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

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

Journal of Neurophysiology ◽

10.1152/jn.2000.84.4.1934 ◽

2000 ◽

Vol 84 (4) ◽

pp. 1934-1941 ◽

Cited By ~ 21

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.

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Muscarinic receptor subtypes in feline tracheal submucosal gland secretion

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.262.2.l223 ◽

1992 ◽

Vol 262 (2) ◽

pp. L223-L228 ◽

Cited By ~ 7

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)

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.6.l1420 ◽

2001 ◽

Vol 281 (6) ◽

pp. L1420-L1424 ◽

Cited By ~ 14

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.

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Activation of the intrinsic-apoptotic pathway in LNCaP prostate cancer cells by genistein- topotecan combination treatments

Functional Foods in Health and Disease ◽

10.31989/ffhd.v3i3.64 ◽

2013 ◽

Vol 3 (3) ◽

pp. 66 ◽

Cited By ~ 2

Author(s):

Vanessa Hörmann ◽

Sivanesan Dhandayuthapani ◽

James Kumi-Diaka ◽

Appu Rathinavelu

Keyword(s):

Prostate Cancer ◽

Cell Death ◽

Cancer Cells ◽

Apoptotic Cell ◽

Treatment Options ◽

Attractive Alternative ◽

Intrinsic Apoptotic Pathway ◽

Prostate Cancer Cells ◽

Apoptotic Pathway ◽

Combination Treatments

Background: Prostate cancer is the second most common cancer in American men. The development of alternative preventative and/or treatment options utilizing a combination of phytochemicals and chemotherapeutic drugs could be an attractive alternative compared to conventional carcinoma treatments. Genistein isoflavone is the primary dietary phytochemical found in soy and has demonstrated anti-tumor activities in LNCaP prostate cancer cells. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy for secondary treatment of lung, ovarian and cervical cancers. The purpose of this study was to detail the potential activation of the intrinsic apoptotic pathway in LNCaP prostate cancer cells through genistein-topotecan combination treatments. Methods: LNCaP cells were cultured in complete RPMI medium in a monolayer (70-80% confluency) at 37ºC and 5% CO2. Treatment consisted of single and combination groups of genistein and topotecan for 24 hours. The treated cells were assayed for i) growth inhibition through trypan blue exclusion assay and microphotography, ii) classification of cellular death through acridine/ ethidium bromide fluorescent staining, and iii) activation of the intrinsic apoptotic pathway through Jc-1: mitochondrial membrane potential assay, cytochrome c release and Bcl-2 protein expression.Results: The overall data indicated that genistein-topotecan combination was significantly more efficacious in reducing the prostate carcinoma’s viability compared to the single treatment options. In all treatment groups, cell death occurred primarily through the activation of the intrinsic apoptotic pathway.Conclusion: The combination of topotecan and genistein has the potential to lead to treatment options with equal therapeutic efficiency as traditional chemo- and radiation therapies, but lower cell cytotoxicity and fewer side effects in patients. Key words: topotecan; genistein; intrinsic apoptotic cell death

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