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Udgivelsesdato: 2008-May 31 Symptoms of posttraumatic stress disorder are often precipitated by sensory cues in the form of visual, auditory, olfactory and gustatory "flashbacks" resulting in enhanced fear-memory consolidation and the characteristic symptoms of re-experiencing, avoidance and hyper-arousal. Single prolonged stress with and without re-stress have been used to explore the neurobiology of this disorder, particularly with respect to contextual conditioning and spatial memory impairment. However, less work has been done regarding associative sensory-related memories linked to aversive events. Although growing evidence supports a role for cholinergic pathways in stress, this has not been studied in the above animal models. We studied the effects of single prolonged stress with and without re-stress on conditioned taste aversion learning in rats, together with differential analysis of frontal cortical and hippocampal [(3)H]-quinuclidinyl benzylate ([(3)H]-QNB) muscarinic receptor binding. Single prolonged stress with and without re-stress both enhanced associative sensory aversion learning 7 days after stressor-taste pairing, although re-stress did not strengthen this response. Increased cortical and hippocampal muscarinic receptor density (B(max)) was found 7 days after single prolonged stress with re-stress, although receptor affinity remained unaltered. Frontal cortical and hippocampal muscarinic receptor changes may thus underlie conditioned taste aversion learning in rats exposed to stress and re-stress. These data suggest that it may be useful to study the role of cholinergic pathways in mediating associative memory in psychiatric disorders such as posttraumatic stress disorder.

Muscarinic receptors mediating contraction of bladder detrusor muscle from female mice were examined. Mice were untreated (A) or treated with oestradiol cypionate (200 microg/kg) 24 h (B) or 96 h (C) before experimentation, or were pregnant (day 17) (D). Saturation radioligand binding experiments using [(3)H]quinuclidinyl benzilate ([(3)H] QNB) indicated similar muscarinic receptor densities and affinities in bladders from groups A and B. Neither oestrogen treatment nor pregnancy altered pD(2) estimates for methacholine. Maximum responses to methacholine and high-K(+) physiological salt solution (KPSS) were significantly greater (P0.05) in tissues from groups C and D than in A and B. Potencies of other muscarinic receptor agonists were similar in groups A and B with an order of acetylcholine plus physostigmine (10 microM) approximately methacholine plus physostigmine (10 microM)methacholine approximately acetylcholinebethanechol. Antagonist pK(B) estimates were similar in bladders from groups A and B with a rank order of: atropine/=4-diphenyl acetoxy-N-methyl piperidine methiodideparafluorohexahydrosiladifenidol approximately pirenzepinehimbacine, implicating muscarinic M(1) and/or M(5) as well as muscarinic M(3) receptors in mediating methacholine-induced bladder contraction.

Since the therapeutic efficacy of Li+ in the treatment of mood disorder is observed only after chronic administration, we examined whether long-term Li+ treatment with a therapeutic concentration affected the elevation of intracellular-free Ca2+ concentration ([Ca2+]i) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Carbachol caused [Ca2+]i elevation through phosphoinositide hydrolysis in a concentration-dependent manner. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for 2 min resulted in a reduction of the carbachol-induced [Ca2+]i elevation. However, PMA did not reduce the carbachol-induced [Ca2+]i elevation in cells treated with PMA for 48 h, reflecting the down-regulation of protein kinase C. Although Li+ at a therapeutic concentration (1 mM) did not affect the carbachol-induced [Ca2+]i elevation in normal cells, it potently inhibited the [Ca2+]i elevation in protein kinase C down-regulated cells. This inhibitory action of Li+ was observed in a concentration- and time-dependent manner. When protein kinase C activity was directly determined, Li+ treatment did not restore protein kinase C activity in protein kinase C down-regulated cells. [3H]Quinuclidinyl benzylate, a muscarinic receptor ligand, bound to membranes derived from normal and protein kinase C down-regulated cells with a similar Kd and Bmax, and Li+ did not affect these parameters of [3H]quinuclidinyl benzylate binding. These results indicated that Li+ at a therapeutic concentration reduced the muscarinic receptor-mediated increased in [Ca2+]i under the protein kinase C-deficient condition without affecting muscarinic receptor or protein kinase C activity.

The identity of the muscarinic receptor subtype in the chick ileum was investigated in functional and binding studies. Preliminary studies [Choo, L.-K., Mitchelson, F., Napier, P. 1988. J. Auton. Pharmacol. 8, 259-266] suggested apparent avian and mammalian family differences in the muscarinic receptor profile of ileal smooth muscle. In the current study, further characterisation was undertaken using a greater range of antagonists exhibiting high affinity for specific muscarinic receptor subtypes. Dissociation constants from functional and binding experiments were compared with published values for antagonists at each of the five muscarinic receptor subtypes. Linear regression and correlation analyses revealed the receptor initiating the contractile response was most likely of the muscarinic M(3) receptor subtype as the slope of the linear regression was 1.01+/-0.14 and the corresponding correlation coefficient (r) was 0.95. The mammalian muscarinic M(5) receptor subtype also showed a high correlation with the data giving a slope of 0.89+/-0.27 and r value of 0.76. These findings were in direct contrast to those from binding experiments in which the single binding site detected was of the muscarinic M(2) receptor subtype. The slope of the linear regression was 1.14+/-0.24 with an r value of 0.87. Thus, these results suggest that there exists a high proportion of the muscarinic M(2) receptor subtype within the tissue that does not contribute to the functional response.

The aim of the study was to reveal general characteristics of the ligand–receptor interaction in the binding and displacement of radiolabeled ligands. The binding and displacement of dl -[ 3 H]propranolol hydrochloride ([ 3 H]propranolol) and l -[propyl-2,3,- 3 H]dihydroalprenolol ([ 3 H]dihydroalprenolol), β-adrenoceptor antagonists, were studied with isolated rat red blood cells, their membranes and ghosts. The binding of [ 3 H]dihydroalprenolol and l -quinuclidinyl-[phenyl-4- 3 H]-benzylate ([ 3 H]quinuclidinyl benzylate), a muscarinic acetylcholine receptor antagonist, was studied with cerebral cortex membranes. The ligand–receptor interaction corresponded to that for a model of two pools of receptors in the same effector system, with the binding of two ligand molecules to the receptors and was described by the following equation: b =[( B m1 A 2 )/( K d1 2 +A 2 )]+[( B m2 A 2 )/( K d2 2 +A 2 )]. The parameters of [ 3 H]propranolol binding to β-adrenoceptors were as follows: K d1 =0.74 nM, K d2 =14.40 nM, B m1 =24 unit/cell, and B m2 =263 unit/cell for native red blood cells from rats; K d1 =0.70 nM, K d2 =19.59 nM, B m1 =9 fmol/mg protein, and B m2 =39 fmol/mg protein for blood ghosts. The parameters of [ 3 H]quinuclidinyl benzylate binding to muscarinic acetylcholine receptors of cerebral cortex membrane were as follows: K d1 =0.43 nM, K d2 =2.83 nM, B m1 =712 fmol/mg, B m2 =677 fmol/mg. The analysis of the equilibrium binding and displacement of [ 3 H]propranolol and [ 3 H]dihydroalprenolol at β-adrenoceptors of membranes, ghosts and native red cells of rats, [ 3 H]dihydroalprenolol at β-adrenoceptors and [ 3 H]quinuclidinyl benzylate at muscarinic acetylcholine receptors of synaptosomal membranes from rat cerebral cortex demonstrated that the receptors bound two ligand molecules each and consisted of two discrete pools of high- and low-affinity receptors. Similar results were obtained for the displacement of [ 3 H]propranolol, [ 3 H]dihydroalprenolol and [ 3 H]quinuclidinyl benzylate by propranolol, dihydroalprenolol and quinuclidinyl benzylate.

We have investigated the pharmacology of the functionally selective muscarinic M1 receptor partial agonist, sabcomeline [SB-202026 (R-(Z)-(+)-alpha-(methoxyamino)-1-azabicyclo[2.2.2] octane-3-acetonitrile)], in rat cortex and heart using radioligand binding and functional studies. The Quinuclidinyl benzilate/Oxotremorine-M acetate ratio from radioligand binding studies suggested that sabcomeline and xanomeline [3(3-hexyloxy-1,25-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridine] are muscarinic receptor partial agonists in cortical and heart membranes. In [35S]GTPgammaS binding studies in rat cortex, carbachol stimulated binding via muscarinic M2/M4 receptors which could be blocked by sabcomeline with a pA2 of 7.2. In rat heart membranes, carbachol also stimulated [35S]GTPgammaS binding studies through muscarinic M2 receptors. Sabcomeline caused a small stimulation of basal [35S]GTPgammaS binding in both rat and heart tissues. Sabcomeline did not stimulate phosphoinositide hydrolysis in rat cortical slices, but did block the muscarinic M1 receptor-mediated response caused by carbachol with apparent pKb of 6.9. Xanomeline and milameline also had no effect on phosphoinositide hydrolysis up to 100 microM. In adenylyl cyclase studies in rat atria, sabcomeline inhibited forskolin-stimulated adenylyl cyclase activity to a similar extent to that of carbachol, xanomeline and milameline. The present study, using the techniques of radioligand binding, supports previous publications which have claimed that sabcomeline is a muscarinic receptor partial agonist. As expected, this study shows that the functional actions of this compound at muscarinic receptor subtypes and in different tissues will depend on receptor reserve.

Asymmetries in muscarinic receptor binding were investigated in the hippocampus of female rats by in vitro autoradiography. Coronal sections from 18 brains were incubated with the muscarinic receptor antagonist [3H]quinuclidinyl benzilate, the muscarinic M1 receptor antagonist [3H]pirenzepine, or the muscarinic M2 receptor antagonist [3H]AF-DX 384. Binding of these radioligands was higher on the right than the left side of CA1, CA3, and dentate gyrus in almost every brain confirming hemispheric asymmetry at the neurochemical level. The ovarian hormone, estradiol, did not alter the asymmetry in muscarinic binding. Neurochemical asymmetries within hippocampal subfields may have implications for physiological and behavioral functions.

Muscarinic receptor subtypes were characterized in fetal (21 day), newborn (3 day), and adult (3 month) rat colon smooth muscle. Saturation binding of the nonselective muscarinic antagonist radioligand [3H]quinuclidinyl benzilate revealed a single class of binding sites in all three age groups. The binding affinities of [3H]quinuclidinyl benzilate were not significantly different among three age groups (KD: 0.19-->0.27 nM). In contrast, the receptor densities (Bmax, fmol/mg protein) showed a significant age-related decrease with fetus (518.9 +/- 7.4) > newborn (480.3 +/- 45.6) >> adult (192.4 +/- 32.8). In both newborn and adult tissues, the muscarinic agonist carbachol bound to two sites with high and low affinities. Although the agonist binding affinities in the newborn tissue were not significantly different from those in the adult tissue, the high-affinity binding sites for carbachol were significantly increased in the later (41%-->61%). Addition of guanosine-5'-O-(3-thio)triphosphate (100 microM) abolished apparent high-affinity binding sites in both newborn and adult tissues. Antagonist competition binding in the newborn tissue indicated a homogeneous population of muscarinic M2 receptors. Unlike in newborn tissues, the heterogeneous binding of pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methobromide in adult tissues revealed coexistence of muscarinic M3 (45%) and M2 (55%) receptors. In accordance, activation of muscarinic receptors in the adult tissue stimulated synthesis of inositol 1,4,5-trisphosphate. These results suggest maturational changes of muscarinic receptor subtypes and their coupling to G proteins in rat colonic smooth muscle. These changes may account, at least in part, for developmental alterations of functional responses in colonic smooth muscle.

The aim of this study was to characterise the muscarinic receptor present in the uterus of the virgin rat. Homogenate binding studies were undertaken using [3H]quinuclidinyl benzilate as the radioligand and atropine (10 microM) to determine non-specific binding. [3H]Quinuclidinyl benzilate binding was saturable with a Kd of 63 pM and a Bmax of 3 fmol/mg protein. The pKi values obtained using antagonists with high affinity for differing muscarinic receptor subtypes were pirenzepine, 6.2; hexahydrosiladifenidol, 6.9; AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]5,11-dihydro-6H - pyrido[2,3-b][1,4]benzodiazepine-6-one), 7.0; and himbacine, 7.8. These findings suggest that muscarinic M2 receptors are present in rat uterus.

This study investigated the influence of diazepam on the binding characteristics of adrenoceptor, muscarinic and benzodiazepine receptors in rat parotid gland membrane using a radioligand binding assay. At a concentration of >10(-6)M, diazepam competed with [(3)H]dihydroalprenolol for β-adrenoceptor, but not [(3)H]prazosin for α-adrenoceptor or [(3)H]quinuclidinyl benzilate for muscarinic receptor. Continuous administration of diazepam at doses of 0.4mg/kg/day, i.p. for 7days in rat significantly decreased pilocarpine (4.0mg/kg, i.p.)-induced parotid salivary flow. Diazepam also produced a significant increase in the dissociation constant (Kd) value for [(3)H]dihydroalprenolol binding, but no change in the maximal binding capacity (Bmax) value, and a decrease in the Kd value for [(3)H]diazepam binding to benzodiazepine receptors, but no change in the Kd or Bmax values for [(3)H]prazosin or [(3)H]quinuclidinyl benzilate binding. These results suggest that continuous administration of diazepam modifies affinity for β-adrenoceptor and benzodiazepine receptor binding sites in parotid gland membrane and that changes in these binding sites may be closely related to diazepam-induced suppression of salivary secretion.

The muscarinic antagonist, quinuclidinyl benzilate (QNB), labeled with carbon 11 was used as a radioligand to visualize in vivo by positron emission tomography (PET) the central muscarinic acetylcholine receptors (mAChR) in baboons (Papio papio). The binding characteristics of [11C]QNB showed its specific binding to central mAChR. [11C]QNB brain uptake was high in cerebral cortex and striatum, areas that are rich in mAChR, whereas it decreased rapidly in cerebellum, evidencing non-specific binding in this structure that is almost devoid of mAChR. These results are consistent with the known cerebral distribution of mAChR in primates. [11C]QNB specific cerebral binding was enhanced by pretreatment with methyl-QNB, a peripherally acting muscarinic antagonist. Specifically labeled binding sites alone were blocked by prior administration of dexetimide, a muscarinic antagonist. Specific radioactivity was driven out from mAChR-rich regions by atropine and dexetimide, drugs with high affinity for mAChR. This competition was stereospecific since only dexetimide, the pharmacologically active isomer of benzetimide, was able to compete with the radioligand on its binding sites. A relationship between the occupancy of [11C]QNB-labeled receptors by atropine or dexetimide and the concomitant induction of a pharmacological effect was also detected by simultaneous PET scanning and electroencephalographic recording. Since mAChR form an important part of choline receptors in the central nervous system, [11C]QNB appears to be a suitable radiotracer to monitor cerebral physiological or pathological phenomena linked to the cholinergic system in living subjects.

The effect of mergocryptine, a new ergot alkaloid, on the cerebral dopaminergic systems was examined using Wistar rats. The administration of mergocryptine (1 and 10 mg/kg i.p.) induced a significant suppression of striatal dopamine (DA) turnover. In vitro addition of mergocryptine (0.01-100 microM) induced a dose-dependent suppression of the release of [3H]DA from striatal slices. Mergocryptine inhibited [3H]apomorphine binding to a striatal synaptosomal fraction, and its IC50 value was found to be 0.23 microM. Pretreatment with apomorphine (100 micrograms/kg s.c.) showed an additive effect on the mergocryptine (10 mg/kg)-induced suppression of DA turnover. These results suggest that mergocryptine may induce the suppression of striatal DA turnover by reducing DA release via the stimulation of presynaptic dopaminergic autoreceptors.

The neurotransmitter acetylcholine is thought to modulate epithelial ion transport in the mammalian small intestine. In this study, the non-selective acetylcholine agonist, carbachol, produced rapid changes in short-circuit current (Isc), an electrical measure of active anion secretion, across isolated sheets of the distal jejunal mucosa-submucosa from swine. The potency of carbachol in elevating Isc was decreased 26-fold in the presence of 10 nM atropine, but remained unchanged by 1-100 microM hexamethonium or 0.1 microM tetrodotoxin. The acetylcholine antagonists produced little or no decrease in spontaneous Isc, whereas tetrodotoxin decreased Isc by 54 microA/cm2. [3H]Quinuclidinyl benzilate (QNB), a muscarinic acetylcholine receptor blocker, bound to a single species of sites within the mucosa-submucosa with a KD = 38 pM and Bmax = 94 fmol/mg protein. Selective muscarinic acetylcholine receptor blockers competed with [3H]QNB for this site with a rank order of affinity indicative of an interaction with a M3-muscarinic acetylcholine receptor. Specific [3H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa and muscularis propria. Transmural electrical stimulation (3-600 pulses/10 s, 0.5 ms, 60 V) of the mucosa-submucosa produced increases in Isc which were dependent upon the number of impulses delivered and did not undergo tachyphylaxis upon repeated stimulation. Responses to electrical transmural stimulation were inhibited by atropine and hexamethonium, as well as the respective neuronal Na+ and Ca2+ channel blockers tetrodotoxin and omega-conotoxin GVIA, suggesting that electrical transmural stimulation depolarizes submucosal cholinergic neurons which terminate on ion-transporting epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats were treated with 10 mg/kg tetrahydroaminoacridine (THA) twice daily for 14 days. THA (10 mg/kg) induced a significant decrease in the number of muscarinic receptors (both M1 and M2) in the cortex and striatum, whereas the number of nicotinic receptors in the cortex and hippocampus increased. Rats treated with physostigmine (0.9 mg/kg) showed a reduced number of muscarinic receptors, but no change in nicotinic receptors. The results indicate that treatment with cholinesterase inhibitors can induce opposite changes in brain muscarinic and nicotinic receptors in vivo.

Membrane preparations of guinea-pig lung (containing multiple muscarinic receptor subtypes) and heart (containing M2 receptors only) were incubated with either neuraminidase, parainfluenza virus (which contains neuraminidase), or virus plus 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a neuraminidase inhibitor. None of these treatments affected [3H]quinuclidinyl benzilate ([3H]QNB) binding. In the lung and heart, carbachol displaced 0.2 nM [3H]QNB from two sites. After treatment with either neuraminidase or virus the high affinity site was shifted to the right, and carbachol displaced QNB from one site with low affinity in the lung. In contrast, neuraminidase or virus decreased the affinity of carbachol for both sites in the heart. The neuraminidase inhibitor completely blocked virus-induced changes in carbachol affinity in both tissues. These results suggest that parainfluenza virus decreases the affinity of agonists for some of the muscarinic receptors in the lung, and for all of the muscarinic receptors in the heart due to its neuraminidase activity, which results in removal of sialic acid. The decreased agonist affinity in the lung may be responsible for the increased vagally induced bronchoconstriction seen in viral respiratory infections.

In human neuroblastoma SH-SY5Y cell preparations, sigma(1) receptor agonists (+)-pentazocine and 1S,2R-(-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) competed for [3H]haloperidol binding with K(i) values of 67+/-10 and 14+/-10 nM, respectively. (+)-Pentazocine or BD737 up to 10 microM did not affect basal levels of intracellular Ca(2+) concentration ([Ca(2+)](i)) in these cells, but they significantly reduced muscarine-induced [Ca(2+)](i) changes in a dose-related manner. However, the reduction by (+)-pentazocine was not reversed by the sigma(1) receptor antagonist haloperidol. Further studies showed (+)-pentazocine, BD737 and haloperidol could compete for [3H]quinuclidinyl benzilate binding in SH-SY5Y cells with K(i) values of 0.51+/-0.06, 0.32+/-0.07 and 4.4+/-2.3 microM, respectively. Thus, the inhibitory effects on muscarine-induced [Ca(2+)](i) changes by (+)-pentazocine and BD737 in SH-SY5Y cells were likely due to blockade of muscarinic receptors, not due to sigma(1) receptor activation by these ligands.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin–cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin–cholate, n-dodecyl-β- d -maltoside or Lubrol-PX. The degree of coprecipitation in digitonin–cholate was unaffected by preincubation of the extract for up to 60 min at 30°C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

A hypotheses that mitoxantrone is a competitive antagonist at muscarinic cholinergic receptors was examined in guinea-pig hearts. In isolated left atrial muscle preparations, electrically paced at 2 Hz, the muscarinic agonist, carbachol, caused a concentration-dependent decrease in developed tension. Mitoxantrone caused a parallel right-ward shift of the concentration–response curve for carbachol. Schild plots for the effect of mitoxantrone on the carbachol concentration–response relationship were linear with a slope of 0.88 which was not significantly different from the unity. The right-ward shift of the carbachol concentration–response relationship by mitoxantrone significantly reversed after an additional incubation with a mitoxantrone-free solution, although the reversal was incomplete after a 2-h incubation in the mitoxantrone-free solution. Mitoxantrone caused a concentration-dependent displacement of specific [3H]quinuclidinyl benzilate binding to membrane preparations obtained from ventricular muscles of guinea-pig hearts. These results indicate that mitoxantrone acts as a competitive antagonist for the muscarinic receptors.

Experimental probing of the model of the muscarinic M2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [3H] N-methylscopolamine and [3H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [3H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (< 10-fold) to the binding site of the M2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M2 receptor, as predicted by the proposed model.

Muscarinic receptor subtypes mediating carbachol-induced contractions of the rat prostatic smooth muscle were determined. The rank order of potency of muscarinic receptor antagonists in blocking the effects of carbachol was (mean pKB estimates in parentheses): atropine (8.90) >> para-fluorohexahydrosiladifenidol (7.75) > or = hexahydrosiladifenidol (7.62) > methoctramine (6.89) > or = pirenzepine (6.68) > or = himbacine (6.67). The specific binding of [3H]quinuclidinyl benzilate to the rat prostatic homogenates was competitively inhibited by (mean pKi values in parentheses): atropine (8.89) >> hexahydrosiladifenidol (7.86) > para-fluorohexahydrosiladifenidol (7.28) > or = himbacine (7.22) > pirenzepine (6.63) > or = methoctramine (6.38). These profiles, whilst different, indicate the probable involvement of muscarinic M3 receptors in the carbachol-induced contraction.

The muscarinic receptor antagonist methyl-quinuclidinyl-benzylate decreased myocardial beta-adrenoceptor density Bmax: 20.4 +/- 2.4 pmol/ml tissue versus 33.3 +/- 4 pmol/ml tissue in control dogs (P < 0.001), as assessed by using [11C]CGP-12177 (((2S)-4-(3-t-butyl-amino-2 hydroxypropoxy)-benzimidazol-2-one)) and positron emission tomography. In contrast, atropine did not induce any change in Bmax: 33.7 +/- 3.6 pmol/ml tissue. We hypothetized that methyl-quinuclidinyl-benzylate induced the release of norepinephrine from sympathetic nerve terminals, an effect which could be blocked by guanethidine. Guanethidine alone (10 mg/kg) did not change Bmax: 35.5 +/- 6 pmol/ml tissue. Guanethidine + methyl-quinuclidinyl-benzylate did not induce any significant change in Bmax: 31.5 +/- 5.1 pmol/ml tissue. Therefore, it seems likely that methyl-quinuclidinyl-benzylate acts at the presynaptic level, probably inducing the release of norepinephrine which then causes a down-regulation of beta-adrenoceptors.

It was previously found that alcuronium increases the binding of [3H]methyl-N-scopolamine to cardiac muscarinic receptors by a positive allosteric action while its effect on the binding of [3H]quinuclidinyl benzilate is negative. The, features of the antagonist's molecule which decide whether its allosteric interaction with alcuronium is positive or negative are not known. In the present work, it was found that alcuronium has a positive allosteric effect also on the binding of [3H]atropine and [3H]methyl-N-piperidinyl benzilate to muscarinic receptors in rat heart atria and that its effect on the binding of [3H]methyl-N-quinuclidinyl benzilate is negative. A comparison of the five radiolabelled antagonists that have been investigated so far indicates that the type of allosteric interaction (positive or negative) is not determined by the presence or absence of the quaternary nitrogen or of the benzilyl moiety in the molecule of the antagonist. Apparently, features of the N-bearing moiety of muscarinic antagonists other than the presence of a charge on nitrogen play a key role in the determination of the type of interaction.

Murine fibroblast cell lines stably transfected with the rat muscarinic m1 or m2 receptor genes were used to study the homologous regulation of the muscarinic M1 or M2 receptors. The cells were pretreated with the muscarinic receptor agonists, (+)-cismethyl-dioxolane, carbachol, 2,8-dimethyl-3-methylene-1-oxa-8-aza-spiro- [4,5]decane ((+) or (-)-YM796) or the muscarinic receptor antagonist atropine for up to 24 h. Our study has demonstrated that the muscarinic receptor nonselective full agonist, (+)-cismethyl-dioxolane, induced the down-regulation of both the muscarinic M1 and the M2 receptors in association with desensitization of the receptor-mediated functions. The muscarinic M1 receptors are down-regulated without significant receptor internalization while the muscarinic M2 receptors are more sensitive to down-regulation than the muscarinic M1 receptors because of significant internalization of the muscarinic M2 receptors in our system. The muscarinic M1 receptor partial agonist, (-)-YM796 induced less down-regulation and no significant desensitization of the muscarinic M1 receptors with no substantial effect on the muscarinic M2 receptor density or function.

The binding of the bisquaternary muscarine receptor antagonist heptane-1,7-bis(dimethyl-3′-phthalimidopropyl)-ammonium bromide (C 7 /3-phth) was investigated at a number of cholinergic binding sites using (−)-[ 3 H]nicotine, [ 3 H]pirenzepine and (−)-[ 3 H]quinuclidinyl benzilate ([ 3 H]QNB) in both central and peripheral tissues. C 7 /3-phth displayed an affinity for muscarine M 2 receptors in rat atria (70.1 nM) which was 1.6-fold greater than for putative M 4 receptors in rabbit lung, and 4- to 5-fold greater than for M 1 receptors in rat cerebral cortex. Its affinity for nicotine receptors in the cortex was low, being 808-fold lower than its affinity for the M 2 receptor. Although the displacement of (−)-[ 3 H]nicotine and [ 3 H]pirenzepine binding in rat cortex by C 7 /3-phth was best described in terms of one-site modelling, low Hill coefficients were observed with C 7 /3-phth in displacement studies using [ 3 H]QNB in this tissue. The possibility of allosteric interactions or multiple receptor subtype interactions is discussed.

The number of muscarinic receptors in the striatum and hippocampus of aged rats is significantly lower than the number measured in young animals. The treatment of aged rats for 30 days with S-adenosyl-L-methionine (SAM) restored the number of muscarinic receptors to levels found in the striatum and hippocampus from young animals. We did not observe a clear-cut difference between the dissociation constants of untreated young and untreated or SAM-trcatcd aged rats, whereas the binding capacity varied. Moreover, in vitro addition of SAM to hippocampal membranes from aged rats resulted in a significant increase in the number of binding sites. This in vitro effect was antagonized by S-adenosyl-L-homocysteine, a specific in vitro inhibitor of methyltransferase activity. The reduction in the muscarinic receptor density could be related to a decrease in neuronal membrane fluidity induced by aging, while its increase after SAM treatment might be ascribed to the ability of this methyl donor to increase the fluidity of cell membranes by stimulating phospholipid synthesis.

While studying the mechanisms of doxorubicin-induced cardiotoxicity, we observed that doxorubicin inhibited the negative inotropic effect of acetylcholine in isolated heart muscle preparations. We therefore examined the effects of doxorubicin on muscarinic acetylcholine receptors. In left atrial muscle preparations isolated from guinea pig heart and stimulated at 2 Hz at 30°C, doxorubicin caused a parallel right-ward shift of the dose-response curves for the negative inotropic effects of acetylcholine. The inhibitory action was reversed by an additional incubation in the absence of doxorubicin. Doxorubicin reversed the carbachol-induced inhibition of developed tension: a high concentration of doxorubicin brought the force back to its original strength. Doxorubicin inhibited specific [3H]quinuclidinyl benzilate (QNB) binding to membrane preparations obtained from ventricular muscle of guinea pig hearts. The pA2 value for doxorubicin obtained in the inotropic study corresponded to the IC50 value for doxorubicin observed in the [3H]QNB binding assay. These results indicate that doxorubicin acts as a weak competitive antagonist on muscarinic acetylcholine receptors.

In human astrocytoma cells (131N1), trifluoperazine potently inhibited histamine-induced phosphoinositide hydrolysis, while it slightly inhibited carbachol-induced phosphoinositide hydrolysis. Trifluoperazine as well as diphenhydramine inhibited [ 3 H]mepyramine binding in astrocytoma cell membranes with K i values of 0.052 μM and 0.005μM, respectively. However, trifluoperazine only slightly inhibited [ 3 H]quinuclidinyl benzilate binding with a K i value of 3 μM. These results indicate that trifluoperazine specifically inhibits histamine-induced phosphoinositide hydrolysis as a histamine H 1 -receptor antagonist in human astrocytoma cells.

The effect of U-50,488H, a selective kappa-opioid agonist, on carbachol-stimulated phosphoinositide (PI) turnover response in rat hippocampal slices was examined. U-50,488H which stimulates PI turnover response in this preparation (Periyasamy and Hoss, 1990, Life Sci. 47, 219), inhibited carbachol-stimulated PI turnover in a concentration-dependent manner with an IC50 value of 33±9.0 μM. The inhibitory effect of U-50,488H was not blocked by the kappa-selective antagonists, e.g., nor-binaltorphimine (10 μM), and MR2266 (10 μM), or tetrodotoxin (1 μM) suggesting that the effect of U-50,488H was mediated neither through the kappa-receptors nor through the release of an endogenous neurotransmitter(s). A Lineweaver-Burke plot of the stimulation of PI turnover by carbachol in the presence and absence of U-50,488H showed that the Km was not changed (11.4±3.4 and 11.5±2.6 μM) whereas the Vmax was reduced from 3849±460 to 1534±31 cpm indicating that the inhibition was non-competitive. U-50,488H also inhibited guanosine 5′-[β,γ-imido]triphosphate (Gpp[NH]p)-stimulated PI turnover in rat hippocampal membranes in a concentration-dependent manner with an IC50 value of 33±12 μM. However, U-50,488H had no effect on exogenously added phospholipase-C-stimulated PI turnover. In receptor binding studies. U-50,488H displaced the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) binding in a concentration-dependent manner with an IC50 value of 4.5±1.5 μM. A Scatchard plot of [3H]QNB binding in the presence and absence of U-50,488H showed that the Kd was changed from 883±250 to 364±147 pM whereas the Bmax remained the same (1.66±0.16 and 1.65±0.18 pmol/mg protein) suggesting that the inhibition was competitive. These data indicate that the inhibitory effect of U-50,488H on carbachol-stimulated PI turnover was mediated through the interaction with the G-protein but not through kappa-receptors or release of neurotransmitter(s).

ACTH-(1-24) decreased the binding of the dopamine D 2 receptor agonist, [ 3 H]N-propylnorapomorphine ([ 3 H]NPA), to rat striatal membranes in a concentration-dependent manner, with a K i of 5 × 10 −7 M. Saturation curves for [ 3 H]NPA binding in the presence of increasing concentrations of ACTH-(1-24) were performed. Scatchard analysis in the presence of ACTH-(1-24) revealed an increased dissociation constant (K d ), while the binding capacity (B max ) was not affected by the peptide, suggesting an apparent competitive interaction between ACTH-(1-24) and [ 3 H]NPA. ACTH-(1-24) also reduced the binding of the dopamine D 2 receptor antagonist [ 3 H]spiperone to striatal membranes, with a K i of 10 −6 M. Much higher concentrations of ACTH-(1-24), up to 10 −4 M, were needed for the displacement of appropriate radiolabelled ligands from dopamine D 1 receptors, serotonin 5-HT 1A , serotonin 5-HT 1B , muscarinic M 1 acetylcholine and histamine H 1 receptors. ACTH-(1-24) also inhibited the binding of [ 3 H]spiperone to dopamine D 2 receptors in membranes of the pituitary gland, the septum and the substantia nigra. ACTH-(1-39) and most ACTH fragments and analogs were less potent than ACTH-(1-24) in displacing [ 3 H]NPA from the dopamine D 2 receptor in striatal membranes. In general there was a relationship between displacing potency and chain length. ACTH-(7-16)-NH 2 and benzyloxycarbonyl-ACTH-(8-16)-NH 2 , however, were more potent than ACTH-(1-24) in reducing the binding of [ 3 H]NPA to dopamine D 2 receptors. ACTH-(7-16)-NH 2 appeared to contain the minimal required amino acid sequence for inhibition of [ 3 H]NPA binding, because a further shortening of the peptide resulted in a marked decrease of inhibitory potency. The present data show that ACTH/MSH-like peptides preferentially interact with dopamine D 2 receptors.

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [ 3 H]Quinuclidinyl benzylate ([ 3 H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (K d ) of 0.41 ± 0.3 nM in WKY rats and of 0.37 ± 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [ 3 H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [ 3 H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.

The effect of a long-term exposure (5 days) to atropine on muscarinic acetylcholine receptors and receptor-coupled second messenger systems was investigated using mouse cerebral cortical neurons in primary culture. The long-term exposure of neurons to atropine (10 nM) induced increases in both the Bmax and Kd values of [3H]quinuclidinyl benzilate (QNB) binding to muscarinic acetylcholine receptors. Alterations in muscarinic receptor-coupled second messenger systems, such as phosphoinositide (PI) hydrolysis and cyclic GMP (cGMP) formation following a long-term exposure to atropine, were also examined. Carbachol-stimulated PI hydrolysis was found to be decreased by the exposure to atropine in spite of the increase of muscarinic receptors. In adduiton, a long-term exposure to atropine had no effect on carbachol-stimulated cGMP formation as well as on the rightward shift of the carbachol competition curve of [3H]QNB binding in the presence of GTP. These results suggest that the up-regulation in muscarinic cholinergic receptors induced by long-term exposure to atropine may involve not only the increase in number of muscarinic receptors but also the decreased responsiveness in muscarinic receptor-coupled second messenger systems.

Muscarinic acetylcholine receptors were studied in human labial salivary glands with (-)-[3H]MQNB. The radioligand bound with a Kd value of 150 pM. The density of muscarinic receptors was 220 fmol/mg protein. Most muscarinic antagonists bound to a homogeneous population of muscarinic receptors in this tissue. However, the inhibition curves of pirenzepine had a Hill slope of 0.57 and could be analyzed by a two site model. These results suggest that the muscarinic receptors in human labial salivary glands are a mixture of M1 and M3 types.

It was of interest to determine the binding characteristics of some neuroleptics too the various muscarinic receptor subtypes to see if a selectivity for or potency at a particular muscarinic subtype might distinguish atypical from typical neuroleptics

In order to explore the possibility of heterogeneity in peripheral muscarine receptors, receptors were characterized in membrane fractions isolated from rabbit urinary bladder and submandibular gland. With [3H]QNB as radioligand, specific binding with very high affinity was found in both preparations. Although the BmaxS for binding in the two preparations were very similar, the mean KD value in the submandibular gland was significantly higher (P less than 0.005) than that in the bladder. Among the anticholinergic drugs, oxybutynin had a significantly lower value for the inhibition constant (Ki) in the submandibular gland whereas Ki for both secoverine and pirenzepine was significantly higher in this tissue than in urinary bladder. The Ki for carbacholine was about 7-fold higher in submandibular gland than in the bladder. Although quinidine and verapamil showed relatively weak binding to the muscarine receptor site, their Ki in the submandibular gland was significantly higher than that in the bladder. The results indicate that although there is a considerable similarity between muscarinic receptors in urinary bladder and submandibular gland, the differences in Ki values for different compounds in the two tissues support the argument favouring heterogeneity of muscarinic acetylcholine receptors in peripheral effector organs.

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 quinuclidinyl benzilate (QNB) binding to membrane fractions of rat and human brain, on the carbamoylcholine-stimulated accumulation of inositol phosphates in rat cerebral cortex and on 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.

Synaptosomal membranes from cat cerebral cortex were labelled with [3H]-quinuclidinyl benzylate ([3H]QNB). There was shown to be a single type of binding site with Kd = 0.34 nM, Bmax = 2.2 nmol/g protein and Hill No. = 1.01. Triton X-100 at 5 x 10(-4)% inhibited the specific binding of [3H]QNB and the inhibition was almost complete at 10(-2)%. Treatment with 2.5 x 10(-6) M atropine, followed by centrifugation washings protected the receptor site from the inhibitory action of the detergent. The protection afforded by other cholinergic drugs was less effective. The use of this technique has confirmed the results of our previous work on the possible pre- and postsynaptic location of central muscarinic receptors. These findings open the possibility for protection of other detergent-sensitive receptors and for their isolation and purification as well-defined macromolecules.

In the present studies, we investigated the binding properties of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) to muscarinic acetylcholine receptors (mAChR) in rat brain homogenates. Our results indicate that the hydrophilic receptor ligand, [3H]NMS, is able to interact with high affinity only with a fraction of the receptor sites available to the lipophilic ligand, [3H]QNB. Furthermore, displacement experiments demonstrated that while both unlabeled QNB and NMS displaced [3H]NMS binding according to the law of mass-action, NMS, but not QNB, displayed binding heterogeneity when [3H]QNB was used as a ligand. Our data suggest that the lipid solubility of a particular mAChR ligand might play an important role in determining its profile of binding to the receptor.

Dispersed acini isolated by collagenase digestion of the rat submandibular gland were used to compare the effects of amiloride and furosemide on the uptake of the isotopic tracer 22Na and on the binding of [3H]quinuclidinyl benzylate ([3H]QNB). In mM concentrations, both inhibitors reduced 22Na uptake in resting cells 34 and 25-29%, respectively. Acetylcholine (1 microM) enhanced uptake 23% and this effect was reduced 45% by amiloride and 26% by furosemide. Amiloride inhibited the binding of [3H]QNB to crude membranes prepared from fresh submandibular glands in a dose-dependent fashion (IC50 = 8 x 10(-6) M). Furosemide (3 x 10(-8) to 10(-3) M) did not inhibit radioligand binding. Na influx into resting salivary acini thus appears to occur by both amiloride-sensitive and furosemide-sensitive transport systems. The similar inhibition by furosemide of unstimulated and stimulated uptake of 22Na suggests that acetylcholine does not significantly activate the cotransport system within the time frame (i.e., 2 min) of the experiments. Acetylcholine appears to activate an amiloride-sensitive Na/H antiport, but amiloride blocks cholinergic receptors and may thus affect Na transport by receptor blockade. Other actions of amiloride, such as its ability to penetrate into cells and to act as a weak base which alters intracellular pH, may also contribute to the inhibition of Na entry into salivary cells.

The effects of a GTP analogue, guanyl-5′-yl imidodiphosphate, Gpp(NH)p on the inhibition of [ 3 H]3-quinuclidinyl benzilate (QNB) binding by some partial agonists were studied with a microsomal fraction of longitudinal muscle of guinea-pig ileum and compared with the intrinsic activity of the partial agonists which was estimated in the longitudinal muscle of guinea-pig ileum. There was a good correlation between the inhibitory effects of Gpp(NH)p on the affinity of the partial agonists to muscarinic receptors and their intrinsic activity.

Using in vitro autoradiography the distribution of [ 3 H]rauwolscine, [ 3 H]prazosin and [ 3 H]quinuclidinyl benzilate binding sites has been demonstrated in cardiac tissue taken from the cat and rat. A similar distribution of both α 1 - and α 2 -adrenoceptor sites was seen but the distribution of muscarinic acetylcholine sites was markedly different. α-Adrenoceptors were present predominantly in ventricular muscle whereas muscarnic acetylcholine receptors exhibited a greater density in atrial tissue compared to ventricular muscle. Opiate receptors were absent from cardiac tissue.

The centrally acting anticholinesterase drug tacrine has been shown to block K + channels in guinea pig left atrium. It competitively blocks the negative inotropic effects of adenosine, 2-chloroadenosine and carbachol. K a values obtained from dose ratio plots were 2.5, 3.5 and 2.9 μM respectively. It was also able to antagonize the shortening of the action potential due to these compounds. Doses of tacrine ranging from 1 to 4 μM restored the AP configuration close to control values. Tacrine also antagonized the binding of 1-quinuclidinyl[phenyl-4- 3 H]benzilate ([ 3 H]QNB) to membranes derived from the atrium and cerebral cortex. K i values of 1.8 ± 0.33 and 1.3 ± 0.47 μ M were obtained respectively. Tacrine was a weak competitor of [ 3 H]phenylisopropyladenosine ([ 3 H]L-PIA) binding in brain membranes. Its diverse pharmacological effects may be relevant to its use in Alzheimer's disease.

The quaternary antagonist gallamine discriminates subpopulations of muscarinic receptors in the forebrain and brainstem of the rat and also modulates the binding of other muscarinic ligands via an allosteric mechanism in both brain regions. The calcium channel antagonist verapamil exhibits a similar allosteric effect in both regions (at 100 μM) but does not differentiate the subpopulations that are defined by gallamine. On the other hand, N-methylatropine does not produce the allosteric effect at 100 μM, but shows a high degree of selectivity (about 1000-fold) for the gallamine-defined subpopulations. These studies indicate that the allosterism and subpopulation specificity of gallamine are separate phenomena and may be examined in isolation by appropriate ligands.

Both the enantiomers of bethanechol displaced R(-)-[3H]quinuclidinyl benzilate from muscarinic binding sites in homogenates from rat jejunum, nasal mucosa, atrial and ventricular myocardium. Stereoselectivity of receptor binding for S(+)- and R(-)-bethanechol was found in all tissues investigated (Ki S(+) less than Ki R(-)). Different proportions of high and low affinity binding sites for bethanechol enantiomers and S(+)-methacholine in jejunal smooth muscle, atrial and ventricular muscle and the presence of only low affinity binding sites in nasal mucosa point to differences in receptor-effector coupling in these tissues. The relative insensitivity of cardiac tissue for bethanechol compared to methacholine might be related to the much smaller proportion of high affinity binding sites found for S(+)-bethanechol in heart ventricular muscle.

The mechanism of agonist-induced degradation of muscarinic cholinergic receptors (mAChRs) was examined by means of [ 3 H]QNB binding by organ-cultured guinea-pig vas deferens. Long-term exposure to ACh decreased the surface mAChR. This so-called ‘down-regulation’ of mAChR was significantly inhibited by anti-endocytotic drugs. Anti-microtubular agents also strongly inhibited the decrease of mAChR remaining on the cell surface. Ammonium chloride and protease inhibitors, which are known to inhibit lysosomal enzymes, had little effect on the decrease and no increase in intracellular mAChR could be detected under these conditions. Chloroquine blocked the decrease in mAChR remaining on the cell surface. Based on these findings, the involvement of clustering and endocytosis in mAChR degradation were considered as possibilities. Furthermore, contraction of the smooth muscle to ACh in long-term desensitization was also examined in relation to the number and nature of the receptors. When the muscles were cultured with ACh and chloroquine or vinblastine, there were indications that the surface mAChR and the contractile system were uncoupled.

The nicotinic acetylcholine receptors in the rat stomach were characterized by means of a radioligand binding assay with (−)-[ 3 H]nicotine as ligand. Saturation binding studies on the gastric fundus membranes revealed the presence of two binding sites with dissociation constant (K D ) values of 3.1 and 289 nM, and maximum binding capacity (B max ) values of 3.6 and 76 fmol/mg protein, respectively. The B max of the high affinity binding site was greatest in the cardia, followed by fundal mucosa, fundal muscle, and, finally antrum. The IC 50 values of cholinergic drugs to inhibit (−)-[ 3 H]nicotine binding in fundus membranes were as follows: (−)-nicotine, 0.12 nM; cytisine, 9.3 nM; acetylcholine, 17.7 nM; carbachol, 700 nM; hexamethonium, 2270 nM. The IC 50 values of α-bungarotoxin, d-tubocurarine and atropine were greater than 100 μM. The muscarinic acetylcholine receptors were also characterized with [ 3 H]quinuclidinyl benzilate and the choline acetyltransferase activity was measured. These results suggest that nicotinic acetylcholine receptors as well as muscarinic acetylcholine receptors are present in the rat stomach and that the regional distribution of these receptors is uneven.

The binding of hexahydrosiladifenidol, procyclidine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) and AF-DX 116 to muscarinic receptors in the heart, ileum, urinary bladder, parotid gland and cerebral cortex from guinea pig was studied in competition experiments with (-)-[3H]QNB. The affinity of AF-DX 116 was higher in the heart than in the cortex and it was extremely low in the parotid gland. The affinities of hexahydrosiladefinidol, procyclidine and 4-DAMP were higher in the cortex and parotid gland than in the heart, bladder and ileum. Hexahydrosiladifenidol and 4-DAMP recognized two classes of muscarinic binding sites in the cortex. However, in contrast to functional data, binding results showed that 4-DAMP hexahydrosiladifenidol and procyclidine did not distinguish between the sites in the smooth muscles and those in the heart. Nevertheless, the present data support the view that the putative M2-receptors are heterogeneous, since the four drugs examined were found to distinguish between the muscarinic binding sites in the parotid gland and those in smooth muscles and heart.

The question of cardiac muscarinic receptor heterogeneity was studied in guinea-pig auricles and ventricle. Radioligand dissociation kinetics were analyzed in the absence and presence of alinidine and AQ-A 39, two muscarinic agents and allosteric modulators of radiotracer dissociation. The dissociation kinetics were monophasic with all 3 radiotracers used and in both auricles and ventricle. [125I]3-Quinuclidinyl 4-iodobenzilate ([125I]QNB) dissociated with an identical half-life (t1/2 off) in ventricle and in left and right auricle, respectively. Alinidine (1 mM) decreased t1/2 off by 50% in ventricle but had no significant influence in auricles. AQ-A 39 (= falipamil) uniformly increased t1/2 off 4-fold in all 3 tissues. The binding of tritium-labelled quinuclidinyl benzilate ([3H]QNB) dissociated equally rapidly from both ventricular and auricular receptors under control conditions but was not affected by alinidine. AQ-A 39 (1 mM) slowed [3H]QNB dissociation 6.5-fold in ventricle but only 3-fold in both auricles. [3H]N-Methyl scopolamine ([3H]NMS) dissociation was uniform in both auricles but was somewhat slower in the ventricle, both in the absence and presence of alinidine. AQ-A 39 was without effect. These results demonstrate differences in ventricular versus auricular receptors detected by [125I]QNB in the presence of alinidine and by [3H]QNB in the presence of AQ-A 39. The quaternary ligand [3H]NMS was unable to detect receptor heterogeneity. No differences were found between right and left auricular receptors. The results presented provide no evidence for the existence of different subtypes of muscarinic receptors for the negative chronotropic and inotropic actions of muscarinic agonists.

Rats were treated with ethanol in the drinking fluid 2 X 1 h daily for 83 weeks. [3H]Muscimol, [3H]quinuclidinyl benzilate ( [3H]QNB) and [3H]nicotine binding was measured in selected brain areas 7, 14 and 21 days after withdrawal of ethanol. A significant increase (P less than 0.05) when compared to controls was found on day 7 in both [3H]-QNB binding in the cortex and high affinity [3H]muscimol binding in the cerebellum. Furthermore, on day 7 of abstinence there was a positive correlation in number of binding sites between [3H]muscimol (high affinity sites; cerebellum) and [3H]QNB (cortex) while a negative correlation was found between [3H]muscimol (low affinity sites; cerebellum) and [3H]nicotine (cortex). The first correlation might indicate a relation between increased excitation (QNB) and increased inhibition (muscimol) in two brain areas.

Humoral (H) endorphin, a novel endogenous factor with opiate-like activity was further characterized using various radioreceptor assays. H-Endorphin displaced both labelled opiates (morphine, ethylketazocine) and opioid peptides (leucine-enkephalin) from their specific binding sites in rat brain membranes. The effect of sodium ions on H-endorphin indicates its agonistic nature which is in agreement with previous pharmacological experiments. However, H-endorphin potentiated the binding of the opiate antagonist naloxone to brain membranes. This peculiar effect of H-endorphin on naloxone, together with the non-conventional interactions observed in various pharmacological assays, clearly distinguishes H-endorphin from other opioid ligands. A multisite model of the opiate receptor is presented. This model, which is compatible with the well documented evidence for receptors heterogeneity also explains non-conventional interactions between various opioid ligands.

Organ cultures of guinea-pig vasa deferentia were used for studying both the decrease in muscarinic cholinergic receptor (mAChR) concentration induced by muscarinic agonists and the simultaneous decrease in contraction to ACh. After a lag period the decrease followed first-order kinetics and was completely blockd by atropine. The decrease was dependent not only on the extent of occupation of mAChR by ACh, but was also related to the efficacy of agonists as deduced from the contractions. Moreover the amount of mAChR was not affected by other contractile agents, norepinephrine and high K+ even under conditions in which mAChR was occupied by atropine or pilocarpine. These findings indicate that the decrease in the amount of mAChR mediated by muscarinic receptors in vas deferens was due to the extent of both receptor occupation and receptor activation by agonists. Furthermore it was shown that the decrease involved energy- and temperature-dependent processes and that cyclic nucleotides did not regulate the quantitative level of mAChR.