Identification of muscarinic receptor subtypes present in cerebellar granule cells: prevention of [3H]propylbenzilyl choline mustard binding with specific antagonists

Subtypes of muscarinic receptors (possible m1 to m5) can be identified by their molecular size, specific effector systems and antagonist specificity. In membranes prepared from primary cultures of cerebellar granule cells, [3H]propylbenzilylcholine mustard [( 3H]PBCM) irreversibly binds to muscarinic receptive proteins, having two major molecular sizes, 92 and 66 kDa. With relatively short periods of incubation (approx. 30 min, 30 degrees C) of [3H]PBCM with atropine, a nonspecific competitive receptor antagonist, the irreversible labeling of these muscarinic proteins by [3H]PBCM could be prevented. Methoctramine, a specific competitive antagonist at muscarinic receptors coupled to inhibition of adenylate cyclase, protected most of the muscarinic receptors having a molecular size of 66 kDa from binding of [3H]PBCM. These 66 kDa receptive proteins are suggested to be muscarinic m2 and m4 subtypes. (-)Quinuclidinyl xanthene-9-carboxylate [(-)QNX], a somewhat specific competitive antagonist at muscarinic receptors coupled to hydrolysis of phosphatidylinositol, prevented the binding of [3H]PBCM to 92 kDa muscarinic receptive proteins and some 66 kDa muscarinic receptive proteins. The 92 kDa receptive proteins are suggested to be the muscarinic m3 subtype and the 66 kDa proteins could be either the m2 or m4 receptor subtype. Lastly, pirenzepine, a nonspecific antagonist at muscarinic receptors mediating inhibition of adenylate cyclase and hydrolysis of PI in these cultures, resembled (-)QNX in preventing binding of [3H]PBCM to the 92 kDa receptive proteins and some 66 kDa receptive proteins. The suggested subtypes of muscarinic receptors, specifically bound by pirenzepine should be the m3 (92 kDa) and the m4 (66 kDa) subtypes, since pirenzepine reportedly exhibits a low affinity for the muscarinic m2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese Hamster Ovary cells.

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca(2+)](i). From 25 to 800 nM MT2 also produced increases in [Ca(2+)](i) by activating m3 receptors, although these increases in [Ca(2+)](i) were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800-1600 nM) also caused significant increases in [Ca(2+)](i) in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 microM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.     

Affinity and efficacy of racemic, (+)-, and (-)-methacholine in muscarinic inhibition of [3H]-noradrenaline release.

The right postganglionic sympathetic nerves of rat isolated perfused hearts (previously loaded with [3H]-noradrenaline) were stimulated electrically with 10 trains of 10 pulses at 10 Hz. The inhibition by methacholine of stimulation-evoked [3H]-noradrenaline overflow into the perfusate (determined in the presence of corticosterone, desipramine, phentolamine, and propranolol) was taken as a measure for activation of presynaptic muscarinic receptors. The evoked [3H]-noradrenaline overflow was inhibited by (+)-, racemic, and (-)-methacholine in a reversible and concentration-dependent manner. The concentration causing 50% inhibition (IC50) was 0.1, 0.26, and 65 microM, respectively, resulting in an isomeric potency ratio IC50 (+)/IC50(-) of 650. The dissociation constant KA of the (+/-)- or (+)-methacholine-presynaptic receptor complex was determined after fractional receptor inactivation according to Furchgott & Bursztyn (1967) with phenoxybenzamine or propylbenzilylcholine mustard as irreversible antagonists of muscarinic receptors. KA for (-)-methacholine was estimated according to Mackay (1966). KA of (+)-, (+/-)-, and (-)-methacholine were 2.5, 4 and 440 microM, resulting in an isomeric affinity ratio KA (+)/KA(-) of 180. The discrepancy between the isomeric IC50 ratio and the isomeric KA ratio is explained by a higher intrinsic efficacy of the (+)-enantiomer compared to the (-)-enantiomer. Thus, (+)-methacholine has to occupy fewer receptors to induce a given inhibition of release than its antipode as revealed by a plot of fractional receptor occupancy vs response. The results show that, in the effector system of presynaptic muscarinic inhibition, methacholine enantiomers differ greatly not only in affinity for the receptor, but also to some extent in the efficiency of signal transmission, and both parameters contribute to the high isomeric potency ratio. The activity of the racemate is fully accounted for by the activity of the (+)-enantiomer.     

The in vitro and in vivo pharmacological activity of Boiga dendrophila (mangrove catsnake) venom

The great taxonomic and prey base diversity of colubrids (non-front-fanged snakes) suggests that their venoms may represent a 'literal gold mine' for scientists eager to find novel pharmacological probes. While pharmacological characterization is lacking for most of these venoms, this is even more so with regard to activity of colubrid venoms on the mammalian autonomic nervous system. This study characterizes the activity of venom from the colubrid, Boiga dendrophila using in vitro smooth muscle preparations and the anaesthetized rat. In the prostatic segment of the rat vas deferens, cumulative additions of venom (1-150 microg ml(-1)) induced concentration-dependent inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches. The inhibitory effect of venom (100 microg ml(-1)) was attenuated by 8-phenyltheophylline (8-PT) (20 microM) and 8-cyclopentyl-1, 3-dipropylxanthine (20 microM) but not idazoxan (1 microM), or a combination of ranitidine (0.2 microM) and thioperamide (10 microM). The inhibitory effect of venom (100 microg ml(-1)) was augmented by dipyridamole (10 microM) but abolished by pretreatment with adenosine deaminase (7.5 units/100 microl) suggesting that it contains components with adenosine A(1) receptor activity, most likely adenosine. In isolated segments of guinea-pig ileum, venom (10-100 microg ml(-1)) caused concentration-dependent contractions which were inhibited by the muscarinic receptor antagonist atropine (0.1 microM) but not by the histamine receptor antagonist mepyramine (0.5 microM). In the anaesthetized rat, venom (5-7.5 mg kg(-1), i.v.) caused a hypotensive effect. Our data suggest that the venom contains components with purinergic and muscarinic receptor activity.     

Expression of a cloned muscarinic receptor in A9 L cells

Using an oligonucleotide based on the sequence of a porcine brain muscarinic receptor cDNA, we recently cloned four distinct muscarinic receptors from the rat and human genomes. In the present study we transfected the rat homolog of the porcine brain muscarinic receptor cDNA into A9 L cells using a mammalian expression vector and a calcium phosphate precipitation procedure. Before transfection, A9 L cells do not bind muscarinic ligands and do not express muscarinic receptor mRNA. After transfection, A9 L cells expressed muscarinic receptor mRNA and saturable, high affinity binding sites for the muscarinic antagonists 3H-quinuclidinyl benzilate and 3H-pirenzepine. The muscarinic receptor antagonists AF DX-116 and pirenzepine displaced bound 3H-quinuclidinyl benzilate with inhibition curves suggestive of a single high affinity binding site. Competition of 3H-quinuclidinyl benzilate-labeled sites with the agonists acetylcholine and carbachol yielded broad inhibition curves, consistent with a heterogeneity of binding sites. In the presence of guanine nucleotide, the agonist inhibition curves were steeper, suggesting the presence of a single low affinity site. The effects of guanine nucleotides on agonist binding are consistent with coupling of these receptors to a guanine nucleotide-binding protein (G-protein) endogenous to A9 L cells. The electrical properties of the transfected A9 L cells were examined using the whole cell patch-clamp technique. Fifty microM acetylcholine induced a conductance which reversed in polarity at -60 mV. This conductance could be reversibly blocked by atropine. These data illustrate the utility of stable transfection of A9 L cells for the characterization of individual cloned muscarinic receptors, their G-protein coupling mechanisms, and resultant physiological responses.     

Tetranitromethane modification of the muscarinic receptors in bovine adrenal medulla

Muscarinic receptor binding was examined in bovine adrenal medullary microsomes following exposure to tetranitromethane (TNM) that modifies tyrosine and cysteine residues in proteins. The TNM (10-100 microM) treatment of adrenal medullary microsomes caused a concentration-dependent and irreversible reduction in the maximum number of binding sites (Bmax) for l-(3H)quinuclidinyl benzilate (QNB), with a slight increase in the equilibrium dissociation constant (KD). Typically, about a 36% decrease and a 1.3-fold increase in the corresponding values were obtained at 50 microM of TNM. The alteration in the Bmax was partially prevented by atropine but not carbamylcholine, and it was not reversed by subsequent treatment with dithiothreitol, a disulfide reducing agent. The change in the KD was unaffected by these agents. The TNM (50 microM) treatment also caused a slight decrease in the affinity of atropine and pirenzepine (for both the high and low affinity sites), and it caused a slight decrease in the affinity of carbamylcholine at the high affinity site, with a large loss of the low affinity site. Thus, the results indicate that TNM causes a loss of muscarinic binding sites and a decrease in the binding affinity of muscarinic receptors in bovine adrenal medulla, probably through modifications of functional groups such as tyrosine residues.     

The determination of presynaptic KA values of methacholine and pilocarpine and of a presynaptic receptor reserve in the rat perfused heart.

Rat isolated perfused hearts with the right sympathetic nerves attached were loaded with [3H]-noradrenaline. The nerves were stimulated with up to 11 trains of 10 pulses at 0.1 Hz. The evoked increases of [3H]-noradrenaline overflow into the perfusate were measured in the presence of cocaine, corticosterone and propranolol. Activation of presynaptic muscarinic receptors by methacholine or pilocarpine inhibited the evoked transmitter release in a reversible and concentration-dependent manner. Preperfusion with phenoxybenzamine (5 microM) for 15 min (followed by a washout of 35 min) changed neither resting nor evoked overflow of [3H]-noradrenaline. The concentration-response curve of methacholine was shifted to the right after exposure of the hearts to phenoxybenzamine (1 microM) without depression of the maximum effect. Pretreatment with phenoxybenzamine (5 microM) reduced the maximum inhibition of release by about 50%. Analysis of the data gave a dissociation constant for the agonist-receptor complex (KA) of 4.0 microM and a receptor reserve of roughly 70%. Half-maximal inhibition of [3H]-noradrenaline release occurred when about 2% of the total receptor population was occupied. Comparison of the concentration-response data for methacholine and pilocarpine revealed a relative efficacy (methacholine/pilocarpine) of 16, a KA of 10 microM for pilocarpine and no receptor reserve for this agonist. The results show that KA values for methacholine and pilocarpine obtained at presynaptic receptors are similar to those obtained at postsynaptic muscarinic receptors. This is in agreement with the idea that muscarinic receptors located on postganglionic adrenergic nerves are not different from those located on effector sites of non-neuronal tissue.     

ELectrophysiologic properties of bretylium tosylate on atrial myocardium

The antifibrillatory property of bretylium tosylate was first observed in experimental atrial fibrillation, yet the cellular basis for this phenomenon has not been explored. The purpose of this study was to determine the electrophysiologic properties of bretylium tosylate on guinea pig atrial myocardium in the presence and absence of cholinergic influence. Bretylium (10(-6) M - 10(-4) M) produced a concentration-dependent prolongation of atrial action potential duration with a threshold concentration of 10(-5) M. This direct effect of bretylium was unaltered by blockade of beta-adrenergic receptors with propranolol (10(-6) M) or blockade of alpha-adrenergic receptors with phentolamine (10(-6) M). In a second series of experiments the muscarinic receptor blocking properties of bretylium were determined. Acetylcholine produced a concentration-dependent shortening of action potential duration in paced (200 ms) left atrial muscle strips. This well-recognized muscarinic effect was unaltered in the presence of bretylium (10(-6) M - 10(-3) M). These data indicate that bretylium tosylate physiologically exerts direct effects on the atrial myocardium to prolong action potential duration. This compound does not appear to physiologically antagonize the effects of acetylcholine and therefore its reported atrial antiarrhythmic properties cannot be explained by muscarinic receptor blockade.     

Changes in muscarinic receptors in the toad urothelial cell line TBM-54 following acrolein treatment

1. In cyclophosphamide-induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM-54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2. The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3. Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration-dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 +/- 6%. Pre-incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M(1) receptor selective), methoctramine (M(2)/M(4) receptor selective) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M(3)/M(1)/M(5) receptor selective)), gave rise to a concentration-dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4. Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M(1)-M(5)) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M(2) receptors was decreased, whereas the expression of muscarinic M(3) receptors was increased. 5. In conclusion, the urothelial cell line TBM-54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide-induced cystitis.     

Direct Involvement of G Protein αq/11n Subunit in Regulation of Muscarinic Receptor-Mediated sAPPα Release

The G(q/11) protein-coupled receptors, such as muscarinic (M1 & M3) receptors, have been shown to regulate the release of a soluble amyloid precursor protein (sAPPalpha) produced from alpha-secretase processing. However, there is no direct evidence for the precise characteristics of G proteins, and the signaling mechanism for the regulation of G(q/11) protein-coupled receptor-mediated sAPPalpha release is not clearly understood. This study examined whether the muscarinic receptor-mediated release of sAPPalpha is directly regulated by Galpha(q/11) proteins. The HEK293 cells were transiently cotransfected with muscarinic M3 receptors and a dominant-negative minigene construct of the G protein alpha subunit. The sAPPalpha release in the media was measured using an antibody specific for sAPP. The sAPPalpha release enhancement induced by muscarinic receptor stimulation was decreased by a G(q/11) minigene construct, whereas it was not blocked by a control minigene construct (the Galpha carboxy peptide in random order, Galpha(q)R) or Galpha(i) constructs. This indicated a direct role of the Galpha(q/11) protein in the regulation of muscarinic M3 receptor-mediated sAPPalpha release. We also investigated whether the transactivation of the epidermal growth factor receptor (EGFR) by a muscarinic agonist could regulate the sAPPalpha release in SH-SY5Y cells. Pretreatment of a specific EGFR kinase inhibitor, tyrophostin AG1478 (250 nM), blocked the EGF-stimulated sAPPalpha release, but did not block the oxoM-stimulated sAPPalpha release. This demonstrated that the transactivation of the EGFR by muscarinic receptor activation was not involved in the muscarinic receptor-mediated sAPPalpha release.     

Endogenous aspartate release in the rat hippocampus is inhibited by M2 'cardiac' muscarinic receptors

The release of endogenous aspartic acid elicited by depolarization of rat hippocampus synaptosomes with 15 mM KCl was totally calcium-dependent. Acetylcholine (ACh) added to the superfusion medium inhibited the K(+)-evoked release of aspartate in a concentration-dependent manner. The effect of ACh was mimicked by oxotremorine and carbachol. It was insensitive to the nicotinic receptor antagonist mecamylamine but blocked by the non-selective muscarinic receptor antagonist atropine. Further pharmacological characterization of the muscarinic receptor involved showed that the ACh effects was insensitive to the M1 selective muscarinic receptor antagonists pirenzepine and dicyclomine. However, the inhibition by ACh of aspartate release was counteracted by 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido-[2-3-b][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 'cardiac' receptor antagonist. The calcium dependence of the release of aspartate and its regulation through presynaptic receptors are suggestive of a transmitter role for this excitatory amino acid. Moreover, the similarities between the present results and those previously obtained with glutamate are compatible with the idea that aspartate and glutamate are co-released in the rat hippocampus.     

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine.

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.     

Regulation of phospholipase Cbeta activity by muscarinic acetylcholine and 5-HT(2) receptors in crude and synaptosomal membranes from human cerebral cortex

Stimulation of phospholipase Cbeta by receptor agonists and G proteins has been characterized in crude cerebral membrane preparations, but little is known about their presynaptic localizations and little information is currently available for human brain tissue. The characteristics of phosphoplipase C transmembrane signaling were studied in crude and synaptosomal plasma membranes from postmortem human prefrontal cortex by measuring the hydrolysis of exogenous [(3)H]phosphatidylinositol4,5bisphosphate(PIP(2)) and the immunoreactive levels of phospholipase C (PLC) and G(alphaq/11) proteins. Regulation of PLC activity by Ca(2+) and the 5-HT(2) receptor agonist 5-methyltryptamine, but not by guanosine 5'-O-[3-thiotriphosphate] and the muscarinic acetylcholine receptor agonist carbachol were different between crude and synaptosomal membranes. KCl (20 mM) stimulation was absent in both preparations. Levels of G(alphaq/11)-protein subunits differed between preparations. The functional inhibition carried out with pirenzepine in crude membranes in order to reverse the carbachol-induced PLC stimulation indicates the existence of a component (53%) of the response that is activated by the M(1) muscarinic acetylcholine receptor subtype, and another component (47%) probably mediated by the M(3) muscarinic acetylcholine receptor subtype. In synaptosomal plasma membranes an increased inhibition of carbachol-induced PLC activation through M(1) was found. The PLC activation by 5-methyltryptamine (ketanserin-sensitive in crude membranes) was absent in synaptosomal plasma membranes suggesting the lack of activity mediated by 5-HT(2)-serotonin receptors.     

Muscarinic receptor agonist-mediated modulation of neuronal activity in rat cerebral cortex.

Multiple cortical neuronal responses were elicited by the iontophoretic application of muscarinic receptor agonists and antagonists in the rat cerebral sensorimotor cortex in vivo. (1) The muscarinic receptor agonist, oxotremorine-M induced a biphasic effect on spontaneous firing. This was evident as an early brief increase in the firing rate over the spontaneous discharge followed by secondary inhibition of spontaneous activity. The excitation could be blocked by the muscarinic receptor non-selective antagonist atropine and by both the M1 receptor antagonist pirenzepine and the M2 receptor antagonists gallamine or methoctramine. Oxotremorine-M inhibition of spontaneous activity was not affected by the M1 receptor antagonist pirenzepine, while evaluation of its sensitivity to gallamine and methoctramine was not possible since these two M2 receptor antagonists also depressed spontaneous activity, unlike pirenzepine. Of the other two muscarinic receptor agonists, oxotremorine had inconsistent and weak excitatory effects whilst McN-A-343 had only weak excitatory or inhibitory effects on spontaneous activity. (2) Oxotremorine-M, oxotremorine and McN-A-343 had a depressant action on neuronal discharges evoked by glutamate or acetylcholine. A depressant effect of oxotremorine-M was also demonstrated on the early excitation evoked by subsequent applications of oxotremorine-M itself. Of the three muscarinic receptor agonists tested, oxotremorine-M was the most potent in evoking a long-term depression of evoked discharges, lasting from several minutes (greater than 5 min) to as long as 40 min. Oxotremorine-M-induced depression of evoked responses was most sensitive to the M2 receptor antagonists, whereas oxotremorine-induced depression was more sensitive to the M1 receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of organophosphorus anticholinesterases on muscarinic receptor-mediated inhibition of acetylcholine release in rat striatum

The aim of the present study was to evaluate the in vitro modulation of muscarinic autoreceptor function by the organophosphorus (OP) anticholinesterases chlorpyrifos oxon, paraoxon, and methyl paraoxon. Acetylcholine (ACh) release was studied by preloading slices from rat striatum with [3H]choline and depolarizing with potassium (20 mM) in perfusion buffer containing hemicholinium-3 (to prevent reuptake of radiolabeled choline). Under these conditions, chlorpyrifos oxon, paraoxon, and methyl paraoxon (0.1-10 microM) all reduced ACh release in a concentration-dependent manner. Addition of the carbamate acetylcholinesterase (AChE) inhibitor physostigmine (20 microM) to the perfusion buffer also decreased ACh release. When physostigmine was present, the three oxons had no additional effect on ACh release. Concentration-dependent inhibition of AChE activity in striatal slices perfused with chlorpyrifos oxon (0.1, 1, and 10 microM) suggested AChE inhibition was responsible for oxon-mediated alterations in ACh release. To differentiate between direct and indirect actions of the OP toxicants on muscarinic autoreceptors, we compared the effects of the oxons on ACh release under two conditions, i.e., tissues were perfused with buffer containing only hemicholinium-3 or with buffer containing hemicholinium-3, physostigmine, and the nonselective muscarinic receptor blocker atropine (100 nM). In the presence of only hemicholinium-3, concentration-dependent inhibition of ACh release was again noted for all oxons, similar to the effects of the muscarinic agonists carbachol and cis-dioxolane. In the presence of physostigmine and atropine, the relative potencies of all agents were markedly reduced. Interestingly, carbachol, cis-dioxolane, paraoxon, and methyl paraoxon all decreased ACh release as before, but chlorpyrifos oxon (100-300 microM) actually increased ACh release. Together, the results suggest that chlorpyrifos oxon, paraoxon, and methyl paraoxon can activate muscarinic autoreceptors indirectly through inhibition of AChE. Both paraoxon and methyl paraoxon also directly activate whereas chlorpyrifos oxon blocks muscarinic autoreceptor function. Qualitative differences in the direct actions of these oxons at this presynaptic regulatory site could contribute to differential toxicity with high-dose exposures.     

Inhibitory effect of lead acetate on contractility of longitudinal smooth muscle from rat ileum

The effect of lead acetate on contractility of ileal longitudinal smooth muscle was determined in vitro. Strips, removed from the ileum of male Wistar rats, were suspended in Tris buffer and isometric tension was recorded. Responses to muscarinic receptor activation with methacholine and membrane depolarization with KCl were measured before and after addition of lead acetate to the tissue bath. Lead concentrations of 1 and 3.2 microM had no effect on methacholine (0.45 microM) or KCl (80 mM) induced contractions. However, at concentrations of 10 to 32 microM lead produced a concentration-dependent decline in the response to both these agonists. Both the receptor affinity and intrinsic activity of methacholine were reduced by 160 microM lead. This concentration of lead also inhibited the contractions induced by cumulative addition of calcium chloride (0.18-4.5 mM) to calcium-depleted, KCl-depolarized tissue. This inhibition could be overcome by increased concentrations of calcium. The decreased response to muscarinic receptor activation by methacholine and to membrane depolarization by KCl could result from the impairment of calcium influx or intracellular function.     

Affinity-labelling of the muscarinic receptor of the guinea-pig ileum

1. The activities of some cholinergic compounds were investigated on the terminal ileum of the guinea-pig and their muscarinic potencies were found to be in the following order: acetylcholine (ACh) greater than diazoacetylcholine (DACh) greater than iodoacetylcholine (IACh) greater than azidoacetylcholine (AACh). 2. Protection experiments with atropine showed a decreased affinity of the four cholinergic compounds for the muscarinic receptor, demonstrating a direct interaction of the drugs and the receptor. 3. The maximal contractions (intrinsic activity) caused by DACh were greater than those casued by ACh, IACh and AACh. 4. Incubation of the ileum with ACh was followed by a reversible loss of its ability to contract (desensitization). The time course of recovery was similiar to that after incubation with AACh. 5. IACh caused a partial (50%), irreversible paralysis of the muscle. 6. The furaziridines seem to react partially irreversibly (30%), whereas paranitrophenyldiazonium fluoborate (p-NDP) caused a complete, irreversible blockade of the muscarinic receptor.     

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.     

Bisquaternary pyridinium oximes as allosteric inhibitors of rat brain muscarinic receptors

The mode of interaction of bisquaternary pyridinium oximes with rat brain muscarinic receptors in cerebral cortex and brain stem preparations was studied by the use of the tritium-labeled antagonist N-methyl-4-piperidyl benzilate ( [3H] 4NMPB). Binding of the labeled muscarinic antagonist was inhibited by these drugs, the most potent inhibitors being 1-(2-hydroxyiminoethylpyridinium)-1-(3-cyclohexylcarboxypyridin ium)dimethyl-ether (HGG-42) and its 3-phenylcarboxypyridinium analog (HGG-12) (apparent KI = 1.3-1.7 and 1.8-2.2 microM, respectively). Analysis of the binding properties suggested that binding of the muscarinic antagonist and the bisquaternary pyridinium oximes was nonexclusive. Kinetic binding data provide evidence that the drugs inhibit binding of muscarinic antagonists in an allosteric manner, with a resulting decrease in the rates of both association of [3H]4NMPB to the receptor and its dissociation from it. These effects were observed both in brain stem and in cortical preparations even after pretreatment and washing out of the inhibitors. The selective natures of HGG-12 and HGG-42 were apparent from their irreversible effects on the number of muscarinic binding sites. In brain stem, the presence of these drugs resulted in a loss of about 30% of binding sites, which accounts in part for the apparent decrease in maximal binding capacity observed in the equilibrium binding of [3H]4NMPB. In the cortex, however, only approximately 10% of the muscarinic receptors were lost upon exposure to these drugs. The decrease in the muscarinic receptor population of the brain stem was dependent on both concentration and time and occurred both in vitro and in vivo following injection of HGG-12 into rats. Unlike the in vitro loss of receptor sites, which was irreversible, the in vivo effect was restored 2 hr after the injection. Taken together, the results suggest that the bisquaternary oximes are allosteric inhibitors of the muscarinic acetylcholine receptor and may be capable of distinguishing between receptor states and inducing specific irreversible effects. Because of these properties, the drugs may prove extremely useful as sensitive probes in studies on the nature of the agonist-receptor-effector relationship.     

Muscarinic M4 receptor inhibition of dopamine D1-like receptor signalling in rat nucleus accumbens

Several studies have indicated the occurrence of an antagonistic interaction between muscarinic and dopamine D1-like receptors in the ventral striatum, but the subtype(s) of muscarinic receptor involved has not been characterized. We show that in membranes of rat nucleus accumbens, carbachol inhibited the stimulation of adenylyl cyclase activity by dopamine and the dopamine D1-like receptor agonist (+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine without affecting the binding properties of dopamine to dopamine D1-like receptors. The carbachol inhibition was competitively counteracted by receptor antagonists with a rank order of potency typical of the involvement of the muscarinic M(4) receptor subtype. Moreover, muscarinic toxin 3, a selective muscarinic M(4) receptor antagonist, completely blocked the carbachol inhibition, whereas muscarinic toxin 7, a selective muscarinic M(1) receptor antagonist, had no effect. The muscarinic inhibition occurred to a similar extent in the core and shell regions. These data demonstrate that in nucleus accumbens, muscarinic M(4) receptors exert a direct inhibitory control on dopamine D1-like receptor signalling.