Cao C, Steinberg T H, Neu H C, Cohen D, Horwitz S B, Hickman S, Silverstein S C
Department of Physiology and Cellular Biophysics, Rover Laboratory of Cellular Physiology, College of Physicians and Surgeons, Columbia University, New York City, New York 10032.
Infect Agents Dis. 1993 Aug;2(4):193-200.
Macrophages possess organic anion transporters that carry membrane-impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxy-fluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug-resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical.(ABSTRACT TRUNCATED AT 250 WORDS)
巨噬细胞拥有有机阴离子转运体，这些转运体可将细胞膜不能通透的荧光染料，如鲁米诺黄（LY）和羧基荧光素，从细胞质转运至内体并排出细胞。丙磺舒是一种有机阴离子转运抑制剂，可阻断这些过程。将J774细胞长时间培养于含有2.5 mM丙磺舒的培养基中最终会杀死大多数细胞。为了鉴定表达增强的有机阴离子转运活性的J774变体，我们通过在含有浓度递增的丙磺舒的培养基中培养J774细胞来筛选丙磺舒抗性（PBR）J774细胞。当通过ATP4通透法使PBR和未筛选的J774细胞负载LY时，PBR细胞积累的LY量约为未筛选细胞的一半。向负载细胞的培养基中添加10 mM丙磺舒可消除这种差异，这表明PBR细胞中LY积累减少是由于LY分泌增强，且PBR细胞表达了增强的有机阴离子转运活性。直接比较J774和PBR J774细胞的LY外排显示，PBR J774细胞中LY的初始分泌速率比未筛选的J774细胞更快。为了确定LY外排是否由P-糖蛋白介导，我们比较了未筛选的J774细胞、PBR J774细胞和多药耐药J774细胞（J7.C1）中的LY外排。J7.C1细胞的LY外排对维拉帕米不敏感，维拉帕米可抑制多药耐药转运体并逆转J7.C1细胞的多药耐药表型。未筛选的J774细胞和J7.C1细胞的LY外排速率几乎相同。（摘要截断于250字）
