M2-Receptor Subtype Does Not Mediate Muscarine-Induced Increases in [Ca2+]i in Nociceptive Neurons of Rat Dorsal Root Ganglia

2000 ◽  
Vol 84 (4) ◽  
pp. 1934-1941 ◽  
Author(s):  
Rainer Haberberger ◽  
Reas Scholz ◽  
Wolfgang Kummer ◽  
Michaela Kress
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Rt Pcr ◽  
Nociceptive Neurons

Multiple muscarinic receptor subtypes are present on sensory neurons that may be involved in the modulation of nociception. In this study we focused on the presence of the muscarinic receptor subtypes, M2 and M3 (M2R, M3R), in adult rat lumbar dorsal root ganglia (DRG) at the functional ([Ca2+]i measurement), transcriptional (RT-PCR), and translational level (immunohistochemistry). After 1 day in culture exposure of dissociated medium-sized neurons (20–35 μm diam) to muscarine was followed by rises in [Ca2+]i in 76% of the neurons. The [Ca2+]i increase was absent after removal of extracellular calcium and did not desensitize after repetitive application of the agonist. This rise in [Ca2+]i may be explained by the expression of M3R, which can induce release of calcium from internal stores via inositoltrisphospate. Indeed the effect was antagonized by the muscarinic receptor antagonist atropine as well as by the M3R antagonist, 4-diphenylacetoxy-N-(2 chloroethyl)-piperidine hydrochloride (4-DAMP). The pharmacological identification of M3R was corroborated by RT-PCR of total RNA and single-cell RT-PCR, which revealed the presence of mRNA for M3R in lumbar DRG and in single sensory neurons. In addition, RT-PCR also revealed the expression of M2R, which did not seem to contribute to the calcium changes since it was not prevented by the M2 receptor antagonist, gallamine. Immunohistochemistry demonstrated the presence of M2R and M3R in medium-sized lumbar DRG neurons that also coexpressed binding sites for the lectin I-B4, a marker for mainly cutaneous nociceptors. The occurrence of muscarinic receptors in putative nociceptive I-B4-positive neurons suggests the involvement of these acetylcholine receptors in the modulation of processing of nociceptive stimuli.

RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Bötzinger complex

2001 ◽  
Vol 281 (6) ◽  
pp. L1420-L1424 ◽  
Author(s):  
Jiunu Lai ◽  
Xuesi M. Shao ◽  
Richard W. Pan ◽  
Edward Dy ◽  
Cindy H. Huang ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Respiratory Rhythm ◽  
Pcr Amplification ◽  
Cholinergic Receptor ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Rt Pcr ◽  
Bötzinger Complex

Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), 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-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M2 and M3 mRNAs were observed in the first round of PCR amplification. All five subtypes, M1–M5, were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M2 and M3, with minor expression of M1, M4, and M5.

Muscarinic receptor subtypes in feline tracheal submucosal gland secretion

1992 ◽  
Vol 262 (2) ◽  
pp. L223-L228 ◽  
Author(s):  
H. Ishihara ◽  
S. Shimura ◽  
M. Satoh ◽  
T. Masuda ◽  
H. Nonaka ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Gland Secretion ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Submucosal Gland ◽  
Muscarinic Receptor Subtypes ◽  
Binding Assays ◽  
Submucosal Glands ◽  
Electrolyte Secretion ◽  
Quinuclidinyl Benzilate

To determine what muscarinic receptor subtype regulates [Ca2+]i mediating airway submucosal gland secretion, we examined the effects of atropine (Atr), pirenzepine (PZ), 11([2-(diethylamino)methyl-1-piperidinyl] acetyl)-5,11-dihydro-6H-pyrido (2,3-b)(1,4)-benzo-diazepin-6-one (AF-DX116) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) on methacholine (MCh)-evoked [Ca2+]i rise in acinar cells, and compared this with mucus glycoprotein (MGP) and electrolyte secretion evoked by MCh from submucosal glands isolated from feline trachea. [Ca2+]i was measured with the Ca(2+)-sensitive fluorescent dye, fura 2. We determined MGP secretion by measuring TCA-precipitable 3H-labeled glycoconjugates and electrolyte secretion by the change in the rate constant of 22Na-efflux from isolated glands. Half-maximal inhibitory concentrations (IC50) of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked [Ca2+]i rise were 10(-7) M, 6 x 10(-6) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. IC50 of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked MGP secretion were 10(-6) M, 2 x 10(-5) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. MCh (10(-5) M)-evoked 22Na efflux was significantly inhibited by 10(-7) M 4-DAMP and 10(-7) M Atr (P less than 0.01, each) but not by 10(-7) M PZ. Receptor binding assays with [3H]quinuclidinyl benzilate showed that the Ki values for PZ, AF-D x 116, 4-DAMP and Atr were 2.2 x 10(-8) M, 6.6 x 10(-7) M, 6.2 x 10(-10) M, and 2.9 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro

1992 ◽  
Vol 73 (5) ◽  
pp. 2069-2073 ◽  
Author(s):  
J. Mullol ◽  
J. N. Baraniuk ◽  
C. Logun ◽  
M. Merida ◽  
J. Hausfeld ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Subtypes ◽  
Muscarinic Antagonists ◽  
Muscarinic Receptor Subtypes ◽  
Mucus Production ◽  
Glandular Secretion ◽  
M3 Receptor

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10–100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1–100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.

The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family

2003 ◽  
Vol 31 (6) ◽  
pp. 1182-1185 ◽  
Author(s):  
A.B. Tobin ◽  
D.C. Budd
Keyword(s):  
Cell Death ◽  
Muscarinic Receptor ◽  
Apoptotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Structural Element ◽  
Apoptotic Pathway ◽  
Receptor Family

It is now clear that G-protein-coupled receptors can regulate programmed cell death (apoptosis) through a variety of mechanisms that are dependent on cell type and receptor subtype. Here we present evidence that the Gq/11-coupled subtypes of the muscarinic receptor family (namely M1, M3 and M5-muscarinic receptor subtypes) are able to protect against apoptotic cell death. In particular we demonstrate that the C-terminal tail of the M3-muscarinic receptor is an essential structural element for signalling to the anti-apoptotic pathway. Removal of the distal portion of the C-terminal tail results in a receptor that is coupled normally to the Gq/11/phospholipase C pathway and the mitogen-activated protein kinase pathway, but is unable to couple to the anti-apoptotic pathway. Furthermore, a poly-basic region conserved within the C-terminal tail of the Gq/11-coupled muscarinic receptor subtypes appears to be the structural determinant of coupling to the anti-apoptotic pathway.

scholarly journals Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing

2005 ◽  
Vol 144 (8) ◽  
pp. 1089-1099 ◽  
Author(s):  
Kylie J Mansfield ◽  
Lu Liu ◽  
Frederick J Mitchelson ◽  
Kate H Moore ◽  
Richard J Millard ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Radioligand Binding ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Rt Pcr ◽  
Human Bladder ◽  
Bladder Detrusor

Spinal Muscarinic and Nicotinic Subtypes Activated by Clonidine in Postincisional Pain

2005 ◽  
Vol 103 (6) ◽  
pp. 1253-1258 ◽  
Author(s):  
Frédéric Duflo ◽  
Emmanuel Boselli ◽  
Philippe Ryvlin ◽  
Dominique Chassard
Keyword(s):  
Postoperative Pain ◽  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Nicotinic Receptor ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Antagonist ◽  
Alpha7 Nicotinic Receptor ◽  
Intrathecal Clonidine ◽  
Nicotinic Receptor Antagonist

Background A recent model of acute incisional pain has been characterized that strongly parallels the postoperative period in patients experiencing evoked pain. In that setting, abundant literature has revealed antihypersensitive effects produced by intrathecally administered alpha2-adrenergic receptor agonists, such as clonidine, in both animals and humans. Recent reports have suggested an obligatory role of spinal acetylcholine receptors in the analgesic action of intrathecal clonidine. The authors sought to determine the involvement of spinal muscarinic and nicotinic receptor subpopulations in the antihypersensitivity effect of intrathecal clonidine in a rodent model for human postoperative pain. Methods After intrathecal catheterization, rats underwent superficial plantar incision. Clonidine or a combination of clonidine and muscarinic receptor subtype antagonists (M1, M2, M3, and M4) or nicotinic receptor subtype antagonists (alpha4beta2 and alpha7) were intrathecally administered, and withdrawal thresholds to mechanical stimuli were examined. Results Spinal clonidine maximally reduced hypersensitivity adjacent to the wound 30 min after its injection. When animals were intrathecally pretreated with the M1 muscarinic antagonist toxin MT-7, the M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine, and the M4 muscarinic antagonist toxin MT-3, clonidine lost its antihypersensitive action. When animals were intrathecally pretreated with the alpha4beta2 nicotinic receptor antagonist dihydro-beta-erythroidine, but not with the alpha7 nicotinic receptor antagonist methyllycaconitine, the antihypersensitivity action of clonidine was abolished. Conclusions These data indicate for the first time that the clonidine-induced increase in punctuate mechanical threshold is mediated via the activation of all but M2 muscarinic receptor subtypes, and via the activation of alpha4beta2 but not alpha7 nicotinic receptor subtypes in a rodent model for human postoperative pain.

Muscarinic receptor subtypes on rat pancreatic acini: secretion and binding studies

1986 ◽  
Vol 251 (2) ◽  
pp. G275-G279 ◽  
Author(s):  
D. S. Louie ◽  
C. Owyang
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Rightward Shift

Characterization of muscarinic receptor subtypes on rat pancreatic acinar cells was examined by using specific muscarinic receptor antagonists to study amylase secretion and binding of [N-methyl-3H]scopolamine ([3H]NMS). Rat pancreatic acini were dispersed in HEPES-Ringer buffer and incubated with acetylcholine +/- 4-diphenylacetoxy-N-methylpiperadine-methiodide (4-DAMP, a specific M2 muscarinic receptor antagonist) or +/- pirenzepine (a specific M1 muscarinic receptor antagonist). 4-DAMP (10(-9) to 10(-6) M) caused a progressive parallel rightward shift in the acetylcholine dose-response curve without a change in maximal amylase release. Only high concentrations of pirenzepine (10(-6) to 10(-4) M) caused a rightward shift in the dose-response curve to acetylcholine. Schild analysis of the data indicated an inhibitory constant (Ki) of 200 pM for 4-DAMP and 183 nM for pirenzepine. The slope of the Schild regression lines was not different from unity, suggesting competitive inhibition. Binding of 50 pM [3H]NMS was specific, rapid, and saturable. [3H]NMS binding was displaced by increasing concentrations of 4-DAMP or pirenzepine with apparent Ki's of 102 pM and 330 nM, respectively, and similar maximal binding levels of 60 fmol/mg prot. We have demonstrated that 4-DAMP has an approximately 1,000-fold greater potency than pirenzepine to inhibit amylase release and binding, indicating that cholinergic-stimulated amylase release from pancreatic acini is mediated by M2 muscarinic receptors.

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