Muscarinic Cholinergic Receptor Antagonist Research Articles

GABA uptake inhibitors produce a greater antinociceptive response in the mouse tail-immersion assay than other types of gabaergic drugs

Antinociception produced by the GABA uptake inhibitors d, 1- SKF-89976A and SKF-100330A was characterized and compared to that produced by other types of GABAergic drugs. Using the mouse tail-immersion assay it was found that the antinociception produced by the uptake inhibitors was antagonized by scopolamine, a cholinergic muscarinic receptor antagonist. However, neither SKF compound demonstrated any significant affinity for muscarinic receptor binding sites suggesting that they are not direct-acting cholinomimetics. In vitro uptake experiments revealed that the SKF compounds selectively inhibit GABA transport, having no effect on the accumulation of aspartic acid, glutamic acid, β - alanine or glycine. Moreover, antinociception and GABA uptake inhibition were stereoselective for SKF-89976A, with the d-isomer being more active in both tests. When comparing antinociceptive responses at maximally effective doses it was also found that the SKF compounds were substantially more efficacious than direct-acting GABA receptor agonists or a GABA transaminase inhibitor. These data suggest that uptake inhibitors may be facilitating GABA transmission in a system that is less affected by other types of GABAergic compounds.

Activation of muscarinic and of alpha 1-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates.

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.

DDT-induced tremor in rats: Effects of pharmacological agents

Rats given a tremorigenic dose of DDT (75 mg/kg, PO) were treated with pharmacological agents either 30 min prior to DDT or 1-2 h prior to testing at the time of peak effect (12 h postdosing). The administration of mephenesin (a centrally acting muscle relaxant) or Dilantin (an anticonvulsant) prior to DDT significantly attenuated tremor. Pretreatment with pizotifen (a serotonergic receptor antagonist) had no significant effect on tremor. Administration of the same agents 1-2 h prior to measurement had minimal effects. Trihexyphenidyl (a muscarinic cholinergic receptor antagonist) exacerbated the tremor produced by DDT. These data suggest that cholinergic neurotransmitter systems may be involved in DDT-induced tremor. That DDT-induced tremor was significantly attenuated by mephenesin and Dilantin is in accord with the conclusion that DDT-induced tremor is a manifestation of repetitive discharge due to interference with ionic conductance.

Interaction of amoxapine with muscarinic cholinergic receptors: An in vitro assessment

Amoxapine, an antidepressant with a rapid onset of therapeutic efficacy and great utility in psychotic depression, has been reported to produce anticholinergic side effects in man similar to those observed with imipramine and amitriptyline. To establish its cholinergic disposition, amoxapine and its metabolites 7-hydroxyamoxapine and 8-hydroxyamoxapine, have been evaluated by determining their effects on (1) quinuclidinyl benzilate (QNB) binding to membrane fractions of rat and human brain, on (2) the carbamoylcholine-stimulated accumulation of inositol phosphates in rat cerebral cortex and on (3) the acetylcholine-induced contraction of the guinea pig ileum. In all three preparations, amoxapine was found to be a considerably weaker antagonist of muscarinic cholinergic receptors than either imipramine (4–27 fold) or amitriptyline (51–300 fold). These results indicate that for amoxapine, no correlation exists between the magnitude of muscarinic receptor inhibition and the extent of ‘anticholinergic’ side effects found in the clinic. Neither the metabolites of amoxapine nor species differences could account for this discrepancy.

Differential effects of d-amphetamine and scopolamine on the ontogeny of rearing

Although rearing is ontogenetically an important behavior, very little is known about the neural bases of rearing. The role development of catecholaminergic and cholinergic neurons play in the ontogeny of rearing was investigated by examining rearing in infant, adolescent, and adult rats following various doses of d-amphetamine (an indirectly acting catecholaminergic agonist) and scopolamine (a cholinergic muscarinic receptor antagonist). d-Amphetamine increased rearing in infants but not in adolescents and adults. These findings suggest that activation of catecholaminergic neurons increases rearing in infants but not in adolescents or adults. Scopolamine increased rearing in adolescents and adults but not in infants, indicating that blocking transmission of cholinergic neurons increases rearing in only older rats.

Central effects of dibutyryl cyclic AMP and GMP on the temperature in conscious rabbits

Intracerebroventricular (i.c.v.) administration of dibutyryl cyclic AMP (Db-cAMP)- and dibutyryl cyclic GMP (Db-cGMP)-induced hyperthermia in rabbits. Central administration of 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711), a selective inhibitor of cAMP phosphodiesterase, only accentuated the hyperthermia due to Db-cAMP whereas a selective inhibitor of cGMP phosphodiesterase, 2-O-proproxyphenyl-8-azapurin-6-one (M and B 22948), only potentiated the hyperthermia caused by Db-cGMP. The hyperthermia due to Db-cAMP and Db-cGMP was not mediated through prostaglandins (PG). In contrast, central administration of an α-adrenergic receptor antagonist, phenoxybenzamine, or a β-adrenergic receptor antagonist, sotalol, only attenuated the hyperthermic response to Db-cAMP while a cholinergic muscarinic receptor antagonist, atropine, specifically antagonized Db-cGMP-induced hyperthermia. I.c.v. administration of a protein synthesis inhibitor, anisomycin, did inhibit the hyperthermia due to Db-cAMP and Db-cGMP. Opiate antagonist, naloxone, did not antagonize Db-cAMP- nd Db-cGMP-induced hyperthermia. These results suggest that a protein mediator is implicated in the induction of hyperthermia by Db-cAMP and Db-cGMP and that cAMP and cGMP may be involved through α/β-adrenergic and cholinergic muscarinic receptors respectively in the central regulation of heat production/conservation in rabbits.

Contraction and [3H]QNB binding in collagenase isolated fundic smooth muscle cells.

Smooth muscle cells from the guinea pig gastric fundus were isolated by successive collagenase digestions. Tritiated quinuclidinyl benzilate [( 3H]QNB) was used to study the binding characteristics of the muscarinic cholinergic receptors on these cells. Each cell bound 8.3 X 10(-19) mol of QNB, and a concentration of QNB of 0.19 nM was required to label one-half of the binding sites. This suggests a concentration of about 500,000 muscarinic cholinergic receptors per smooth muscle cell. The muscarinic cholinergic receptor antagonists atropine and scopolamine inhibited QNB binding with a 50% inhibiting concentration (IC50) in the nanomolar range, whereas the agonists acetylcholine (ACh), oxotremorine, and carbamylcholine had IC50S in the micromolar range. Hill coefficients (nH) for antagonists approached unity, but agonists displayed fractional nH. Exposure of cells to cholinergic muscarinic agonists resulted in dose-dependent decreases in cell length. The concentration of agonist required to induce half-maximal contractions (ED50) was 8.3 X 10(-12) M for ACh and 6.3 X 10(-13) M for oxotremorine. Atropine (10(-9) M) decreased the sensitivity to ACh, increasing the ED50 for ACh-induced contractions to 1.2 X 10(-10) M. These results suggest the existence of muscarinic receptor heterogeneity for cholinergic agonists but not for antagonists.

The actions of tetraethylammonium on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3′:5′ monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 μM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1–2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.

The effect of aging on cholinergic receptor binding in the rat urinary bladder

AbstractBinding sites with high affinity and specificity for [3H]quinuclidinyl benzilate (QNB), a cholinergic muscarinic receptor antagonist, were found in homogenates of rat urinary bladder. Atropine and scopolamine inhibited 50% of the specifically bound [3H]QNB (IC50) at a concentration of approximately 2 nM, whereas the agonist oxotremorine had an IC50 of 580 nM. By contrast, the IC50 for the ganglionic nicotinic antagonist mecamylamine was greater than 100,000 nM. These values are similar to those reported for [3H]QNB binding sites in rat brain.[3H]QNB binding was characterized in rat bladder obtained from animals aged 6, 16, and 26 months. No differences in either the number of binding sites or the affinity constant for the radioligand were noted between these groups, suggesting that there are no agerelated alterations in muscarinic receptor recognition sites in this organ.

Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

Intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP), induced hyperthermia in guinea-pigs which was not mediated through prostaglandins (PG) or norepinephrine since a prostaglandin synthesis inhibitor, indomethacin, and an α-adrenergic receptor blocking agent, phenoxybenzamine did not antagonize the hyperthermia. In contrast, the hyperthermic response to dibutyryl cyclic AMP was attenuated by central administration of a β-adrenergic receptor antagonist, sotalol, indicating that cyclic AMP may be involved, through β-adrenergic receptors, in the central regulation of heat production/conservation. Central administration of dibutyryl cyclic GMP (Db-cGMP) produced hypothermia which was not mediated via histamine H 1- or H 2-receptors and serotonin since the H 1-receptor antagonist, mepyramine, the H 2-receptor antagonist, cimetidine, and the serotonin antagonist, methysergide, had no antagonistic effects. The antagonism of hypothermia induced by dibutyryl cyclic GMP and acetylcholine + physostigmine, by central administration of a cholinergic muscarinic receptor antagonist, atropine, and not a cholinergic nicotinic receptor antagonist, d-tubocurarine, suggests that cholinoceptive neurons and endogenous cyclic GMP may regulate heat loss through cholinergic muscarinic receptors. These results support a regulatory role in thermoregulation provided by a balance between opposing actions of cyclic AMP and cyclic GMP in guinea-pigs.

Muscarinic Cholinergic Receptor Binding

An attempt was made to characterize, in vivo, specific binding to the muscarinic cholinergic receptor in the calf using the radioiodinated ligand quinuclidinyl benzilate (123I-OH-QNB) and single photon detection emission computed tomography (SPECT). The supratentorial brain activity was significantly increased after the intravenous infusion of 123I-OH-QNB as compared to free 123I. Scopolamine, a muscarinic cholinergic receptor antagonist, decreased the measured brain activity when infused prior to 123I-OH-QNB consistent with pharmacologic blockade of specific receptor binding. Quantitative in vitro tissue distribution studies obtained following SPECT imaging were consistent with regionally distinct specific receptor binding in the striatum and cortical gray matter, nonspecific binding in the cerebellum, and pharmacologic blockade of specific binding sites with scopolamine. Although 123I-OH-QNB is not the ideal radioligand, our limited success will hopefully encourage the development of improved binding probes for SPECT imaging and quantitation.

The effect of nicotinic and muscarinic receptor blockade on cholera toxin induced intestinal secretion in rats and cats.

The effects of hexamethonium (cholinergic nicotinic receptor antagonist) and atropine (cholinergic muscarinic receptor antagonist) on cholera toxin induced secretion were investigated in denervated segments of the small intestine of rats and cats. While there was no effect of atropine, hexamethonium markedly inhibited choleraic secretion and turned it into a net fluid absorption in many animals. This observation further strengthens our hypothesis that the enteric nervous system is involved in cholera secretion.

A role for dopamine in growth hormone regulation in the dog.

The role of dopamine (DA) in the secretion of GH in most animal species is still controversial. We examined in the dog the effect on GH release of nomifensine, a drug which activates both noradrenergic and dopaminergic neurotransmission. Nomifensine (0.4-2.8 mg/kg, iv), administered into 12 unanesthetized male and female beagles, induced short-dose-related rises in canine GH (cGH) levels. Blockade of alpha-adrenergic receptors by phentolamine prevented the GH stimulatory effect of 2.8 mg/kg nomifensine, and an iv bolus injection of haloperidol (a neuroleptic which antagonizes both norepinephrine and DA receptor function) given 45 min before was equally effective. Selective blockade of DA receptors by pimozide significantly reduced the GH-releasing effect of nomifensine. In sum, these data indicated that the effect of nomifensine was the consequence of an enhanced noradrenergic and dopaminergic-neurotransmission. Pretreatment with domperidone, a DA receptor blocker unable to cross the blood-brain barrier, failed to modify the GH-releasing effect of nomifensine, suggesting that the DA component subserving the neuroendocrine effect of the drug lies within the blood-brain barrier. Further evidence for a stimulatory role of DA on GH release was the fact that apomorphine, a direct stimulant of DA receptors, induced a rise in cGH levels when administered to dogs pretreated with domperidone. The latter drug was used to prevent emesis and distress due to activation of peripheral DA receptors by apomorphine. However, apomorphine was only active in the dog at doses (250 and 500 microgram/kg, sc) greatly exceeding those active in releasing GH in man, suggesting that the role of DA in cGH regulation is ancillary to that exerted by noradrenergic neurotransmission. In a final study, atropine, a muscarinic cholinergic receptor antagonist, abolished the neuroendocrine effect of nomifensine, a finding which suggests that cholinergic medication plays an important role in cGH regulation.

Characterization of muscarinic cholinergic receptors on intact rat anterior pituitary cells

[ 3H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [ 3H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K d) of 1.5 nM was observed when the [ 3H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K d 20 nM) was observed when [ 3H] QNB concentration was above 20 nM. Using 10 nM [ 3H] QNB for binding, the second order association rate constant (k 1) of 0.064 nM −1 min −1 and first order dissociation rate constant (k 2) of 0.078 min −1 ( T 1 2 8 min) were observed. k 2/k 1 = K d of 1.22 nM is in good agreement with K d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [ 3H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [ 3H] QNB binding than the muscarinic agonist.

C5a induced tracheal contraction: a histamine independent mechanism.

C5a, a peptide derived from the fifth component of complement, caused significant prolonged smooth muscle contraction in isolated guinea pig trachea. Diphenhydramine, a histamine receptor antagonist of the H1 type, had no effect on either the rate, amplitude or duration of C5a induced tracheal contraction, whereas it significantly inhibited the tracheal response to exogenous histamine. Diphenhydramine also caused a significant delay in the response to compound 48/80 in normal guinea pig trachea and to antigen in actively sensitized trachea, indicating that the antihistamine was capable of inhibiting tracheal contractions to endogenously released histamine. C5a induced tracheal smooth muscle contraction was also unaffected by antagonists of H2, muscarinic cholinergic and alpha adrenergic receptors. These results indicate that C5a induced tracheal contraction is independent of histamine and is potentially a mediator of allergic bronchospasm.

Cholinergic and adrenergic receptors on mouse cardiocytes in vitro

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2–14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via α- and β-adrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [ 3H]Alprenolol, a potent β-adrenergic receptor antagonist, and [ 3H]quinuclidinyl benzilate ([ 3H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize β-adrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave ∼800 sites/μm 2, a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells.