Dobrydneva Yuliya, Wilson Emily, Abelt Christopher J, Blackmore Peter F
Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23501-1980, USA.
J Cardiovasc Pharmacol. 2009 Mar;53(3):231-40. doi: 10.1097/FJC.0b013e31819b5494.
Thrombin increases intracellular free Ca ([Ca]i) in human platelets by 2 mechanisms: internal mobilization and the influx of Ca via store-operated Ca entry (SOCE). 2-Aminoethoxydiphenyl borate (2-APB) is an inhibitor of SOCE. In search for nonboron analogues of 2-APB, we identified a well-known compound, phenolphthalein, structurally related to 2-APB. Many phenolphthalein analogues inhibited the ability of thrombin and thapsigargin (a specific activator of SOCE) to increase [Ca]i. Phenolphthalein has an IC50 approximately 10 microM to inhibit thrombin-induced [Ca]i elevation, its active analogues have a similar potency. Several phenolphthalein analogues also inhibited thrombin-induced intracellular Ca mobilization, which indicates action on inositol 1,4,5-trisphosphate receptors. We identified structural features among active and inactive phenolphthalein analogues that are responsible for the activity. Opening of the 5-membered lactone ring of phenolphthalein resulted in a total loss of activity. If the diphenyl rings possessed primary amine, dimethyl amine, or a cyano group, there was no activity. Modifications to the diphenyl groups that were tolerated include phosphate, sulfate, iodine, bromine, methyl, nitrite, and methoxy. Inhibition of thrombin-induced [Ca]i increase by phenolphthalein was not mediated by an increase in cyclic adenosine monophosphate because the inhibitor of cyclic adenosine monophosphate-dependent protein kinase A, 4-cyano-3-methylisoquinoline, did not affect the inhibitory action of phenolphthalein. The inhibitory effect of phenolphthalein was not mediated by an increase in NO/cyclic guanosine monophosphate (cGMP) because the inhibitors of NO-sensitive soluble guanylyl cyclase, methylene blue, and ODQ did not affect the inhibition. Phytohemagglutinin and thapsigargin-induced SOCE in Jurkat cells was also inhibited by phenolphthalein and 2-APB to the same extent as seen in platelets. Therefore, phenolphthalein and its derivatives structurally similar to 2-APB inhibit SOCE in platelets and other cells.
凝血酶通过两种机制增加人血小板内的游离钙（[Ca]i）：内部动员和通过储存-操作性钙内流（SOCE）使钙流入。2-氨基乙氧基二苯基硼酸酯（2-APB）是SOCE的抑制剂。为了寻找2-APB的非硼类似物，我们鉴定出一种与2-APB结构相关的知名化合物酚酞。许多酚酞类似物抑制了凝血酶和毒胡萝卜素（一种SOCE的特异性激活剂）增加[Ca]i的能力。酚酞抑制凝血酶诱导的[Ca]i升高的IC50约为10微摩尔，其活性类似物具有相似的效力。几种酚酞类似物还抑制了凝血酶诱导的细胞内钙动员，这表明其作用于肌醇1,4,5-三磷酸受体。我们确定了活性和非活性酚酞类似物中负责活性的结构特征。酚酞五元内酯环的打开导致活性完全丧失。如果二苯基环带有伯胺、二甲胺或氰基，则没有活性。对二苯基进行的可耐受修饰包括磷酸、硫酸、碘、溴、甲基、亚硝酸盐和甲氧基。酚酞对凝血酶诱导的[Ca]i增加的抑制作用不是由环磷酸腺苷增加介导的，因为环磷酸腺苷依赖性蛋白激酶A的抑制剂4-氰基-3-甲基异喹啉不影响酚酞的抑制作用。酚酞的抑制作用不是由一氧化氮/环磷酸鸟苷（cGMP）增加介导的，因为一氧化氮敏感的可溶性鸟苷酸环化酶的抑制剂亚甲蓝和ODQ不影响这种抑制作用。酚酞和2-APB对植物血凝素和毒胡萝卜素诱导的Jurkat细胞中的SOCE的抑制程度与在血小板中所见相同。因此，酚酞及其与2-APB结构相似的衍生物抑制血小板和其他细胞中的SOCE。
