从培养的垂体细胞制备的质膜囊泡中Ca2+转运机制。I. Na+/Ca2+交换活性的表征

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. I. Characterization of Na+/Ca2+ exchange activity.

作者信息

Kaczorowski G J, Costello L, Dethmers J, Trumble M J, Vandlen R L

出版信息

J Biol Chem. 1984 Aug 10;259(15):9395-403.

Abstract

GH3 rat anterior pituitary cells possess a Na+/Ca2+ exchange transport mechanism which is present in purified plasma membrane vesicles prepared from these cells. Imposition of an outwardly directed Na+ gradient in vesicles results in a marked concentrative uptake of Ca2+ which is abolished by the Ca2+ ionophore A23187. Transport activity depends on a sustained Na+ gradient. Dissipation of the driving force by treatment with Na+ ionophores or by passive gradient collapse abolished transport activity. The exchange reaction is completely reversible since addition of extravesicular Na+ enhances Ca2+ efflux from Ca2+ loaded vesicles. A kinetic analysis of Na+/Ca2+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 microM, respectively, and a Vmax of about 8 nmol/min/mg of protein for Ca2+ uptake at 25 degrees C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 microM and Vmax of 16 nmol/min/mg of protein for Ca2+ influx. Na+/Ca2+ exchange is not inhibited by protonophores indicating that Ca2+ flux does not occur via coupled Na+/H+, Ca2+/H+ exchange. Transport is inhibited by derivatives of the pyrazine diuretic amiloride. The pH dependency of Ca2+ uptake displays a sigmoidal relationship with stimulation of activity at alkaline pH and inhibition at acid pH. Furthermore, the reaction is electrogenic (i.e. more than 2 Na+ transported per Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artificially imposed membrane potentials on the rate of Ca2+ transport. Plasma membrane vesicles prepared from bovine anterior pituitary glands also display Na+/Ca2+ exchange with many of the same characteristics. These results support the notion that Na+/Ca2+ exchange functions in Ca2+ homeostasis in pituitary cells.

摘要

GH3大鼠垂体前叶细胞具有一种Na⁺/Ca²⁺交换转运机制，该机制存在于从这些细胞制备的纯化质膜囊泡中。在囊泡中施加外向性Na⁺梯度会导致Ca²⁺的显著浓缩摄取，而Ca²⁺离子载体A23187可消除这种摄取。转运活性依赖于持续的Na⁺梯度。用Na⁺离子载体处理或被动梯度崩溃使驱动力耗散会消除转运活性。交换反应是完全可逆的，因为添加细胞外Na⁺会增强Ca²⁺从负载Ca²⁺的囊泡中的流出。对Na⁺/Ca²⁺交换的动力学分析表明，两种底物均呈现饱和动力学，Na⁺和Ca²⁺的表观Km值分别为17 mM和5 μM，在25℃时Ca²⁺摄取的Vmax约为8 nmol/min/mg蛋白质。除了Na⁺/Ca²⁺交换外，该转运体还以Ca²⁺/Ca²⁺交换模式发挥作用，Ca²⁺流入的表观Km为20 μM，Vmax为16 nmol/min/mg蛋白质。Na⁺/Ca²⁺交换不受质子载体的抑制，这表明Ca²⁺通量不是通过耦合Na⁺/H⁺、Ca²⁺/H⁺交换发生的。转运受到吡嗪类利尿剂阿米洛利衍生物的抑制。Ca²⁺摄取的pH依赖性呈现S形关系，在碱性pH下活性受到刺激，在酸性pH下受到抑制。此外，该反应是生电的（即每转运1个Ca²⁺会转运超过2个Na⁺），这在交换过程中亲脂性阳离子的刺激摄取以及人工施加的膜电位对Ca²⁺转运速率的影响中得到了证明。从牛垂体前叶制备的质膜囊泡也表现出具有许多相同特征的Na⁺/Ca²⁺交换。这些结果支持了Na⁺/Ca²⁺交换在垂体细胞Ca²⁺稳态中发挥作用的观点。