Shahidullah M, Wilson W S
Ocular Pharmacology Laboratory, Institute of Biomedical and Life Sciences, Glasgow University, Scotland, UK.
Curr Eye Res. 1997 Oct;16(10):1006-16. doi: 10.1076/ceyr.16.10.1006.9018.
To examine extracellular ATP for its ability to mobilise intracellular calcium in bovine ciliary epithelial cells; to establish and characterise P2Y2 receptor-mediated signal transduction in this tissue.
Bovine ciliary epithelial cells were isolated and cultured until confluence. The cells were reseeded on sterile coverslips and grown to obtain monolayers, then loaded with fura-2. Fluorescence was measured by a computer-controlled spectrofluorimeter and values calculated for intracellular calcium concentration. ATP, its analogues and other drugs were tested for their ability to mobilise intracellular calcium by adding them to the bathing solution.
Basal cytosolic calcium in bovine ciliary epithelium was 138.4 +/- 0.8 nM (n = 274). In the presence of extracellular Ca2+, ATP, UTP or ADP induced a transient dose-dependent increase in intracellular calcium (maximum approx. 400%), which declined rapidly. The agonist potency order was UTP = ATP > ADP > AMP. Adenosine, alpha, beta-methylene-ATP and 2-methylthio-ATP were ineffective in mobilising intracellular calcium, as were adrenaline, noradrenaline, acetylcholine and carbachol. The response to ATP and UTP remained, in the absence of extracellular calcium or the presence of nickel. Desensitisation of the calcium response by repeated exposure to ATP was augmented by phorbol-myristate-acetate and abolished by staurosporine. The ATP response was abolished by preincubation with pertussis toxin. Microfluorimetric measurements on single cells established that both pigmented and non-pigmented epithelia responded to ATP or UTP similarly.
In the bovine ciliary epithelium, ATP stimulates P2Y2 receptors coupled to a pertussis toxin-sensitive G protein. The results also suggest that this receptor activates phospholipase C, leading to mobilisation of calcium from intracellular stores.
研究细胞外ATP动员牛睫状上皮细胞内钙的能力；建立并表征该组织中P2Y2受体介导的信号转导。
分离牛睫状上皮细胞并培养至汇合。将细胞重新接种于无菌盖玻片上并生长形成单层，然后用fura-2进行负载。通过计算机控制的荧光分光光度计测量荧光，并计算细胞内钙浓度值。通过将ATP及其类似物和其他药物添加到浴液中来测试它们动员细胞内钙的能力。
牛睫状上皮细胞的基础胞质钙为138.4±0.8 nM（n = 274）。在细胞外Ca2+存在的情况下，ATP、UTP或ADP可诱导细胞内钙出现短暂的剂量依赖性增加（最大约400%），随后迅速下降。激动剂效力顺序为UTP = ATP > ADP > AMP。腺苷、α,β-亚甲基-ATP和2-甲硫基-ATP在动员细胞内钙方面无效，肾上腺素、去甲肾上腺素、乙酰胆碱和卡巴胆碱也无效。在无细胞外钙或存在镍的情况下，对ATP和UTP的反应仍然存在。反复暴露于ATP导致的钙反应脱敏被佛波醇-肉豆蔻酸酯-乙酸增强，并被星形孢菌素消除。ATP反应在与百日咳毒素预孵育后被消除。对单个细胞的显微荧光测量表明，色素上皮和非色素上皮对ATP或UTP的反应相似。
在牛睫状上皮中，ATP刺激与百日咳毒素敏感G蛋白偶联的P2Y2受体。结果还表明，该受体激活磷脂酶C，导致从细胞内储存中动员钙。
