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    The M(1) muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

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    Author
    Mei, Lin.
    Issue Date
    1989
    Advisor
    Yamamura, Henry I.
    Roeske, William R.
    
    Metadata
    Show full item record
    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Abstract
    The data of this study indicate that pirenzepine(PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [³H]IP₁ accumulation in the SH-SY5Y cells was decreased in the presence of 1 μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M₁ mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m₁ gene. The transfected B82 cells (cTB10) showed specific [³H](-)QNB binding activity. The mAChRs in these cells are of the M₁ type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M₁ mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M₁ mAChR densities in these cells characterized by [³H](-)MQNB binding ranged from 12 fmol/10⁶ cells in LK3-1 cells to 260 fmol/10⁶ cells in the LK3-8 cells. The Hill coefficients of the CCh/[³H](-)MQNB competition curves were close to unity for the LK3-1 cells and were less than one in the higher receptor clones. The percentage of the M₁ mAChRs which had high affinity for CCh decreased as the receptor density increased, suggesting the presence of endogenous factors in these cells which may be important for the agonist affinity state of the receptor. A significant correlation was observed between the density of the M₁ mAChR with high affinity for CCh and the maximum [³H]IP₁ accumulation in these cells. There is no significant difference among the CCh EC₅₀ values, its K(A) values and the K(H) values of the CCh/[³H](-)MQNB competition curves. These results suggest that the high affinity state for CCh may be the functional state of the M₁ mAChRs in these cells. Although a linear correlation between the total M₁ mAChR density and the maximum [³H]IP₁ accumulation was observed, there was evidence for the existence of spare M₁ receptors in the clones with high receptor densities but not in those with low receptor densities.
    Type
    text
    Dissertation-Reproduction (electronic)
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Pharmacology & Toxicology
    Graduate College
    Degree Grantor
    University of Arizona
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