Inhibition of acetylcholine muscarinic M1 receptor function by the M1 -selective ligand muscarinic toxin 7 (MT-7) (original) (raw)
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Inhibition of acetylcholine muscarinic M(1) receptor function 2000
1 MT-7 (1 ± 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M 1 receptor, inhibited the acetylcholine (ACh)-stimulated [ 35 S]-guanosine-5'-O-(3-thio)triphosphate ([ 35 S]-GTPgS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M 1 receptor subtype. 2 MT-7 failed to aect the ACh-stimulated [ 35 S]-GTPgS binding in membranes of CHO cells expressing either the M 2 , M 3 or M 4 receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M 1 and M 4 receptor subtypes, MT-7 (0.3 ± 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to aect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. 4 In both CHO/M 1 and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist eect with minimal changes in the agonist EC 50 value. 5 In CHO/M 1 cell membranes, MT-7 (0.05 ± 25 nM) reduced the speci®c binding of 0.05, 1.0 and 15 nM [ 3 H]-N-methylscopolamine ([ 3 H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [ 3 H]-NMS by about 5 fold.
Action of the muscarinic toxin MT7 on agonist-bound muscarinic 2004
The muscarinic toxin MT7 is the most selective ligand for the muscarinic M 1 receptors. Previous studies have shown that the toxin interacts with the antagonist -receptor complex and slows the antagonist dissociation rate, possibly by binding to an allosteric site and impeding the access to and egress from the orthosteric binding pocket. In the present study, we investigated the action of MT7 on agonistoccupied receptors in functional and radioligand binding assays of the cloned human muscarinic M 1 receptor expressed in Chinese hamster ovary cells. In time-course experiments, the addition of MT7 rapidly blocked the acetylcholine-stimulated guanosine-5V-O-(3-[ 35 S]thio)triphosphate binding to membrane G proteins. Similarly, in acetylcholine-treated cells MT7 completely stopped the agoniststimulated [ 3 H]inositol phosphate accumulation. In dissociation experiments using membranes pre-equilibrated with [ 3 H]acetylcholine, the addition of MT7 increased the rate of radioligand dissociation. The data indicate that MT7, while partially stabilizing the antagonist -receptor complex, effectively destabilizes the agonist-occupied muscarinic M 1 receptors. D
Localization of M1 muscarinic receptors in rat brain using selective muscarinic 1997
Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M 1 receptors in homogenates of rat cerebral cortex. Iodination of the toxin, 125 I-muscarinic toxin-1 ( 125 I-MT-1), renders the toxin selective for M 1 muscarinic receptors. Quantitative measurement of 125 I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of 125 I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord. © 1997 Elsevier Science Inc.
Immunochemical Studies of the Muscarinic Acetylcholine Receptor
Journal of Receptors and Signal Transduction, 1987
Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and Nmethylscopolamine showed that the human embryonic lung fibroblast CCLl37 possesses approximately 2 x lo5 muscarinic receptors/cell, i. e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.
British Journal of Pharmacology, 1996
1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin-and dopamine DI receptorstimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Kd but did not change the Bm value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3HJ-NMS binding sites with a Ki of 8.0 nM. 3 In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4 In rat cerebral cortical membranes prelabelled with [3H]-myo-inositol, MT3 counteracted the methacholine stimulation of [3H]-inositol phosphates formation with a Ki value of 113 nM.
Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system
Toxicon, 2000
Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are ®ve subtypes that are all expressed in the brain of mammals (m1±m5). There are regional dierences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been dicult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3 H±NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo eects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents. #
Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity 1996
1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin-and dopamine DI receptorstimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Kd but did not change the Bm value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3HJ-NMS binding sites with a Ki of 8.0 nM. 3 In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4 In rat cerebral cortical membranes prelabelled with [3H]-myo-inositol, MT3 counteracted the methacholine stimulation of [3H]-inositol phosphates formation with a Ki value of 113 nM.
Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line
Neurochemical Research, 1992
Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (CHAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10 -5 M retinoic acid (R_A) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a Bma x of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An ICso of 2.5 x 10 -8 M and 0.9 x 10 -8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7 x 10 -z M and 3 x 10 -e M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC5o:110 p~M) thus suggesting the presence of an M3 receptor. Acetylcholine (100 p~M) plus eserine (50 ~M) and BW284c55 (1 IxM), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 raM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 txM), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of c~-bungarotoxin (c~-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M~ receptor.
Biochemical and Biophysical Research Communications, 1998
1, 2, 10, 11). Such a mechanism may thus affect all Muscarinic receptors M 1 , M 3 and M 5 were expressed fast or transient responses mediated by receptors. Bein Sf9 cells. Three different patterns of inhibition of cause of its kinetic character this type of inhibition has Ca 2/ elevations could be resolved for the subtype nonbeen called pseudo-noncompetitive (2) or insurmountselective muscarinic receptor antagonists: (i) a right able (11). Ca 2/ elevation is the best known rapid and shift of the agonist dose-response curve, (ii) a right transient second-messenger signal due to a rapid of shift of the agonist dose-response curve and a depres-IP 3 production and Ca 2/ store depletion as well as and sion of the maximum signal, and (iii) an intermediate removal of Ca 2/ from the cytosol. Consequently, an appattern where the antagonist apparently behaved parent noncompetitive inhibition has been observed in more competitively at higher concentrations. A simumuscarinic receptor mediated Ca 2/ and IP 3 responses lation performed assuming that these differences are (1-4). previous results have suggested that pseudo-nondue to differences in the dissociation rates of the ancompetitive antagonist may show receptor subtype setagonists reproduced all three different modes of inhilectivity (7). We have therefore in this study aimed to bition; the novel intermediate pattern (iii) is suggested scrutinize these previous observations by comparing to be caused by an intermediate antagonist dissocia-
British Journal of Pharmacology, 1995
1 Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2 m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 /M for half maximal stimulation. 3 The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 gIM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 ,M and 5.92 ,UM respectively) but not with rapid agonist-mediated calcium elevation (EC5o = 0.32 gM) measured in fura-2-AM loaded cells. 4 These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.