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.     

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.     

Presynaptic M2-muscarinic receptors on noradrenergic nerve endings and endothelium-derived M3 receptors in cat cerebral arteries.

The muscarinic (M) receptors involved in the vasodilation elicited by acetylcholine (ACh) and in the carbachol inhibition in electrically induced [3H]noradrenaline (NA) release in cat cerebral arteries was investigated. For this, atropine, pirenzepine, AF-DX 116, 4-DAMP, non-specific, M1, M2 and M3 receptor antagonists, respectively, were used. ACh elicited concentration-dependent relaxations up to 10(-6) M which were attenuated by these antagonists; the order of potency (pA2 values) to inhibit the ACh-induced relaxation was: atropine (10.1) 4-DAMP (8.9) greater than pirenzepine (7.6) greater than AF-DX 116 (5.9). The electrical stimulation (200 mA, 0.3 ms, 2 Hz, during 1 min) of these arteries preincubated with [3H]NA caused tritium release which was inhibited by carbachol (10(-6) M). The 4 antagonists attenuated the action of the M agonist; the order of potency (pIC50 values) was: atropine (8.7) greater than 4-DAMP (8.1) greater than AF-DX 116 (7.9) greater than pirenzepine (5.8). The action of McN-A-343, a putative M1 agonist, was also investigated. This agent produced small vasodilator responses and elevated concentrations (5 x 10(-5) M) inhibited the stimulated NA release, which was partially antagonized by atropine (10(-7) M) and pirenzepine (10(-8) and 10(-7) M). These results suggest the existence of M3 and M2 receptors mediating the relaxation induced by ACh and the NA release inhibition evoked by carbachol, respectively.     

Competitive inhibition of phosphoinositide hydrolysis by the muscarinic receptor antagonist AF-DX 116 is of low affinity in mouse cerebral cortex

Carbamylcholine stimulated [³H]inositol phosphate accumulation in mouse cerebral cortical slices with an ED₅₀ value of approximately 70 μM. Increasing concentrations of the M₂ selective muscarinic cholinergic receptor antagonist, AF-DX 116 (0.3–3.0 μM), produced parallel shifts to the right for concentration-response curves to carbamylcholine. A pA₂ value for AF-DX 116 of 6.5 (low affinity) was obtained frommSchild plot analysis. It is concluded that the M₂ muscarinic receptor subtype, as defined by high affinity [³H]AF-DX 116 radioligand binding, is not appreciably coupled to polyphosphoinositide hydrolysis in the mouse cerebral cortex.     

Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography

The muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M1 (pirenzepine), M2 (AF-DX 116), and M3 (4-DAMP) and the classical nonselective antagonist atropine. pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.50, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M3.     

Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites

The main objective of the present work was to determine whether the regulation of ACh release by nicotinic and muscarinic autoreceptors is compromised in the aged rat brain. For this, the effects of the nicotinic agonist N-methylcarbamylcholine (MCC) and the muscarinic-M2 antagonist AF-DX 116 on ACh release from brain slices of young (3-month-old), adult (9-month-old), and aged (27-month-old) rats were tested. The ability of MCC to enhance spontaneous ACh release in hippocampal, cerebral cortical, and cerebellar slices was only modestly altered with age. In contrast, the sensitivity of muscarinic autoreceptors in the aged hippocampus and cerebral cortex, but not the striatum, to blockade by the muscarinic-M2 antagonist AF-DX 116 was severely attenuated. To assess whether the age-related changes in cholinergic autoreceptor function may be due to deficits in presynaptic cholinergic markers, we tested whether choline acetyltransferase (ChAT) activity, basal and evoked ACh release, and nicotinic and muscarinic binding sites are altered in the aged rats. ChAT activity in forebrain regions was decreased in the aged compared to the young and mature adult rats. Furthermore, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in striatal, hippocampal, and cortical slices of aged rats. The densities of nicotinic and muscarinic-M2 binding sites, assessed using 3H-MCC and 3H-AF-DX 116 as selective ligands, respectively, were markedly reduced in homogenates of the striatum, hippocampus, cerebral cortex, and thalamus of aged rats. In contrast, muscarinic-M1 sites, selectively labeled with 3H-pirenzepine, were not affected. Therefore, it appears that age-related decrements in ChAT activity and in muscarinic-M2, but not nicotinic, binding sites in the rat brain are reflected in a decreased function of muscarinic-M2 autoreceptors. However, the positive correlation between loss of ChAT activity, decreased muscarinic-M2 binding sites, and impaired muscarinic autoreceptor function is clearly tissue dependent.     

Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex

The in vitro receptor binding properties of a muscarinic antagonist himbacine have been studied in rat cerebral cortical, cardiac and ileal membranes. Himbacine displayed high affinity (KH = 2.94 nM) for 19%, and low affinity (KL = 71.2 nM) for the remaining muscarinic receptors in rat cerebral cortex. This high affinity of himbacine agrees with its affinity for the 62% of cerebral cortical [3H]AF-DX 116 binding sites (KH = 2.30 nM). The affinity of himbacine for cardiac receptors (Ki = 9.06 nM) and ileal receptors (Ki = 12.4 nM) was the same. Therefore, himbacine appears to be a high-affinity M2-selective ligand which recognizes a subtype of M2 receptors in the cerebral cortex.     

Muscarinic and nicotinic modulation of cortical acetylcholine release monitored by in vivo microdialysis in freely moving adult rats

The aim of the present study was to investigate, using in vivo dialysis, the existence of muscarinic and nicotinic receptors controlling acetylcholine release in the cortex of freely behaving rats. Various muscarinic receptor antagonists, including the nonselective blocker atropine, and a variety of M₂ drugs (AF-DX 116, AF-DX 384, AQ-RA 741) potently stimulated, in a concentration-dependent manner, the in vivo release of acetylcholine in the rat cortex. The effects of all these antagonists were long lasting. The nature of these putative muscarinic autoreceptors is likely of the pharmacologically defined M₂subtype on the basis of the high potency of the antagonists of the AF-DX series and the variability and shorter duration of action of the effects of the prototypic M₁ blocker, pirenzepine. 4-DAMP, a purported M₃ blocker, also potently stimulated in vivo cortical acetylcholine release, but this likely relates to its now established, rather limited selectivity for any given muscarinic receptor subtypes. Peripheral and central injections of nicotine also induced the in vivo release of acetylcholine in the rat cortex, albeit with a lower potency and shorter duration of action than muscarinic antagonists. Interestingly, the combination of a muscarinic antagonist, such as atropine, AF-DX 116, or AF-DX 384, in the presence of nicotine, induced tremendous releases of cortical acetylcholine up to 8-to 10-fold over basal values. This is clearly more than a simply additive effect, and it reveals the great capacity of cortical cholinergic nerve terminals to synthesize and release acetylcholine. Optimal pharmacological manipulations of these putative muscarinic and nicotinic autoreceptors could thus be useful in disorders in which the activity of cholinergic inputs is decreased, such as in Alzheimer's disease. © 1994 Wiley-Liss, Inc.     

Characterization of muscarinic receptor subtypes in Alzheimer and control brain cortices by selective muscarinic antagonists.

Subtypes of muscarinic receptors were characterized in the frontal cortices of control and Alzheimer brains, with labelled quinuclidinyl benzilate [3H]QNB and the unlabelled muscarinic antagonists pirenzepine, AF-DX 116, hexahydro-sila-diphenidol (HHSiD), para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and himbacine. High and low affinity sites were observed for both pirenzepine and AF-DX 116 in human control frontal cortices. The majority (76%) of the pirenzepine binding sites showed high affinity to the muscarinic receptors (M1), while the rest of the binding sites had an affinity that was 40 times less. AF-DX 116 displayed two sets of binding sites where the high affinity AF-DX 116 (M2) sites constituted 27%, while the low affinity AF-DX 116 (non-M2 site) was 73%. A single class of binding sites was observed for HHSiD, p-F-HHSiD and himbacine in human frontal cortices. HHSiD showed an affinity in the frontal cortices that was comparable to that of the pirenzepine high affinity binding (M1) sites. The affinity of p-F-HHSiD was three times lower than that of HHSiD but similar to himbacine. A significant increase in the affinity (+ 40%) as well as in the Bmax (+ 99%) value was observed for the pirenzepine high affinity binding sites (M1) in the frontal cortices of Alzheimer brains compared to controls. Similarly, a significant increase was observed in the Bmax value (+ 60%) for the AF-DX 116 low affinity binding sites (non-M2), while no change was found for the high affinity binding sites (M2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiographic distribution of [3H]AF-DX 116 muscarinic-M2 receptor binding sites in rat brain

The distribution of muscarinic-M2 receptors in rat brain was investigated by in vitro autoradiography using [3H]AF-DX 116, a putative probe for the muscarinic-M2 receptor subtype. Incubation of rat brain coronal sections with 10 nM [3H]AF-DX 116 showed highest binding site densities in discrete areas such as the superior colliculus and certain thalamic and brainstem nuclei, similar to the distribution reported for [2H]acetylcholine/M2 sites. [3H]AF-DX 116 site densities were markedly lower in forebrain areas such as cortex, striatum, and hippocampus, in contrast to the distribution seen for [3H]pirenzepine-M1 binding sites, which were concentrated in these forebrain areas; however, differential patterns of labeling were observed for the two muscarinic-M2 probes, [3H]AF-DX 116 and [3H]acetylcholine, in the hippocampal formation. Although [3H]AF-DX 116 binding was broadly distributed in multiple subfields of the hippocampus, [3H]acetylcholine binding was discretely distributed in a manner resembling that of acetylcholinesterase staining. This suggests the existence of muscarinic-M2 subtypes in the CNS, especially in the hippocampal formation.     

Muscarinic receptors and second-messenger responses of neurons in primary culture

The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were maintained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [3H]N-methylscopolamine (NMS) bound specifically and with high affinity (Kd = 70 pM) to a homogeneous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [3H]NMS by pirenzepine indicated the presence of heterogeneous sites (81% high affinity sites, Kh = 51 nM, K1 = 1.5 microM); AF-DX 116 showed the opposite selectivity (15% high affinity sites, Kh = 56 nM, K1 = 1.3 microM). The dopamine agonist SKF-38393 (1 microM) enhanced the accumulation of cyclic adenosine monophosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC50, 1.7 microM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC50, 61 microM). Both responses were antagonized by pirenzepine (apparent Ki of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent Ki 540 nM and 160 nM, respectively). In binding studies on brainstem cultures, AF-DX 116 indicated the presence of two sites of approximately equal abundance (Kh = 170 nM, K1 = 2.9 microM); data for pirenzepine were adequately fit by a one-site model (Kd = 630 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1-5 min) and the second phase (7-30 min) after the injection. The muscarinic antagonist atropine (0.1-10 ng, i.t.) and the M(3) receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.1-20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20-100 ng, i.t.) did not cause inhibition. The M(1) muscarinic receptor antagonist pirenzepine (10-100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M(2) muscarinic receptor antagonist 11-?(2-[(diethylamino)methyl]-1-piperidinyl?acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AF-DX116; 10-1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 microg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M(3) muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.     

Selectivity of pirenzepine in the central nervous system. I. Direct autoradiographic comparison of the regional distribution of pirenzepine and carbamylcholine binding sites

The binding capacities of the novel antagonist pirenzepine and the agonist carbamylcholine were examined autoradiographically to compare their abilities to reduce the binding of 1-[3H]quinuclidinyl benzilate ([3H]-1-QNB). This technique, which is applicable to any muscarinic ligand, permits a direct comparison between the binding of carbamylcholine and pirenzepine in the same assay. Analysis of the binding curves generated by standard scintillation counting of whole-brain slices indicated that the ligands bound heterogeneously to muscarinic receptors in the brain. Following apposition of slides to tritium-sensitive film, the binding profile for each ligand was examined visually and by microdensitometry. Regional analyses indicated that the agonist carbamylcholine displayed highest potency for thalamic nuclei, lower potency for cortical regions, and the lowest affinity for layers of the hippocampus. The M1-selective ligand pirenzepine displayed the highest potency for the dentate gyrus of the hippocampus, with lower inhibition levels in the cortex, and the lowest levels of inhibition found in the thalamus. The distribution of high affinity agonist sites was found to be distinct from the distribution of high-affinity antagonist binding sites. In a separate assay, the regional inhibition of pirenzepine and scopolamine was compared for the hippocampus and the forebrain. While scopolamine did not distinguish between muscarinic receptor sites in the hippocampus and cortex, pirenzepine inhibited [3H]-1-QNB labeling in the hippocampus significantly greater than in the cerebral cortex, providing additional evidence for the hypothesis that pirenzepine is a selective antagonist.     

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.     

Presynaptic inhibitory receptors mediate the depression of synaptic transmission upon hypoxia in rat hippocampal slices

Hypoxia markedly depresses synaptic transmission in hippocampal slices of the rat. This depression is attributed to presynaptic inhibition of glutamate release and is largely mediated by adenosine released during hypoxia acting through presynaptic adenosine A(1) receptors. Paired pulse facilitation studies allowed us to confirm the presynaptic nature of the depression of synaptic transmission during hypoxia. We tested the hypothesis that activation of heterosynaptic inhibitory receptors localized in glutamatergic presynaptic terminals in the hippocampus, namely gamma-aminobutyric acid subtype B (GABA(B)) receptors, alpha(2)-adrenergic receptors, and muscarinic receptors might contribute to the hypoxia-induced depression of synaptic transmission. Field excitatory postsynaptic potentials were recorded in the CA1 area of hippocampal slices from young adult (5-6 weeks) Wistar rats. Neither the selective antagonist for alpha(2)-adrenergic receptors, rauwolscine (10 microM), nor the antagonist for the GABA(B) receptors, CGP 55845 (10 microM), modified the response to hypoxia. The selective adenosine A(1) receptor antagonist, DPCPX (50 nM), reduced the hypoxia-induced depression of synaptic transmission to 59.2+/-9.6%, and the muscarinic receptor antagonist, atropine (10 microM), in the presence of DPCPX (50 nM), further attenuated the depression of synaptic transmission to 49.4+/-8.0%. In the same experimental conditions, in the presence of DPCPX (50 nM), the muscarinic M(2) receptor antagonist AF-DX 116 (10 microM), but not the M(1) receptor antagonist pirenzepine (1 microM), also attenuated the hypoxia-induced depression to 41.6+/-6.6%. Activation of muscarinic M(2) receptors contributes to the depression of synaptic transmission upon hypoxia. This effect should assume particular relevance during prolonged periods of hypoxia when other mechanisms may become less efficient.     

Antisense oligodeoxynucleotides against the muscarinic m2, but not m4, receptor supports its role as autoreceptors in the rat hippocampus

Antisense oligodeoxynucleotides against muscarinic m2 and m4 receptors were used to investigate the role of these receptor subtypes as negative autoreceptors in the regulation of acetylcholine (ACh) release in the rat hippocampus. Following the continuous infusion of antisenses into the third ventricle (1 microgram microliter-1 h-1, 3 days), 3H-AF-DX 384/muscarinic M2-like binding was significantly decreased in the medial septum by the antisense against the m2 receptor whereas M2-like binding in the dorsal striatum was decreased by the antisense against the m4 receptor. In contrast, 3H-pirenzepine/muscarinic M1-like binding was unaffected by either antisense treatment in any of the brain areas investigated. When perfused into the hippocampus via a dialysis probe, the purported muscarinic M2 receptor antagonist AF-DX 384 (100 nM) increased hippocampal ACh release in freely moving rats. This effect of AF-DX 384 was significantly attenuated by the m2, but not the m4, receptor antisense treatment. Hippocampal choline acetyltransferase activity was not affected by either antisense treatments. Taken together, these results suggest that the molecularly defined muscarinic m2 receptor regulates hippocampal ACh release by acting as a negative autoreceptor. In contrast, the molecularly defined m4 receptor is unlikely to be directly involved in the negative regulation of ACh release in the rat hippocampus. Therefore, inhibiting muscarinic m2 receptor function may be an alternative approach to regulate the release of ACh in neurodegenerative diseases associated with impaired cholinergic functions.     

Quantitative autoradiography of M2 muscarinic receptors in the rat brain identified by using a selective radioligand [3H]AF-DX 116

Muscarinic receptors of the M2 type have been studied in the rat brain using quantitative autoradiography with the selective ligand, [3H]AF-DX 116. High specific binding of [3H]AF-DX 116 was found in areas such as laminae IV and V of the parietal cerebral cortex, thalamus and hypothalamus and dentate gyrus. Intermediate [3H]AF-DX 116 binding was found in the frontal cortex, hippocampus, caudate-putamen, nucleus accumbens, and claustrum as well as in certain brainstem nuclei such as the nucleus of the solitary tract and the dorsal motor nuclei of the vagus. In contrast, the accessory olfactory nucleus, globus pallidus and cerebellum contained very low concentrations of M2 receptors. The present study demonstrates a unique regional distribution of M2 receptors in the rat brain.     

Distribution and function of cholinergic receptors in the sheep detrusor muscle

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.     

The effect of subtype-selective muscarinic receptor antagonists on the cholinergic current in motoneurons of the lobster cardiac ganglion

Muscarinic agonists evoke a voltage-dependent inward current in motoneurons of the lobster cardiac ganglion. In this study, a number of drugs, known to show muscarinic receptor subtype selectivity in mammals, were used to determine the pharmacological profile of the muscarinic receptor on lobster motoneurons. The neurons were held under voltage-clamp, and various concentrations of the antagonists were applied in the presence of 1 mM methacholine. From competition curves plotting agonist-induced current against antagonist concentration, the inhibitor affinity constant and the slope factor were determined. The rank order of potencies of antagonists having an effect was: atropine greater than pirenzepine greater than 4-DAMP greater than methoctramine greater than HHSiD = (R)-HHD greater than (S)-HHD. Neither AF-DX 116 nor gallamine were effective at concentrations as high as 10 mM. The M1-selective agonist McN-A-343 had no effect. Although this crustacean muscarinic receptor resembles the mammalian M1 muscarinic receptor because of its relatively high affinity for pirenzepine, the rank order of other subtype-specific antagonists does not otherwise resemble that of any of the pharmacologically defined muscarinic receptors in mammals. It may be preferable, therefore, to use a term such as 'pirenzepine-sensitive' muscarinic receptor rather than M1 or 'M1-like' for invertebrate muscarinic receptors with pharmacological characteristics like those reported here.