兔肾皮质内体中的H⁺/Ca²⁺交换

H+/Ca2+ exchange in rabbit renal cortical endosomes.

作者信息

Hilden S A, Madias N E

机构信息

Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts.

出版信息

J Membr Biol. 1989 Dec;112(2):131-8. doi: 10.1007/BF01871274.

DOI:10.1007/BF01871274

PMID:2533622

Abstract

We have examined the effect of second messengers on ATP-driven H+ transport in an H+ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1 mM) had no effect on H+ transport. Acridine orange fluorescence in the presence of 0.5 mM Ca2+ (+1 mM EGTA) was 19 +/- 6% of control. Inhibition of ATP-driven H+ transport by Ca2+ was concentration dependent; 0.25 and 0.5 mM Ca2+ (+1 mM EGTA) inhibited acridine orange fluorescence by approximately 50 and approximately 80%, respectively. Ca2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca2+ did not affect ATP hydrolysis. ATP-dependent Br- uptake was virtually unchanged in the presence of 0.5 mM Ca2+ (+1 mM EGTA). These vesicles were also shown to transport Ca2+ in an ATP-dependent mode. Inositol 1,4,5-trisphosphate had no effect on ATP-dependent Ca2+ uptake. These results are consistent with the co-existence of an H+ ATPase and an H+/Ca2+ exchanger on these endosomes, the latter transport system using the H+ gradient to energize Ca2+ uptake. Attempts to demonstrate an H+/Ca2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca2+ uptake could be demonstrated. A Ca2(+)-dependent increase in H+ permeability and an ATP-dependent Ca2+ uptake might have important implications for the regulation of vacuolar H+ ATPase activity as well as the homeostasis of cytosolic Ca2+ concentration.

摘要

我们研究了第二信使对从兔肾皮质分离的含H⁺ATP酶的内体组分中ATP驱动的H⁺转运的影响。环磷酸腺苷（0.1 mM）对H⁺转运无影响。在0.5 mM Ca²⁺（+1 mM乙二醇双乙醚二胺四乙酸）存在下，吖啶橙荧光为对照的19±6%。Ca²⁺对ATP驱动的H⁺转运的抑制作用呈浓度依赖性；0.25 mM和0.5 mM Ca²⁺（+1 mM乙二醇双乙醚二胺四乙酸）分别使吖啶橙荧光抑制约50%和约80%。Ca²⁺还使pH梯度消散速率呈浓度依赖性增加。Ca²⁺不影响ATP水解。在0.5 mM Ca²⁺（+1 mM乙二醇双乙醚二胺四乙酸）存在下，ATP依赖性Br⁻摄取实际上未改变。这些囊泡还显示以ATP依赖模式转运Ca²⁺。肌醇1,4,5 -三磷酸对ATP依赖性Ca²⁺摄取无影响。这些结果与这些内体上存在H⁺ATP酶和H⁺/Ca²⁺交换器一致，后者利用H⁺梯度为Ca²⁺摄取提供能量。在没有ATP的情况下试图证明H⁺/Ca²⁺反向转运体未成功。然而，当通过与ATP预孵育建立pH梯度并随后用己糖激酶+葡萄糖去除残留ATP时，可以证明Ca²⁺摄取受到刺激。Ca²⁺依赖性的H⁺通透性增加和ATP依赖性的Ca²⁺摄取可能对液泡H⁺ATP酶活性的调节以及胞质Ca²⁺浓度的稳态具有重要意义。