Cavallini L, Coassin M, Borean A, Alexandre A
Dipartimento di Chimica Biologica, C. N. R. Centro di Studio delle Biomembrane, Università di Padova, Italy.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):567-74. doi: 10.1042/bj3190567.
The treatment of aspirinated platelets with the endomembrane Ca(2+)-ATPase inhibitor thapsigargin (Tg) induces a large increase in cytosolic pH (pH1), as measured with the intracellular fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. In contrast, Tg induces a decrease in pH1 in the presence of the Na+/H+ exchanger inhibitor 5-(N,N-hexamethylene)-amiloride (NHA). Both effects are inhibited if the cytosolic free Ca2+ concentration ([Ca2+]1) is chelated by loading with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxymethyl ester (BAPTA-AM). Without BAPTA, the pH effects are inhibited in the presence of BSA or the phospholipase A2 inhibitor oleoyloxyethylphosphocholine. These observations are consistent with the Tg-induced pH effects being mediated at least in part by the release of arachidonic acid (ArA) on activation of phospholipase A2 by the increased [Ca2+]1. Exogenous ArA promotes a rapid decrease in pH1 in platelets suspended in a high-[Na+] medium, and an increase in pH1 if platelets are depolarized by suspension in a high-[K+] medium in the presence of valinomycin and the external pH is increased to 7.9. The protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) behaves like ArA, although ArA is not a protonophore. It is concluded that ArA activates a proton conductance across the plasma membrane. The latter is inhibited by La3+. In high-[Na+] media, the pH1 previously decreased by ArA recovers rapidly on removal of ArA with BSA. The effect is prevented by NHA. The recovery after BSA is much slower if FCCP rather than ArA is used to decrease pH1, but it is fast again with both ArA and FCCP. Furthermore, pH1 previously decreased by ArA also recovers readily on inhibition of the ArA-activated H+ conductance with La3+, and the effect is NHA-sensitive. When pH1 is decreased with the K+/H+ ionophore nigericin, a rapid recovery is activated by ArA followed by BSA (but not by BSA alone). The effect is independent of Ca2+ and protein kinase C. It is concluded that ArA, besides activating the H+ conductance, also acts as an activator of the Na+/H+ exchanger.
用内膜Ca(2+)-ATP酶抑制剂毒胡萝卜素（Tg）处理抽吸的血小板，会导致胞质pH（pH1）大幅升高，这是用细胞内荧光指示剂2',7'-双（2-羧乙基）-5（6）-羧基荧光素测量得出的。相比之下，在存在Na+/H+交换抑制剂5-（N,N-六亚甲基）-氨氯吡脒（NHA）的情况下，Tg会导致pH1降低。如果通过加载双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰氧甲酯（BAPTA-AM）螯合胞质游离Ca2+浓度（[Ca2+]1），则这两种效应均会受到抑制。在没有BAPTA的情况下，在存在牛血清白蛋白（BSA）或磷脂酶A2抑制剂油酰氧乙基磷脂酰胆碱的情况下，pH效应会受到抑制。这些观察结果与Tg诱导的pH效应至少部分是由[Ca2+]1增加激活磷脂酶A2后释放花生四烯酸（ArA）介导的一致。外源性ArA会使悬浮在高[Na+]培养基中的血小板的pH1迅速降低，如果在缬氨霉素存在下将血小板悬浮在高[K+]培养基中并将外部pH提高到7.9，则pH1会升高。质子载体羰基氰化物对三氟甲氧基苯腙（FCCP）的作用与ArA相似，尽管ArA不是质子载体。得出的结论是，ArA激活了跨质膜的质子传导。后者受到La3+的抑制。在高[Na+]培养基中，先前因ArA而降低的pH1在用BSA去除ArA后会迅速恢复。NHA可阻止这种效应。如果使用FCCP而不是ArA来降低pH1，则BSA后的恢复要慢得多，但使用ArA和FCCP时恢复又会很快。此外，先前因ArA而降低的pH1在用La3+抑制ArA激活的H+传导后也很容易恢复，并且该效应对NHA敏感。当用K+/H+离子载体尼日利亚菌素降低pH1时，ArA随后加BSA（但单独用BSA不行）会激活快速恢复。该效应与Ca2+和蛋白激酶C无关。得出的结论是，ArA除了激活H+传导外，还作为Na+/H+交换器的激活剂起作用。
