(Received for publication, September 23, 1996, and in revised form, January 6, 1997)
From the Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
The mechanisms of cellular lead uptake were characterized using a fluorescence method in cells loaded with indo-1. Pb2+ bound to intracellular indo-1 with much higher affinity than Ca2+ and quenched fluorescence at all wavelengths. Pb2+ uptake into pituitary GH3 cells, glial C6 cells, and a subclone of HEK293 cells was assessed by fluorescence quench at a Ca2+-insensitive emission wavelength. Pb2+ uptake was concentration- and time-dependent. Pb2+ uptake in all three cell types occurred at a much faster rate when intracellular Ca2+ stores were depleted by two different methods: addition of drugs that inhibit the endoplasmic reticulum Ca2+ pump (thapsigargin, cyclopiazonic acid, and tert-butylhydroquinone), and prolonged incubation of cells in Ca2+-free media. Application of receptor agonists, which deplete intracellular Ca2+ stores via inositol trisphosphate-sensitive channels, did not activate Pb2+ uptake. Agonists were just as effective as thapsigargin in stimulating uptake of Ca2+ but less so in stimulating uptake of Mn2+. Basal and stimulated Pb2+ uptake were partially reduced by 1 mM extracellular Ca2+ and strongly inhibited by 10 mM Ca2+. Pb2+ entry in GH3 cells was inhibited by two drugs that block capacitative Ca2+ entry, La3+ and SK&F 96365. Depolarization of electrically excitable GH3 cells increased the initial rate of Pb2+ uptake 1.6-fold, whereas thapsigargin increased uptake 12-fold. In conclusion, Pb2+ crosses the plasma membrane of GH3, C6, and HEK293 cells via channels that are activated by profound depletion of intracellular Ca2+ stores.
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.
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).
Determination of Pb2+-Indo-1 Equilibrium ConstantThe 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),
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(Eq. 1) |
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.
Cell CultureCells 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).
Measurement of Pb2+ Uptake with Fluorimetric SpectrophotometerCells 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+ (Keq for
Pb2+ = 1021.8 versus
1010.6 M1 for Ca2+)
(13). A cell-permeant metal chelator, TPEN, was added 1 min later
(Keq = 1013.98
M
1 for Pb2+ and 104.4
M
1 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.
Measurement of Intracellular Free Ca2+ Concentration, [Ca2+]i[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
Fmax was obtained; EGTA (10 mM, pH
8.3) was then added and Fmin was obtained.
[Ca2+]i was calculated from the relationship
[Ca2+]i = Kd × (F Fmin)/(Fmax
F) and a Kd value of 254 nM
(12).
Indo-1 and fura-2 are ratio dyes commonly used to measure
intracellular Ca2+. We found that the
Keq for the binding of Pb2+ to
indo-1 is 1010.46 M1, almost 4 orders of magnitude higher than the reported Keq
for the Ca2+-indo-1 complex, 106.6
M
1 (12). The affinity of Pb2+ for
fura-2 is also very high, Keq = 1012.38 M
1 (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.
Indo-1 as an Intracellular Pb2+ IndicatorThe
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
(Keq = 1021.8
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 (Keq for Pb2+ = 1014
M
1 (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.
Role of Voltage-sensitive Ca2+ Channels in Pb2+ Transport
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).
Pb2+ Transport through Store-operated Ca2+ Channels
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.
Inhibition of Pb2+ Entry by Ca2+
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).
Inhibition by La3+ and SK&F 96365
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.
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 ICRAC (Ca2+ release-activated Ca2+ current). ICRAC 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, C6, and 301 cells via voltage-insensitive cation channels. This previously unrecognized pathway for Pb2+ uptake is activated by the emptying of intracellular Ca2+ stores.