Miyamoto Y, Marczin N, Catravas J D, Del Monte M A
School of Optometry, University of California at Berkeley, 94720, USA.
Curr Eye Res. 1996 Mar;15(3):229-36. doi: 10.3109/02713689609007616.
Taurine uptake into cultures of human retinal pigment epithelial (HRPE) cells was monitored for 7 days after seeding. A culture medium containing 16% fetal bovine serum (FBS) was used for 2 days and switched to one with 8% FBS. Uptake of taurine (25 nM) was approximately 1.5 pmol/mg protein/15 min for 3 days, then decreased by 45% and was maintained at a decreased level till the 7th day. When the 16% FBS medium was used for the entire culture period, a similar profile of taurine uptake was observed but decrease of the uptake started on the 3rd day. Treatment of cells with 100 ng/ml cholera toxin (CT) for 24 h between the 6th and 7th days returned taurine uptake to its high level observed at the beginning of the cell culture. A similar CT treatment of cells between the 2nd and 3rd days enhanced taurine uptake significantly but this enhancement was much smaller. CT increased taurine uptake in treatment-time and dose dependent manners. Forskolin (FSK) (10 mM) and 8-Bromocyclic adenosine 3',5'-monophosphate (1 mM) also increased taurine uptake. KT5720 at 1 microM, a selective inhibitor of cAMP-dependent protein kinase (PKA), partially blocked CT-induced enhancement of taurine uptake. The level of cAMP was higher on the 3rd day than the 7th day but its response to 3-isobutyl-1-methyl-xanthine, FSK and CT was similar on both days. A kinetic analysis revealed that CT treatment decreases the apparent Michaelis-Menten constant of the taurine transporter while the drastic reduction of taurine uptake during the cell culture period is due to a decrease in the maximal velocity. The results show that cAMP elevated by CT treatment enhances taurine uptake via an increase in the affinity of the transporter. The decrease of taurine uptake during the culture period seems to be related to a decrease in the amount of the transporter.
在接种后人视网膜色素上皮（HRPE）细胞培养物中监测牛磺酸摄取7天。含有16%胎牛血清（FBS）的培养基使用2天，然后换成含8%FBS的培养基。牛磺酸（25 nM）的摄取在3天内约为1.5 pmol/mg蛋白质/15分钟，然后下降45%，并维持在较低水平直至第7天。当在整个培养期使用16%FBS培养基时，观察到类似的牛磺酸摄取情况，但摄取量在第3天开始下降。在第6天和第7天之间用100 ng/ml霍乱毒素（CT）处理细胞24小时，可使牛磺酸摄取恢复到细胞培养开始时观察到的高水平。在第2天和第3天之间对细胞进行类似的CT处理可显著增强牛磺酸摄取，但这种增强要小得多。CT以处理时间和剂量依赖的方式增加牛磺酸摄取。福斯高林（FSK）（10 mM）和8-溴环腺苷3',5'-单磷酸（1 mM）也增加牛磺酸摄取。1 microM的KT5720，一种环磷酸腺苷依赖性蛋白激酶（PKA）的选择性抑制剂，部分阻断了CT诱导的牛磺酸摄取增强。第3天的环磷酸腺苷水平高于第7天，但两天对3-异丁基-1-甲基黄嘌呤、FSK和CT的反应相似。动力学分析表明，CT处理降低了牛磺酸转运体的表观米氏常数，而细胞培养期间牛磺酸摄取的急剧减少是由于最大速度的降低所致。结果表明，CT处理升高的环磷酸腺苷通过增加转运体的亲和力来增强牛磺酸摄取。培养期间牛磺酸摄取的减少似乎与转运体数量的减少有关。
