Cellular Uptake of Lead Is Activated by Depletion of Intracellular Calcium Stores

Abstract

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...

Ionomycin, a Carboxylic Acid Ionophore, Transports Pb2+ with High Selectivity

“…This is in contrast to A23187 (Fig. 4B) (5,6,29) and because competition between these cations at intracellular sites is important in mechanisms of Pb 2ϩ toxicity (30,31). Using Equation 2 and the data in Fig.…”

“…The identification of mechanisms leading to lead toxicity is therefore of considerable interest and actively investigated. Work of this type conducted at the cellular level has been facilitated by the recent demonstration that entrapped Indo-1 can be used to monitor Pb 2ϩ transport into cells (5,6), even though this fluorescent compound is responsive to changes in the intracellular free Ca 2ϩ concentration (7). Transport studies utilizing Indo-1-loaded cells of several types have shown that Pb 2ϩ enters via plasma membrane Ca 2ϩ channels that are activated upon depletion of internal Ca 2ϩ stores (5,6), as is also true for other toxic cations (8,9).…”

Effect of Chemotherapy on Zn, Fe, Mg, Pb, Ca and Se in the Serum

“…Pb has an adverse effect on Fe deficiency, and Fe deficiency increases the absorption of Pb [17]. The absorption of Pb is positively correlated by the lower presence of Ca [18]. Fe is an important part of hemoglobin and myoglobin.…”

Molecular and ionic mimicry and the transport of toxic metals

“…Hinkle (1997a, 1997b) also suggested that Pb 2+ is transported into cells via Ca 2+ channels. Experiments in cultured rat pituitary GH 3 cells, rat C 6 glioma cells, 301 cells (a subclone of Human Embryonic Kidney (HEK) 293 cells; Kerper and Hinkle, 1997a) and bovine brain capillary endothelial cells (Kerper and Hinkle, 1997b) indicate that Pb 2+ enters these cells via channels that are activated by the reduction of intracellular stores of Ca 2+ . These experiments also showed that the uptake of Pb 2+ was a time-and concentrationdependent process and was inhibited significantly by the addition of Ca 2+ .…”