilate and (AMPA) binding were decreased in anterior cingulate and piriform cortex, and in accumbens and striatum. Piperidyl-3,4-3H(N)-TCP binding was decreased in most hippocampal regions. Many of these long-term alterations would not have been predicted by prior studies of the neurotoxic effects of continuous phencyclidine, and these results do not suggest a unitary source for the neurotoxicity. Whereas retrosplenial cortex, the structure which degenerates earliest, showed minimal alterations, some of the most consistent, long term alterations were in structures which evidence no immediate signs of neural degeneration, such as anterior cingulate cortex and caudate nucleus. In these structures, some of the receptor changes appeared to develop gradually (they were not present immediately after cessation of drug administration), and thus were perhaps due to changed input from regions evidencing neurotoxicity. Some of these findings, particularly in anterior cingulate, may have implications for models of schizophrenia.

Anandamides Inhibit Binding to the Muscarinic Acetylcholine Receptor

Loss of memory and cholinergic transmission are associated with both Alzheimer's disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [3H]quinuclidinyl benzilate ([3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 microM). Binding of the more polar antagonist [N-methyl-3H]scopolamine ([3H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 microM). R-methanandamide inhibits more than 90% of both [3H]QNB binding (I50 = 34 microM) and [3H]NMS binding (I50 = 15 microM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [3H]oxotremorine-M at low concentrations (25-75 microM), but significantly inhibit agonist binding at higher concentrations (I50 = 150 microM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [3H]NMS binding to the mAChR, even at concentrations as high as 1 microM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function.

Depletion of dietary n-3 fatty acid affects the level of cyclic AMP in rat hippocampus.

Prolonged depletion of dietary n-3 fatty acid induces a neurological disturbance. To ascertain the deficit of neurotransmission at the time of n-3 deficiency, the concentrations of cAMP and inositol triphosphate, and the activities of protein kinases A and C were examined in vitro in rat hippocampus. Furthermore, the saturation binding study of [3H]quinuclidinyl benzilate, a specific antagonist to muscarinic cholinergic receptor, was performed. Rats were fed a safflower oil diet as the deficient group and a soybean oil diet as the control group. Hippocampi were obtained from rats in the 3rd generation in the deficient group and in the 2nd generation in the control group. Dietary effect was not observed in the parameters except for the concentration of cAMP, which was significantly higher in the deficient group than in the control group.

Brain soluble fractions which modulate Na+, K+-ATPase activity likewise modify muscarinic receptor.

Two brain soluble fractions, named peaks I and II, which respectively stimulate and inhibit neuronal Na+, K+-ATPase activity, have been isolated by gel filtration in Sephadex G-50. Since cholinergic transmission seems related to such enzyme activity, in this study we evaluated the effect of brain peak I, peak II, a more purified fraction II-E and commercial ouabain, on specific binding of the muscarinic antagonist [3H]quinuclidinyl benzilate to membranes from rat cerebellum, hippocampus and cerebral cortex. We found that binding was increased by peak I and decreased by peak II, II-E and ouabain, all effects proving concentration-dependent. Since the changes exerted on the muscarinic receptor followed a pattern similar to the one already described for synaptosomal membrane Na+, K+-ATPase activity, both systems seem to interact at a functional level.

Ionizing radiation stimulates muscarinic regulation of rat intestinal mucosal function.

The aim of this study was to determine whether ionizing radiation modifies muscarinic regulation of intestinal mucosal function. Rats exposed to total body 8-Gy gamma-irradiation or sham irradiated were studied up to 21 days after irradiation. Basal and carbachol-stimulated short-circuit current (Isc) and transepithelial conductance (Gt) of stripped ileum were determined in Ussing chambers. Muscarinic receptor characteristics using the muscarinic antagonist [3H]quinuclidinyl benzilate and three unlabeled antagonists were measured in small intestinal plasma membranes together with two marker enzyme activities (sucrase, Na+-K+-ATPase). Enzyme activities were decreased 4 days after irradiation (day 4). Basal electrical parameters were unchanged. Maximal carbachol-induced changes in Isc and Gt were increased at day 4 (maximal DeltaIsc = 195.8 +/- 14.7 microA/cm2, n = 19, vs. 115.4 +/- 8.2 microA/cm2, n = 63, for control rats) and unchanged at day 7. Dissociation constant was decreased at day 4 (0.73 +/- 0.29 nM, n = 10, vs. 2.14 +/- 0.39 nM, n = 13, for control rats) but unchanged at day 7, without change in binding site number. Thus total body irradiation induces a temporary stimulation of cholinergic regulation of mucosal intestinal function that may result in radiation-induced diarrhea.

Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors. Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes. Carbachol and xanomeline stimulated basal [35S]-GTPgammaS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPgammaS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 microM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors. In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5-HT2A, h5-HT2B and h5-HT2c receptors with potencies similar to its affinity at these receptors. These studies indicate that xanomeline is a potent agonist at 5-HT1A and 5-HT1B receptors and an antagonist at 5-HT2 receptor subtypes.

Effect of NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, on neurotransmitter receptor systems in aged rats.

In order to examine the effect of age and nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), we studied the changes on major neurotransmitter receptor systems in 6 (adult) and 24-month-old (aged) Fischer male rats using receptor autoradiography. L-NAME was administrated intraperitoneally in aged rats once a day for 4 weeks. [3H]QNB (quinuclidinyl benzilate), [3H]HC (hemicholinium-3), [3H]muscimol, [3H]SCH 23390 ([N-methyl-3H]R[+]-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-7-ol-benzazepine), [3H]nemonapride and [3H]mazindol were used as markers of muscarinic acetylcholine receptors, high-affinity choline uptake sites, GABAA (gamma-aminobutyric acidA) receptors, dopamine D1 receptors, dopamine D2 receptors and dopamine uptake sites, respectively. The age-related change in [3H]muscimol binding in the brain was more pronounced than that in [3H]QNB, [3H]HC, [3H]SCH 23390, [3H]nemonapride and [3H]mazindol binding. Chronic treatment (4 weeks) with L-NAME caused no significant changes in [3H]QNB, [3H]muscimol, [3H]SCH 23390 and [3H]nemonapride binding in most areas of aged rat brain, as compared with vehicle-treated aged animals. However, chronic treatment with L-NAME caused a significant reduction in [3H]HC and [3H]mazindol binding in any brain regions of aged rats in comparison with the vehicle-treated aged animals. These results demonstrate that the GABAergic system is more susceptible to aging processes than cholinergic and dopaminergic systems in the brain. Furthermore, our findings suggest that nitric oxide may play some role in the regulation of choline uptake and dopamine uptake systems during aging processes. Copyright 1998 Lippincott Williams & Wilkins

NMR spectral analysis and molecular dynamics study of 3-quinuclidinol and 3-quinuclidinyl benzilate

Proton chemical shifts and coupling constants of 3-quinuclidinol and 3-quinuclidinyl benzilate were determined by computer analysis. The relatively broad lines of the quinuclidine protons have their origin in numerous non-resolved transitions arising from the extensively coupled spin system of 12 nuclei. The signs of the long-range coupling constants were determined by COSY-45 and E.COSY. Vicinal coupling constants were converted into proton–proton torsion angles by applying the Altona–Haasnoot equation. Some geometrical ambiguities were revealed, which were studied by molecular dynamics calculations and simulated annealing. The torsion angles and the results from molecular dynamics calculations indicated significant flexibility of the quinuclidine part. For the sake of completeness the carbon chemical shifts of both compounds are presented. © John Wiley & Sons, Ltd.

Use of a spreadsheet to quantitate the equilibrium binding of an allosteric modulator

Using the program Microsoft excel, a spreadsheet was developed for constrained, simultaneous analysis of multiple datasets obtained from equilibrium binding experiments, according to an allosteric model of interaction. This approach was used to quantitate the interaction between the modulator (heptane-1,7-bis (dimethyl 3′-phthalimidopropyl) ammonium bromide) (C 7/3-phth) and the radioligands [ 3 H]N- methylscopolamine and [ 3 H] quinuclidinyl benzilate at cortical and atrial muscarinic receptors. The interaction between various concentrations of the radioligands and C 7/3-phth, in the guinea pig atrium and in the rat cerebral cortex, could be well described by the allosteric model. The affinity of C 7/3-phth for unoccupied atrial receptors was significantly higher than for cortical receptors. The negative cooperativity between [ 3 H] quinuclidinyl benzilate and the modulator was higher in cortex than that between the modulator and [ 3 H]N- methylscopolamine . It is suggested that the described method has wide applicability because of the extensive availability of spreadsheet programs, the analytical advantages offered by constrained, simultaneous nonlinear regression and the ability to adapt the spreadsheet to almost any model of ligand–receptor interaction.

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1. Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [3H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2. [3H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (Kd) values of [3H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (Bmax) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3. The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4. This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.