Sandoval Alvaro J, Riquelme Jeanette P, Carretta María D, Hancke Juan L, Hidalgo María A, Burgos Rafael A
Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile.
J Leukoc Biol. 2007 Nov;82(5):1266-77. doi: 10.1189/jlb.0307196. Epub 2007 Aug 7.
Neutrophil's responses to G protein-coupled chemoattractants are highly dependent on store-operated calcium (Ca(2+)) entry (SOCE). Platelet-activating factor (PAF), a primary chemoattractant, simultaneously increases cytosolic-free Ca(2+), intracellular pH (pH(i)), ERK1/2, and Akt/protein kinase B (PKB) phosphorylation. In this study, we looked at the efficacy of several putative SOCE inhibitors and whether SOCE mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils. We demonstrated that the absence of external Ca(2+) and the presence of EGTA reduced the intracellular alkalinization and ERK1/2 phosphorylation induced by PAF, apparently via SOCE influx inhibition. Next, we tested the efficacy of several putative SOCE inhibitors such as 2-aminoethoxydiphenyl borate (2-APB), capsaicin, flufenamic acid, 1-{beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365), and N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2) on Ca(2+) entry induced by PAF or thapsigargin. 2-APB was the most potent SOCE inhibitor, followed by capsaicin and flufenamic acid. Conversely, SK&F 96365 reduced an intracellular calcium (Ca(2+)) peak but SOCE partially. BTP2 did not show an inhibitory effect on Ca(2+) following PAF stimuli. 2-APB strongly reduced the pH(i) recovery, whereas the effect of flufenamic acid and SK&F 96365 was partial. Capsaicin and BTP2 did not affect the pH(i) changes induced by PAF. Finally, we observed that 2-APB reduced the ERK1/2 and Akt phosphorylation completely, whereas the inhibition with flufenamic acid was partial. The results suggest that 2-APB is the most potent SOCE inhibitor and support a key role of SOCE in pH alkalinization and PI-3K-ERK1/2 pathway control. Finally, 2-APB could be an important tool to characterize Ca(2+) signaling in neutrophils.
中性粒细胞对G蛋白偶联趋化因子的反应高度依赖于储存式钙（Ca(2+)）内流（SOCE）。血小板活化因子（PAF）作为一种主要的趋化因子，可同时增加胞质游离钙（Ca(2+)）、细胞内pH值（pH(i)）、细胞外调节蛋白激酶1/2（ERK1/2）以及Akt/蛋白激酶B（PKB）的磷酸化水平。在本研究中，我们探究了几种假定的SOCE抑制剂的效果，以及SOCE是否介导牛中性粒细胞内的碱化、ERK1/2和Akt/PKB磷酸化。我们发现，缺乏细胞外Ca(2+)以及存在乙二醇双四乙酸（EGTA）时，PAF诱导的细胞内碱化和ERK1/2磷酸化会减少，这显然是通过抑制SOCE内流实现的。接下来，我们测试了几种假定的SOCE抑制剂的效果，如2-氨基乙氧基二苯硼酸盐（2-APB）、辣椒素、氟芬那酸、1-{β-[3-(4-甲氧基苯基)丙氧基]-4-甲氧基苯乙基}-1H-咪唑盐酸盐（SK&F 96365）以及N-(4-[3,5-双(三氟甲基)-1H-吡唑-1-基]苯基)-4-甲基-1,2,3-噻二唑-5-甲酰胺（BTP2）对PAF或毒胡萝卜素诱导的Ca(2+)内流的影响。2-APB是最有效的SOCE抑制剂，其次是辣椒素和氟芬那酸。相反，SK&F 96365可降低细胞内钙（Ca(2+)）峰值，但对SOCE的抑制作用不完全。BTP2对PAF刺激后的Ca(2+)没有抑制作用。2-APB强烈降低pH(i)的恢复，而氟芬那酸和SK&F 96365的作用则是部分性的。辣椒素和BTP2不影响PAF诱导的pH(i)变化。最后，我们观察到2-APB完全降低了ERK1/2和Akt的磷酸化，而氟芬那酸的抑制作用是部分性的。结果表明，2-APB是最有效的SOCE抑制剂，并支持SOCE在pH碱化和PI-3K-ERK1/2信号通路调控中起关键作用。最后，2-APB可能是表征中性粒细胞中Ca(2+)信号传导的重要工具。
