Effects of extracellular ATP on Ca^<2+> movement, and phosphoinositide metabolism, leading to a functional change of EGF receptor

我们利用人表皮癌细胞系a -431，通过测量P_2嘌呤受体动员和提高细胞内钙（[Ca^<2+>]i和内流^<45>Ca^<2+>）的能力，研究了P_2嘌呤受体的配体特异性。我们还研究了细胞外ATP对肌磷酸代谢、钙动员、^<45>Ca^<2+>内流、EGF受体蛋白磷酸化的影响，并通过Scatchard图分析了EGF受体。研究了外atp酶的底物特异性。结果表明：(1)[Ca^<2+>]i升高非常快，在ATP刺激后30 s内达到最大值。随后触发了^<45>Ca^<2+>内流，最大流速在大约2分钟内达到。(2) [Ca^<2+>]i升高和^<45>Ca^<2+>内流不仅受到ATP的刺激，还受到GTP、UTP、ADP和UDP的刺激。其他核苷酸或核苷是无效的。6种ATP/ADP类似物，ATP- γ S， AM…更多的P- PNP， AMPPCP， AMPCPP, ADP- β S， AMPCP，这些化合物不抑制上述活性。其中两种，atp - γ S和AMPPNP反而刺激了Ca^<2+>和Ca^<2+>内流的动员。(3)从配体特异性来看，激发这种细胞活性的受体可能是p_2型嘌呤受体。一对叠氮化合物的检查，叠氮- amppnp衍生物被发现能够光亲和标记目前的受体。(4)细胞内储存的钙离子是产生IP_3所必需的，因此当细胞受到ATP刺激时，[Ca^<2+>]i和[Ca^< 45>] Ca^<2+>升高。(5)与A431细胞相关的ecto- atp酶的特异性与P_2受体的配体特异性存在较大差异，表明两者参与的系统不同。(6) ATP作用下IP_3的形成和[Ca^<2+>]i的升高提示DG的增加，这有望刺激蛋白激酶c。事实上，我们观察到EGF受体蛋白丝氨酸和苏氨酸残基磷酸化增强，同时EGF受体对EGF的亲和力也发生了变化（亲和力降低）。(7) ATP刺激[^3H]-肌醇进入细胞和/或膜蛋白。对PCA不溶性膜磷脂的分析表明，ATP刺激形成的主要化合物是磷脂酰肌醇。磷脂酰肌醇的合成需要细胞外Ca^<2+>的存在。少

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 … More 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