INTRODUCTION

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 PbCO₃ (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⁺ (2). Pb²⁺ has been reported to enter electrically excitable bovine chromaffin cells via L-type voltage-sensitive Ca²⁺ channels (VSCCs)¹ (3, 4).

Pb²⁺ is known to substitute for Ca²⁺ in many cellular processes and to interfere with reactions that require Ca²⁺. The present investigation was carried out to determine whether Pb²⁺ can enter cells through Ca²⁺ channels. We studied the involvement of both VSCCs and voltage-insensitive channels responsible for the Ca²⁺ uptake that occurs in response to depletion of intracellular Ca²⁺ stores, which is referred to as capacitative or store-operated Ca²⁺ uptake (5-8). Three different cell lines were used. Electrically excitable GH₃ cells, a line derived from rat pituitary, were chosen because they have very well characterized L-type VSCCs (9). C₆, a nonexcitable rat glioma line, was used because it is derived from brain, one of the principal target organs for Pb²⁺ toxicity. Nonexcitable 301 cells, a subline of HEK293 that is stably transfected with the G protein-coupled, Ca²⁺-mobilizing receptor for thyrotropin-releasing hormone (TRH) (10), were used because they display a large capacitative Ca²⁺ uptake in response to TRH.

A new method for monitoring the uptake of Pb²⁺ into cells, using the fluorescent dye indo-1, is also described. This commonly used Ca²⁺ indicator has a very high affinity for Pb²⁺. Binding to Pb²⁺ quenches indo-1 fluorescence at all wavelengths, and fluorescence can be monitored using an emission wavelength at which fluorescence is insensitive to changes in Ca²⁺ concentration. This method offers the advantages of convenience, sensitivity, rapidity, and the opportunity to monitor Pb²⁺ uptake in real time by fluorescence spectroscopy or microscopy. Using this fluorimetric method, we have found a novel pathway for Pb²⁺ influx into cells. We report here that Pb²⁺ enters cells via voltage-insensitive cation channels that are activated by the depletion of intracellular Ca²⁺ stores.

EXPERIMENTAL PROCEDURES

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-pyridylmethyl)ethylenediamine (TPEN) were from Molecular Probes (Eugene, OR). Bovine serum albumin, cyclopiazonic acid, t-butylhydroquinone, endothelin, verapamil, and LaCl₃ were from Sigma. Cyclosporin A was from Sandoz Pharmaceuticals (East Hanover, NJ), TRH from Calbiochem, thapsigargin from Research Biochemicals International (Natick, MA), SK&F 96365 from Biomol (Plymouth Meeting, PA), and Pb(NO₃)₂ from Baker (Phillipsburg, NJ).

The affinity of indo-1 for Pb²⁺ was measured in a buffer mimicking the cytosolic ionic composition (130 mM KCl, 20 mM NaCl, and 15 mM HEPES) at 37 °C using 336 nm excitation and 405 nm emission wavelengths. The fluorescence of 0.25 µM indo-1 pentapotassium salt was measured following addition of 10 mM CaCl₂, which saturates the dye with Ca²⁺, and then after addition of 0-6 µM Pb(NO₃)₂, which displaces Ca²⁺ and quenches fluorescence. The concentration of Pb(NO₃)₂ causing a half-maximal decrease in fluorescence was determined, and the affinity constant for Pb²⁺-indo-1 was calculated from the relationship (11),

(Eq. 1)

where K_Pb²⁺ and K_Ca²⁺ are the equilibrium association constants for the Pb²⁺ and Ca²⁺ complexes of indo-1, respectively, and [Pb²⁺]₅₀ is the concentration of Pb²⁺ causing half-maximal quench of Ca²⁺-indo-1 fluorescence at a Ca²⁺ concentration of [Ca²⁺]₅₀. A value of 10^6.6 M¹ was used for K_Ca²⁺ (12).

Indo-1 pentapotassium salt or fura-2 pentapotassium salt (0.25 µM) in 2 ml of a cytosol-like buffer were placed in a cuvette at room temperature. To this was added consecutively 1 mM CaCl₂, 10 µM Pb(NO₃)₂, and 10 mM EGTA, with spectra obtained after each addition. Fura-2 fluorescence was measured at an emission wavelength of 493 nm during scans of excitation wavelengths from 270 to 420 nm. Indo-1 fluorescence was measured with an excitation wavelength of 336 nm during scans at emission wavelengths from 370 to 560 nm. Slit widths were 10 nm for both excitation and emission.

Cells were maintained as monolayer cultures at 37 °C in a humidified 95% air, 5% CO₂ environment. GH₃ cells were grown in F-10 medium supplemented with 15% horse serum and 2.5% fetal calf serum. C₆ and 301 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. 301 cells are a clone of HEK293 cells stably transfected with cDNA encoding the mouse TRH receptor (10).

Cells were grown on 60-mm plastic culture dishes to near confluence, gently washed or scraped off the dish in Hank's balanced salt solution containing 10 mM HEPES, pH 7.4 (HBSS), centrifuged, and resuspended in 1 ml of HBSS; normal HBSS contains 1.26 mM CaCl₂. To this suspension were added 4 µM indo-1 acetoxymethyl ester, 1 µg/ml cyclosporin A, and 0.1% bovine serum albumin. Cells were incubated for 30 min at 37 °C in the dark, then diluted 10-fold in either HBSS, nominally Ca²⁺-free HBSS, or buffer solution containing 150 mM NaCl, 10 mM HEPES, and 1 g/liter glucose (HEPES-buffered saline) and centrifuged. Cells were resuspended in 2 ml of HEPES-buffered saline (unless otherwise noted) at a density of 2-5 × 10⁶ cells/ml and placed in a cuvette with a stir bar, and fluorescence was monitored with a Perkin-Elmer LS-5B fluorimetric spectrometer at 37 °C with 10 nm excitation and 20-nm emission slit widths. KCl (5 mM) was added to the HEPES-buffered saline in the experiment measuring the effect of depolarization on Pb²⁺ uptake in GH₃ cells. When GH₃ cells were maintained in nominally K⁺-free medium, Ca²⁺ uptake via VSCCs was abnormal (data not shown). The rates of Pb²⁺ uptake by nonexcitable cells, with or without thapsigargin pretreatment, were the same whether cells were maintained in nominally K⁺-free buffers or buffers containing 5 mM KCl. Unless specifically noted, fluorescence was measured at the Ca²⁺-insensitive wavelength, with excitation at 336 nm and emission at 450 nm.

Lead was added to the cells as Pb(NO₃)₂ in HEPES-buffered saline for 1-5 min. Uptake was stopped by the addition of DTPA, an extracellular chelator with high affinity for heavy metals and lower affinity for Ca²⁺ (K_eq for Pb²⁺ = 10^21.8 versus 10^10.6 M¹ for Ca²⁺) (13). A cell-permeant metal chelator, TPEN, was added 1 min later (K_eq = 10^13.98 M¹ for Pb²⁺ and 10^4.4 M¹ for Ca²⁺) (14).

All results shown are representative experiments from at least three trials in which control and treated cells were from parallel dishes plated on the same day at the same density. Initial rates of fluorescence quench were measured after addition of Pb²⁺ (5 or 10 µM for C₆ and 301 cells or 1 µM for GH₃ cells) immediately after any sharp drop in fluorescence due to Pb²⁺ quench of extracellular dye. The rates of fluorescence quench following drug treatment are expressed relative to the rates of fluorescence quench in control reactions run on replicate cultures in the same experiment. Values shown are the mean and standard error of the ratio of treated to control fluorescence quench.

[Ca²⁺]_i of indo-1-loaded cells suspended in HBSS was measured at Ca²⁺-sensitive wavelengths, 336 nm excitation and 405 nm emission. Cells were lysed with 50 µM digitonin and F_max was obtained; EGTA (10 mM, pH 8.3) was then added and F_min was obtained. [Ca²⁺]_i was calculated from the relationship [Ca²⁺]_i = K_d × (F  F_min)/(F_max  F) and a K_d value of 254 nM (12).

RESULTS

Indo-1 and fura-2 are ratio dyes commonly used to measure intracellular Ca²⁺. We found that the K_eq for the binding of Pb²⁺ to indo-1 is 10^10.46 M¹, almost 4 orders of magnitude higher than the reported K_eq for the Ca²⁺-indo-1 complex, 10^6.6 M¹ (12). The affinity of Pb²⁺ for fura-2 is also very high, K_eq = 10^12.38 M¹ (4).

Fig. 1 depicts the excitation spectra of fura-2 and the emission spectra of indo-1. Curves labeled a show the spectra of the Ca²⁺ complexes of the dyes, b the spectra of the Pb²⁺ complexes, and c the spectra of free fura-2 and indo-1. The spectra of the Ca²⁺- and Pb²⁺ complexes of fura-2 are very similar. The Pb²⁺-indo-1 spectrum, however, is vastly different from the spectrum of either free indo-1 or the Ca²⁺-indo-1 complex. Pb²⁺ quenches the fluorescence of indo-1 and Ca²⁺-indo-1 at all wavelengths.

We have taken advantage of the spectral properties of the Pb²⁺ complex of indo-1 and measured the fluorescence of indo-1-loaded cells at 450 nm, the Ca²⁺ isosbestic point, where fluorescence is unaffected by Ca²⁺ but almost completely quenched by Pb²⁺. The high affinity of Pb²⁺ for indo-1 means that Pb²⁺ taken up by a cell will bind almost quantitatively to the probe.

The traces in Fig. 2 show time- and concentration-dependent uptake of Pb²⁺ by indo-1-loaded GH₃, C₆, and 301 cells. Addition of Pb(NO₃)₂ resulted in a decrease in fluorescence as Pb²⁺ entered the cells and quenched indo-1 fluorescence. Quenching was stopped when DTPA, a membrane-impermeant metal chelator, was added to chelate extracellular Pb²⁺; DTPA binds Pb²⁺ with extremely high affinity (K_eq = 10^21.8 M¹ (13)). Since DTPA does not enter cells, the small, immediate jump in fluorescence that occurred when it was added was probably due to extracellular indo-1 that had leaked out of the cells during the experiment. Typically, this was a small fraction of the total change in fluorescence caused by Pb²⁺. Fluorescence was recovered to near baseline values by addition of membrane permeant TPEN, which chelates intracellular metals (K_eq for Pb²⁺ = 10¹⁴ M¹ (14), yielding a measure of the change in fluorescence due to cellular uptake of Pb²⁺. In these and all of the following Pb²⁺ uptake traces, addition of Pb(NO₃)₂ caused an immediate small drop in fluorescence followed by a slower decline over the next 3 min. The initial fast drop was likely due to Pb²⁺ binding to a small amount of dye which was outside the cells. This initial decrease was roughly equivalent to the fluorescence increase caused by DTPA, the extracellular chelator. Qualitative changes in rates of uptake were readily apparent with the fluorescence quench approach, and results were quantitatively reproducible when trials were performed on replicate dishes.

Addition of DTPA and TPEN to indo-1-loaded cells suspended in HBSS alone had little or no effect on fluorescence in C₆ or GH₃ cells. In 301 cells, addition of TPEN increased fluorescence of cells in HBSS and caused fluorescence levels to increase to values above base line after Pb²⁺ uptake had occurred (see Fig. 2). This may indicate that in 301 cells, fluorescence of some of the intracellularly trapped indo-1 is quenched by endogenous metals.

We estimated that the intracellular concentration of indo-1 in 301 cells was 10-20 µM after 30-min loading (data not shown). When Pb²⁺ uptake reactions were extended for long enough, fluorescence reached a stable minimum, suggesting that all of the intracellular indo-1 was quenched (see for example thapsigargin traces in Figs. 4 and 6). Assuming that the Pb²⁺/indo-1 molar binding ratio is 1:1, then the total intracellular Pb²⁺ concentrations can reach at least 10-20 µM when total extracellular Pb²⁺ is 1 µM.

To investigate whether Pb²⁺ enters GH₃ cells via VSCCs, we monitored Pb²⁺ uptake during cell depolarization. Pb²⁺ uptake was increased 1.6 ± 0.2-fold (n = 13) when the cells were depolarized by the addition of high extracellular K⁺. When an L-channel antagonist, verapamil, was added, uptake was slowed (Fig. 3, right panel). Excess Pb²⁺ was then added to show that the dye was not already mostly quenched when KCl and verapamil were added. Under the same conditions, high K⁺ caused a large increase in [Ca²⁺]_i that was fully reversed by verapamil (Fig. 3, left panel). These results suggest that following a strong depolarization, there is some uptake of Pb²⁺ ions through VSCCs in GH₃ cells. However, the effect of depolarization was slight compared with the 12-fold stimulation caused by store depletion in GH₃ cells (see below), and the effects of depolarization on Pb²⁺ uptake were much less than the effects on Ca²⁺ uptake. High K⁺ did not increase Pb²⁺ uptake by nonexcitable 301 cells (data not shown).

Uptake of extracellular Ca²⁺ can be activated by the emptying of intracellular Ca²⁺ stores, located in the endoplasmic reticulum (5-8). This store-operated Ca²⁺ entry, also referred to as capacitative Ca²⁺ uptake, presumably occurs via Ca²⁺-conducting channels that are not voltage-gated and have not been fully characterized. We next assessed the contribution of these channels to Pb²⁺ transport using several paradigms that have been shown to activate Ca²⁺ transport via store-operated channels.

Thapsigargin, cyclopiazonic acid, and tert-butylhydroquinone are structurally unrelated drugs that inhibit the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), an enzyme responsible for pumping Ca²⁺ into the intracellular stores (15). When the ATPase is inhibited, the stores are emptied by an endogenous Ca²⁺ leak. The three SERCA inhibitors deplete Ca²⁺ stores and activate the entry of extracellular Ca²⁺ through store-operated channels in many cell types. We added 1 µM thapsigargin, 10 µM cyclopiazonic acid, or 10 µM tert-butylhydroquinone to cells, waited 5-10 min for the Ca²⁺ stores to empty, and then added 1-10 µM Pb(NO₃)₂. These conditions were sufficient to deplete intracellular Ca²⁺ stores and stimulate uptake of extracellular Ca²⁺ in GH₃, C₆, and 301 cells (Ref. 16 and data not shown). Thapsigargin, cyclopiazonic acid, and tert-butylhydroquinone also stimulated Pb²⁺ entry in GH₃, C₆, and 301 cells (results shown for thapsigargin only in Fig. 4, top). Thapsigargin-stimulated increases in the initial rates of Pb²⁺ uptake, compared with untreated cells, were 11.6 ± 2.0-fold for GH₃, 16.3 ± 5.8-fold for C₆, and 3.1 ± 0.4-fold for 301 cells.

Intracellular Ca²⁺ stores can be emptied without the use of SERCA inhibitors by incubating cells in medium with low Ca²⁺ for an extended period. C₆ or 301 cells were maintained in either normal medium or in nominally Ca²⁺-free medium for a total of ~45 min. Ca²⁺-depleted C₆ and 301 cells took up Pb²⁺ at a faster rate than cells in Ca²⁺-replete medium (Fig. 4, bottom).

Another way to stimulate store-operated Ca²⁺ uptake is by activating Ca²⁺-mobilizing, G protein-coupled receptors (5-8). This releases intracellular Ca²⁺ by initiating an intracellular signaling pathway leading to the production of inositol 1,4,5-trisphosphate (IP₃); IP₃ binds to and activates the IP₃ receptor, a Ca²⁺ channel in the endoplasmic reticulum membrane, causing efflux of Ca²⁺ from the stores. The resultant store depletion stimulates entry of extracellular Ca²⁺. The effect of receptor activation on Ca²⁺ and Pb²⁺ uptake was tested using the transfected TRH receptor in 301 cells and the endogenous endothelin receptor in C₆ cells. When indo-1 fluorescence was followed at a Ca²⁺-sensitive wavelength, TRH and endothelin could be seen to evoke large [Ca²⁺]_i transients in 301 (Fig. 5A) and C₆ cells (Fig. 5C), respectively. Since the cells were suspended in Ca²⁺-free medium, the [Ca²⁺]_i increases resulted from the release of intracellular Ca²⁺. The rates of Ca²⁺ influx after re-addition of extracellular Ca²⁺ were much greater if cells had been treated with agonists first; this result is a hallmark of store-operated, or capacitative, Ca²⁺ influx (5-8). However, the same agonists did not appear to stimulate Pb²⁺ influx. As shown in Fig. 5, B and D, the rates of Pb²⁺ uptake by 301 and C₆ cells were not changed by prior treatment with TRH or endothelin, respectively.

Since Mn²⁺ quench of intracellular dye is frequently used to monitor the activity of store-operated Ca²⁺ channels, we compared the uptake of Ca²⁺, Pb²⁺, and Mn²⁺ in 301 cells following depletion of intracellular Ca²⁺ stores with maximally effective concentrations of TRH or thapsigargin (Fig. 6). Thapsigargin and TRH were equally effective at stimulating Ca²⁺ uptake; thapsigargin increased [Ca²⁺]_i 2.9 ± 0.2-fold and TRH 3.2 ± 0.4-fold. In contrast, TRH was not as effective as thapsigargin at stimulating uptake of Pb²⁺ or Mn²⁺. The rate of Pb²⁺ quench was stimulated 3.1 ± 0.4-fold by thapsigargin, but was not significantly increased by TRH (1.1 ± 0.1-fold). Thapsigargin increased Mn²⁺ uptake 2.0 ± 0.2-fold and TRH increased it 1.2 ± 0.2-fold.

To determine how completely TRH depleted Ca²⁺ stores, we compared the extent of store depletion caused by different drugs. 301 cells were exposed to 1 µM thapsigargin or TRH and 5 min later challenged with a second store-depleting agent (Fig. 7). Thapsigargin depleted the Ca²⁺ stores almost completely, as evidenced by the very small amount of Ca²⁺ released in response to the second drug (TRH). TRH caused substantial but not complete store depletion, since Ca²⁺ was released upon addition of thapsigargin after TRH.

To determine whether extracellular Ca²⁺ competes with Pb²⁺ for uptake in GH₃ cells, Pb²⁺ uptake was measured in the presence of 0, 1, and 10 mM extracellular Ca²⁺. In both unstimulated and thapsigargin-treated cells, Pb²⁺ uptake was partially inhibited by a physiological concentration of Ca²⁺ (1 mM) and strongly inhibited by 10 mM extracellular Ca²⁺ (Fig. 8). Similar results were obtained with 301 cells (data not shown).

Uptake of Pb²⁺ in GH₃ cells was inhibited by addition of 25 µM extracellular La³⁺, a broad spectrum Ca²⁺ channel antagonist (Fig. 9, left panel). SK&F 96365 is an imidazole derivative that inhibits both voltage-sensitive and store-operated Ca²⁺ influx (18). GH₃ cells were incubated in 5 µM SK&F 96365 during dye loading, washing, and measurement of fluorescence. In the presence of 5 µM SK&F 96365, cyclopiazonic acid-stimulated Pb²⁺ uptake was virtually eliminated (Fig. 9, right panel), as was stimulated Ca²⁺ uptake (data not shown). Effects of 5 µM SK&F 96365 on basal Pb²⁺ uptake by GH₃ cells were inconsistent, probably because SK&F 96365 causes a dose-dependent release of intracellular Ca²⁺ by itself (18-20); in the absence of a store-depleting agent, SK&F 96365 can activate capacitative influx by causing store depletion as well as inhibit the uptake process.² In C₆ and 301 cells, 5-20 µM SK&F 96365 had no effect on basal, thapsigargin- or cyclopiazonic acid-stimulated uptake of Pb²⁺. In 301 cells, 5 µM SK&F 96365 did not block capacitative uptake of Ca²⁺ but 20 µM did.

DISCUSSION

Although environmental Pb²⁺ is a major public health concern and toxic effects of Pb²⁺ have been well documented, it is not known how Pb²⁺ crosses cell membranes. The experiments presented here demonstrate that Pb²⁺ enters electrically excitable and nonexcitable cells by a previously unrecognized pathway involving voltage-insensitive, store-operated cation channels. According to the capacitative Ca²⁺ entry model originally proposed by Putney (5), voltage-insensitive Ca²⁺ channels are stimulated by the emptying of intracellular Ca²⁺ stores. The influx of extracellular Ca²⁺ through these store-operated channels allows subsequent refilling of the stores. Although the basic findings have been verified in many cell types (5-8), the nature of the intracellular signal is not well understood. Hoth and Penner (21) described a Ca²⁺ current in rat mast cells that is dependent on release of intracellular Ca²⁺, which they have termed I_CRAC (Ca²⁺ release-activated Ca²⁺ current). I_CRAC is dependent on extracellular Ca²⁺ and is inhibited by other divalent cations. This current may account for some or all of the capacitative Ca²⁺ uptake described above. Several recently cloned mammalian homologues of the Drosophila trp protein may be components of channels involved in capacitative Ca²⁺ uptake (22); it is not known whether such channels are permeable to Pb²⁺.

In GH₃, C₆, and 301 cells, uptake of Pb²⁺ occurs via a route that has some characteristics in common with store-operated Ca²⁺ influx. Two Ca²⁺ channel antagonists that inhibit store-operated Ca²⁺ influx, La³⁺ and SK&F 96365, both inhibited influx of Pb²⁺ in GH₃ cells. Emptying of intracellular Ca²⁺ stores by two different mechanisms (SERCA-inhibiting drugs and incubation in Ca²⁺-free medium) also caused enhanced uptake of Pb²⁺. These features are generally considered diagnostic for capacitative Ca²⁺ influx. Finally, Pb²⁺ uptake by store-operated channels was inhibited by high concentrations of extracellular Ca²⁺, suggesting that Ca²⁺ and Pb²⁺ share an uptake mechanism.

However, there were several differences between the Ca²⁺ and Pb²⁺ responses to store depletion. In C₆ and 301 cells, SK&F 96365 did not inhibit Pb²⁺ uptake but did inhibit capacitative Ca²⁺ uptake, and agonist binding did not activate Pb²⁺ uptake but did stimulate Ca²⁺ uptake. Uptake of Mn²⁺ and Ca²⁺ in 301 cells, unlike that of Pb²⁺, was stimulated by TRH, but for Mn²⁺ the stimulation was not as great as with thapsigargin. The reasons for these differences are not known. However, differences in the capacitative Ca²⁺ uptake response evoked by SERCA inhibitors and receptor agonists have been found in many systems (5-8). One explanation may be that SERCA inhibitors deplete intracellular stores more thoroughly than agonists (see Fig. 7 for 301 cells and Ref. 23 for C₆ cells). Another explanation may be the presence of multiple intracellular Ca²⁺ stores. There is evidence for multiple intracellular, nonmitochondrial Ca²⁺ stores in GH₃ cells (24). Rat hepatocytes have separate IP₃-sensitive and GTP-sensitive Ca²⁺ stores, both of which are released by thapsigargin (25). Thapsigargin also releases separate IP₃-sensitive and -insensitive Ca²⁺ stores in neuronal cell lines (26) and in bovine aortic endothelial cells (27). A final possibility is that activation of the channels responsible for Pb²⁺ transport is a localized reaction, requiring Ca²⁺ release at sites that are unaffected by agonist binding.

There is considerable evidence that multiple channels are involved in the transport of divalent cations and that some of these channels are stimulated by emptying of intracellular Ca²⁺ stores. In mouse lacrimal acinar cells, addition of methacholine, a muscarinic receptor agonist, stimulates entry of Ca²⁺, Sr²⁺, and Ba²⁺, but not of Mn²⁺ or Co²⁺ (28). Ba²⁺, Mn²⁺, and Co²⁺, but not Sr²⁺, are taken up by unstimulated cells, presumably by a different pathway. There appear to be at least four different channels for divalent cations in A7r5 cells (a vascular smooth muscle cell line), two of which are stimulated by a receptor agonist, vasopressin (29). Channels that conduct multiple cations have also been reported in human umbilical vein endothelial cells (20, 30) and bovine artery endothelial cells (31).

GH₃ cells have well characterized, L-type VSCCs that have been shown previously to be responsible for the increase in Ca²⁺ uptake following depolarization (9) and to be a major route of uptake for Cd²⁺ (32) and Zn²⁺ (33). In the present study, strong depolarization of GH₃ cells increased Pb²⁺ entry 1.6-fold, in agreement with previous data indicating that some Pb²⁺ uptake can occur through VSCCs (4, 5, 34). However, the 1.6-fold increase in Pb²⁺ uptake caused by depolarization was much less than the 12-fold stimulation caused by store depletion in the same cells. The relative contributions of store-operated and voltage-gated Ca²⁺ channels to basal Pb²⁺ entry under physiological conditions are not known. The Ca²⁺ channel inhibitor SK&F 96365 did not consistently reduce basal Pb²⁺ uptake, but the effects of this drug are difficult to interpret because it can deplete intracellular Ca²⁺ stores as well as block cation channels. In this regard, the contributions of different cation channels to constitutive uptake of Ca²⁺ under physiological conditions are not clear.

These experiments show that indo-1 is a useful indicator for intracellular Pb²⁺. Measuring the quenching of intracellularly trapped indo-1 monitors Pb²⁺ transport in real time and offers a sensitive, convenient, and inexpensive alternative to other methods for Pb²⁺ measurement. Use of the radioisotope ²⁰³Pb can be difficult due to its short half-life (52 h). Atomic absorption can yield accurate Pb²⁺ measurements, but requires specialized equipment and a delay before results can be obtained. Fura-2, another fluorescent Ca²⁺ indicator, has been used previously (4) to measure intracellular Pb²⁺ in bovine chromaffin cells, but the spectra of the Pb²⁺ and Ca²⁺ complexes of fura-2 are so similar that it is difficult to distinguish Pb²⁺ uptake from an increase in intracellular Ca²⁺ (Fig. 1). Using indo-1, it is possible to monitor Pb²⁺ at an emission wavelength at which fluorescence is insensitive to changes in Ca²⁺ concentration. The method described here is analogous to the use of fluorescence quench as a measure of Mn²⁺ uptake.

The amounts of lead used in these experiments are physiologically relevant. Observed clinical effects of chronic lead poisoning occur at a blood concentration as low as 10 µg/dl (approximately 0.5 µM), but concentrations in exposed individuals can be much higher (for review, see Ref. 35). Although >90% of blood lead is in red blood cells, the concentration of free lead in plasma of exposed individuals may be within the limits of sensitivity of the indo-1 assay. Furthermore, the uptake mechanisms described here function when cells are maintained in buffers containing physiological concentrations of Ca²⁺ ion.

In summary, we have developed a novel fluorimetric assay with indo-1 to measure Pb²⁺ entry into cells and demonstrated that Pb²⁺ enters GH₃, C_6, and 301 cells via voltage-insensitive cation channels. This previously unrecognized pathway for Pb²⁺ uptake is activated by the emptying of intracellular Ca²⁺ stores.