Mechanism Underlying Levofloxacin Uptake by Human Polymorphonuclear Neutrophils

Abstract

ABSTRACT 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.