Zharikov S I, Block E R
Research Service, VA Medical Center, Gainesville, FL, USA.
Biochim Biophys Acta. 1998 Feb 2;1369(1):173-83. doi: 10.1016/s0005-2736(97)00191-0.
We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.
我们使用源自培养的肺动脉内皮细胞的质膜囊泡，研究了通过Y⁺系统转运[³H]-L-精氨酸的机制。质膜囊泡对[³H]-L-精氨酸的摄取不依赖于钠离子，对反式刺激敏感，不受质子传导离子载体的影响，并被阳离子氨基酸选择性抑制。在广泛的底物浓度范围内进行的动力学实验表明，只有一群L-精氨酸转运体，其Km = 130 μM。为了阐明L-精氨酸转运的驱动力，我们在不同的跨膜离子梯度下测量了质膜囊泡对[³H]-L-精氨酸的摄取。只有当质膜囊泡两侧施加膜电位时，质膜囊泡才会积累[³H]-L-精氨酸，并且摄取速度与所产生的膜电位大小呈线性相关。囊泡内存在钾离子对于L-精氨酸摄取到囊泡中不是必需的，但对于L-精氨酸转运的反式刺激是必需的。质膜囊泡中积累的[³H]-L-精氨酸可被耗散跨膜钾梯度的试剂（如皂角苷、短杆菌肽和尼日利亚菌素）释放。钾通道激活剂二氮嗪和平滑肌松弛素对[³H]-L-精氨酸摄取没有显著影响，而钾通道抑制剂四乙铵、4-氨基吡啶和格列本脲则导致质膜囊泡对[³H]-L-精氨酸的转运减少。本研究首次证明了钾离子在L-精氨酸转运机制中的特定作用，特别是在反式刺激现象中。
