Muscarinic Agents Research Articles

Synthesis and biological evaluation of iodine‐125 iodocaramiphen. A potential M1 muscarinic imaging agent for SPECT

AbstractIodocaramiphen (2) is a selective muscarinic antagonist which binds in vitro with high affinity and selectivity to the M1 subtype of the muscarinic receptor. We report the synthesis of iodine‐125 labeled iodocaramiphen ([125I]‐2) via a tributylstannyl intermediate (3) in 50% radiochemical yield with a specific activity greater than 1000 mCi/μmol. Biodistribution studies in female Fischer rats demonstrated that [125I]‐2 had significant cerebral localization (0.7% injected dose/gram) at 60 minutes post injection. The uptake of activity washed out rapidly from the brain, however, and did not demonstrate specific uptake in those cerebral regions rich in muscarinic receptors. In addition, preinjection with (±)‐QNB (5 mg/kg) blocked uptake of approximately 25% of the injected radiolabel in the brain at 2 hours. The non‐selectivity of 2 toward muscarinic receptors in vivo may result from the metabolism of 2 by various esterases or the affinity of 2 for sigma sites in the brain.

M 2-type Muscarinic Receptors Mediate Prejunctional Inhibition of Norepinephrine Release in the Human Iris-Ciliary Body

Human muscarinic receptors comprise a family of five separate gene products, three of which (designated as M 1, M 2 and M 3 subtypes) can be distinguished pharmacologically. Previous work indicates that sympathetic nerve terminals in the anterior uvea contain prejunctional muscarinic receptors that, upon activation by agonists, inhibit the neural release of norepinephrine. The aim of this study was to characterize the prejunctional effects of muscarinic agents on the electrically-evoked secretion of 3H-norepinephrine in isolated, superfused human iris-ciliary body tissue segments. Stimulation-evoked 3H-norepinephrine release was inhibited > 80% by carbachol and muscarine, but was unaffected by the M 1-selective agonist McN A-343. Pilocarpine behaved as a partial agonist in this system, producing less than 40% of the maximum inhibition. The rank order of potency of selective antagonists at prejunctional muscarinic receptors was methoctramine (M 2) > AF-DX 116 (M 2) > pirenzepine (M 1) ≥ para-fluoro hexahydro-sila-definidol (M 3). These data suggest that prejunctional muscarinic receptors in the human iris-ciliary body correspond to the M 2 subtype. No evidence for age-related differences in prejunctional muscarinic receptor activity was seen in tissues obtained from 13 human donors, aged 10-83 years. Prejunctional muscarinic receptors may play a role in mediating the inhibitory effects of parasympathetic nerve stimulation or cholinomimetic drugs on ocular sympathetic neurotransmission in vivo.

Interaction of cholinergic-dopaminergic systems in the regulation of memory storage in aversively motivated learning tasks

These experiments examined the interaction between muscarinic cholinergic and dopaminergic systems in the modulation of memory storage. Male CD1 mice (25–30 g) were trained in an inhibitory avoidance (IA) and a Y-maze discrimination (YMD) task. The first experiment examined the dose-response effects, on retention, of agonists and antagonists specific for either D 1- or D 2-receptors. Immediately posttraining mice were given i.p. injections of saline, the D 1-receptor agonists SKF 38393 (3.0, 10.0 or 30.0 mg/kg) or SKF 77434 (3.0, 10.0 or 30.0 mg/kg), the D 1-receptor antagonists SCH 23390 (0.03 (0.03, 0.1, 0.3 or 1.0 mg/kg), the D 2-receptor agonist quinpirole (0.3, 1.0 or 3.0 mg/kg) or the D 2-receptor antagonist sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg). Retention was tested 48 h later. The drugs affecting D 1-receptors did not affect retention. In contrast, in both tasks quinpirole enhanced retention and sulpiride impaired retention. In the IA task, quinpirole (3.0 mg/kg) blocked the retention impairing effects of the muscarinic cholinergic antagonist atropine (10.0 mg/kg), and sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg) significantly attenuated the memory enhancing effects of the muscarinic cholinergic agonist oxotremorine (35.0 or 70.0 μg/kg). D 1-receptor agents did not modify the effects of either atropine or oxotremorine on retention of the IA response. These findings suggest that the effects of cholinergic muscarinic agents on retention of the IA response are mediated by influences involving D 2-dopaminergic mechanisms. In the YMD task, atropine (10.0 mg/kg) blocked the memory-enhancing effects of quinpirole (3.0 mg/kg) and oxotremorine (35.0 or 70.0 μg/kg) attenuated the memory impairing effect of sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg). D 1-receptor agents did not modify the effects of either atropine or oxotremorine on retention of the YMD task. These results suggest that the effects of the D 2-dopaminergic agents on retention of the YMD are mediated by muscarinic cholinergic mechanisms. Although retention of both the IA and the YMD are modulated by interactions between D 2-dopaminergic and muscarinic cholinergic systems, the ways in which these mechanisms interact are task-dependent.

Muscarinic modulation of neurotransmission: The effects of some agonists and antagonists

1. 1. Functional studies were performed to evaluate the effects of some muscarinic agents at the neuromuscular junction of the mouse. 2. 2. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analyzed by means of extracellular recording. The amplitude of spontaneous and of evoked acetylcholine release, the frequency of spontaneous acetylcholine release and the time course of the quantal release have been measured by means of an EPC7 patch clamp amplifier. 3. 3. This electrophysiological method revealed multiple dose-related effects of some agonists and antagonists on the above parameters. Concentration-response curves related to the parameters underlying the function of this cholinergic synapse were obtained and the apparent EC 50 values calculated. 4. 4. Many of the interactions of the agonists and antagonists could inhibit neuromuscular transmission. The rank order potencies related to the inhibition of the evoked signals were carbachol > oxotremorine > d, l-muscarine for the agonists and methoctramine > 4-DAMP > l-hyoscyamine > AFDX-116 > ipratropium > pirenzepine for the antagonists. 5. 5. These findings suggest a more complicated pattern related to the muscarinic action at the mouse neuromuscular junction with the involvement of some post-synaptic located sites.

Postsynaptic effects of methoctramine at the mouse neuromuscular junction

Functional studies were performed to evaluate the effects of methoctramine at the neuromuscular junction of the mouse. The presynaptic control of acetylcholine release and the postsynaptic activation of the nicotinic receptor have been analysed by means of the extracellular recording with an EPC7 Patch Clamp amplifier. This electrophysiological method revealed a dose-related inhibitory effect of methoctramine on the studied parameters. The dramatic reduction of the kinetics of the quantal conductance change indicates an action at the postsynaptic level. The effects of methoctramine have been compared with those of the muscarinic agonist oxotremorine. Concentration/response curves for the two drugs were obtained and the apparent ec 50 values calculated. The effects of oxotremorine were not antagonized by 1 μM methoctramine. These findings suggest an interaction of some muscarinic agents on the postsynaptic receptor-ion-channel complex at the mouse neuromuscular junction.

Synthesis of a new series of N‐substituted‐3‐[l‐alkyl(aryl)‐ 4‐piperidyl]azetidin‐2‐ones as possible muscarinic agents

AbstractA new series of N‐substituted‐3‐[1‐alkyl(aryl)‐4‐piperidyl]azetidin‐2‐ones 2a‐j structurally related to the previously reported 2,7‐diazaspiro[3.5]nonan‐1‐ones 1 have been synthesized and tested for their activity as muscarinic agents, both in vitro and in vivo. Preliminary pharmacological results showed that compounds 2 are devoid of significant cholinergic properties.

Synthesis of a new series of 8‐substituted‐2,8‐diazaspiro[4.5]decan‐3‐ones

AbstractA new series of 8‐substituted‐2,8‐diazaspiro[4.5]decan‐3‐ones has been synthesized and tested in in vitro and in vivo assays predictive for cholinergic activity, in comparison with the muscarinic agent RS‐86. Preliminary pharmacological data indicate that the compounds are devoid of significant cholinergic properties.

Muscarinic potentiation of excitatory amino acid-evoked dopamine release in mesencephalic cells: specificity for the NMDA response and role of intracellular messengers.

Of the five cloned muscarinic receptor subtypes, dopamine (DA) neurons in the substantia nigra and ventral tegmental areas have been shown to be selectively enriched with the mRNA for the m5 subtype, suggesting that muscarinic modulation of DA neurons may have a distinct pharmacology. In the present study we have used dissociated cell cultures of fetal rat ventral mesencephalon to characterize muscarinic modulation of DA neurons. [3H]DA release stimulated by activation of N-methyl-D-aspartate (NMDA) receptors was potentiated by carbachol, a mixed muscarinic-nicotinic agonist, and by oxotremorine-M, a muscarinic agonist. Neither carbachol nor oxotremorine-M had an effect on [3H]DA release evoked by the non-NMDA agonists, kainate or quisqualate. A nicotinic agonist, DMPP, had no effect on NMDA-stimulated release. Potentiation of NMDA-stimulated [3H]DA release by oxotremorine-M was inhibited by the broad spectrum muscarinic antagonist, QNB, and by low concentrations of a putative M1 antagonist, pirenzepine, while much higher concentrations of a purported M2 antagonist, AF-DX 384, were required to reverse the oxotremorine-M effect. The muscarinic antagonist, 4-DAMP, was active in a concentration range between that required for pirenzepine and AF-DX 384. Further experiments examined intracellular messenger mechanisms coupled to the muscarinic receptors modulating NMDA-stimulated [3H]DA release. In contrast to oxotremorine-M, two muscarinic agents with only weak partial agonism with respect to phosphoinositide turnover, pilocarpine and arecoline, had no effect on NMDA-stimulated [3H]DA release.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid response of striatal muscarinic M 1-receptor mRNA to muscarinic cholinergic agents in rat brain

The effects of a single administration of muscarinic cholinergic agents on the level of muscarinic M 1-receptor messenger RNA (M 1-R mRNA) in the rat striatum were studied. Carbachol increased the M 1-R mRNA expression rapidly and transiently, while trihexyphenidyl decreased it. These results suggest that muscarinic cholinergic agents participate in the positive regulation of muscarinic receptor mRNA in the early stage after treatment, contrary to the negative regulation in the chronic stage.

Deficits in working but not reference memory in adult rats in which limbic seizures had been induced before weaning: Implications for early brain injuries

Male adult rats that displayed limbic seizures between postnatal days 18 and 21 after a single s.c. injection of Li followed 4 h later by a muscarinic agent were trained in a radial arm maze; they were compared with rats that had received the Li-pilocarpine (but had not displayed overt seizures) and to nonhandled controls. Only the rats that had displayed the (preweaned) seizures displayed significant impairment for working memory but not for reference memory. Light microscopy demonstrated histological evidence of earlier damage only within select thalamic structures that are directly associated with the amygdaloid-hippocampal complex. The results are compatible with the hypothesis that early seizures during the time of CA1 hippocampal maturation can produce long-term changes in the efficacy of short-term memory.

Cocultures of adult ventricular myocytes with stellate ganglia or intrinsic cardiac neurones from guinea pigs: spontaneous activity and pharmacological properties.

The aim was to develop long term primary cocultures of adult ventricular myocytes with autonomic neurones dissociated from stellate or intrinsic cardiac ganglia. This was to provide an experimental model for the investigation of the regulatory role of autonomic neurones with respect to cardiac myocyte function. Ventricular myocytes and stellate and intrinsic cardiac neurones were enzymatically dissociated from adult male guinea pigs and plated together on 13 mm cover slips; the cultures were maintained in an incubator for two to 10 weeks. The electrical properties of cultured myocytes and neurones were investigated by means of a conventional microelectrode technique and the spontaneous contractile activity of the myocytes was recorded by a video system. The electrical, contractile, and pharmacological properties of myocyte-neuronal networks were investigated by superfusing the cultures with various neuromodulators and blockers. The electrical properties of the cultured myocytes and neurones were similar to those reported in other in vitro studies. Innervated and non-innervated cardiomyocyte cultures responded differently, however, to various pharmacological interventions. Spontaneous contractions were attenuated by tetrodotoxin (4 x 10(-7) M), beta adrenergic blockade, and nicotinic blockade more in cocultures than in cardiac myocyte cultures alone. On the other hand, the beta agonist isoproterenol increased the spontaneous beating in both types of cocultures more than in myocytes alone. The effects of the muscarinic agonist bethanechol and the muscarinic blocker atropine were not significantly different in innervated and non-innervated cultures. Nicotine induced either an increase or decrease in contractile rates of both cocultures and did not affect non-innervated myocyte cultures. Cocultures of myocytes and autonomic neurones alter the responses of myocytes relative to cultures without neurones. The results suggest that functional contacts can be established between adult myocytes and dissociated neurones in primary cultures. Contractile rates of such myocyte cultures are influenced by the presence of neurones. Neurones innervating these cultures were modified by beta adrenergic, muscarinic, and nicotinic agents.

High Affinity Binding of [3H]Imidacloprid in the Insect Acetylcholine Receptor

l-(6-Chloronicotinyl)-2-nitroimino-imidazolidine (imidacloprid or IMI) is a potent insecticide of a new chemical class considered from earlier studies to act at the insect nicotinic acetylcholine receptor. In a direct approach to the mode of action of IMI, it was radiolabeled and [3H]IMI was examined in binding studies to elucidate its pharmacological profile. [3H]IMI undergoes high affinity specific binding in house fly head P2 membranes with 95% specific binding, a dissociation constant of 1.2 nM, and a maximal binding site capacity of 853 fmol/mg protein. The standard binding assay consisted of 1 nM [3H]IMI and 200 μg membrane protein in 50 mM NaCl, 10 mM sodium phosphate (pH 7.4) containing 0.1% Triton X-100 with incubation for 60 min at 22°C prior to filtration. The radioligand undergoes rapid biphasic association and dissociation consistent with a two-stage sequential reaction in each case. [3H]IMI binding is very sensitive to carbachol (IC50 1.9 μM) and other choline esters (IC50s 0.2 to 0.5 μM for acetylcholine, propionylcholine, and butyrylcholine in the presence of paraoxon as a cholinesterase inhibitor). The pharmacological profile for [3H]IMI binding indicates inhibition by both nicotinic and muscarinic agents with IC50s of 0.6 μM for (-)-nicotine, 2.2 μM for α-bungarotoxin, 30 μM for D-tubocurarine, 90 μM for atropine, 275 μM for quinuclidinyl benzilate, and 288 μM for dexetimide. Lineweaver-Burk plots establish competitive inhibition kinetics for [3H]IMI with acetylcholine, α-bungarotoxin, and quinuclidinyl benzilate. Detection of [3H]IMI binding in membrane preparations from several insects but not from the vertebrates examined is consistent with the selective toxicity of the nitromethylene and nitroimine insecticides.

Cholinergic responsiveness of neurons in the ventroposterior thalamus of the anesthetized rat

Acetylcholine has been implicated as an important neurotransmitter in the mechanisms of thalamic activation. Cholinergic mechanisms are thought to directly underlie the high level of excitability observed in thalamic relay neurons during waking and rapid eye movement sleep. We sought to determine if the cholinergic responsiveness of neurons in the ventroposterior nuclei of the thalamus in rat is consistent with this view. Neurons in the chloral hydrate-anesthetized rat were studied with extracellular recording and microiontophoretic application of cholinergic agents. In most cases (63% of 63 cells), the ejection of the agonist, carbachol, had no observable effect on spontaneous activity. Facilitation (25%), inhibition (8%) and inhibition followed by facilitation (3%) were also observed. Carbachol ejections that by themselves were ineffective in altering spontaneous activity proved capable, in 93% of 28 cells, of antagonizing the uniformly facilitatory responses produced by glutamate ejection. The putative M 1-selective, cholinergic agonist, McN-A-343, was also ineffective alone in altering spontaneous activity in the majority of cases (74% of 27 cells) and produced only inhibitory responses in the remaining seven neurons studied. Interacting applications of McN-A-343 and glutamate resulted, in all cases, in antagonism of glutamate facilitation ( N = 12). The various responses to applied cholinergic agonists were all capable of being antagonized by muscarinic receptor-blocking agents. Both the high proportion of inhibitory responses and the antagonism of glutamate facilitatory responses suggest that ventroposterior neurons in the rat differ from other thalamocortical relay neurons in the rat and cat with regard to cholinergic responsiveness. Additionally, the lack of predominantly facilitatory responding renders it unlikely that cholinergic mechanisms directly underlie increases in excitability of ventroposterior neurons observed during waking and rapid eye movement sleep.

Reply to klemm

A very large number of reports have accumulated in the field relating to dopamine receptors and connected issues. For example, more than 3000 reports relating to "dopamine" are cited in MEDLINE, the database of the US National Library of Medicine (1992). Even when limited to "dopamine receptor", the number of the reports is still over 800. In such a situation, we feel it is important that we select only effective and important reports to achieve good citations. Redundant sentences should be avoided and the number of references should be kept to a reasonable minimum. A great number of papers illustrate that D-I blockade causes catalepsy. The report of Christensen et al. (1984) is recognized as the pioneer, and therefore we referred to their paper in the context of our own (Wanibuchi and Usuda 1990). Numerous reports agree with the fact that cholinergic systems in the brain are involved in catalepsy induced by dopamine antagonists. This is generally accepted. Although this cholinergic involvement was not the main theme in our paper, we wanted to state that research relating to the cholinergic system in catalepsy has started to take a new turn. In recent years, five genes that encode muscarinic acetylcholine receptor subtypes have been found to be present in the brain and they have been isolated. Evidence is accumulating that distributions of their mRNAs are abundant in specific areas of the brain (Mei et al. 1989). Immunocytochemistry has demonstrated that m1-, m2or m4-receptor proteins are distributed in various regions in the brain including striaturn and substantia nigra (Levey et al. 1991). It is likely, therefore, that these receptor subtypes could play important roles in the interaction between the cholinergic and dopaminergic systems. Although selective agents for each of the five mnscarinic receptor subtypes are not available, complex interactions among these muscarinic and dopaminergic receptors may be clarified by using the recently reported selective muscarinic agents in combination (Fifth International Symposium on Subtypes of Muscarinic Receptors 1992).

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons.

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d-tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic-like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.

Modulation of the voltage-dependent Ca2+ channels of smooth muscle by phosphorylation

The voltage-dependent Ca2+ channels (VDC) in smooth muscle cell membranes are the major pathway by which Ca2+ enters the cell during contraction. It has been reported that VDC can be modulated by reversible channel protein phosphorylation and dephosphorylation reactions. In intestinal smooth muscle cell, muscarinic agents have been reported to increase the break-down of phosphatidylinositol 4,5-bisphosphate, which indicates that inositol-1,4,5-trisphosphate and diacylglycerol (DG) can be generated. DG activates protein kinase C. Carbachol (CCh), phorbol 12,13-dibutyrate and phosphatase inhibitors, okadaic acid and calyculin A increased the inward currents passing through the L-type VDCs. These effects were inhibited by protein kinase inhibitors, H-7 and staurosporine. The CCh effect was also inhibited by GDP beta S. Therefore, it seems possible that DG, a product of phosphatidylinositol break-down might mediate muscarinic effects on L-type VDC, the last via stimulation of protein kinase C, and that L-type VDC activity might be modulated by protein kinase C-mediated phosphorylation and protein phosphatase type-1-mediated dephosphorylation of the channel or related protein(s) in guinea pig taenia coli smooth muscle cells.

Halothane Myocardial Depression

The involvement of the adenylate cyclase system in myocardial depression by halothane was investigated in an isolated, electrically stimulated, rat left atrial preparation. The twitch tension dose-response curve to the muscarinic agent, carbachol, was unaltered by 0.27 mM (0.8%) halothane. Pertussis toxin irreversibly blocks the inhibition of adenylate cyclase by muscarinic agonists. Atria from animals pretreated with pertussis toxin were insensitive to the negative inotropic effect of carbachol, but the depression in twitch tension by halothane was unaltered. Halothane shifted the dose-response curve to isoproterenol downward and to the right. However, the depression in twitch tension by 0.27 mM halothane was similar in preparations incubated with the nonhydrolyzable cAMP analogue, dibutyryl-cAMP, compared to control atria stimulated with isoproterenol. We conclude that halothane attenuates the response to beta-adrenergic stimulation in myocardial tissue, and alterations in adenylate cyclase activity do not contribute significantly to this process.

Severity of experimental allergic encephalomyelitis in rats depends upon the temporal contiguity between limbic seizures and inoculation

Limbic seizures (forepaw clonus) were induced in Lewis rats by subcutaneous injections of lithium (3 mEq/kg) followed 24 h later by a muscarinic agent. Either 7 days before, 7 days after, or on the day of seizures, rats were inoculated with a spinal cord preparation. Other groups received these preparations but served as treatment (no seizure) controls. In three separate experiments, rats in which seizures had been induced at the time of inoculation displayed significant increases in the severity of clinical symptoms 14–20 days layer relative to controls while rats in which seizures had been induced 7 days after inoculation displayed less severe symptoms; the latter effect was partially simulated by multiple injections of 1 mg/kg dezamethasone. The immunofacilitation effect was much stronger that the immunosuppression and explained 25–30% of the variance in the clinical severity scores.

Hormone signaling systems in inner medullary collecting ducts.

The inner medullary collecting duct is a complex tissue that exhibits a variety of hormone signaling systems. These include the following: adenylyl cyclase activity stimulated by vasopressin (AVP), beta-adrenergic agonists, or prostanoids and inhibited by alpha 2-adrenergic agents or adenosine; guanylate cyclase activity in response to atrial natriuretic peptide (ANP); phospholipase C activity stimulated by ANP, AVP, bradykinin, endothelin, epidermal growth factor (EGF), and muscarinic cholinergic agents; and phospholipase A2 activity stimulated by AVP, bradykinin, EGF, and endothelin. The signal transduction mechanisms for each of these hormone signaling systems is succinctly reviewed, and the interactions between different signaling pathways are discussed. Central to this interaction is the mutually inhibitory relationship between activation of adenylyl cyclase and phospholipases. Increasing cellular adenosine 3',5'-cyclic monophosphate content impairs activation of phospholipases A2 and C; conversely, stimulation of phospholipase C impairs AVP-stimulated adenylyl cyclase activity via activation of protein kinase C.

Inhibition of the nicotinic acetylcholine response by serotonergic and muscarinic agents in chick ciliary ganglion neurones

Chick ciliary ganglion neurones were investigated by whole cell voltage clamp recordings. The ACh- or nicotine-induced inward current was partially inhibited by perfusing the neurones with 5-HT. This effect was rapid (⩽ 1 min), dose-dependent (50–1000 μM) and quickly reversible. The selective 5-HT 1A agonist 8-OH-DPAT (10 μM) reduced the nicotinic ACh response more potently, irrespective of the absence or presence of propranolol (1 μM), a known 5-HT 1A antagonist. Other serotonergic antagonists, like ICS 205–930 (1 μM), mianserin (10 μM) and methysergide (10 μM), also failed to antagonize the 5-HT-mediated decrease in the nicotinic response. Muscarine (50 μM) did not affect the nicotine-induced inward current but the muscarinic agonist oxotremorine (10 μM) also decreased the nicotine-induced inward current. Atropine, at small concentrations failed to block this effect but caused some reduction of the ACh response itself at larger (1–10 μM) concentrations. It is suggested that 5-HT may modulate synaptic transmission in ciliary ganglion neurones in vivo. The site of action of 5-HT, oxotremorine and atropine might be at or close to the ACh receptor complex, because of the fast onset and reversibility of the effects and lack of specificity for structurally different drugs.