Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO-K1 cells.
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1. Affinity estimates were obtained for several muscarinic antagonists against carbachol-stimulated [3H]-inositol phosphates accumulation in Chinese hamster ovary (CHO-KI) cells stably expressing either human muscarinic M3 or M5 receptor subtypes. The rationale for these studies was to generate a functional antagonist affinity profile for the M5 receptor subtype and compare this with that of the M3 receptor, in order to identify compounds which discriminate between these two subtypes. 2. The rank order of antagonist apparent affinities (pK(B)) at the muscarinic M5 receptor was atropine (8.7) > or =tolterodine (8.6) = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.6)> darifenacin (7.7) > or =zamifenacin (7.6)>oxybutynin (6.6)= para-fluorohexahydrosiladifenidol (p-F-HHSiD, 6.6)>pirenzepine (6.4) > or = methoctramine (6.3)=himbacine (6.3)>AQ-RA 741 (6.1). 3. Antagonist apparent affinities for both receptor subtypes compare well with published binding affinity estimates. No antagonist displayed greater selectivity for the muscarinic M5 subtype over the M3 subtype, but himbacine, AQ-RA 741, p-F-HHSiD, darifenacin and oxybutynin displayed between 9- and 60 fold greater selectivity for the muscarinic M3 over the M5 subtype. 4. This study highlights the similarity in pharmacological profiles of M3 and M5 receptor subtypes and identifies five antagonists that may represent useful tools for discriminating between these two subtypes. Collectively, these data show that in the absence of a high affinity M5 selective antagonist, affinity data for a large range of antagonists is critical to define operationally the M5 receptor subtype. (+info)
Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells.
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We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[(3)H]methylscopolamine demonstrated that mAChRs are approximately 2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca(2+) concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M(4) and M(5) receptor mRNAs. These data suggest that M(4) receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M(5) receptor may reside in the basolateral membrane of a subset of cells. (+info)
Muscarinic--but not nicotinic--acetylcholine receptors mediate a nitric oxide-dependent dilation in brain cortical arterioles: a possible role for the M5 receptor subtype.
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Increases in cortical cerebral blood flow are induced by stimulation of basal forebrain cholinergic neurons. This response is mediated in part by nitric oxide (NO) and reportedly involves both nicotinic and muscarinic receptors, some of which are possibly located in the vessel wall. In the present study, the vasomotor response(s) elicited by acetylcholine (ACh) on isolated and pressurized bovine and/or human intracortical penetrating arterioles were investigated, and pharmacological characterization of the receptor involved in this response was carried out. Acetylcholine (10(-11) to 10(-4) mol/L) dose dependently dilated bovine and human intracortical arterioles at spontaneous tone (respective pD2 values of 6.4+/-0.3 and 7.2+/-0.3 and E(Amax) of 65.0+/-26.8 and 43.2+/-30.1% of the maximal dilation obtained with papaverine) and bovine arterioles after preconstriction with serotonin (pD2 = 6.3+/-0.1, E(Amax) = 80.0+/-17.9% of induced tone). In contrast, nicotine (10(-8) to 10(-4) mol/L) failed to induce any vasomotor response in bovine vessels whether at spontaneous or at pharmacologically induced tone. Application of the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-5) mol/L) elicited a gradual constriction (approximately 20%) of the arterioles, indicating the presence of constitutive NO release in these vessels. Nomega-Nitro-L-argigine (10(-5) to 10(-4) mol/L) also significantly blocked the dilation induced by ACh. The muscarinic ACh receptor (mAChR) antagonists pirenzepine, 4-DAMP, and AF-DX 384 dose dependently inhibited the dilatation induced by ACh (10(-5) mol/L) with the following rank order of potency: 4-DAMP (pIC50 = 9.2+/-0.3) >> pirenzepine (pIC50 = 6.7+/-0.4) > AF-DX 384 (pIC50 = 5.9+/-0.2). These results suggest that ACh can induce a potent, dose-dependent, and NO-mediated dilation of bovine and/or human intracortical arterioles via interaction with an mAChR that best corresponds to the M5 subtype. (+info)
Human esophageal smooth muscle cells express muscarinic receptor subtypes M(1) through M(5).
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Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10-14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific alpha-actin. mRNA encoding muscarinic receptor subtypes M(1)-M(5) was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura 2 fluorescence. Basal [Ca(2+)](i), which was 135 +/- 22 nM, increased transiently to 543 +/- 29 nM in response to 10 microM ACh in CM cells (n = 8). This response was decreased <95% by 0.01 microM 4-diphenylacetoxy-N-methylpiperidine, a M(1)/M(3)-selective antagonist, whereas 0.1 microM methoctramine, a M(2)/M(4)-selective antagonist, and 0.1 microM pirenzepine, a M(1)-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca(2+)](i) mediated primarily by the M(3) receptor and involving release of Ca(2+) from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology. (+info)
Brain-stimulation reward thresholds raised by an antisense oligonucleotide for the M5 muscarinic receptor infused near dopamine cells.
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Oligonucleotides targeting M5 muscarinic receptor mRNA were infused for 6 d into the ventral tegmental area of freely behaving rats trained to bar-press for lateral hypothalamic stimulation. The bar-pressing rate was determined at a range of frequencies each day to evaluate the effects of infusions on reward. M5 antisense oligonucleotide (oligo) infusions increased the frequency required for bar pressing by 48% over baseline levels, with the largest increases occurring after 4-6 d of infusion. Two control oligos had only slight effects (means of 5 and 11% for missense and sense oligos, respectively). After the infusion, the required frequency shifted back to baseline levels gradually over 1-5 d. Antisense oligo infusions decreased M5 receptors on the ipsilateral, but not the contralateral, side of the ventral tegmentum, as compared with a missense oligo. Therefore, M5 muscarinic receptors associated with mesolimbic dopamine neurons seem to be important in brain-stimulation reward. (+info)
Acetylcholine increases the free intracellular calcium concentration in podocytes in intact rat glomeruli via muscarinic M(5) receptors.
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The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca2+](i)) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca2+](i) increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca2+ concentrations. The [Ca2+](i) plateau was not affected by the L-type Ca2+ channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 microM) blocked the plateau. Diphenylacetoxy-N:-methylpiperidine methiodide (100 nM), an M(3)/M(5) receptor antagonist, and pirenzepine (1 microM), an M(1) receptor antagonist, completely inhibited the effect of ACh. [Ca2+](i) measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca2+](i) was increased in single podocytes. Immunohistochemical studies did not demonstrate M(3) receptor expression in glomerular cells. M(1) receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M(5) receptors were found only in podocytes. The data show that ACh increases [Ca2+](i) in podocytes of intact glomeruli, most likely via muscarinic M(5) receptors. (+info)
Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization.
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In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca(2+) channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors encoding rTRP6A or rTRP6B increased Ca(2+) influx and gave rise to a novel Ba(2+) influx after activation of M(5) muscarinic acetylcholine receptors. By contrast, passive depletion of intracellular Ca(2+) stores with thapsigargin did not induce Ba(2+) influx in cells expressing rTRP6 isoforms. Ba(2+) influx was also stimulated in rTRP6A-expressing cells after exposure to the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing cells failed to show OAG-induced Ba(2+) influx. Expression of a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked M(5) muscarinic acetylcholine receptor-dependent Ba(2+) influx in COS cells that were transfected with rTRP6 cDNAs. Together these results suggest that rTRP6 participates in the formation of Ca(2+) channels that are regulated by a G-protein-coupled receptor, but not by intracellular Ca(2+) stores. In contrast to the results we obtained with rTRP6A and rTRP6B, cells expressing rTRP6C showed no increased Ca(2+) or Ba(2+) influxes after stimulation with carbachol and also did not show OAG-induced Ba(2+) influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein. (+info)
RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Botzinger complex.
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Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Botzinger complex (pre-BotC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BotC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BotC taken from rat medullary slices. Cholinergic receptor subtype M(2) and M(3) mRNAs were observed in the first round of PCR amplification. All five subtypes, M(1)-M(5), were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BotC are M(2) and M(3), with minor expression of M(1), M(4), and M(5). (+info)