L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic false transmitters: physiological and biochemical actions in central and peripheral systems

Three N-ethyl substituted analogs of acetylcholine (ACh) were evaluated for potential use as false neurotransmitters to decrease cholinergic transmission. This evaluation included (1) the elevation of arterial blood pressure upon central administration, (2) depression of blood pressure upon intravenous injection and (3) interactions with central muscarinic and peripheral nicotinic receptors. With respect to the central pressor response, ACh, acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) were full agonists of decreasing potency; acetyltriethylcholine (ATECh) was a partial agonist of considerably lower potency. The duration of response paralleled potency. With respect to the peripheral depressor response, ACh and AMECh were full agonists of equal potency, and ADECh and ATECh were partial agonists of at least 100-fold lower potency. In terms of their affinity for central muscarinic receptors (brainstem and cerebral cortex), the following series was obtained: ACh greater than AMECh much greater than ADECh = ATECh. All of the agents had a greater affinity for muscarinic receptors in the brainstem compared to cortex. Acetylcholine and AMECh recognized multiple receptor binding conformations; the binding of ADECh and ATECh indicated interaction with a single set of equivalent sites. The affinity for nicotinic ACh receptors from the Torpedo electric organ was ACh = AMECh much greater than ADECh; ATECh had little affinity for these receptors. Acetylcholine, AMECh and ADECh stimulated the binding of [3H]phencyclidine to the ion channel of nicotinic receptor (potency series = ACh greater than AMECh = ADECh); ATECh was inactive. Acetylcholine, AMECh and ADECh also induced receptor conversion to a desensitized conformation; ATECh did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

[3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [3H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [3H]-(+/-)-L-364,718 correlated with their ability to displace [125I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [125I]CCK-8 than [3H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [3H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl2. [3H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [3H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [3H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions.     

On the involvement of multiple muscarinic receptor subtypes in the activation of phosphoinositide metabolism in rat cerebral cortex

We have examined the activation of phosphoinositide metabolism by muscarinic agonists in rat cerebral cortex, in an attempt to delineate the mechanisms by means of which some selective antagonists inhibit this response in a manner that deviates from simple mass action law. The accumulation of [3H]inositol phosphates induced by the full agonist carbamylcholine in cell aggregates preparations was inhibited by muscarinic antagonists with the following order of potency: telenzepine greater than atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than pirenzepine greater than hexahydro-sila-difenidol greater than AF-DX 116. The same order of potency was found for the competition of these antagonists with [3H]telenzepine binding to M1 muscarinic receptors. The inhibition of the formation of [3H]inositol phosphates activated by acetylcholine, carbamylcholine, and oxotremorine-M by pirenzepine and telenzepine showed biphasic curves, with 62-73% of the response being inhibited with high affinity. Atropine, AF-DX 116, and pirenzepine shifted the concentration-response curves of oxotremorine-M to the right in a parallel manner. However, pirenzepine at micromolar concentrations showed deviation from linearity of the Schild regression. The blockade by high concentrations of pirenzepine and telenzepine showed less than additive dose ratios when assayed in the presence of atropine, suggesting deviation of their antagonism from simple competition. However, after alkylation with propylbenzilylcholine mustard in the presence of low concentrations of pirenzepine, the response to carbamylcholine and oxotremorine-M showed monophasic inhibition curves by pirenzepine and linear Schild regression for this antagonist. These results support the interpretation that the formation of [3H]inositol phosphates is activated by multiple muscarinic receptor subtypes in rat cerebral cortex. The profile of affinities of muscarinic antagonists indicates that a major component of the response is activated by an M1 receptor subtype and a minor component is probably mediated by M3 muscarinic receptors when acetylcholine, carbamylcholine, or oxotremorine-M are used to stimulate the response. Conversely, pirenzepine inhibited the response induced by methacholine and bethanechol in a monophasic manner with high affinity (Ki = 13 nM), suggesting that these agonists can selectively stimulate phosphoinositide metabolism through activation of M1 muscarinic receptors in rat cerebral cortex.     

In vivo characterisation of novel efficacious muscarinic receptor agonists

Although a number of muscarinic agonists have been used in clinical trials for Alzheimer's Disease, many of these compounds are low in potency and have only limited intrinsic efficacy. The present study describes four non-quaternary oxadiazole based muscarinic agonists from a quinuclidine and a 1-azanorbornane series. These displayed up to 1000 fold higher affinity than arecoline and were efficacious muscarinic agonists at cortical receptors. All four compounds produced peripherally mediated salivation and centrally mediated hypothermia at doses 50-50,000 fold lower than arecoline. The most potent was L-670,548, the methyl oxadiazole in the 1-azanorbornane series, which had an ED50 of 0.0016 mg/kg on the hypothermia model. This derivative was also the most potent compound in ex vivo binding studies (ED50 0.0069 mg/kg) and showed excellent brain penetration (3.8% of the administered dose). These derivatives are the first non quaternary efficacious agonists which show good penetration into the CNS (central nervous system), and will prove useful tools in understanding the role of muscarinic receptors in CNS function.     

Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo-M ratio as an index of efficacy at cortical muscarinic receptors.

1. Radioligand binding assays using [3H]-N-methylscopolamine (NMS) and [3H]-oxotremorine M (Oxo-M) have been devised to predict the efficacy of test compounds at muscarinic receptors in rat cerebral cortex. 2. Muscarinic antagonists, including non-selective and both M1- and M2-selective compounds, displayed similar affinity for both binding assays. 3. Full agonists such as carbachol and muscarine possessed a ratio of potencies against the antagonist versus the agonist ligand (NMS/Oxo-M ratio) of greater than 4000. 4. Compounds which have been shown previously to display partial agonist activity in functional assays e.g. pilocarpine and RS86 had intermediate NMS/Oxo-M ratios of 100-150. A second group of compounds which included oxotremorine had somewhat higher ratios (500-1400). 5. The ratio of affinity constants for the two assays predicted the ability of agonists to stimulate cortical phosphatidyl-inositol turnover. 6. These results suggest that the NMS/Oxo-M ratio may be a useful prediction of efficacy for novel compounds acting at cortical muscarinic receptors.     

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。     

Muscarinic receptor heterogeneity in rat central nervous system. II. Brain receptors labeled by [3H]oxotremorine-M correspond to heterogeneous M2 receptors with very high affinity for agonists

We compared the binding characteristics of muscarinic receptors labeled by [3H]oxotremorine-M ([3H]oxo-M) in homogenates of brain cortex and heart from rat. In both tissues [3H]oxo-M bound, with the same KD (6.5 nM), to a fraction of the receptors labeled by [3H]-N-methylscopolamine ([3H]NMS). This [3H]oxo-M receptor population represented, respectively, 15-20% and 35-40% of the total number of [3H]NMS receptors in cortex and heart. The three unlabeled agonists oxotremorine, carbamylcholine, and pilocarpine, when tested in competition with [3H]oxo-M, displayed a homogeneous super high affinity toward [3H]oxo-M-labeled receptors, and were unable to discriminate between brain and heart receptors labeled by [3H]oxo-M. By contrast, selective muscarinic antagonists showed some selectivity for either brain or heart [3H]oxo-M-labeled receptors. We analyzed competition curves between [3H]oxo-M and secoverine, pirenzepine, AF-DX 116, dicyclomine, or gallamine, assuming the existence of one or two receptor subclasses. Heart muscarinic receptors labeled by [3H]oxo-M were homogeneous M2 receptors of the C type with very low affinity for pirenzepine (Ki = 400 nM). Brain [3H]oxo-M-labeled receptors were heterogeneous receptors, with 30% (the B type) having a higher affinity for dicyclomine and a lower affinity for AF-DX 116 and gallamine than cardiac receptors, whereas the remaining 70% (the C type) showed "cardiac-like" binding properties. Both [3H]oxo-M-labeled subtypes in cortex homogenates had a low affinity for pirenzepine, indicating that [3H]oxo-M labeled only B and C (M2) receptors in this tissue. GTP inhibited completely [3H]oxo-M binding in heart homogenates with an IC50 at 300 nM. In cortex homogenates, GTP showed the same potency, but its efficacy was much lower (with only 30% maximal inhibition). [3H]oxo-M dissociation kinetics were monophasic in heart homogenates and biphasic in cortex homogenates. [3H]oxo-M dissociation from both tissues was slowed by gallamine and d-tubocurarine and accelerated by GTP. We found no correlation between B versus C [3H]oxo-M receptors, GTP-sensitive versus GTP-insensitive receptors, and rapidly versus slowly dissociating receptors, suggesting that [3H] oxo-M labeled a large variety of muscarinic receptor-regulatory protein complexes, all having an SH affinity for agonists.     

Agonist binding to M1 muscarinic receptors is sensitive to guanine nucleotides

Putative M1 (high-affinity pirenzepine) muscarinic receptors in rabbit hippocampal membranes, treated with 0.1 mM N-ethylmaleimide (NEM), were selectively labeled with [3H]pirenzepine. A single class of binding sites was labeled with a Kd of 3.4 nM, consistent with the pharmacologically-defined M1 subtype of muscarinic receptors. While full muscarinic agonists bound to high- and low-affinity states of [3H]pirenzepine-labeled M1 sites with a KL/KH ratio of approximately 100, the ratio for partial muscarinic agonists was approximately 10. The high-affinity binding of all agonists tested required divalent cations, and was interconverted to low-affinity binding in the presence of the non-hydrolyzable GTP analogue, guanylyl imidodiphosphate (GppNHp). Direct labeling of the high-affinity agonist state of M1 receptors was achieved with 5 nM [3H]oxotremorine-M by selectively uncoupling the high-affinity agonist state of M2 (low-affinity pirenzepine) receptors with NEM. The rate of dissociation of [3H]Pxotremorine-M from M1 receptors was accelerated 6-fold by GppNHp. These results provide further evidence which suggests that putative M1 muscarinic receptors activate second messenger systems by coupling to NEM-insensitive guanine nucleotide-binding proteins.     

Autoradiographic analyses of agonist binding to muscarinic receptor subtypes

The binding of four muscarinic receptor agonists to regions of rat brain was examined through quantitative autoradiographic techniques. Oxotremorine, arecoline, pilocarpine and bethanechol were chosen based on their different potencies and efficacies in muscarinic second messenger systems. Overall, the order of potency for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain slices was oxotremorine greater than pilocarpine = arecoline much greater than bethanechol. Regional assays of agonist potency indicated that all agonists were more selective for brainstem and thalamic regions than for hippocampal and cortical regions. The high selectivity of agonists for areas such as the paraventricular thalamus and the superior colliculus, which also display low affinity for pirenzepine, suggests that muscarinic agonists bind with higher affinity to M2 receptors. Of the four agonists examined, pilocarpine displayed the lowest selectivity for M2 receptors in that IC50 values for pilocarpine were only 3-fold higher in the hippocampal and striatal regions (e.g. CA3: 40.6 +/- 9.4 microM) than in thalamic and brainstem regions (e.g. paraventricular thalamus: 14.9 +/- 6.2 microM). Oxotremorine was 8-fold more potent in the brainstem and thalamus, while arecoline and bethanechol were, respectively, 19- and 100-fold more selective for brainstem and thalamic receptors. Scatchard analyses revealed heterogeneous binding profiles for some agonists within single brain regions, suggesting that multiple agonist sites exist even within regions of predominantly M1 or M2 receptors. For example, arecoline displayed curved Scatchard plots within the external layers of the cerebral cortex, layer CA1 of the hippocampus (predominantly M1 subtype), and the paraventricular thalamus (predominantly M2 subtype). The ability of agonists to recognize multiple sites within a single region may reflect the ability to recognize receptors coupled or uncoupled to second messenger systems through G-proteins.     

Kinetic studies of [3H]-N-methylscopolamine binding to muscarinic receptors in the rat central nervous system: evidence for the existence of three classes of binding sites

We compared the binding of [N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) and pirenzepine to muscarinic receptors in four regions of the rat central nervous system (cortex, hippocampus, striatum, and cerebellum) and in rat heart. Equilibrium binding studies suggested the existence of three classes of receptors: A, receptors with high affinity for pirenzepine and [3H] NMS (in cortex, hippocampus, and striatum); B, receptors with intermediate affinity for pirenzepine and high affinity for [3H]NMS (in the same brain regions); and C, receptors with low affinity for pirenzepine and [3H]NMS (in cerebellum and heart). Dissociation kinetic studies indicated that the receptor types A, B, and C had different koff values allowing, therefore, a separate study of their binding properties. We observed that: [3H]NMS recognized muscarinic receptors A, B, and C with the following order of potency: B greater than A much greater than C; and pirenzepine recognized these receptors with a different order of potency: A much greater than B greater than C. Thus, dissociation kinetics provide a useful tool to identify muscarinic receptor types.     

Interaction of deglycosylated muscarinic receptors with ligands and G proteins

Endoglycosidase F was used to investigate the role of the carbohydrate moiety of muscarinic acetylcholine receptors in antagonist and agonist binding, and the interaction with G proteins. The receptors were purified from porcine cerebrum, treated with endoglycosidase F and then covalently labeled with [3H]propylbenzilylcholine mustard [( 3H]PrBCM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]PrBCM-labeled receptors showed that the endoglycosidase F treatment caused a decrease in apparent Mr from 70 to 51 kDa, the Mr predicted for the peptide portions of M1, M2 and M4 subtypes. Endoglycosidase F-treated receptors had essentially the same affinities for both agonists and antagonists as those of control receptors. In addition, treated receptors that had been reconstituted in lipid vesicles with G proteins showed guanine nucleotide-sensitive high affinity for agonists. These results suggest that the carbohydrate moiety of muscarinic acetylcholine receptors is not involved in their interaction with muscarinic ligands and G proteins.     

Characterization of the binding of [3H]L-158,809: a new potent and selective nonpeptide angiotensin II receptor (AT1) antagonist radioligand.

[3H]L-158,809, a new potent and AT1-selective nonpeptide angiotensin II receptor antagonist, bound saturably and reversibly to rat adrenal membranes. Scatchard and Hill plot analyses indicated a single class of high affinity (Kd = 0.66 nM) binding sites. The relative potencies of various angiotensin II-related peptide and nonpeptide antagonists in displacing [3H]L-158,809 binding correlated with their potencies in displacing the binding of 125I-Sar1,Ile8-angiotensin II to adrenal AT1 receptors. [3H]L-158,809 binding to adrenal membranes was not affected by addition of guanosine-5'-(beta,gamma-imido)triphosphate or various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. The potencies of angiotensin II receptor agonists, but not antagonists, in inhibiting specific [3H]L-158,809 binding were decreased in the presence of guanosine-5'-(beta,gamma-imido)triphosphate. Specific [3H]L-158,809 binding was also observed in rat liver and kidney. Collectively, the data indicate that [3H]L-158,809 represents a new, potent, nonpeptide, antagonist radioligand suitable for the study of angiotensin II AT1 receptors.     

Mu opiate receptors are selectively labelled by [3H]carfentanil in human and rat brain

[11C]Carfentanil is a potent opioid agonist currently in use as a specific PET (position emission tomography) scan radioligand for brain mu opioid receptors. In order to investigate the receptor interactions of carfentanil in detail [3H]carfentanil was used as a radioligand for labelling receptors in rat and human brain tissue homogenates. [3H]Carfentanil was found to bind saturably and with high affinity (KD = 0.08 +/- 0.01 nM) to membranes prepared from human cortical (Bmax = 42 +/- 3 fmol/mg) and thalamic (Bmax = 84 +/- 3 fmol/mg) tissues and rat cortex (Bmax = 82 +/- 4 fmol/mg) and diencephalon (Bmax = 105 +/- 5 fmol/mg). Association (1.23 +/- 0.19 X 10(10) Mol-1 X min-1 and dissociation rate (0.19 +/- 0.03 min-1) constants were determined in human cortical tissues; results from studies in rat cortical, and rat diencephalon tissue homogenates produced similar kinetic rate constants. Competition studies with a variety of drugs indicated that [3H]carfentanil interacts primarily with mu opioid receptors in the four tissues studied; the affinities of a series of non-radioactive opioid ligands were essentially identical in the four tissues (correlation coefficients = 0.88-0.93). Naloxone, morphine, DAGO [( D-Ala2-MePhe4-Gly-ol5]enkephalin), DADL [( D-Ala2-D-Leu5]enkephalin) and EKC (ehtylketazocine) potently displaced specific [3H]carfentanil binding with nM potency while the kappa agonist U-69593, the sigma agonists (+)-SKF 10047, (+)-3-PPP [3-hydroxyphenyl)-N-propylpiperidine) and haloperidol and PCP (phencyclidine) were less potent displacing agents. The higher affinities of DAGO and morphine versus DADL for the [3H]carfentanil binding sites indicates that delta opioid receptors are not being labelled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from brainstem of rat

The influence of halothane on muscarinic receptors with a high affinity for agonists was studied using [3H]oxotremorine-M. [3H]Oxotremorine-M bound with high affinity (KD = 2.8 nM) to a subpopulation of muscarinic receptors in the brainstem of rat, representing 32% of the total receptor pool. Agonist affinity for binding sites for [3H]oxotremorine-M was not affected by a guanine nucleotide (5'-guanylylimidodidiphosphate; Gpp(NH)p), although the level of binding was decreased, presumably due to the conversion of receptors to lower affinity conformations. However, only 58% of 3 nM binding of [3H]oxotremorine-M was sensitive to Gpp(NH)p. Halothane had two effects on the binding of [3H]oxotremorine-M: halothane (1) decreased the level of binding of [3H]oxotremorine-M without affecting agonist affinity for the surviving sites, and (2) lowered the sensitivity of the binding of [3H]oxotremorine-M to Gpp(NH)p by a factor of 120. The decrease in binding of [3H]oxotremorine-M binding was nonselective with regard to the sensitivity of the receptors to the guanine nucleotide, insofar as Gpp(NH)p inhibited the binding of [3H]oxotremorine-M to the same extent in the presence and absence of halothane. These results suggest that halothane (1) converts both G protein-coupled and -uncoupled muscarinic receptors to states of lower agonist affinity and (2) lowers the affinity of receptor-G protein complexes for guanine nucleotides.     

Medium supplementation with zinc enables detection of imipramine-induced adaptation in glycine/NMDA receptors labeled with [3H]L-689,560

Antidepressant drugs after chronic administration induce adaptive changes in the NMDA receptor complex. Radioligand-receptorbinding studies using [3H]5,7-dichlorokynurenic acid demonstrated a "down-regulation" of the glycine site/NMDA receptor following chronic treatment with antidepressants and electroconvulsive shock. However, binding procedure using this radioligand is time consuming because it requires the use of centrifugation method in the separation process. The introduction of a new radioligand of glycine/NMDA receptor, [3H]L-689,560 enables the application of a rapid filtration method. In the present study we demonstrate that 2-week treatment with imipramine (15 mg/kg ip) did not evoke alterations in specific [3H]L-689,560 binding and in IC50 value of glycine in displacing [3H]L-689,560 binding in the mouse or rat cortex. However, longer, a 4-week treatment with imipramine induced a significant 71% increase in IC50 value in displacing [3H]L-689.560 binding in the mouse cortex. Moreover, the presence of zinc in the incubation media, dose-dependently enhances detection of imipramine-induced increase in IC50 value of glycine in displacing [3H]L-689,560 binding in the rat cortex. The present data indicate that: (1) [3H]L-689,560 may be a suitable ligand for assessing adaptation of the glycine/NMDA sites and (2) the presence of zinc enhances detection of imipramine-induced reduction of glycine affinity for glycine/NMDA receptors labeled with [3H]L-689,560 which further indicates a significance of zinc in the mechanism of antidepressant treatment.     

Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [3H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC50 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [3H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (Ki 85 nM) for [3H]-oxotremorine-M-labelled muscarinic receptors in rat brain.     

Novel alkoxy-oxazolyl-tetrahydropyridine muscarinic cholinergic receptor antagonists

The purpose of the present studies was to compare a novel series of alkoxy-oxazolyl-tetrahydropyridines (A-OXTPs) as muscarinic receptor antagonists. The affinity of these compounds for muscarinic receptors was determined by inhibition of [³H]pirenzepine to M1 receptors in hippocampus, [³H]QNB to M2 receptors in brainstem, and [³H]oxotremorine-M to high affinity muscarinic agonist binding sites in cortex. All of the compounds had higher affinity for [³H]pirenzepine than for [³H]QNB or [³H]oxotremorine-M labeled receptors, consistent with an interpretation that they are relatively selective M1 receptor antagonists, although none were as selective as pirenzepine. In addition, dose-response curves were determined for antagonism of oxotremorine-induced salivation (mediated by M3 receptors) and tremor (mediated by non-M1 receptors) in mice. In general, the A-OXTPs were equipotent and equieffective in antagonizing both salivation and tremor, although there were modest differences for some compounds. Dose-response curves also were determined on behavior maintained under a spatial-alternation schedule of food presentation in rats as a measure of effects on working memory. The A-OXTPs produced dose-related decreases in percent correct responding at doses three- to ten-fold lower than those which decreased rates of responding. However, only one compound, MB-OXTP, produced effects on percent correct responding consistent with a selective effect on memory as opposed to non-memory variables. The present results provide evidence that these alkoxy-oxazolyltetrahydropyridines are a novel series of modestly M1-selective muscarinic receptor antagonists, and that one member of the series, MB-OXTP, appears to be more selective in its effects on memory than previously studied muscarinic antagonists.     

N(alpha)-imidazolylalkyl and pyridylalkyl derivatives of histaprodifen: synthesis and in vitro evaluation of highly potent histamine H(1)-receptor agonists

A novel series of N(alpha)()-imidazolylalkyl and pyridylalkyl derivatives of histaprodifen (6, 2-[2-(3,3-diphenylpropyl)imidazol-4-yl]ethanamine) was synthesized and evaluated as histamine H(1)-receptor agonists. The title compounds displayed partial agonism at contractile H(1)-receptors of guinea pig ileum and were at least equipotent with histamine. Agonist effects of the new derivatives were susceptible to blockade by the H(1)-receptor antagonist mepyramine (2-100 nM). In the imidazole series, suprahistaprodifen (51, [2-[2-(3,3-diphenylpropyl)-1H-imidazol-4-yl]ethyl]-[2-(1H-imidazol-4-yl)ethyl]amine, N(alpha)-2-[(1H-imidazol-4-yl)ethyl]histaprodifen) showed the highest H(1)-receptor agonist potency ever reported in the literature (pEC(50) 8.26, efficacy E(max) 96%). Elongation of the alkyl spacer from ethyl to butyl decreased activity from 3630% (ethyl, 51) to 163% (butyl, 53) of histamine potency. The exchange of the terminal imidazole nucleus for a pyridine ring resulted in compounds with comparably high potency. A decrease in agonist potency and efficacy was observed when the attachment of the alkyl spacer was consecutively changed from the ortho to the meta and the para position, respectively, of the pyridine ring. The pyridine series that contained a butyl chain possessed the highest potency and affinity. N(alpha)-[4-(2-pyridyl)butyl]histaprodifen (56) emerged as a strong partial agonist, being almost equipotent with 51 (pEC(50) 8.16, E(max) 89%). Compounds 51 and 56 also showed potent partial agonism at contractile H(1) receptors in guinea pig aorta and potently activated H(1)-receptor-mediated endothelium-dependent relaxation in the rat aorta. Compounds 51-65 displayed low to moderate affinity at H(2), H(3), and M(3) receptors in functional models of guinea pig. Collectively, N(alpha)-imidazolylalkyl- and N(alpha)-pyridylalkyl-substituted histaprodifens represent a novel class of potent H(1)-receptor agonists. These compounds may be useful to define the (patho)physiological role of the H(1)-receptor and refine molecular models of H(1)-receptor activation.     

Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.