The mechanisms of cellular lead uptake were characterized using a fluorescence method in cells loaded with indo-1. Pb 2؉ bound to intracellular indo-1 with much higher affinity than Ca 2؉ Lead is a ubiquitous environmental contaminant that can damage various organs, including those of the neurological, hematological, renal, and reproductive systems. Although it is readily taken up by many tissues, very little is known about the mechanisms of lead transport into cells. Lead is taken up in human red blood cells via anion exchange, probably as PbCO 3 (1). Based on in vivo studies, it has been suggested that lead transport across the rat blood-brain barrier is passive and pH-dependent and that the transported species is PbOH ϩ (2). Pb 2ϩ has been reported to enter electrically excitable bovine chromaffin cells via L-type voltage-sensitive Ca 2ϩ channels (VSCCs) 1 (3, 4). Pb 2ϩ is known to substitute for Ca 2ϩ in many cellular processes and to interfere with reactions that require Ca 2ϩ . The present investigation was carried out to determine whether Pb 2ϩ can enter cells through Ca 2ϩ channels. We studied the involvement of both VSCCs and voltage-insensitive channels responsible for the Ca 2ϩ uptake that occurs in response to depletion of intracellular Ca 2ϩ stores, which is referred to as capacitative or store-operated Ca 2ϩ uptake (5-8). Three different cell lines were used. Electrically excitable GH 3 cells, a line derived from rat pituitary, were chosen because they have very well characterized L-type VSCCs (9). C 6 , a nonexcitable rat glioma line, was used because it is derived from brain, one of the principal target organs for Pb 2ϩ toxicity. Nonexcitable 301 cells, a subline of HEK293 that is stably transfected with the G protein-coupled, Ca 2ϩ -mobilizing receptor for thyrotropin-releasing hormone (TRH) (10), were used because they display a large capacitative Ca 2ϩ uptake in response to TRH. A new method for monitoring the uptake of Pb 2ϩ into cells, using the fluorescent dye indo-1, is also described. This commonly used Ca 2ϩ indicator has a very high affinity for Pb 2ϩ . Binding to Pb 2ϩ quenches indo-1 fluorescence at all wavelengths, and fluorescence can be monitored using an emission wavelength at which fluorescence is insensitive to changes in Ca 2ϩ concentration. This method offers the advantages of convenience, sensitivity, rapidity, and the opportunity to monitor Pb 2ϩ uptake in real time by fluorescence spectroscopy or microscopy. Using this fluorimetric method, we have found a novel pathway for Pb 2ϩ influx into cells. We report here that Pb 2ϩ enters cells via voltage-insensitive cation channels that are activated by the depletion of intracellular Ca 2ϩ stores.
EXPERIMENTAL PROCEDURESMaterials-Cell culture reagents and physiological salt solutions were obtained from Life Technologies, Inc. Tissue culture plasticware was from Corning. Indo-1 acetoxymethyl ester, indo-1 pentapotassium salt, fura-2 pentapotassium salt, diethylenetriamine-pentaacetic anhydride (DTPA), and tetrakis-(2-pyridylmethy...