| Co-Investigator(Kenkyū-buntansha) |
SUGITA Kenji Meikai University, School of Dentistry, Department of Oral Physiology, Instructo, 歯学部, 助手 (90171157)
KURIHARA Kinji Meikai University, School of Dentistry, Department of Oral Physiology, Instructo, 歯学部, 助手 (10170086)
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| Research Abstract |
Using a human epidemoidal carcinoma cell line, A-431, we have investigated ligand specificity of P_2 purinoceptor by measuring the ability to mobilize and elevate intracellular calcium, [Ca^<2+>]i and to influx ^<45>Ca^<2+>. We also investigated the effects of extracellular ATP on inositolphosphate metabolism, calcium mobilization, ^<45>Ca^<2+> influx, phosphorylation of EGF receptor protein, analysis of EGF receptor by Scatchard plotting. The substrate specificity of ecto-ATPase was also studied. The following results were obtained:
(1)[Ca^<2+>]i elevation was very rapid and maximum [Ca^<2+>]i concentration was obtained within 30 s after the stimulation with ATP. The onset of ^<45>Ca^<2+> influx was triggered thereafter with the maximum velocity being achieved in approximately 2 min.
(2)The [Ca^<2+>]i elevation and ^<45>Ca^<2+> influx were stimulated by not only ATP but also GTP, UTP, ADP, and UDP. Other nucleotides or nucleosides were ineffective. Six ATP/ADP analogues, ATP-gamma S, AM
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P- PNP, AMPPCP, AMPCPP, ADP-beta S, AMPCP, were found that these compounds do not inhibit the activity described above. Two of them, ATP-gamma S and AMPPNP rather stimulated the mobilization of Ca^<2+> and ^<45>Ca^<2+> influx.
(3)From the ligand specificity, the receptors responsible in provoking such cellular activity were suggested to be P_2-type purinoceptors. Of a couple of azido-compounds examined, an azido-AMPPNP derivative was found to be able to photoaffinity label the present receptors.
(4)The calcium ion stored in the cells were essential to produce IP_3 and hence elevate [Ca^<2+>]i and influx ^<45>Ca^<2+> when cells were stimulated with ATP.
(5)The specificity of ecto-ATPase associated with A431 cells were quite different from the ligand specifity of P_2 prinoceptor, indicating that the two proteins were involved in different systems.
(6)IP_3 formation and [Ca^<2+>]i elevation by ATP suggest the increase of DG, which is expected to stimulate protein kinase C. As a matter of fact, we observed the enhanced phosphorylation of serine and threonine residues of EGF receptor protein, and also the changes of affinity (affinity decreased) of the receptor for EGF.
(7)The incorporation of [^3H]-inositol into cells and/or membrane protein were stimulated with ATP. The analysis of PCA insoluble membrane phospholipids showed that the main compound formed by stimulation with ATP was phosphatidylinositol. The synthesis of phosphatidylinositol required the presence of extracellular Ca^<2+>. Less
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