Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells.
The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.
Di Virgilio
F
F
C.N.R. Unit for the Study of the Physiology of Mitochondria, Padova, Italy.
Fasolato
C
C
Steinberg
T H
TH
D000818:Animals; D001572:Benzofurans / Q000493:pharmacokinetics*; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D016257:Fura-2; D006706:Homeostasis; D009447:Neuroblastoma / Q000378:metabolism*; D010673:Pheochromocytoma / Q000378:metabolism*; D013442:Sulfinpyrazone / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D001572:Benzofurans; D005456:Fluorescent Dyes; D002118:Calcium; D016257:Fura-2; D013442:Sulfinpyrazone