Behrman H R, Ohkawa R, Preston S L, MacDonald G J
Endocrinology. 1983 Sep;113(3):1132-40. doi: 10.1210/endo-113-3-1132.
The objective of the present studies was to examine adenosine uptake in the rat luteal cell, to characterize the cellular products after uptake, and to assess the role of adenosine transport and conversion to cAMP in amplification of LH-stimulated cAMP accumulation. Adenosine uptake showed an apparent Km of 7.3 +/- 0.6 microM, and a maximum velocity of 2.2 +/- 1.4 pmol/min X 10(5) cells at 24 C; uptake was temperature dependent (Q10 = approximately 3) and inhibited by dipyridamole (IC50 = 7 microM). Radiolabeled adenosine uptake was inhibited by AMP (IC50 = 14 microM), ATP (IC50 = 16 microM), guanosine (IC50 = 20 microM), inosine (IC50 = 22 microM), ADP (IC50 = 26 microM), and theophylline (IC50 = 5 mM); no inhibition by adenine, hypoxanthine, xanthine, prostaglandin F2 alpha (PGF2 alpha), PGE2, or LH was seen. Cellular products of radiolabeled adenosine uptake were found primarily in the trichloroacetic acid-soluble fraction (88%), and 90% of the radioactivity in this fraction comigrated with adenine nucleotides on electrophoresis; time-dependent incorporation of radioactivity into RNA, DNA, and protein was also seen. Adenosine transport did not appear to be related to the functional state of the luteal cell; for example, no change in the characteristics of uptake was seen in cells obtained from hypophysectomized animals or in cells incubated directly with PGF2 alpha or LH. Adenosine increased cell ATP levels in a dose-dependent manner in parallel with amplification of LH-stimulated cAMP accumulation. A substantial proportion of the total cAMP produced by the cells was derived from extracellular adenosine (40-90%). This response was directly related to the concentration of adenosine, and LH increased the magnitude of cAMP derived from adenosine by about 2-fold. Based on the present studies, adenosine uptake in the luteal cell appears to occur by a dipyridamole-sensitive, phosphorylation-dependent transport system which is independent of pituitary hormones or PG regulation. Moreover, amplification of LH-dependent cAMP accumulation by adenosine appears to be primarily a mass effect due chiefly to utilization of extracellular adenosine by the cell as a prosubstrate for conversion into cAMP by adenylate cyclase.
本研究的目的是检测大鼠黄体细胞中的腺苷摄取情况，鉴定摄取后的细胞产物，并评估腺苷转运及转化为环磷酸腺苷（cAMP）在促黄体生成素（LH）刺激的cAMP积累放大过程中的作用。腺苷摄取的表观米氏常数（Km）为7.3±0.6微摩尔，在24℃时最大速度为2.2±1.4皮摩尔/分钟×10⁵个细胞；摄取具有温度依赖性（温度系数Q10约为3），并被双嘧达莫抑制（半数抑制浓度IC50 = 7微摩尔）。放射性标记的腺苷摄取受到AMP（IC50 = 14微摩尔）、ATP（IC50 = 16微摩尔）、鸟苷（IC50 = 20微摩尔）、肌苷（IC50 = 22微摩尔）、ADP（IC50 = 26微摩尔）和茶碱（IC50 = 5毫摩尔）的抑制；未观察到腺嘌呤、次黄嘌呤、黄嘌呤、前列腺素F2α（PGF2α）、前列腺素E2或LH的抑制作用。放射性标记的腺苷摄取的细胞产物主要存在于三氯乙酸可溶部分（88%），该部分90%的放射性在电泳时与腺嘌呤核苷酸迁移率相同；还观察到放射性随时间掺入RNA、DNA和蛋白质中。腺苷转运似乎与黄体细胞的功能状态无关；例如，从垂体切除动物获得的细胞或直接用PGF2α或LH孵育的细胞中，摄取特征没有变化。腺苷以剂量依赖性方式增加细胞ATP水平，同时LH刺激的cAMP积累也相应放大。细胞产生的总cAMP中有很大一部分来自细胞外腺苷（40 - 90%）。这种反应与腺苷浓度直接相关，LH使源自腺苷的cAMP量增加约2倍。基于本研究，黄体细胞中的腺苷摄取似乎通过一种对双嘧达莫敏感、依赖磷酸化的转运系统进行，该系统独立于垂体激素或前列腺素调节。此外，腺苷对LH依赖性cAMP积累的放大作用似乎主要是一种质量效应，主要是由于细胞将细胞外腺苷作为前体底物利用，通过腺苷酸环化酶转化为cAMP。
