Selectivity of pirenzepine in the central nervous system. III. Differential effects of multiple pirenzepine and scopolamine administrations on muscarinic receptors as measured autoradiographically

The effects of intrahippocampal injections of scopolamine and pirenzepine on muscarinic receptor binding were examined by quantitative autoradiographic techniques. Brain slices from animals which had received 7 injections of either scopolamine (n = 5) or pirenzepine (n = 5) over a 22-day injection schedule were compared with slices from 5 saline-injected controls for receptor binding to the whole slice and within selected regions of the brain as measured autoradiographically. The total number of receptors was determined from direct binding assays with 1-[3H]quinuclidinyl-benzilate ([3H]-1-QNB), while the binding of the selective ligands pirenzepine, carbamylcholine, and scopolamine was examined through inhibition studies. The data from the whole slices indicated that pirenzepine-treated animals contained more receptors for [3H]-1-QNB than either saline- or scopolamine-injected controls. Slices from the same animals also displayed a lower affinity for pirenzepine. Slices from scopolamine-injected animals revealed neither an increase in receptor number nor a decrease in antagonist affinity, although the binding of the agonist carbamylcholine was increased. Quantitative analysis of the autoradiograms generated from the slices indicated that the increase in receptor number for pirenzepine-injected animals was predominantly within the cerebral and cingulate cortices. The inhibition by pirenzepine was also lower in these areas in the same group of animals. Agonist inhibition was altered in the central layers of the cerebral cortex and in the pretectal area in scopolamine-treated animals. The results suggest separate mechanisms of drug action and adaptation for pirenzepine and scopolamine.     

Regulation of muscarinic receptors by intrahippocampal injections of gallamine.

Multiple intrahippocampal injections of gallamine impair performance of a representational memory task in rats. The binding of [3H]-(-)-quinuclidinyl benzilate (QNB) to rat brain sections was measured to determine if changes in receptor binding were associated with the deleterious effects of gallamine. [3H]-(-)-QNB binding to sections taken from gallamine-injected animals was compared with binding in saline-injected control animals. Autoradiographic analyses indicated an increase in [3H]-(-)-QNB binding sites within all layers of the cerebral cortex and in the superior colliculus in gallamine-treated animals as compared to saline-injected controls. Significant increases were noted in cortical layers IV and V (P less than 0.025) in gallamine-treated animals. No significant changes (P greater than 0.05) in the number of binding sites were observed in the hippocampus, neostriatum or various thalamic nuclei. The ability of unlabeled pirenzepine, gallamine and carbamylcholine to inhibit 0.2 nM [3H]-(-)-QNB binding also was measured to determine changes in the distribution of receptor subtypes. No significant changes were observed in any brain region for the binding of the selective antagonists pirenzepine and gallamine or the agonist carbamyl-choline. Although other possibilities are considered, the data suggest that an increase in the number of muscarinic receptors may contribute to the observed behavioral deficits associated with long-term gallamine treatment.     

Effects of serotonergic denervation on the density and plasticity of brain muscarinic receptors in the rat.

This study investigated whether serotonergic lesion may affect density, sensitivity, and plasticity of muscarinic receptors in hippocampus and cerebral cortex. Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) in rats produced a 90% reduction in cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents. In these brain areas, the 5,7-DHT lesion did not affect the overall density of muscarinic receptors or those of M1 and non-M1 muscarinic receptor subtypes as assayed using [3H]N-methylscopolamine ([3H]NMS), [3H]pirenzepine, and [3H]NMS in the presence of pirenzepine, respectively. In addition, the binding of the muscarinic agonist [3H]oxotremorine-M (OXO-M), taken as an indirect index of coupling efficiency of non-M1 receptors with G-proteins, did not change significantly in cortex and hippocampus of 5,7-DHT-lesioned rats. Similarly, carbachol-induced accumulation of [3H]inositol phosphates (InPs) in hippocampal miniprisms showed no significant differences between tissues from 5,7-DHT-lesioned and sham-operated rats. In sham-operated rats, an intraperitoneal (i.p.) injection of scopolamine (10 mg/kg once daily) during 21 days caused an increased density of [3H]NMS binding sites in cortex (+20%) and hippocampus (+26%). This up-regulation was restricted to non-M1 receptors subtypes. In 5,7-DHT-lesioned rats, chronic scopolamine failed to modify significantly the density of cortical or hippocampal M1 or non-M1 receptors. These results suggest 1) that 5-HT denervation did not affect the density and sensitivity of muscarinic receptors and 2) that the ability of cortical and hippocampal non-M1 receptors to up-regulate following repeated injection of scopolamine requires the integrity of 5-HT neurons terminating in these brain structures.     

Sensory deafferentation fails to modify muscarinic receptor binding in raccoon somatosensory cortex

The characteristics and distribution of muscarinic acetylcholine (mACh) receptor binding in primary somatosensory (SI) cortex and the caudate nucleus of raccoons were studied using [3H]-QNB, a muscarinic antagonist. The binding characteristics were similar to reported values in rat and cat. Autoradiographs produced from tissue sections labeled with [3H]-QNB showed the distribution of mACh receptors in the forebrain of the raccoon. [3H]-QNB binding was highest in cerebral cortex, neostriatum and hippocampus. Within SI cortex, binding was high in layers I-III and VI and relatively low in layers IV and V. Autoradiographs obtained from animals that had undergone peripheral deafferentation of part of the forepaw revealed no changes in [3H]-QNB binding in the affected cortical region during the time that physiological reorganization is known to occur.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[3H]methyldioxolane

(+)-cis-[3H]Methyldioxolane ((+)-[3H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[3H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[3H]CD states and binding sites for the muscarinic antagonists [3H]pirenzepine (r = -0.79) and (-)-[3H]quinuclidinyl benzilate (r = -0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.     

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.     

Muscarinic cholinergic receptor subtypes in normal human brain and Alzheimer's presenile dementia

Brain muscarinic M1 and M2 binding sites, as defined by their affinity for pirenzepine, were studied in 24 regions of 3 normal control brains. For each region, the binding of [3H]QNB, a non-subtype selective antagonist, was saturable with a mean KD of 0.117 +/- 0.066 nM. Computer-assisted analysis of the pirenzepine competition binding curves yielded the amount of high affinity (M1) and low affinity sites (M2) as well as their respective Ki values for this ligand. The neocortex contained a mixed population of 67% M1 and 33% M2 sites, without locoregional heterogeneity. The muscarinic receptors of the caudate nucleus, putamen, pallidum, hippocampus and nucleus amygdalis were predominantly of the M1 type as well. The cerebellum and the brain-stem are examples of regions containing over 90% M2 sites. White matter structures like the centrum ovale appeared to contain low concentrations of [3H]QNB binding sites, predominantly of the M1 subtype. The data for the frontal cortex were compared with data obtained in 3 patients who died from Alzheimer's disease with very early onset: the muscarinic receptor concentrations were somewhat lower but their M1/M2 ratio remained unchanged. GTP caused an appreciable rightward shift and steepening of the carbachol competition binding curve in M2 predominant regions such as the pons, whereas only a slight shift was observed in the cortex. In the presence of GTP, the alkylating reagent N-ethylmaleimide caused a 35-fold increase of the affinity for carbachol in M2 predominant regions. In contrast, regions where the receptors are predominantly of the M1 type, N-ethylmaleimide caused only a 5-fold increase in agonist affinity. These findings confirm our previously formulated hypothesis that the ability of N-ethylmaleimide to modulate the agonist affinity is an additional criterion for the characterization of M1 and M2-type receptors.     

Muscarinic receptors and receptor-mediated actions on rat thymocytes

Rat thymocytes possess a single class of saturable, high affinity binding sites for muscarinic antagonists of the benzilate type such as [3H]3-quinuclidinyl benzilate ([3H]3-QNB). The average number of receptors per cell is 3000 and the equilibrium dissociation constant of [3H]3-QNB on intact cells is 7.5 nM. In the work reported here we found that perturbation of the thymocyte membrane by addition of phytohemagglutinin (4 micrograms/ml) caused a transient increase in muscarinic antagonist binding, and hydrocortisone (100 mg/kg s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of muscarinic receptor sites on thymocytes by 100%. Atropine treatment, which in other tissues causes increased muscarinic receptor concentration, did not alter the receptor number on thymocytes. Binding of carbachol to the receptor on intact cells resulted in inhibition of cAMP synthesis and stimulation of cGMP synthesis. These muscarinic agonist effects were each inhibited by the simultaneous addition of the muscarinic antagonist atropine (5 X 10(-5) M). No stimulation of phosphatidylinositol turnover by muscarinic agonists was observed.     

Muscarinic cholinergic receptor subtypes in the rat brain. I. Quantitative autoradiographic studies

The binding characteristics of N[³H]methylscopolamine (³H]NMS) to slide-mounted tissue sections were studies using quantitative autoradiography. Binding of [³H]NMS was saturable, reversible of high affinity (K_d = 0.26nM). The inhibition of [³H]NMS binding produced by several muscarinic agonists and antagonists was analyzed in 29 discrete brain regions by constructing complete displacement curves. Comparison of IC₅₀ values obtained both biochemically and by autoradiography demonstrated to a very close agreement, supporting the validity of the autoradiographic approach. The competition curves for the agonists carbachol, oxotremorine and 2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion-hydrobromide (RS 86) fitted to a two-site model, with comparable affinity values from region to regions, although different proportions of high- and low-affinity sites were seen in the different areas studied. The distribution of high- and low-affinity sites was similar for the three agonists. Atropine showed monophasic curves presenting similar affinities in all regions studied. In contrast, pirenzepine differentiated between high- and low-affinity sites which showed a distribution opposite to that observed for the agonists. Gallamine, a ligand for a putative regulatory site in the muscarinic receptor, inhibited [³H]NMS binding in a biphasic manner. The calculated IC₅₀ values for the gallamine high- and low-affinity sites did not vary from region to region and the distribution of these sites correlated well with that observed for the agonists. High-affinity pirenzepine sites (also called M₁ sites) were localized mainly in forebrain areas, such as striatum, hippocampus and cortex, and their regional distribution correlated with that of the low-affinity sites for the agonists and gallamine. On the other hand, low-affinity sites for pirenzepine (named M₂ sites) were mainly found in the brainstem and parts of the thalamus. A good correlation was found between pirenzepine low-affinity sites and agonist and gallamine high-affinity sites. The significance of these findings is discussed in relation to the known and possible effects of selective M₁ and M₂ centrally acting agents.     

Characterization of [3H]AF-DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic-M2 receptor sites

This study shows that [³H]AF-DX 116 binds specifically, saturably, and with high affinity to putative muscarinic-M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [³H]AF-DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (K_d < 4.0 nM) and one class of lower affinity sites (K_d > 50 nM, except in the cerebellum). The apparent maximal capacities (B_max) of [³H]AF-DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [³H]AF-DX 116 binding sites was 10.4 ± 0.4 (K_d = 1.9 ± 0.2 nM) and 39.1 ± 2.6 (K_d = 26 ± 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the K_d for the higher and lower affinity [³H]AF-DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF-DX 116 is more potent than (–)QNB > atropine > methoctramine > 4-DAMP > gallamine > NMS > carbamylcholine > oxotremorine > pirenzepine > > nicotine in competing for the higher affinity [³H]AF-DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites. For example, (–)QNB was more potent than AF-DX 116 and pirenzepine was more potent than either oxotremorine or 4-DAMP at the lower affinity [³H]AF-DX 116 sites. In addition, pirenzepine was modestly more potent at the lower compared to the higher affinity sites. Neither the higher nor the lower affinity [³H]AF-DX 116 sites were sensitive to the effects of Gpp(NH)p or N-ethylmaleimide. In addition, the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding to the higher and the lower affinity sites was altered by Gpp(NH)p and NEM, to a similar extent. However, Gpp(NH)p decreased the affinity of carbamylcholine (i.e., increased the IC₅₀), whereas N-ethylmaleimide had the opposite effect. Furthermore, N-ethylmaleimide also appeared to steepen the curve for the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding, as evidenced by the increased n^H. Thus it appears that [³H]AF-DX 116 binds to two subsets of muscarinic-M2 receptors in the rat brain, which can be differentiated by their affinity for certain agonists and antagonists.     

Human M1-, M2- and M3-muscarinic cholinergic receptors: binding characteristics of agonists and antagonists

The muscarinic acetylcholine receptors were identified in membrane preparations from human tissues by the specific binding of 1-[benzilic-4,4'-3H] quinuclidinyl benzilate. Saturation binding isotherms of this radioligand yielded a total amount of receptors of 435 +/- 208, 159 +/- 65 and 913 +/- 89 fmol/mg protein, respectively, in the hippocampus, pons and submandibular gland. Non linear least squares analysis of competition binding studies with the antagonists pirenzepine and AF-DX 116 indicates that the majority of receptors are of the M1-type in the hippocampus (83%, high affinity for pirenzepine, intermediate affinity for AF-DX 116), the M2-type in the pons (low affinity for pirenzepine and high affinity for AF-DX 116), and the M3-type in the submandibular gland (low affinity for pirenzepine and AF-DX 116). Competition binding parameters of the agonists carbachol, arecoline, oxotremorine, pilocarpine and MCN-A-343 were compared for M1, M2 and M3 receptors in the human hippocampus, pons and submandibular gland. GTP caused a shift to the right and a steepening of the shallow agonist competition curves in the 3 tissues but did not affect the initially steep ones. This effect is explained by a GTP-mediated conversion of high- to low-agonist affinity sites. The extent of the nucleotide shift was much greater for M2 receptors as compared with M1 and M3 receptors. The GTP effect was impaired by the sulphydryl reagent N-ethylmaleimide, probably due to alkylation of GTP-binding proteins. Moreover, the reagent provoked also an increase of the agonist affinity for the uncoupled muscarinic receptors. For all agonists, this increase was more pronounced for the M2 receptors than for the M1 and M3 receptors. These findings suggest structural differences between the agonist binding sites of M1 and M3 receptors versus the M2 receptors.     

Characterization of muscarinic cholinergic receptors in the submandibular gland of the rat

The specific muscarinic ligand [3H]quinuclidinyl benzilate ([3H]QNB) was used to label acetylcholine receptors in the submandibular gland of the rat. Specific binding of [3H]QNB increased linearly with tissue concentration in the range of 0.02-0.3 mg of protein/ml. Kinetic analysis of [3H]QNB binding revealed the presence of a single population of high affinity binding sites, with a dissociation constant of 87.2 pM and a Hill coefficient of 0.95. The binding was saturable and the receptor density was 214 fmol/mg of protein. The rate constants at 37 degrees C for association and dissociation of the [3H]QNB-receptor complex were 5.98 X 10(-8) M-1 X min-1 and 6.6 X 10(-3) X min-1, respectively. The ratio k-1/k+1 gave a Kd value of 11.1 pM, similar to the Kd value (13.1 pM) determined by kinetic parameters when extrapolated at infinitely low receptor concentration. Muscarinic antagonists displaced [3H]QNB from muscarinic receptors with a Hill coefficient near to 1.0. Displacement curves for muscarinic agonists and for the atypical antagonist pirenzepine had Hill values significantly less than one. In the presence of 0.1 mM GPP(NH)P, the potency of agonists but not antagonists in displacing [3H]QNB binding decreased 2 to 3-fold. The [3H]QNB binding site was sensitive to the inhibitory effect of various sulfhydryl reagents. Repeated treatments of rats with an acetylcholinesterase inhibitor led to a decreased density of muscarinic receptors in the submandibular gland. This alteration was specific for the muscarinic recognition site and was paralleled by a reduced sensitivity to carbachol.     

Facilitation of amphetamine-induced rotation by muscarinic antagonists is correlated with M2 receptor affinity

This study examined the relationship between the affinity of cholinergic drugs for muscarinic receptor subtypes and their potency in potentiating or inhibiting amphetamine-induced rotation. The ascending nigrostriatal dopaminergic pathway was unilaterally lesioned in male Wistar rats using 6-hydroxydopamine. In these rats, ipsiversive rotation induced by amphetamine sulphate (1 mg/kg, s.c.) was dose-dependently inhibited by the cholinergic agonists oxotremorine, RS86 and pilocarpine and by the acetylcholinesterase inhibitor physostigmine. In contrast the cholinergic antagonists scopolamine, secoverine and dicyclomine facilitated amphetamine-induced rotation. Agonist and antagonist potencies were then compared with M1 and M2 binding site affinities estimated by displacing [3H]pirenzepine from forebrain and [3H]QNB from brainstem homogenates. The data suggest a relationship between antagonist potency and M2 binding site affinity.     

Autoradiographic localization of peripheral M1 muscarinic receptors using [H]pirenzepine

A novel antimuscarinic agent, pirenzepine, has been proposed to distinguish at least two subtypes of muscarinic receptor. M1 receptors have been designated as those displaying a high affinity for pirenzepine. Both functional and binding studies have revealed a prevalence of M1 receptors in sympathetic ganglia while autonomic effector tissues have only low densities of M1 receptors. In the present study, in vitro autoradiographic procedures have been used to localize specifically high affinity binding sites for pirenzepine (M1 receptors) in sections of guinea-pig ileum, rat superior cervical ganglion and rat submaxillary gland. The overall localization of muscarinic receptors was also studied using the non-selective antagonist, [³H]N-methylscopolamine. The highest densities of M1 receptors were found in superior cervical ganglion, sympathetic nerve bundles, myenteric ganglia and mucous secreting cells of the submaxillary gland, while lower densities were found in smooth muscle and serous secreting cells of the submaxillary gland. No area found to possess muscarinic receptors was devoid of M1 receptors.     

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[H]methyldioxolane

(+)-cis-[³H]Methyldioxolane ((+)-[³H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[³H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[³H]CD states and binding sites for the muscarinic antagonists [³H]pirenzepine (r = −0.79) and (-)-[³H]quinuclidinyl benzilate (r = −0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.     

Autoradiographic localization of peripheral M1 muscarinic receptors using [3H]pirenzepine

A novel antimuscarinic agent, pirenzepine, has been proposed to distinguish at least two subtypes of muscarinic receptor. M1 receptors have been designated as those displaying a high affinity for pirenzepine. Both functional and binding studies have revealed a prevalence of M1 receptors in sympathetic ganglia while autonomic effector tissues have only low densities of M1 receptors. In the present study, in vitro autoradiographic procedures have been used to localize specifically high affinity binding sites for pirenzepine (M1 receptors) in sections of guinea-pig ileum, rat superior cervical ganglion and rat submaxillary gland. The overall localization of muscarinic receptors was also studied using the non-selective antagonist, [3H]N-methylscopolamine. The highest densities of M1 receptors were found in superior cervical ganglion, sympathetic nerve bundles, myenteric ganglia and mucous secreting cells of the submaxillary gland, while lower densities were found in smooth muscle and serous secreting cells of the submaxillary gland. No area found to possess muscarinic receptors was devoid of M1 receptors.     

Anandamides inhibit binding to the muscarinic acetylcholine receptor

Loss of memory and cholinergic transmission are associated with both Alzheimer’s disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [³H]quinuclidinyl benzilate ([^3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 μM). Binding of the more polar antagonist [N-methyl-³H] scopolamine ([³H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 μM). R-methanandamide inhibits more than 90% of both [³H]QNB binding (I₅₀=34 μM) and [³H]NMS binding (I₅₀=15 μM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [³H]oxotremorine-M at low concentrations (25–75 μM), but significantly inhibit agonist binding at higher concentrations (I₅₀=150 μM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [³H]NMS binding to the mAChR, even at concentrations as high as 1 μM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function. An abstract of some of these findings was published in FASEB J., 12 (4) (1998) #882.     

Selectivity of pirenzepine in the central nervous system. II. Differential effects of pirenzepine and scopolamine on performance of a representational memory task

The behavioral effects of the two muscarinic antagonists scopolamine and pirenzepine were examined using a representational memory task for rats in a T-maze. Rats were pretrained to a criterion of 100% correct responses for daily sessions of 10 paired-run trials. The training procedures eliminated all neophobic or wary responses, and response times were invariably short (less than 3 s). Following the initial training sessions, guide cannulae were surgically implanted bilaterally over the hippocampus of each animal. Following recovery from surgery, animals were injected with saline (0.5 microliter to each hippocampus), scopolamine hydrobromide (0.5 microliter of a 60 mg/ml solution (30 micrograms) to each side), or pirenzepine (0.5 microliter of a 69.1 mg/ml solution (34.6 micrograms) to each side) according to a fixed schedule. Saline injections aimed between the blades of the dorsal dentate gyrus failed to produce any change in the performance of the memory task. Initial doses of scopolamine, applied to the same area, produced a decrease in the percentage of correct responses as did the initial dose of pirenzepine. In contrast to pirenzepine, scopolamine also produced increases in response times even to the point of defaulted trials (response times greater than 90 s) in some animals following drug injections. Saline injections failed to produce significant impairments on the days following scopolamine injections, although animals receiving pirenzepine injections were still impaired on the two days immediately following the initial pirenzepine injection. Subsequent doses of pirenzepine were ineffective in producing an impairment of performance while scopolamine injections were less effective than the initial dose.(ABSTRACT TRUNCATED AT 250 WORDS)