Pirenzepine Binding Research Articles

Laminar Alterations in γ‐Aminobutyric AcidA, Muscarinic, and β Adrenoceptors and Neuron Degeneration in Cingulate Cortex in Alzheimer's Disease

The laminar distribution of binding to a number of postsynaptic neurotransmitter receptors was assessed autoradiographically in postmortem samples of area 23a in posterior cingulate cortex from 13 Alzheimer and nine age-matched control cases. Specific binding in all Alzheimer cases was compared to that in control cases, and the following alterations were observed: reduced muscimol binding in most layers; no changes in pirenzepine binding; and elevated cyanopindolol binding in layers Ic, IIIc, and IV. The Alzheimer cases were classified further on the basis of neuronal degeneration: class 1, no neuron loss; class 2, greatest losses in layer II or III; class 3, greatest losses in layer IV; and class 4, greatest losses in layer V or VI. This classification uncovered further alterations in ligand binding patterns. First, muscimol binding was reduced in layers II and III only in class 2 cases and in layers V and VI only in class 4 cases. Second, pirenzepine binding was reduced in layers Ic, IIIa-b, and VI of class 1 cases and layers Va and VI of class 4 cases. In spite of neuron degeneration in classes 2 and 3, there was no change in pirenzepine binding in these classes. Third, elevated cyanopindolol binding occurred in classes 3 and 4, whereas classes 1 and 2 had normal levels of binding. These results suggest that cases of Alzheimer's disease express heterogeneities in neocortical pathology which are reflected in the laminar patterns of binding to postsynaptic receptors. Reductions in muscimol binding to the gamma-aminobutyric acidA receptor had the closest relationship with neuron degeneration, whereas pirenzepine binding appeared to reflect a compensation in muscarinic receptors for changes in neuron densities.

Muscarinic receptor-mediated inhibition of voltage-activated Ca current in neuroblastoma × glioma hybrid (NG 108-15) cells — reduction of muscarinic agonist and antagonist potency by tetraethylammonium (TEA)

Patch clamp techniques were used to record voltage-activated Ca currents from NG 108-15 cells. The muscarinic agonists acetylcholine (ACh) and oxotremorine-M partially inhibited the Ca current (mean inhibition 37%) with EC 50 values of 0.32 μM and 0.14 μM, respectively. The EC 50 for ACh in the presence of neostigmine (1 μM) was 0.11 μM. Tetraethylammonium (TEA) (10 mM) shifted ACh (+ neostigmine) and oxotremorine dose-response curves to the right by 36- and 20-fold, respectively. TEA also inhibited muscarinic receptor binding ( K i values in brackets) of (i) N-methyl scopolamine ([ 3H]NMS) to homogenates of rat heart (86 μM) and rat submandibular gland (178 μM) and to intact NG 108-15 cells (358 μM) and (ii) [ 3H]pirenzepine binding to rat cerebral cortex homogenates (111 μM). Atropine antagonized muscarinic agonist responses and was found to have a pK B of 9.8 ± 0.39, but 10 mM TEA reduced the potency of atropine by 69-fold, probably because of the weak antagonist action of TEA.

Laminar postnatal development of muscarinic cholinergic receptors in rat visual cortex and the effect of monocular deprivation

Abstract Quantitative receptor autoradiography was used to study the postnatal development of the laminar pattern of muscarinic acetylcholine receptors (including the M 1 -receptor subtype), in the visual cortex of both normal (control) and monocularly deprived rats (one eyelid sutured at the age of 11 days). In control rats an age-dependent change, between postnatal days 15 and 25, occurred in the pattern of [ 3 H]quinuclidinyl benzilate (QNB) binding to the total population of muscarinic acetylcholine receptors density, with a tetramodal distribution altering to a trimodal distribution. However, for [ 3 H]pirenzepine binding, a developmental change from a trimodal distribution to a bimodal distribution in M 1 -receptor density was observed after postnatal day 25. Monocular deprivation until postnatal day 25 results in a laminar pattern of [ 3 H]QNB binding sites which is qualitatively similar to that found in the visual cortex of normal (i.e. non-visually deprived) 15-day-old rats, suggesting a retardation in the development of binding sites following visual deprivation. In contrast, the visual cortical laminar pattern of [ 3 H]pirenzepine binding sites was not qualitatively altered following monocular deprivation until 25 days postnatal. However, a higher density of M 1 -receptor binding sites was detected in cortical layers IV to VI of the non-deprived (ipsilateral to the closed eye) visual cortex, compared to age-matched controls. The developmental changes in the laminar pattern of muscarinic receptors might be a consequence of an adaptive process related to the onset of visual information processing at the cortical level, which is supported by the finding that inadequate visual experience alters the laminar maturation of muscarinic receptors.

The Na +, K +-ATPase inhibitor ouabain and the Na + ionophore monensin have opposite effects upon carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K +. Enhancement of the carbachol reponse at 6 mM, but not at 18 mM K +, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [ 3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Quabain did not affect [ 3H]pirenzepine binding at any of the concentrations tested (6–600 μM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.

M 1 muscarinic antagonists interact with σ recognition sites

The M 1-selective muscarinic antagonists aprophen, caramiphen, carbetapentane, 2-DAEX, dicyclomine, hexahydrosiladifenidol, iodocaramiphen, nitrocaramiphen, oxybutynin and trihexyphenidyl potently inhibited binding to σ sites in brain. Both basic ester and non-ester structural type compounds which exhibit affinity for the muscarinic receptor also demonstrated affinity for the σ site, while the classical antimuscarinic agents atropine and QNB, and the tricyclic pirenzepine, were ineffective in binding to this site. We also observed a significant correlation between the K i values for σ compounds to inhibit [ 3H]pirenzepine binding and their IC 50 values to inhibit carbachol-stimulated phosphoinsitide turnover. These observations may aid in elucidating the relationship of σ binding to inhibition of phosphoinositide turnover stimulated by cholinergic agonists.

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways > trachea > parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M 1 receptor antagonist, pirenzepine, and M 2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro . Schild plot pA 2 values were compatible with low potency antagonism, thereby favoring the presence of M 3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg −1 day −1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (K d 3H-QNB), population density (B max in fmol mg −1 protein), or function (methacholine EC 50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M 3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.

Pharmacological studies on novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 and YM954

We have investigated the pharmacological profiles of the novel muscarinic agonists, 1-oxa-8-azaspiro[4.5]decane derivatives, YM796 (2,8-dimethyl-3-methylene) and YM954 (2-ethyl-8-methyl-3-oxo). These compounds, like the putative M 1 agonists, RS86 and AF102B, inhibited [ 3H]pirenzepine binding to cerebral cortical membranes in the micromolar range and weakly inhibited [ 3H]quinuclidinyl benzylate binding to cerebellar membranes. Their (-) isomers had Hill coefficients lower than 1.0 (±)-YM796, (±)-YM954 and RS86, but not AF102B, stimulated phosphoinositide hydrolysis in hippocampal slices, an effect which is mainly linked to M 1 receptors. (±)-YM796 (0.031 mg/kg p.o.) and (±)-YM954 (0.016 mg/kg p.o.) reversed the cognitive impairment in nucleus basalis magnocellularis-lesioned rats in a passive avoidance task more effectively than did RS86 and AF102B. Similar results were obtained in scopolamine-treated rats. Finally, (±)-YM796 was weaker than (±)-YM954 and RS86 in the induction of tremor, hypothermia and contraction of isolated ileum, which are mainly mediated by M 2 and/or M 3 receptors. These results suggest that (±)-YM796, (±)-YM954 and RS86 have M 1 agonistic activity in central nervous system and that (±)-YM796 has relatively weak M 2 and/or M 3 agonistic activity.

Intrastriatal grafts derived from fetal striatal primordia: II. Reconstitution of cholinergic and dopaminergic systems

Reconstitution of striatal cholinergic and dopaminergic systems was studied in intrastriatal grafts derived from embryonic day 15 rat striatal primordia and implanted into adult host rats in which unilateral ibotenic acid lesions had previously been made in the striatum. Histochemical, immunohistochemical, and ligand binding autoradiographic techniques were applied to analyze different constituents of these two systems and to study their locations relative to each other in grafts allowed to grow for 9-17 months following transplantation. For the cholinergic system, a modular organization was found in the striatal grafts with stains for choline acetyltransferase and acetylcholinesterase, respectively the synthetic and degradative enzymes for cholinergic neurons; by autoradiographic [3H]hemicholinium binding, specific for high affinity choline uptake sites associated with cholinergic terminals; and by autoradiographic [3H]pirenzepine binding, selective for M1 receptors. For the dopaminergic system, a comparable modular organization was found in the grafts by immunostaining for tyrosine hydroxylase, the catecholamine synthetic enzyme; by autoradiographic [3H]mazindol binding for dopamine uptake sites; and by [3H]SCH23390 binding for dopamine D1 receptors and [3H]sulpiride binding for dopamine D2 receptors. The results indicate that the distributions of the cholinergic and dopaminergic markers in striatal grafts are in close anatomical register. These markers for intracellular and membrane-associated components of the cholinergic and dopaminergic systems were preferentially localized in the acetylcholesterase-rich patches of the grafts in which cortical and thalamic fibers have also been found in striatal grafts, and in which output neurons projecting to the pallidum are located. This anatomical correlation suggests that the substrates for cholinergic-dopaminergic interactions typical of the normal striatum may be reinstated in the grafts both in relation to efferent neurons establishing connections with the host brain that are typical of normal striatofugal connections, and in relation to major afferent fiber systems from the host brain originating in regions known to project densely to the normal striatum. Accordingly, the cholinergic and dopaminergic systems in such grafts may regulate the functional influence of the grafts on the behavior of host animals.

Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal M1, atrial M2 and ileal M3 receptors

1. Affinity constants for 15 non-selective or putatively selective muscarinic antagonists were determined at muscarinic autoreceptors and postsynaptic receptors (linked to phosphatidylinositol (PI) hydrolysis) in rat hippocampal slices, at muscarinic receptors mediating contractility in guinea-pig atria or ileal smooth muscle and at binding sites in rat cerebral cortical membranes labelled with [3H]-1-quinuclidinyl benzilate or [3H]-pirenzepine. 2. Comparison of the affinities of these antagonists at central M1 receptors (inositol-monophosphate formation in rat hippocampal slices) with their affinities at peripheral M1 receptors (inhibition by McN-A-343 of electrically stimulated twitches in rabbit vas deferens) provides support for the suggestion that these receptors may differ pharmacologically. 3. Comparison of affinity constants obtained by displacement of specifically bound [3H]-pirenzepine from rat cerebral cortical membranes with those obtained in functional tests showed poor correlations between affinities for binding sites and for functional atrial receptors or for hippocampal autoreceptors. A significant correlation was found between affinities for [3H]-pirenzepine binding and those determined at muscarinic receptors linked to PI turnover in rat hippocampus. A significant correlation was also obtained between the affinities for specific [3H]-pirenzepine binding sites in cortical membranes and the affinities at ileal receptors. 4. Comparison of the affinity values for muscarinic autoreceptors in rat hippocampus with affinity values obtained from in vitro models of muscarinic receptor subtypes showed no significant correlations between these autoreceptors and either M1 or M3 receptors. A significant correlation was found between antagonist affinities for hippocampal autoreceptors and muscarinic receptors in the heart. Therefore, muscarinic autoreceptors in rat hippocampus are pharmacologically similar to the M2 (cardiac) muscarinic receptor subtype.

Effects of WEB 1881 FU, a Novel Nootropic, on Cholinergic and Adrenergic Receptors in the Rat Brain: Action on M1-Muscarinic Receptors

AbstractEffects of 4-aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate (WEB 1881 FU), a novel pyrrolldinone nootropic, on acetylcholine (ACh) receptors and adrenoceptors were investigated using crude membranes of the rat brain. The affinity order of WEB 1881 FU was: M1-muscarinic (m) ACh receptor>M2-mACh receptor>α2-adrenoceptor>β- adrenoceptor>α1-adrenoceptor>nicotinic ACh re-ceptor. The WEB 1881 FU-competition curve for [3H]pirenzepine binding in hippocampal membranes was rightward-shifted by GTPγS; such behavior was also observed in the case of oxotremorine but not with scopolamine. The effects of long-term administration of WEB 1881 FU(30 mg/kg/day, I.p.) for 21 days resulted in a significant decrease in the Bmix of [3H]quinuclidinyl benzilate binding in the cerebral cortex, hippocampusandstriatum. In addition, the Bmax of [3H] pirenzepi ne binding to hippocampal and striatal membranes and that of [3H]AF-DX 116 binding to cerebellar membranes were significantly decreased as well. From these results, WEB 1881 FU seems to act on M1-mACh receptors, and its long-term administration probably induces the down-regulation of mACh receptors, mainly M1-mACh receptors in the hippocampus and striatum and M2-mACh receptors In the cerebellum

Effects of WEB 1881 FU, a novel nootropic, on cholinergic and adrenergic receptors in the rat brain: Action on M1-muscarinic receptors.

Effects of 4-aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate (WEB 1881 FU), a novel pyrrolidinone nootropic, on acetylcholine (ACh) receptors and adrenoceptors were investigated using crude membranes of the rat brain. The affinity order of WEB 1881 FU was: M1-muscarinic (m) ACh receptor greater than M2-mACh receptor greater than alpha 2-adrenoceptor greater than beta-adrenoceptor greater than alpha 1-adrenoceptor greater than nicotinic ACh receptor. The WEB 1881 FU-competition curve for [3H]pirenzepine binding in hippocampal membranes was rightward-shifted by GTP gamma S; such behavior was also observed in the case of oxotremorine but not with scopolamine. The effects of long-term administration of WEB 1881 FU (30 mg/kg/day, i.p.) for 21 days resulted in a significant decrease in the Bmax of [3H]quinuclidinyl benzilate binding in the cerebral cortex, hippocampus and striatum. In addition, the Bmax of [3H]pirenzepine binding to hippocampal and striatal membranes and that of [3H]AF-DX 116 binding to cerebellar membranes were significantly decreased as well. From these results, WEB 1881 FU seems to act on M1-mACh receptors, and its long-term administration probably induces the down-regulation of mACh receptors, mainly M1-mACh receptors in the hippocampus and striatum and M2-mACh receptors in the cerebellum.

Laminar distributions of muscarinic acetylcholine, serotonin, GABA and opioid receptors in human posterior cingulate cortex

Experimental animal studies have demonstrated a number of receptor localizations on specific cortical afferents and neurons. The present study of human posterior cingulate cortex evaluates the laminar distributions of particular receptors and their likely association with components of the neuropil. Coverslip autoradiographic and single grain counting techniques were used followed by heterogeneity analysis in which the layer of peak binding and an index of heterogeneity were determined for each ligand. The index was calculated by determining specific binding by layer as a percentage of binding in all layers. The differences from an absolutely homogeneous distribution, i.e. 11.1% for each of nine layers, were subtracted and the absolute laminar differences summed to form the index. High indices of over 15 reflected heterogeneous binding patterns in neocortex. The binding of ligands for muscarinic acetylcholine, serotonin, opioid, GABA and beta adrenoceptors was evaluated. Pirenzepine binding peaked in layer II of area 23a but was extremely homogeneous with an index of heterogeneity of 8.9. In contrast, oxotremorine-M binding had a peak in layer IIIc and an index of 16.4, while AF-DX 116 binding peaked in layer IIIa-b and had an index of 30.6. Of the ligands for serotonin uptake and receptor binding paroxetine binding was evenly distributed in layers I–III and had a low index of heterogeneity of 9.8. Ketanserin binding was also homogeneous and, since it had an index of 8.9, this pattern was virtually the same as that for paroxetine. In contrast, serotonin and 8-hydroxy-2-(di- n-propylamino)tetralin binding peaked in layer II and had very high indices of 20.8 and 50.3, respectively, suggesting only a limited association with that of the paroxetine distribution. Finally, there were three layers which contained peaks in binding for ligands for opioid, GABA and beta adrenoceptors. Firstly, layer Ia had peak dynorphin-A binding, the latter of which had an index of 22.6. Secondly, Tyr- d-Ala-Gly-MePhe-Gly-ol and 2- d-penicillamine-5- d-penicillamine-enkephalin binding peaked in layer II and had indices of 8.6 and 17.4, respectively. Thirdly, muscimol and (−)-cyanopindolol binding peaked in layer IIIa-b and had indices of 29.6 and 11.1, respectively. When viewed in the context of experimental animal studies, it is likely that heterogeneities in oxotremorine-M and paroxetine binding are associated with the termination of the thalamic and raphe nuclei, respectively. While serotonin, receptors are co-distributed with serotonin uptake sites, serotonin 1A receptors have a significant mismatch with these sites. Finally, postsynaptic receptors preferentially peak in different layers including kappa opioid receptors in layer Ia, M 1 acetylcholine, serotonin 1A, mu and delta opioid receptors in layer II, GABA A and beta adrenoceptors in layer IIIa-b and serotonin 2 receptors in layer IIIc. Thus, ligand binding analyses provide new insights into the structure and connections of human cerebral cortex.

Unilateral decortication affects muscarinic cholinergic receptor binding in rat basal forebrain

The distribution of muscarinic acetylcholine receptors (including the M(1)-receptor subtype), beta-adreno receptors, and noradrenaline uptake sites was studied in cholinergic basal forebrain nuclei (medial and lateral septum, ventral and dorsal limb of the diagonal band, substantia innominata and nucleus basalis of Meynert) of adult rats 25 days after unilateral decortication, using quantitative receptor autoradiography. Unilateral neocortex suction lesion resulted 25 days postoperatively in significant enhancements of [(3)H]quinuclidinyl benzilate binding to the total population of muscarinic acetylcholine receptors in both sides of the substantia innominata (> 55%) and of the nucleus basalis of Meynert (> 45%) compared to sham-operated control rats. Additionally, increased binding levels in the medial septum (59%) and dorsal limb of the diagonal band (48%) were found in unilaterally decorticated rats. [(3)H]pirenzepine binding to M(1)-receptors was increased only in the nucleus basalis of Meynert (by 35%), in both sides, as a consequence of hemi-decortication. Unilateral decortication did not affect either [(3)H]dihydroalprenolol binding to beta-adreno receptors, or [(3)H]desipramine binding to noradrenaline uptake sites, in any of the basal forebrain regions studied. It is suggested that the enhanced binding to muscarinic acetylcholine receptors in the substantia innominata, medial septum and dorsal limb of the diagonal band following cortical damage may reflect an adaptive response to the morphological changes seen in cholinergic neurons of the nucleus basalis of Meynert after cortical lesion, whereas the receptor binding changes in the nucleus basalis of Meynert itself cannot be easily related to the morphological alterations observed there.

Effects of repeated administration of tetrahydroaminoacridine (THA) on muscarinic receptor subtypes in the rat brain

The effects of a chronic treatment (21 days) with the acetylcholinesterase (AChE) inhibitor tetrahydroaminoacridine (THA) on muscarinic receptors subtypes were investigated at various times after the last administration of the drug, in various brain areas including cortex, striatum, hippocampus and cerebellum. Forty eight hours after the end of chronic THA treatment, the number of muscarinic receptors, labelled with [3H]NMS, was significantly lowered in the cortex and the striatum but not in the hippocampus or cerebellum. High affinity pirenzepine binding sites (M1 receptors), directly assayed using [3H]pirenzepine saturation assays or estimated by pirenzepine [3H]NMS competition, were lowered only in the cortex and in the striatum of THA-treated rats. In contrast, the number of low affinity pirenzepine sites (M2 receptors), was not significantly modified. At shorter wash-out period (18 h), the density of M1 receptors decreased by 26, 46 and 52% in the hippocampus, cerebral cortex and striatum, respectively. In all cases, Kd values remained unchanged suggesting that the loss of M1 sites was not due to a modification of radioligand affinity for the receptors. Although THA displayed a micromolar affinity for M1 and M2 receptors in vitro, this AChE inhibitor did not interfere with the receptor assays since no trace of residual free THA was detected in rat brain at 48 h post-treatment.These results suggest that chronic treatment with THA produced a selective down-regulation of M1 receptors; they also indicate that these receptors may be regulated differently in cortical, striatal, hippocampal or cerebellar regions.

Bilateral changes in neocortical [ 3H]pirenzepine and [ 3H]oxotremorine-M binding following unilateral lesions of the rat nucleus basalis magnocellularis: an autoradiographic study

Neocortical choline acetyltransferase (ChAT) activity and muscarinic [ 3H]pirenzepine, [ 3H]oxotremorine-M, [ 3H] N-methylscopolamine ([ 3H]NMS; both high- and low-affinity agonist (carbachol) sites) and nicotinic [ 3H]acetylcholine binding were assessed both ipsi- and contralaterally 1 week and 13 weeks after unilateral ibotenic acid lesions of the rat nucleus basalis magnocellularies (NBM). Ipsilateral ChAT activity was reduced to 49% of control values 1 week postlesion but by 13 weeks had recovered to 80% of control values. Contralateral ChAT activity did not change significantly at either 1 week or 13 weeks postlesion. At 1 week postlesion, [ 3H]oxotremorine-M binding was increased by 33% and 54% in ipsilateral and contralateral neocortex, respectively. By week 13, both ipsi- and contralateral [ 3H]oxotremorine-M binding had returned to normal but [ 3H]pirenzepine binding was significantly decreased by 31% and 39% in the ipsilateral and contralateral hemispheres, respectively. The binding of [ 3H]NMS and [ 3H]acetylcholine did not differ significantly from control values at either 1 week or 13 weeks postlesion. These data suggest that none of the cholinergic binding sites studied is preferentially localized presynaptically and that there may be interhemispheric regulation of neocortical cholinergic binding sites.

Muscarinic receptor subtype specificity of 5′-(isobutylthio)-adenosine (SIBA) and its analogs

1. 1. 5′-(Isobutylthio)-adenosine (SIBA) and its analogs, at 100 μM, inhibited [ 3 H]N- methylscopolamine binding to homogenates of whole brain and cortex (mainly M 1 subtype receptors) by 11–30% and to cerebellum (mainly M 2 subtype receptors) by 20–39%. 2. 2. At 0.01–1.0 μM, stimulation of [ 3H]QNB and NMS-inaccessible [ 3H]QNB binding was observed, with the most induced by 1 μM 3-deaza-SIBA. 3. 3. In contrast, [ 3H]pirenzepine ([ 3H]PZ) binding to whole brain and cortex was inhibited in a dose-dependent manner with K i values in the μM range. 4. 4. As antagonists of acetylcholine-induced contraction of guinea pig ileum (mainly M 2 subtype receptors), the analogs were slightly more potent than pirenzepine, but several orders of magnitude less than atropine; the order of potency was opposite that determined for the binding of [ 3H]PZ to cortex. 5. 5. Thus, SIBA and its analogs may have differential effects on muscarinic receptor subtypes and show some specificity for the M 1 receptor subtype.

Muscarinic receptor subtype (M1) identification on rabbit pulmonary vascular endothelium in vivo.

We have investigated the presence of M1-subtype muscarinic receptors on pulmonary vascular endothelium in vivo. Utilizing multiple indicator-dilution techniques, we studied [3H]-pirenzepine ([3H]-PNZ; a selective M1 receptor antagonist) binding during a single transpulmonary pass in anesthetized, artificially ventilated rabbits, before and after administration of 4.3 mumol/kg of the nonselective muscarinic receptor antagonist atropine. [14C]-Dextran (MW = 70,000-90,000) served as the intravascular indicator. Before atropine administration, approximately 20% of [3H]-PNZ was lost (i.e., presumed bound to PNZ-specific and nonspecific sites) during a single pass through the pulmonary microvasculature. A significant decrease in [3H]-PNZ binding occurred 90 min after atropine (to approximately 60% of total initial binding), but not after saline, reflecting the loss of specific binding to M1 receptors. These data are in support of previous findings from our laboratory indicating a pressor response to M1 receptor stimulation in the rabbit pulmonary circulation as well as an endothelium-dependent contractile response to M1 receptor stimulation in vitro.

Muscarinic receptor coupling to inositol phospholipid metabolism in guinea-pig cerebral cortex, parotid gland and ileal smooth muscle

Inositol phospholipid hydrolysis induced by agonist-stimulation of muscarinic receptors has been examined in slices of guinea-pig cerebral cortex, parotid gland and ileal smooth muscle. An assay measuring 3H-inositol phosphate formation from prelabelled lipids in the presence of LiCl, allowed marked stimulation by agonists to be followed. The pD 2-value of carbachol differed markedly, between tissues being more than 10-fold lower in cerebral cortex than in parotid gland. The partial agonist oxotremorine showed the largest relative maximal responsiveness in parotid gland, followed by ileum and cortex. Atropine suppressed the phosphoinositide response to carbachol with an almost similar affinity in each tissue, but pirenzepine was found to have a 20-fold higher affinity in cerebral cortex, p K i = 7.7 than in parotid gland, p K i = 6.3. Carbachol, even in the presence of guanosine triphosphate (GTP), displayed complex binding against 3H-AT-methylscopolamine ( 3H-NMS) in cortical and ileal membranes, though in membranes from the parotid gland a single homogeneous population was found. Atropine inhibition of 3H-NMS parallelled its suppression of the phosphoinositide response, the affinities in each tissue studied being similar. Pirenzepine inhibited binding from two components in cerebral cortex, the high affinity value being similar to that obtained in the phosphoinositide assay. In parotid gland, however, only low affinity pirenzepine binding sites were observed, closely resembling the affinity found for this antagonist in the functional assay. These experiments suggest (a) that there are differences between agonist occupation of muscarinic receptors and phosphoinositide hydrolysis within the different tissues, (b) that both high and low affinity pirenzepine binding sites appear to be linked to phosphoinositide metabolism, and (c) that low affinity pirenzepine sites may be more efficiently coupled to the hydrolysis of phosphoinositides.

Evidence for an endogenous factor involved in maintenance of pirenzepine high-affinity binding in rat brain stem

Sucrose gradient centrifugation was used to isolate membranes enriched in muscarinic receptors from bovine brain stem. Unlike the receptors in crude synaptosomal preparations of this tissue, the enriched preparations displayed only low-affinity pirenzepine binding. Similar results were obtained when purified preparations were preincubated for 1 hr in pH 7.0 buffer at 37 degrees C; however, preincubation in a pH 5.0 buffer partially restored the high-affinity pirenzepine binding. These results suggest that an endogenous factor, which is present in the crude synaptosomes of the brain stem and is removed by sucrose gradient centrifugation, is involved in maintenance of the high-affinity pirenzepine binding of the muscarinic receptors.

Chronic, oral aluminum administration to rats: Cognition and cholinergic parameters

Administration of aluminum sulfate in the drinking water of male Sprague-Dawley rats for thirty days resulted in an impairment of both consolidation and extinction of a passive avoidance task. No impairment of performance was observed on an active avoidance task, radial arm maze or open field activity measure. Biochemical analysis indicated a slight (<10%) but significant increase in hippocampal muscarinic receptor number after aluminum treatment as determined by tritiated quinuclidinyl benzilate ( 3H-QNB) binding. No changes were found in choline acetyltransferase (ChAT) activity, phosphoinositide hydrolysis, 3H-QNB binding in the cortex or tritiated pirenzepine ( 3H-PZ) binding in the hippocampus or cortex. These results indicate that cholinergic degeneration was not the cause of the observed cognitive impairments.