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首页> 外文期刊>Biochimica et biophysica acta. Biomembranes >Leukotriene C_4 (LTC_4) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes
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Leukotriene C_4 (LTC_4) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

机译:白三烯C_4(LTC_4)不与cGMP共享细胞外排机制:通过摄取人红细胞由内而外的囊泡来表征cGMP转运

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摘要

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C_4 (LTC_4), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC_(50) = 110 ± 20 nM), but cGMP was unable to inhibit LTC_4 uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [~3H]cGMP uptake with IC_(50) of 2200 ± 700 μM for GSSG, 410 ± 210 μM for S-(p-nitrobenzyl)glutathione and 37 ± 16 μM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 μM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 ℃. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC_(50) of 43 ± 12 μM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn~(2+) and Co~(2+), were unable to substitute Mg~(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 μM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC_4. However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.
机译:cGMP从细胞外转运需要能量,并且具有与ATP供电的阴离子泵兼容的特性。在本研究中,具有人红细胞内外囊泡的模型用于cGMP转运蛋白的进一步表征。白三烯拮抗剂MK571(IC_(50)= 110±20 nM)浓度依赖性抑制白三烯C_4(LTC_4)(一种多药耐药蛋白(MRP)的底物)的摄取,但是cGMP无法抑制LTC_4的摄取。氧化型谷胱甘肽(GSSG)和谷胱甘肽S-共轭物引起[〜3H] cGMP吸收的浓度依赖性抑制,GSSG的IC_(50)为2200±700μM,S-(对硝基苄基)谷胱甘肽的410±210μM。 S-癸基谷胱甘肽分别为37±16μM。抗氧化剂,例如还原型谷胱甘肽和二硫苏糖醇,在浓度高达100μM时,不影响转运,但都抑制了cGMP的吸收,最大浓度约为100μM。 1 mM时为25%。 cGMP泵对温度敏感,没有低于20℃的活动。 cGMP的运输取决于pH,其最大活性介于pH 8.0和8.5之间。钙引起浓度依赖性抑制,IC_(50)为43±12μM。镁在1至20 mM的范围内具有明显的活化作用,在10 mM时具有最大作用。其他二价阳离子Mn〜(2+)和Co〜(2+)不能替代Mg〜(2+),但在1 mM时引起一定​​的活化。 EDTA和EGTA分别独立地刺激cGMP转运浓度,在100μM时分别比对照高50%和100%。本研究表明,cGMP泵具有与有机阴离子转运ATPase兼容的特性,而对MRP底物LTC_4没有亲和力。但是,谷胱甘肽S-结合物对cGMP转运蛋白的阻断表明它是几种GS-X泵之一。

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