• The Korean Journal of Pharmacology
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• v.23 no.2
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• pp.101-111
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• 1987

We investigated the binding properties of $(^3H)$ QNB and $(^3H)$ NMS to mAchR to elucidate the characterstics of mAchR in rat brain by using two different preparations (homogemates & intact brain cell aggregates). The binding properties of both ligands demonstrated high affinity and saturability in both experiments, however $(^3H)$ QNB showed a significantly higher maximal binding capacity than tha ot $(^3H)$ NMS 1. In rat brain homogenates; Displacement of both lignands with several mAchR antagonists resulted in competition curves in accoradnce with the law of massaction for QNB, atropine & scopolamine in thie preparation, also a similar profile was found for the quaternary ammonium analogs of atropine & scopolamine (methyl atropine & methylscopolamine) when $(^3H)$ NMS was used to label the receptors in rat brain. But when these hydrophillic antagonists were used to displace $(^3H)$ QNB, they showed interaction with high- and low-affinity binding sites in brain homogenates. Pirenzepine, the nonclassical mAchR antagonist, was able to displace both ligands from binding sites in this preparation. 2. In intact rat brain cell aggregates; Intact bain cell aggregates were used to elucidate the binding characteristics of $(^3H)$ NMS to mAchR in rat. The magnitude of binding of this ligand was related linearly to the amount of cell protein in the binding assay with a high ratio of total to nonspecific binding. mAchR antagonists displaced specific $(^3H)$ NMS binding according to the law of mass-action, while it was possible to resolve displacement curves using mAchR agonist into high-& low-affinity component. 3. Our results indicate that more hydrophilic receptor ligand $(^3H)$ QNB, displacement experiments in both tissues demonstrated that the lipid solubility of a particulr mAchR ligand might play an important role in determining its profile of binding to the mAchR, and the concentrations of mAchR in rat brain are both on the cell surface (membrane-bound receptor) and in the intracelluar membrane (intermembrane-bound receptor). 4. The results are discussed in terms of the usefulness of dissociated intact rat brain cells in studying mAchR in central nervous system.

• Archives of Pharmacal Research
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• v.10 no.4
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• pp.212-218
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• 1987

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This proparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity ($B_{max}$) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37.deg.C but was not apparent inincubation at $15^{\circ}}C$In addition, it was abolished when the receptors were blocked by atropine. The decline in ($^3H$)N-methylscopolamine (($^3H$)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that 'true' receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist ($^3H$) quinuclidinyl benzilate (($^3H$) QNB) insted of the hydrophilic ligand ($^3H$)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggested that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.

• Journal of Korean Neurosurgical Society
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• v.30 no.sup2
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• pp.189-196
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• 2001

Objective : We tested the hypothesis that photodynamic therapy(PDT) with Photofrin inhibits tumor invasion of U87 human glioma cells using several in vitro assay to measure tumor invasiveness. The effects of PDT on cell growth, directional migration and cell invasion were investigated. Material and Method : Tumor cells were treated with Photofrin at various doses and at a fixed optical(632nm) dose of $100mJ/cm^2$. Cytotoxicity was tested using the MTT method. Invasion assays including the matrigelartificial basement membrane barrier migration and spheroid confrontation with confocal microscopic analysis were used to study the relationship between PDT and invasiveness. Result : U87 cells showed a dose dependent cytotoxic response to increasing Photofrin dose. Data from the matrigel artificial basement membrane assay indicate that PDT inhibits the U87 cell migration dose dependently. Low doses of subcytotoxic PDT treatment, such as 2.5ug/ml Photofrin dose, also appeared to significantly inhibit migration of U87 cells(p<0.05). In co-cultures between U87 cell spheroids and brain aggregates, progressive invasion with destruction of the brain aggregate occurs. The extent of tumor cell infiltration and proportion or intact brain aggregate remaining after 24h differs in Photofrin PDT treated versus Photofrin only control, with changes suggestive of a dose-response effect. Conclusion : our data indicate that PDT with Photofrin significantly inhibits the invasiveness of U87 cells, and this inhibition is dose dependent.