Effects of an irreversible muscarinic agonist (BM123) on avoidance and spontaneous alternation performance

The present study sought to assess whether the compound N-[4-(2-chloro-ethylmethylamine)-2-butynyl]-2-pyrrolidone (BM123), a potent muscarinic agonist that binds irreversibly to the muscarinic receptor (mAChR), has long-lasting functional effects which may be related to a reduction in functional mAChRs. Passive (inhibitory) avoidance performance, one-way active avoidance learning, and spontaneous alternation behavior were studied in rats. The results confirmed the acute muscarinic stimulating effects of BM123, including tremor, salivation, chromodacryorrhea and hypothermia. In addition, when measured 3-4 days after administration, rats treated with BM123 had disrupted spontaneous alternation performance and tended to have impaired performance for the inhibitory avoidance task with facilitated acquisition of active avoidance. This spectrum of effects is consistent with previous reports showing a 20-40% reduction in mAChRs at these times after BM123. The reversible muscarinic agonist, oxotremorine, was without significant effect. In a further experiment, it was found that pretreatment with methyl atropine did not prevent the disruption of spontaneous alternation behavior by BM123, whereas pretreatment with atropine did. Thus, these long-lasting behavioral effects of BM123 are related to its alkylation of and subsequent reduction in central mAChRs.     

中枢N受体对M受体介导体温降低作用的调节

在清醒大鼠上，可进中枢的Ｎ受体拮抗剂美加明可对抗Ｎ受体激动剂烟碱降低体温的作用，增强Ｍ受体激动剂氧化震颤素降低体温的作用；不进中枢的外周Ｎ受体拮抗剂六甲溴铵不影响烟碱和氧化震颤素的上述作用．每天３次ｓｃ烟碱２．５ｍｇ·ｋｇ－１，连续７ｄ，大鼠对烟碱降低体温的作用产生慢性耐受后，氧化震颤素降低体温的作用无显著变化．提示以体温变化为指标，美加明封闭中枢Ｎ受体功能后，中枢Ｍ受体对其激动剂的敏感性增强；反复给予烟碱诱导中枢Ｎ受体功能失敏后，中枢Ｍ受体对其激动剂的敏感性无显著变化．     

Activity of N-methyl-alpha- and -beta-funaltrexamine at opioid receptors

The N-methyl analogues (2a, 2b) of the nonequilibrium mu opioid receptor antagonist beta-funaltrexamine (1b) were synthesized and evaluated in the guinea pig ileum preparation (GPI). These analogues are highly potent, reversible opioid agonists and possess no nonequilibrium antagonist activity. The ineffectiveness of 2b in protecting against irreversible blockage of mu opioid receptors by 1b and the fivefold lower reactivity of 2b with cysteine suggest that N-methyl substitution adversely affects both the first and second recognition steps that are essential for effective covalent blockage of opioid receptors.     

Neuronal muscarinic receptors attenuate vagally-induced contraction of feline bronchial smooth muscle.

In anaesthetized cats, stimulation of the vagus nerves produced bradycardia and a bronchoconstriction which was measured as an increase in lung resistance (RL) and a fall in dynamic lung compliance (Cdyn); these effects were abolished by atropine. Gallamine potentiated vagally-mediated changes in RL and Cdyn at doses that blocked muscarinic receptors in the heart and inhibited neuromuscular transmission. (+)-Tubocurarine and suxamethonium did not affect the response of the lung or the heart to vagal stimulation. Bronchoconstriction induced by intravenous acetylcholine was not potentiated by gallamine, indicating that postsynaptic muscarinic receptors in the lung and changes in muscle tone were not involved. Potentiation of vagally-induced bronchoconstriction appears to be due to blockade of inhibitory muscarinic receptors located in the pulmonary parasympathetic nerves innervating both central and peripheral airways. Pilocarpine was an agonist for these neuronal receptors as it inhibited vagally-induced bronchoconstriction at low doses (10 ng to 1 microgram kg-1). The results demonstrate that gallamine is an antagonist and pilocarpine an agonist at neuronal muscarinic receptors which attenuate parasympathetic nerve activity in feline lung.     

Behavioral effects of some diphenyl-substituted antimuscarinics: comparison with cocaine and atropine.

To more fully characterize the behavioral excitatory effects observed with certain diphenyl-substituted antimuscarinics, various behavioral effects of benactyzine, a prototype excitatory antimuscarinic, was evaluated in rats. These effects were compared to those of cocaine, atropine, and azaprophen, a muscarinic antagonist that contains both the diphenyl substituents of benactyzine and a ring isomeric with the tropane ring of atropine. Under a fixed-interval 5-min schedule of food presentation, cocaine and benactyzine increased response rates. Atropine and azaprophen only decreased responding. The muscarinic agonist oxotremorine attenuated the rate-increasing effects but did not alter the disruptions in the temporal patterning produced by benactyzine or shift the dose-effect function to the right. In rats discriminating 10 mg/kg cocaine from saline, benactyzine partially substituted for cocaine, producing a maximum of 50% cocaine-appropriate responses. Benactyzine fully substituted for scopolamine in rats discriminating 0.056 mg/kg scopolamine from saline. All antimuscarinics increased locomotor activity when activity levels were low in control animals, but the increases were less than those produced by cocaine. Cocaine increased both locomotor activity and fixed-interval responding at comparable doses, whereas 10-fold higher doses of benactyzine were required to increase locomotor activity. These results support the following conclusions: 1) In addition to its classical antimuscarinic behavioral profile, benactyzine has behavioral excitatory actions similar in some respects to those of cocaine; 2) the behavioral excitatory effects of benactyzine do not appear to be due solely to antagonism of muscarinic receptors; and 3) the alkyl-ester may be an important structural feature of diphenyl-substituted antimuscarinics for the induction of behavioral stimulation.     

Interactions of atropine with heterologously expressed and native alpha 3 subunit-containing nicotinic acetylcholine receptors

(1) Atropine, a classical muscarinic antagonist, has been reported previously to inhibit neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, the action of atropine has been examined on alpha3beta4 receptors expressed heterologously in Xenopus oocytes and native nAChRs in medial habenula neurons. (2) At concentrations of atropine often used to inhibit muscarinic receptors (1 micro M), responses induced by near-maximal nicotine concentrations (100 micro M) at negative holding potentials (-65 mV) are inhibited (14-30%) in a reversible manner in both alpha4 and alpha3 subunit-containing heteromeric nAChRs. Half-maximal effective concentrations (IC(50) values) for atropine inhibition are similar for the four classes of heteromeric receptors studied (4-13 micro M). (3) For alpha3beta4 nAChRs in oocytes, inhibition by atropine (10 micro M) is not overcome at higher concentrations of agonist, and is increased with membrane hyperpolarization. These results are consistent with non-competitive antagonism--possibly ion channel block. (4) At low concentrations of both nicotine (10 micro M) and atropine (<10 micro M), potentiation ( approximately 25%) of alpha3beta4 nAChR responses in oocytes is observed. The relative balance between potentiation and inhibition is dependent upon membrane potential. (5) In rat medial habenula (MHb) neurons, atropine (0.3-3.0 micro M) inhibited nicotine-induced responses in both a concentration and membrane potential-dependent manner (at -40 mV, IC(50)=4 micro M), similar to the effects on alpha3beta4-nAChRs in oocytes. However, unlike heterologously expressed receptors, potentiation was barely detectable at depolarized membrane potentials using low concentrations of nicotine (3-10 micro M). Conversely, the weak agonist, choline (1-3 mM) was observed to augment responses of MHb nAChRs.     

A comparison of the central and peripheral effects of atropine on force lever performance

The central and peripheral effects of atropine, a muscarinic antagonist, on motor control were tested in a force lever apparatus. Three rhesus monkeys were trained to extend their arms through a tube and press a manipulandum with between 25 and 40 grams of force for 3 continuous seconds. Responding was maintained by the delivery of 1.5 ml of water. Single injections of atropine sulfate or atropine methylnitrate (a quaternary derivative of atropine which does not cross the blood brain barrier) were given 30 min prior to the session. Atropine sulfate disrupted force lever performance in a those related fashion. Atropine methylnitrate had little or no effect on responding. The effects of atropine on motor control as measured in this procedure, therefore, appear to be centrally mediated.     

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123)

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [³H](–)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3–4 days. Learned responses and those requiring temporal discrimination took 8–11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.     

The role of spinal muscarinic acetylcholine receptors in clonidine-induced anti-nociceptive effects in rats

We have examined the effects of intrathecal (i.t.) injection of the muscarinic acetylcholine receptor antagonist atropine on the clonidine-induced nociceptive effect in formalin-induced nociception in rats. The injection of 5% formalin into the hind paw caused biphasic nociceptive responses, and i.t. injection of clonidine inhibited both phases of the nociceptive response in a dose-dependent manner. Pretreatment with atropine (i.t.) only partially inhibited the nociceptive effect of clonidine. These results suggest that the nociceptive effect of clonidine in the rat formalin model may be at least partly mediated by muscarinic acetylcholine receptors in the spinal cord.     

Effects of intravenous mu and kappa opioid receptor agonists on sensory responses of convergent neurones in the dorsal horn of spinalized rats.

1. Electrophysiological experiments have been performed to assess the effects of intravenously administered mu and kappa opioid agonists on the responses to noxious thermal and mechanical and non-noxious tactile stimuli of single convergent neurones in laminae III-VI of the dorsal horn of spinalized rats anaesthetized with alpha-chloralose. 2. The mu receptor agonists tested were fentanyl (1-16 micrograms kg-1) and morphine (0.5-16 mg kg-1) and the kappa-receptor agonists U-50,488 (1-16 mg kg-1) and tifluadom (0.1-1.6 mg kg-1). Multiple drug tests were made on each cell so that compounds could be compared under closely comparable conditions. 3. In one protocol, thermal and mechanical nociceptive responses of matched amplitudes were elicited alternately. Both mu and kappa agonists dose-dependently reduce the neuronal responses. Thermal nociceptive responses were as sensitive to the kappa agents as were the mechanical nociceptive responses; the mu agonists similarly reduced both types of response in parallel. 4. In another protocol, nociceptive and non-nociceptive responses were elicited alternately to permit the degree of selective antinociception to be assessed. The mu agonists were scarcely selective, fentanyl reducing nociceptive only slightly (but significantly at 4-16 micrograms kg-1) more than non-nociceptive responses. The kappa-opioid agonist U50,488 reduced tactile responses somewhat more than nociceptive responses. 5. The spontaneous discharge of these cells with ongoing activity was reduced to a significantly greater degree than the evoked responses; this is likely to have contributed to the non-selectivity of the reduction of the evoked responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of a 2-chloroethylamine derivative of oxotremorine (BM 123) to muscarinic receptors in the rat cerebral cortex

The interaction of a mustard analogue of oxotremorine, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), with muscarinic receptors in the rat cerebral cortex was investigated using 3H-ligand-binding methods. When cortical homogenates were preincubated with BM 123 (1.0 mM), washed extensively, and then assayed for the binding of the specific muscarinic antagonist, [3H](-)-N-methylscopolamine, a decrease in binding capacity was noted without an accompanying change in affinity. The rate at which BM 123 alkylated muscarinic receptors was sensitive to temperature, with little or no receptor alkylation occurring at 0 degree. Thus, it was possible to estimate the affinity of BM 123 and its transformation products for muscarinic receptors by measuring their ability to competitively inhibit 3H-ligand binding to cortical homogenates at 0 degree. When measured by competitive inhibition of [3H]oxotremorine-M and [3H](-)-N-methylscopolamine binding, the concentrations of the aziridinium ion of BM 123 required to displace 50% of specific 3H-ligand binding were 3.5 nM and 4.5 microM, respectively. In contrast, the parent 2-chloroethylamine and its alcoholic hydrolysis product were much less active. The kinetics of the alkylation of muscarinic receptors by BM 123 were consistent with a model in which the aziridinium ion rapidly forms reversible complexes with superhigh high and low affinity sites which slowly convert to covalent complexes. The rate of alkylation of the superhigh affinity site was slowest whereas the converse was true for the low affinity site. It was possible to alkylate the high and low affinity sites selectively with BM 123 by taking advantage of kinetic differences in the rates of alkylation of these two sites. Atropine, oxotremorine, and oxotremorine-M antagonized the rate of alkylation of muscarinic receptors in a manner that was consistent with competitive inhibition.     

Effects of subcutaneous and intracerebroventricular injection of physostigmine on the acute corneal nociception in rats

The present study investigated the effects of subcutaneous (sc) and intracerebroventricular (icv) injections of physostigmine (a cholinesterase inhibitor), atropine (an antagonist of muscarinic cholinergic receptors) and hexamethonium (an antagonist of nicotinic cholinergic receptors) on the acute corneal nociception in rats. Local application of 5 M NaCl solution on the corneal surface of the eye produced a significant nociceptive behavior, characterized by eye wiping. The number of eye wipes was counted during the first 30 s. The sc (0.25, 0.5 and 1 mg/kg) and icv (1.25, 2.5, 5 and 10 μg) injections of physostigmine significantly (p < 0.05) decreased the number of eye wipes. Atropine and hexamethonium at (2 mg/kg, sc and 20 μg, icv) had no effects when used alone, however, atropine, but not hexamethonium prevented the antinociception induced by physostigmine (sc and icv). The results of this study indicate that the central muscarinic, but not nicotinic receptors might be involved in the antinociceptive effect of physostigmine in the acute corneal model of pain in rats.     

The dose-related effects of paracetamol on hyperalgesia and nociception in the rat.

1. We have studied the effects of 3 low doses of paracetamol (25, 50 and 100 mg kg-1 p.o.) on inflammatory hyperalgesia, inflammatory oedema, and nociceptive thresholds in rats. 2. At the lower dose (25 mg kg-1), paracetamol reduces only central hyperalgesia. 3. At the doses of 50 and 100 mg kg-1, paracetamol reduces also peripheral hyperalgesia; moreover, it enhances nociceptive thresholds to a mechanical stimulus in the non-inflamed paws. 4. Neither paw inflammatory oedema nor tail nociceptive thresholds to a thermal stimulus were modified by paracetamol administration. 5. Our results suggest that paracetamol can reduce hyperalgesia without affecting physiological nociception and inflammation.     

Synthesis and evaluation of melphalan-containing N,N-dialkylenkephalin analogues as irreversible antagonists of the delta opioid receptor

N,N-Dialkylated leucine enkephalin analogues containing melphalan (Mel) in place of Phe4 were synthesized as potentially irreversible antagonists of the delta opioid receptor. These compounds, along with the corresponding Phe4 peptides, were tested for both agonist and antagonist activity in the GPI and MVD smooth muscle preparations. All but two of the eight compounds showed antagonist activity at 1 microM against [D-Ala2,D-Leu5]enkephalin (DADLE) in the MVD when tested under reversible conditions; in all cases the Mel4 peptide had lower activity against DADLE than did the corresponding Phe4 peptide. At higher concentrations (10 microM) the two active Mel4 analogues, (benzyl)2Tyr-Gly-Gly-Mel-Leu (2a) and (allyl)2Tyr-Aib-Aib-Mel-Leu (3a), both showed weak irreversible antagonism at the delta receptor. Compound 2a was a selective irreversible delta opioid antagonist while 3a was an irreversible antagonist at both the mu and delta opioid receptors.     

Muscarinic receptor binding and behavioral effects of atropine following chronic catecholamine depletion or acetylcholinesterase inhibition in rats

Rats were subjected to one of two experimental treatments: (1) intraventricular infusion of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), known to permanently reduce brain dopamine and norepinephrine levels, or (2) chronic administration of the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). Both treatments are believed to produce relative overactivity of cholinergic systems and to suppress forward locomotion. The anticholinergic agent atropine sulfate yielded excessive forward walking in otherwise chronically akinetic 6-OHDA-treated rats, whereas atropine slightly decreased locomotion in controls. The hypothesis that such supersensitivity to atropine may be related to a reduction in the density of muscarinic cholinergic receptors was not supported: First, ³H-quinuclidinyl benzilate (QNB) binding to membrane preparations was not decreased in the 6-OHDA-treated rats; secondly, atropine did not induce excessive forward locomotion in the DFP-treated rats in which ³H-QNB binding was decreased. There were other changes in the DFP-treated rats consistent with muscarinic receptor alteration, including tolerance to the locomotor suppressive effects of DFP, cross tolerance to the cholinergic agonist pilocarpine, and exaggerated atropine-induced increases in core temperature and stereotypy. It is concluded that 6-OHDA and DFP produce different long-term changes in cholinergic brain systems and atropine-sensitive behaviors.     

The assessment of antagonist potency under conditions of transient response kinetics.

The muscarinic acetylcholine receptor antagonists, atropine and pirenzepine, produced an apparent insurmountable antagonism of muscarinic M(1) receptor-mediated intracellular Ca(2+) mobilization in Chinese hamster ovary (CHO) cells when tested against the agonists carbachol or xanomeline. Each antagonist caused a dextral shift of the agonist concentration-response curves with depression of the maximum response that was incomplete (i.e., saturated) and which varied with the pairs of agonist and antagonist. Equilibrium competition binding assays found no deviation from simple, reversible competitive behavior for either antagonist. The relative rates of dissociation of unlabeled atropine and pirenzepine were also assessed in radioligand kinetic studies and it was found that atropine dissociated from the receptor approximately 8-fold slower than pirenzepine. Numerical dynamic simulations suggested that the insurmountability of antagonism observed in the present study was probably a kinetic artifact related to the measurement of transient responses to a non-equilibrated agonist in the presence of a slowly dissociating antagonist. Importantly, the patterns of antagonism observed included a saturable depression of agonist maximal response, a mode of antagonism that is incompatible with the previously described phenomenon of hemi-equilibrium states. Monte Carlo simulations indicated that reasonable, semi-quantitative estimates of antagonist potency could be determined by a minor modification of standard methods, where equieffective agonist concentrations, rather than EC(50) values, are compared in the absence and presence of antagonist. Application of the latter approach to the functional data yielded estimates of antagonist potency that were in excellent agreement with those derived from the equilibrium binding assays, thus indicating that the present method can be useful for quantifying antagonist potency under non-equilibrium conditions.     

Presynaptic muscarinic receptors inhibiting endogenous noradrenaline release in the portal vein of the freely moving rat.

1. In the portal vein of the freely moving unanaesthetized rat, the existence of presynaptically located inhibitory muscarinic receptors was investigated by use of the muscarinic agonist methacholine (MCh) 2. Infusion of MCh (0.3 micrograms min-1) did not significantly inhibit the endogenous noradrenaline (NA) overflow in portal plasma. However, after inducing high intra-synaptic concentrations of NA by blocking the presynaptic alpha 2-adrenoceptors with yohimbine (1 mg kg-1), MCh (0.3 microgram min-1) was able to reduce the yohimbine-induced enhanced NA overflow by 38%. 3. The MCh-induced inhibition was almost completely abolished after blockade of the presynaptic muscarinic receptors with atropine (0.6 mg kg-1). 4. During electrical stimulation of the portal vein nervous plexus the evoked NA overflow was strongly inhibited (95%) during MCh-infusion (0.3 microgram min-1). Again atropine (0.6 mg kg-1) was able to reverse this inhibition. 5. These results show the existence of presynaptic muscarinic receptors inhibiting endogenous NA overflow from the portal vein nervous plexus under conditions of enhanced sympathetic activity in the freely moving rat.     

Cholinergic control of male mating behavior in hamsters: effects of systemic agonist or antagonist treatment

Sexual behavior in male rats is thought to depend in part on central cholinergic activity. In particular, previous studies of responses to systemically administered cholinergic drugs suggest that male rat behavior can be facilitated by the muscarinic agonist oxotremorine but is disrupted by the muscarinic antagonist scopolamine. However, it is not clear how broadly these effects generalize across species. To address this issue, we observed the impact on sexual behavior in male hamsters of systemic treatment with oxotremorine or scopolamine. In each case, the peripheral muscarinic antagonist methylscopolamine was used as an auxiliary or control treatment to better isolate central cholinergic effects. Both oxotremorine and scopolamine disrupted male behavior in hamsters. For example, both increased the likelihood of failure to achieve intromission or ejaculation. Further, even on completed tests oxotremorine treatment led to changes including increases in mount latency and postejaculatory interval while scopolamine treatment caused changes including increases in ejaculation latency and intromission frequency. The many changes caused by these treatments suggest that acetylcholine helps to control many elements of male behavior, probably by acting at multiple brain sites. The generally similar responses to a cholinergic agonist and antagonist suggest the dependence of efficient mating behavior on optimal levels of central cholinergic activity.     

Physostigmine-insensitive behavioral excitatory effects of atropine in squirrel monkeys

Lever press responses of squirrel monkeys were maintained under a multiple schedule in which the first response after 3 min produced either food or electric shock depending on the prevailing stimulus. Atropine sulfate (0.3-3 mg/kg, IM) given immediately before experimental sessions disrupted the temporal pattern of responding and produced dose-related decrease in rates of food- and shock-maintained responding. Increases in responding occurred when 1 mg/kg atropine was given 1 to 12 hr prior to experimental sessions. A maximal increase of 200% of control rates was seen following the 2-hr pretreatment. Qualitatively similar effects were obtained with scopolamine suggesting that the time-dependent increases may be a general consequence of muscarinic receptor blockade. Response patterning changes and response rate increases were also produced by coadministration of atropine and physostigmine both given immediately before the session. Increases in rates of responding have also been observed previously after administration of atropine with rate-decreasing doses of the direct-acting muscarinic agonist oxotremorine. Physostigmine did not reverse the rate increases or the alteration in temporal patterning produced by the 2-hr atropine pretreatment; rate decreases induced by immediate pretreatment with atropine were blocked by physostigmine. Thus, the response rate-decreasing effects of atropine were distinct from its rate-increasing effects. Whereas the rate-decreasing effects of atropine appear to involve muscarinic receptors, increases in responding may not. Such nonmuscarinic behavioral excitatory actions of atropine may be expressed when the muscarinic-related decreases are blocked by physostigmine or oxotremorine, or when the decreases are overridden by excessive nonmuscarinic stimulation, perhaps triggered by time-dependent changes in acetylcholine turnover associated with atropine.     

Derivatives of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide

A series of tertiary and quaternary analogues (acyclic imides, sulfonimides, N-acetyl sulfonamides, and trifluoroacetamides) of the selective partial muscarinic agonist N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM 5,35) was synthesized. The compounds were found to be muscarinic agonists, partial agonists, or antagonists in the isolated guinea pig ileum. Replacement of the acetyl group or the N-methyl group of 35 and its analogues by a methanesulfonyl group abolished efficacy and decreased affinity at ileal muscarinic receptors. Trifluoroacetamide analogues of 35 also had lower affinity and efficacy than 35. Substitution of an acetyl group for the N-methyl group in compounds related to 35 decreased efficacy, but had no appreciable effect on affinity. Most of the tertiary amines showed central antimuscarinic activity as they antagonized oxotremorine-induced tremors in mice.