Mechanism Underlying Levofloxacin Uptake by Human Polymorphonuclear Neutrophils

The mechanism of radiolabeled levofloxacin ([(3)H]levofloxacin) uptake by human polymorphonuclear neutrophils (PMNs) was investigated by a classical velocity centrifugation technique. PMNs were incubated with levofloxacin for 5 to 180 min under various conditions before centrifugation through an oil cushion. Radioactivity was measured in the cell pellet to determine the amount of cell-associated drug. The uptake of levofloxacin was moderate with a cellular concentration/extracellular concentration ratio of about 4 to 6. Levofloxacin accumulated in PMNs parallel to the extracellular concentration, without saturation, over the range of 2.5 to 200 mg/liter (linear regression analysis: r = 0.92; P < 0.001). The activation energy was low (36 ± 7.2 kJ/mol). Levofloxacin uptake was increased in Ca(2+)-depleted, EGTA-containing medium by approximately 33% (P = 0.022), while Ni(2+), a Ca(2+) channel inhibitor, inhibited it in a concentration-dependent manner, with the concentration that inhibited 50% of control uptake being approximately 2.65 mM. Verapamil (an l-type Ca(2+) channel inhibitor) and other pharmacologic agents which modify Ca(2+) homeostasis did not modify levofloxacin uptake. Interestingly, Ca(2+) and Mg(2+) inhibited levofloxacin uptake in a concentration-dependent manner. EGTA, Ni(2+), and verapamil did not modify levofloxacin efflux; thapsigargin, a Ca(2+) pool-releasing agent, modestly increased the intracellular retention of levofloxacin. In addition, contrary to other fluoroquinolones, probenecid at 1 to 10 mM did not modify either levofloxacin uptake or efflux. These data are consistent with a mechanism of passive accumulation of levofloxacin in PMNs. Extracellular Ca(2+) and Mg(2+) may influence the structural conformation of levofloxacin or the lipophilicity of PMN membranes, thus explaining their effect on levofloxacin uptake.

本研究采用经典速度离心技术，探究了人类多形核中性粒细胞（polymorphonuclear neutrophils, PMNs）摄取放射性标记左氧氟沙星（[(3)H]levofloxacin）的分子机制。研究人员将多形核中性粒细胞与左氧氟沙星在不同实验条件下孵育5至180分钟，随后通过油垫法完成离心。收集细胞沉淀并检测其放射性活度，以定量计算与细胞结合的药物总量。左氧氟沙星的细胞摄取水平较为适中，细胞内药物浓度与细胞外药物浓度的比值约为4至6。在2.5至200 mg/L的浓度范围内，左氧氟沙星在多形核中性粒细胞内的蓄积量与细胞外浓度呈线性正相关，未出现饱和现象（线性回归分析：r=0.92；P<0.001）。该摄取过程的活化能较低，为36±7.2 kJ/mol。在钙离子缺失、添加乙二醇双(氨基乙基醚)四乙酸（EGTA）的培养基中，左氧氟沙星的摄取量提升约33%（P=0.022）；而钙离子通道抑制剂Ni²+则以浓度依赖性方式抑制该摄取过程，其半抑制浓度约为2.65 mM。维拉帕米（l-type Ca(2+) channel inhibitor，L型钙离子通道抑制剂）及其他调控钙离子稳态的药理学试剂，均未对左氧氟沙星的摄取产生显著影响。值得注意的是，钙离子与镁离子可通过浓度依赖性方式抑制左氧氟沙星的摄取。乙二醇双(氨基乙基醚)四乙酸（EGTA）、Ni²+及维拉帕米均未改变左氧氟沙星的外排过程；而钙池释放剂毒胡萝卜素（thapsigargin）则可轻度增加细胞内左氧氟沙星的滞留量。此外，与其他氟喹诺酮类（fluoroquinolones）药物不同，1至10 mM浓度的丙磺舒（probenecid）既未影响左氧氟沙星的摄取，也未改变其外排过程。上述实验结果与左氧氟沙星在多形核中性粒细胞内以被动蓄积方式转运的结论一致。细胞外钙离子与镁离子可能通过改变左氧氟沙星的分子构象或多形核中性粒细胞细胞膜的脂溶性，进而影响其摄取过程。