Program in Neuroscience, Department of Biological Sciences, Ohio University, Athens, Ohio 45701
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ABSTRACT |
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In this study, Zn2+ transport in rat cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of 65Zn2+ with an apparent affinity of 15-20 µM. 65Zn2+ transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn2+ was assessed with the Zn2+-selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn2+. Depletion of intracellular Zn2+ with the membrane-permeant chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn2+ depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 µM Zn2+, pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 µM Zn2+, pH 6.0. These data were confirmed in parallel 65Zn2+ transport experiments.
pH; zinquin; carboxyseminaphthorhodofluor-1 fluorescence; ion transport; transition elements; primary culture
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INTRODUCTION |
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IT IS KNOWN THAT
Zn2+ can enter neurons by two distinct pathways. The first
pathway is transporter mediated and slow, occurs under resting
conditions, and is observed at concentrations of Zn2+ well
below 100 µM; relatively small amounts of Zn2+ enter the
cell by this pathway (5, 8, 9). The second pathway is
channel mediated and rapid, requires depolarization or receptor
activation, and is seen at concentrations of Zn2+ >100
µM; greater amounts of Zn2+ enter the cell by this
pathway (29, 30, 32). The transporter-mediated pathway can
be easily distinguished from channel pathways by its insensitivity to
various channel blockers (5, 9) (e.g., MK801, GYKI 53466, nifedipine, FTX-3.3, -conotoxin GVIA, and CNQX). Previously, the
transporter-mediated pathway was studied in synaptosomes
(34) and hippocampal slices (19), but the mechanisms of Zn2+ transport were never elucidated. More
recently, this laboratory has studied (6, 8, 9) the
mechanisms of 65Zn2+ transport in rat brain
plasma membrane vesicles. 65Zn2+ transport
showed saturation with increasing concentrations of 65Zn2+, and 65Zn2+
influx was inhibited when extravesicular pH was lowered. The transport
mechanism appeared reversible in that both pH-dependent Zn2+ influx and efflux were observed. Together, these
studies led to a working hypothesis: plasma membrane transport of
Zn2+ is pH dependent because it depends on a
Zn2+/nH+ antiport mechanism.
The recent cloning of cDNA coding for several proteins associated with
plasma membrane Zn2+ transport function, i.e., DMT1
(17), hZIP1 (15), hZIP2 (14), and ZnT-1 (28), has provided new molecular and genetic
tools with which to address the mechanism of plasma membrane
Zn2+ transport. All the cloned Zn2+
transporters are apparently independent of cellular energy stores, show
no dependence on Na+, K+, or Cl
concentrations, and show an apparent affinity for Zn2+ in
the micromolar range (14, 15, 17, 28). Expression studies
have revealed interesting pH effects on Zn2+ transport
mediated by either DMT1 (17, 35) or hZIP1 and hZIP2 (14, 15). DMT1 shows nonspecific divalent metal transport (most notably iron transport in the intestine) coupled with protons (i.e., low extracellular pH stimulates Fe2+ uptake when the
protein is expressed in Xenopus oocytes; Ref. 17). When DMT1 was transiently expressed in COS-7 cells,
Fe2+ uptake was greatly increased and showed a dependence
on extracellular pH (35). In contrast to expression
studies in Xenopus oocytes, Fe2+ uptake was
inhibited in transfected COS-7 cells by extracellular acidification,
and transport had an optimal pH of 6.8. Thus it is still not clear what
effect changes in extracellular or intracellular pH should have on DMT1
function. hZIP is the human homolog of the ZIP family of
Zn2+ transporters cloned from yeast and plants
(16). Three human ZIP gene products have been identified
(hZIP1-3); both hZIP1 and hZIP2 have been functionally
characterized (14, 15). Zn2+ transport
observed in K562 erythroleukemia cells expressing either hZIP1 or hZIP2
was inhibited by lowering extracellular pH (14). The
inhibition at low pH was reversed in cells expressing hZIP2 by addition
of HCO
Although Zn2+ is an abundant element in the brain, its cellular homeostasis and compartmentalization are poorly understood. Once inside the neuron, Zn2+ is thought to exist in at least four distinct cellular pools (13). The first pool is composed of metalloproteins (e.g., metalloenzymes and transcription factors) that use tightly bound Zn2+ as a required cofactor to carry out their cellular functions. The second pool constitutes Zn2+ bound to cytoplasmic metallothionein-III (MT-III), which is thought to be a reservoir and buffer of cytosolic Zn2+ (12, 22, 27). The third pool, cytosolic free Zn2+, is maintained at very low levels probably well below 100 nM (1, 5, 23, 29), presumably by the actions of cytosolic MT-III. The fourth pool is compartmentalized Zn2+. Experimental evidence shows that many eukaryotic cell types contain compartmentalized Zn2+ and that cytoplasmic organelles can sequester Zn2+ (10, 25). A well-characterized compartment containing Zn2+ in neurons is synaptic vesicles of glutamatergic neurons of the cerebral cortex and in particular the hippocampal formation (13).
The present studies were designed to address some of the gaps in our knowledge of Zn2+ transport and homeostasis in cortical neurons. The results provide convincing evidence of a pH-dependent transport mechanism for Zn2+ in the plasma membrane of cortical neurons in primary culture. Zn2+ influx was favored when 1) the flow of ions was down a concentration gradient and 2) the cell interior was acidic with respect to the extracellular medium. In addition, evidence was obtained of the extrasynaptic compartmentalization of newly transported Zn2+.
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MATERIALS AND METHODS |
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Primary culture of cortical neurons. Primary culture of embryonic (embryonic day 18) cortical neurons was performed as described previously (7, 24). Brains were removed from the skulls and kept moist in Hanks' balanced salt solution (HBSS; without Mg2+ and Ca2+) for further dissection. With a dissecting microscope, the cerebral cortex was carefully separated by blunt dissection from the brain stem diencephalon, olfactory bulbs, and cerebellum, which was discarded. Next the meninges and choroid plexus were stripped away. The cerebral hemispheres were cut into small pieces (about 4 pieces for each hemisphere) and trypsinized in HBSS at room temperature. After trypsinization, nerve cells were dissociated by gentle trituration through the narrow opening of a fire-polished Pasteur pipette. The dissociated neurons suspended in HBSS were plated on 24-well culture plates (Falcon) or culture plates containing sterilized coverslips coated with polyethylenimine (50% solution; Sigma, St. Louis, MO), which was diluted 1:1,000 in borate buffer. The cortical neurons were allowed to attach to the plates or coverslips at 37°C and 5% CO2 in 1 ml of MEM solution (GIBCO BRL) supplemented with 10 mM sodium bicarbonate, 2 mM L-glutamine, 1 mM pyruvate, 20 mM KCl, 10% glucose, and 10% (vol/vol) heat-inactivated fetal bovine serum. The desired cell density was obtained by adjusting the volume of cell suspension added to each plate (final plating density was 5 × 105 cells/ml). The medium was replaced with fresh supplemented MEM after 3-6 h and 24 h later switched to 1 ml of Neurobasal medium (GIBCO BRL) supplemented with 0.5 mM glutamine and 2% B27 (GIBCO BRL). Use of serum-free culture medium, which does not favor glial proliferation, allows the culture of cortical neurons in the near absence of glial cells.
Measurement of 65Zn2+
transport.
Cortical neurons (4-7 days in vitro) attached to 24-well plates
were assayed for 65Zn2+ transport as follows
(all buffers were at 37°C). Each well of a 24-well plate was
first washed with Locke's buffer (in mM: 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1.0 MgCl2, 5 HEPES, and 10 glucose, pH
7.4). Various pretreatments (e.g.,
NH3/NH4 prepulse) were performed at this
point to prepare the cells for subsequent
65Zn2+ transport assay. The transport
experiment was initiated by switching to Locke's buffer (pH 7.4 or 6.0 or various other conditions) containing 65Zn2+
(NEN, Boston, MA). Depending on the experiment,
65Zn2+ was mixed with nonradioactive
Zn2+ to obtain a final concentration in the range of
0.001-0.005 µCi/µl. The specific activity of
65Zn2+ (expressed as cpm/nmol Zn2+)
was determined for each experiment by assaying an aliquot of each
solution containing 65Zn2+ for radioactivity.
The cells were incubated for various times at 37°C. Normally, 5%
of the total Zn2+ in the reaction buffer was taken up by
the cells. To terminate a transport reaction, the buffer containing
65Zn2+ was rapidly removed by aspiration and
replaced with ice-cold Locke's buffer (pH 7.4) without
Zn2+ added. Next, the wells were washed three times with
0.5 ml of ice-cold wash buffer containing Locke's buffer (without
Ca2+ and Mg2+ added) and 1 mM EGTA (pH 7.4).
The cells were lysed by freeze thawing (
70°C). The lysed cells in
each well were resuspended in 250 µl of buffer, of which 200 µl
were used to assay radioactivity and 40 µl were used to determine
protein. Protein concentration was determined by a Bio-Rad method with
bovine serum albumin as a standard, and 65Zn2+
was determined in a gamma counter. With the specific activity and
protein concentration, counts per minute were converted to nanomoles
per milligram for each well. In experiments using
65Zn2+, parallel experiments were done in pH 6 Locke's buffer containing 1 mM Cd2+. The results from
these experiments (nmol/mg) were subtracted from "total"
65Zn2+ uptake to obtain
65Zn2+ transport and nonspecific
65Zn2+ binding. Cortical cells are firmly
attached to the culture plates and can easily withstand several buffer
washes. This was verified by viewing the cells in each well before and
after the washing procedure was complete.
MTT+ assay. Many manipulations described in these experiments have the potential of causing cell death, and therefore assessments of acute cell viability were performed. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT+) conversion was determined colorometrically as described elsewhere (24). Parallel cultures to be used for MTT+ measurements received the same experimental manipulations but were not exposed to 65Zn2+ used for transport. Instead, these cultures were allowed to remain in fresh Locke's buffer for an additional 24 h to allow time for acute effects on cell survival to occur. After 24 h the cells were assayed for MTT+ cleavage and conversion. Activities obtained after experimental manipulations were compared with cells that were switched only to fresh Locke's buffer for 24 h. Negligible cell death was observed in high-density cortical cultures exposed only to fresh Locke's buffer for 24 h, as judged by MTT+ assay.
Microscopy and zinquin labeling. Cells were cultured as described in Primary culture of cortical neurons in culture plates containing sterilized glass coverslips. For visualization of intracellular Zn2+ with zinquin, the medium was discarded and replaced with fresh Locke's buffer before an experiment was started. After various experimental settings at 37°C, the medium was discarded and the glass coverslips were washed twice with 1 ml of Locke's buffer. One milliliter of Locke's buffer was left in the culture well to prevent the cells from drying out. A 5 mM concentrate of zinquin ester (freshly dissolved in DMSO) was diluted directly into the wells to produce a final concentration of 25 µM. Although zinquin is highly permeable to lipid bilayers, the ester form was used so that only intracellular zinquin would contribute to the observed signal. Zinquin experiments were also performed at 12.5 µM with similar results. The culture plates were reincubated at 37°C for 30 min to allow zinquin to bind specifically to intracellular Zn2+. The cells were washed three times with fresh Locke's buffer (pH 7.4) and then immediately mounted on glass slides with Aquatex (for immediate viewing) to prevent leakage of zinquin. Coverslips inverted on microscope slides were examined using a Nikon Eclipse 600 with epifluorescence and differential interference contrast (DIC) optics, equipped with a SPOT RT digital camera for image capture. Zinquin fluorescence was observed using an ultraviolet filter block (EF-4 UV 2E/C DAPI filter block, EX 330-380). Zinquin labeling experiments were repeated in at least three different cultures. The digital images presented are qualitatively similar and representative of what was observed in all three experiments. Zinquin ester was obtained from Luminus (Adelaide, SA, Australia).
Carboxyseminaphthorhodofluor-1 fluorescence. Cortical neurons attached to glass coverslips were loaded with carboxyseminaphthorhodofluor (SNARF)-1 by incubation for 30 min in Locke's buffer (pH 7.4) at 37°C containing 5 µM 5- (and 6)-carboxy SNARF-1 acetoxymethyl ester, acetate (Molecular Probes, Eugene, OR). The cells were then washed with 1 ml of Locke's buffer before pH measurement. Cells attached to coverslips were held in a cuvette at an ~45° angle to the incident light beam by a coverslip holder (Hitachi Instruments, San Jose, CA). To switch buffer solutions, the coverslip and holder were lifted out of the cuvette and quickly placed into a waiting cuvette containing the next desired buffer. SNARF-1 fluorescence was measured in a fluorescence spectrophotometer (Hitachi F-2000) with excitation at 514 nm and emission wavelengths of 585 and 630 nm. The fluorescence ratio F585/F630 was calibrated in separate experiments with cells treated with 10 µM nigericin in Locke's buffers of various pH containing 120 mM KCl (2). With the calibration data, intracellular pH was calculated directly from F585/F630. F585/F630 was a linear function of pH over pH values between 6.4 and 8.0. In each experiment, to correct for scattered light/autofluorescence, data obtained from cells treated as above but without incubation with SNARF-1 were subtracted from the data obtained with SNARF-1.
Buffer preparation.
All buffers were pH adjusted on the day of the experiment. HEPES
buffers without HCO
Statistical analysis. Each preparation of cortical neurons (1 pregnant rat) normally yields three to four 24-well plates at the density used for these experiments. The data presented (except data in Fig. 6, which include up to 4 different preparations) are the means of multiple determinations with wells from the same 24-well plate. All the data presented were repeated in at least two and normally at least three different preparations of cortical neurons with similar results, although variation did exist when different preparations were compared. Potential sources of variation include differences in cell density and animal variation. Statistical analysis and nonlinear curve fitting were accomplished with GraphPad Prism software (San Diego, CA). Data were analyzed by t-test, one-way ANOVA with Tukey's post test, or, where appropriate, two-way ANOVA.
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RESULTS |
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Previous studies from this laboratory (6, 8) characterized a robust, saturable, pH-dependent 65Zn2+ transport in plasma membrane vesicles isolated from adult rat brain. The present study used cortical neurons in primary culture derived from fetal rat cortex to characterize 65Zn2+ transport. Because it is well known that high concentrations of heavy metals are toxic to neurons, determining the toxicity of the various conditions to be used in this study was necessary. To accomplish this, cells were exposed to various experimental conditions for a brief exposure (5 min) or a long exposure (60 min), and then cell death was determined 24 h later with the MTT+ conversion assay. It was found that exposure of cells to concentrations of Zn2+ as high as 300 µM in Locke's buffer for as long as 60 min did not result in significant cell death when assayed 24 h later, similar to results recently reported by Sheline et al. (31). Likewise, changing buffer pH to either 6 or 8 from 7.4 did not result in evidence of increased cell death. La3+ (1 mM) was used in plasma membrane vesicle experiments to inhibit 65Zn2+ transport; however, La3+ was quite toxic to neurons even after a brief exposure. Cd2+ (1 mM) was much less toxic to the neurons than La3+. No cell death was observed after a brief exposure, although after the 60-min exposure, some cell death was evident 24 h later. Various other transition elements (Co2+, Mn2+, Cu2+, and Fe2+) showed intermediate toxicity less than La3+ but more than Cd2+. Nickel was found to be similar to Cd2+ in toxicity.
In this study, 65Zn2+ uptake was measured under
conditions in which transport-mediated pathways predominate over
channel-mediated pathways for 65Zn2+ influx
(i.e., Zn2+ was <100 µM, extracellular Ca2+
and Mg2+ were at physiological concentrations, and
nondepolarizing conditions were used). Under these conditions,
65Zn2+ transport is insensitive to calcium and
glutamate channel blockers (9). The effect of various
transition elements on 65Zn2+ uptake is shown
in Fig. 1. Neurons in culture were
exposed to various transition elements (Cd2+,
Co2+, Mn2+, Cu2+, Fe2+,
Ni2+) in Locke's buffer for 5 min with 30 µM
65Zn2+. The transition elements were included
at a concentration of 300 µM (conditions that for the most part do
not produce acute toxicity). All the transition elements produced
significant inhibition of 65Zn2+ accumulation.
However, inhibition was incomplete. Increasing the concentration of the
transition elements to 1 mM produced a greater level of inhibition,
albeit still incomplete. The greatest inhibition was seen with
Cd2+. The reduction of extracellular pH to 6.0 reduced the
total 65Zn2+ uptake by >50%. Inhibition by
transition elements was still evident (except Mn2+), even
at pH 6.0. In this experiment, the inhibitory effects of transition
elements and extracellular acidification appeared to be additive.
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The 65Zn2+ uptake measured under the above
conditions probably included 65Zn2+ binding to
the surface of neurons and culture plates, which was resistant to
removal by EGTA. To characterize 65Zn2+ binding
better, the time course of 65Zn2+ uptake was
determined (see Fig. 2, which illustrates
the effects of pH and Cd2+ inhibition). Incubation of
neurons with 30 µM 65Zn2+ pH 7.4 resulted in
a slow accumulation of 65Zn2+, which began to
approach a plateau after 30-60 min. Changing extracellular pH to 8 enhanced 65Zn2+ uptake (most noticeably after
30 min of exposure), whereas reduction of extracellular pH to 6 inhibited 65Zn2+ uptake (Fig. 2A).
The addition of 1 mM Cd2+ at pH 7.4 reduced
65Zn2+ uptake to a level similar to that seen
at pH 6. The addition of 1 mM Cd2+ at pH 6 further reduced
65Zn2+ uptake to nearly that seen after isotope
dilution by addition of 1 mM nonradioactive Zn2+ (Fig.
2B). Again, the effects of Cd2+ and
extracellular acidification appeared to be additive. In the presence of
1 mM Cd2+ and pH 6.0, the 65Zn2+
signal closely approximates nonspecific binding, as defined by isotope
dilution, and suggests near-complete inhibition of transport without
acute toxicity, particularly when exposures are kept brief.
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Figure 3 shows
65Zn2+ uptake as a function of increasing
65Zn2+ concentration.
65Zn2+ uptake showed saturation with respect to
increasing 65Zn2+ concentration, and the
Michaelis-Menten constant (Km) and the maximum rate of 65Zn2+ transport
(Vmax) were estimated by computer-assisted
nonlinear curve fitting to a rectangular hyperbola (Fig. 3).
The Km obtained (15-20 µM) was similar to
that obtained with plasma membrane vesicles when La3+ was
used as an inhibitor (6). In the presence of 1 mM
Cd2+ and pH 6.0, a small but measurable component of
nonspecific 65Zn2+ binding remained that was
resistant to removal by EGTA (see also Fig. 2). Decreasing
extracellular pH (in the absence of Cd2+) decreased the
estimated Vmax without changing the estimated Km (consistent with this finding, vesicle
studies have shown that lowering pH produces noncompetitive inhibition
of 65Zn2+ influx). The dashed line in Fig. 3
shows the curve fit for the corresponding data with 1 mM
Cd2+-pH 6.0 subtracted from pH 7.4 (no effect was observed
by subtracting the 1 mM Cd2+-pH 6.0 data on the estimate of
Km, confirming that at low concentrations of
Zn2+ the contribution of nonspecific binding to measured
Zn2+ transport was small and insignificant). A Hill plot of
these data yielded a slope for the Hill coefficient 1, consistent
with the transport of a single zinc ion per turnover of the transport mechanism(s), similar to that obtained in plasma membrane vesicles (6). The inhibition seen by lowering extracellular pH was
completely reversible. After a brief exposure (5 min) to pH 6.0 Locke's buffer, 65Zn2+ transport measured in
pH 7.4 Locke's buffer was similar to that seen in cells preincubated
in pH 7.4 buffer (data not shown).
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The effect of changing extracellular pH on Zn2+ transport
in cortical neurons is shown in Fig. 4.
Zn2+ transport in cortical neurons showed a clear
monophasic dependence on extracellular pH (between 6 and 8), increasing
with increasing pH and approaching a maximum at pH 8. The data were fit
to a sigmoidal dose-response curve with an estimated EC50
of pH 6.77. Next, cortical neurons were preincubated for 20 min in
Locke's buffer containing 1 mM iodoacetate and KCN (without glucose)
and 65Zn2+ accumulation was assayed for an
additional 5 min under these same conditions. Metabolic inhibition
failed to inhibit 65Zn2+ transport; on the
contrary, a small increase in 65Zn2+ was
observed (control with glucose: 2.25 ± 0.17 nmol · mg1 · 5 min
1;
without glucose, 1 mM iodoaccetate, and KCN: 2.81 ± 0.19 nmol · mg
1 · 5 min
1;
means ± SE; n = 6; P > 0.05).
The small increase in 65Zn2+ transport during
metabolic inhibition may be the result of intracellular acidification
(see below). Finally, it was determined whether Na+,
Cl
, Ca2+, or Mg2+ was required
for 65Zn2+ transport. Removal of
Ca2+ and Mg2+ from the Locke's buffer did not
affect 65Zn2+ transport (providing additional
support for the lack of influence of channel-mediated pathways of
Zn2+ influx), and substitution of either thiocyanate for
Cl
or choline or Li+ for Na+
caused a small but statistically insignificant increase in
65Zn2+ transport (data not shown). Thus
cortical neurons in primary culture exhibit
65Zn2+ transport with properties that closely
mirror results obtained with plasma membrane vesicle preparations
including 1) saturation with respect to increasing
Zn2+ concentrations in the micromolar range, 2)
extracellular pH dependence, 3) inhibition by other
transition elements, and 4) transport independent of
cellular energy stores.
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On the basis of our published data (6, 8), we hypothesized
that pH effects are fundamental to the transport mechanism, perhaps
mediated by an antiport process. Therefore, it was important to show in
cortical neurons, consistent with the antiporter hypothesis, that
intracellular acidification would enhance plasma membrane 65Zn2+ transport. To test this hypothesis,
intracellular acidification was induced in cortical neurons by the
NH3/NH4+ prepulse method
(2). To confirm that intracellular acidification occurred,
intracellular pH was monitored by SNARF-1 fluorescence. The
intracellular pH of resting cells in Locke's buffer (nominally HCO1 · 3 min
1;
means ± SE; n = 4). Thus convincing evidence has
been presented for the enhancement of 65Zn2+
transport by intracellular acidification.
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A possible candidate that could mediate the Zn2+ transport
activity seen in the present studies is hZIP2 (14, 15),
which shows pH dependence similar to that observed in the present study and appears to be a Zn2+-HCO
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To provide even more convincing evidence of pH dependent
Zn2+ transport in cortical neurons, proof of changes in
intracellular Zn2+ after Zn2+ transport was
obtained with zinquin and fluorescence microscopy. Digital fluorescent
images of cortical cultures labeled with zinquin paired with the
corresponding DIC image of the same field are shown in Fig.
8.
Fig. 8A2 shows resting cortical neurons labeled for 30 min
at 37°C with 25 µM zinquin after a 25-min preincubation in Locke's
buffer pH 7.4. Cultures were plated at high density similar to
conditions used for 65Zn2+ transport assays.
Zinquin labeling was punctate rather than diffuse. This suggests that
zinquin was unable to report the presence of free cytosolic
Zn2+. This finding is not surprising, because zinquin is
known to partition into bilayers (33), and is consistent
with the notion that in resting cortical neurons, free cytosolic
Zn2+ is maintained at submicromolar levels. A uniform
punctate labeling pattern was observed in nearly all cortical neurons,
which included the soma and cell processes. Zinquin labeling was not
observed in the nucleus. Punctate zinquin labeling was observed within regions of neurons that were devoid of any visible apposition between
cells (putative synaptic contacts).
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It was next determined whether zinquin labeling would be altered when the neurons were briefly incubated with Zn2+ under conditions used for measuring 65Zn2+ transport. Figure 8B2 shows zinquin labeling in cortical neurons after a 20-min preincubation in Locke's buffer pH 7.4 followed by incubation with 30 µM Zn2+ for 5 min (conditions identical to those used for 65Zn2+ transport assays). No discernable difference could be seen in zinquin labeling when comparing resting neurons with or without prior Zn2+ exposure. Apparently, free cytosolic Zn2+ rose slightly, if at all, under these conditions, well below the detection limit of zinquin. Similarly, the pattern of punctate labeling was unaltered by incubation with Zn2+.
Although attempts at observing changes in intracellular Zn2+ in resting cells were unsuccessful, it was thought likely that such changes should be easier to detect after cellular Zn2+ depletion. In Fig. 8C2, neurons were exposed to 25 µM N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), a cell-permeant Zn2+ chelator, for 20 min. This buffer was removed, and then fresh Locke's buffer pH 7.4 without Zn2+ was added for a 5-min incubation. Finally, this buffer was removed, and fresh Locke's buffer pH 7.4 containing 25 µM zinquin was added. Punctate zinquin labeling was metal dependent, as evidenced by its TPEN sensitivity. Neurons labeled with zinquin after exposure to TPEN followed by a 5-min exposure to 30 µM Zn2+ in Locke's buffer pH 7.4 are shown in Fig. 8D2. The uniform punctate labeling returned (compare Fig. 8D2 with C2) within neuronal processes and cell bodies of neurons (although it might appear that the intensity of zinquin labeling was slightly greater in Fig. 8D2 than in either A2 or B2, such quantitative assessments are not possible with zinquin). This recovery of punctate labeling after TPEN-induced depletion was clearly pH dependent as shown in Fig. 8E2, which shows neurons exposed to TPEN followed by a 5-min exposure to 30 µM Zn2+ in Locke's buffer pH 6.0. Zinquin labeling was nearly absent, similar to TPEN treatment alone. These data provide convincing evidence that pH-dependent transport of extracellular Zn2+ results in the loading of the same cytoplasmic organelles that are labeled with zinquin.
Finally, as a positive control, zinquin labeling was observed after Zn2+ depletion with TPEN followed by exposure to 20 µM pyrithione (a well-characterized Zn2+ ionophore) and 30 µM Zn2+ (in Locke's buffer, pH 7.4) for 5 min. Pyrithione treatment should result in large increases in free cytosolic Zn2+ that should be detected by zinquin. Indeed, a diffuse labeling was observable in the cell bodies, which was not seen previously. This suggested that in the presence of pyrithione and Zn2+, free cytosolic Zn2+ increased to micromolar levels that were detectable by zinquin. These data indicate that, although zinquin partitions into bilayers, significant amounts remain in the cytosol. Additional punctate labeling was clearly seen in neuronal processes and cell bodies (compare Fig. 8F2 with B2). However, additional punctate labeling may result from the ionophoric activity of zinquin (see DISCUSSION). These data provide additional evidence that zinquin labeling was Zn2+ dependent and can detect new Zn2+ that enters cortical neurons.
To strengthen this argument, parallel experiments were performed that
show the effects of TPEN and pyrithione on
65Zn2+ transport in cortical neurons (Fig.
9). Each bar represents subtracted data,
i.e., 65Zn2+ transport under the conditions
indicated after subtraction of 65Zn2+ binding
in Locke's buffer pH 6 with 1 mM Cd2+ added. A negative
value indicates that the 65Zn2+ binding
measured under those conditions was less than that obtained in Locke's
buffer pH 6 with 1 mM Cd2+ added. The first bar shows
control cells exposed to Locke's buffer pH 7.4 for 20 min and then
exposed to the same buffer containing 30 µM
65Zn2+ for an additional 5 min. If 20 µM
pyrithione was included with the 65Zn2+, a
significant increase in uptake was observed. Data obtained with
fluorescent indicators for Zn2+ (Ref. 23; Fig.
8) suggest that cytosolic concentrations of Zn2+ reach
micromolar levels after pyrithione treatment. Therefore, it was
expected that the increase in 65Zn2+ uptake
would be much greater than what was observed. It is likely that during
the washes with EGTA, pyrithione remaining in the membrane provided an
efficient efflux channel releasing significant amounts of the
65Zn2+ that was accumulated during the previous
pyrithione incubation when 65Zn2+ was present.
Brief Zn2+ depletion induced by TPEN treatment (20 min) was
not toxic to the neurons, because they were able to take up
65Zn2+ in Locke's buffer pH 7.4 to nearly the
same level as neurons without prior exposure to TPEN and pyrithione had
its expected large increase in 65Zn2+ uptake as
well. These results are consistent with zinquin labeling experiments
(Fig. 8F2). In contrast, cells exposed to 30 µM
65Zn2+ in pH 6.0 buffer were unable to take up
significant 65Zn2+ after TPEN-induced
Zn2+ depletion. Again, these results are consistent with
zinquin labeling (Fig. 8E2). When TPEN was present in the
Locke's buffer used for 65Zn2+ transport,
65Zn2+ binding levels were near zero, lower
than that obtained in Locke's buffer pH 6 with 1 mM Cd2+
added. It should be noted that although 1 mM Cd2+-pH 6 was
used to define nonspecific 65Zn2+ binding,
Cd2+ was not used with zinquin. This is because zinquin
fluorescence is increased by Cd2+ (albeit to a much lesser
degree than Zn2+).
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DISCUSSION |
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pH-dependent Zn2+ binding or Zn2+ transport? Radionuclide accumulation, when used to directly measure Zn2+ fluxes in cultured cells, offers the advantages of 1) absolute certainty that only Zn2+ is being measured and 2) unequaled sensitivity and precision. Unfortunately, it is often difficult to distinguish true influx/efflux from changes in cell surface binding. Therefore, when radionuclide accumulation is used, the data must satisfy several criteria associated with transport phenomena. 1) The process must occur under experimental conditions that mimic the physiological concentrations of major ions found in vivo and show saturation with respect to increasing Zn2+ concentrations. 65Zn2+ uptake occurred under physiological ionic conditions and showed saturation kinetics with respect to increasing concentrations of 65Zn2+. The calculated affinity for the putative transport process was similar to that reported for known mammalian Zn2+ transporters (14, 15, 17, 28). 2) Transported 65Zn2+ must be resistant to repeated extracellular chelation. Transported 65Zn2+ was resistant to repeated extracellular chelation by the high-affinity membrane-impermeant chelator EGTA. 3) The transport process must independently depend on both intracellular and extracellular conditions. The transport of 65Zn2+ independently depended on both intracellular and extracellular pH. That is, 65Zn2+ transport was inhibited by extracellular acidification and stimulated by intracellular acidification. Each effect was observed when the corresponding intracellular or extracellular pH was normal. 4) Experiments must provide evidence that intracellular Zn2+ levels change as a result of Zn2+ transport. Under resting conditions, incubation with Zn2+ resulted in little if any change in intracellular Zn2+ as evidenced by zinquin fluorescence. This was not surprising because Marin and coworkers (23) showed only small increases in N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ) fluorescence in cortical neurons after exposure to 10 µM Zn2+. In contrast, 65Zn2+ uptake can readily be observed under these same conditions that roughly equates to 1 fmol Zn2+ taken up per cell. Studies in plasma membrane vesicles (8) suggest that Zn2+/Zn2+ exchange does occur under these conditions. Thus the net uptake of Zn2+ under these conditions is probably quite small. On the other hand, changes in zinquin labeling after TPEN depletion showed good agreement with 65Zn2+ transport studies, including pH dependence. After cellular Zn2+ depletion, newly transported Zn2+ appeared to be directed to preexisting cytoplasmic organelles. Because all four of these criteria have been met, these data provide convincing evidence of a pH-dependent Zn2+ transport process in cortical neurons. Even so, the possibility exists that a portion of the 65Zn2+ signal could result from covalent bonds with protein functional groups on the membrane surface. However, neither Zn2+ nor Cd2+ is particularly redox active (e.g., Ca2+ is a much more powerful reducing agent than either Zn2+ or Cd2+), making it unlikely that covalent bonds between Zn2+ and amino acid functional groups would readily form at neutral pH.
Zn2+/nH+
antiport mechanism and identity of the transporter.
An appealing mechanistic explanation for these data is that the pH
gradient across the plasma membrane, in particular its direction being
opposite to the direction of Zn2+ flux, is a principal
determinant of the extent of 65Zn2+ transport.
These data are clearly consistent with a
Zn2+/nH+ antiport mechanism (8)
but do not provide direct evidence that such a mechanism exists. A
direct test of this hypothesis will be to determine whether
Zn2+ transport is associated with H+ flux as
evidenced, for example, by changes in pHi. The addition of
extracellular micromolar Zn2+ (at pH 7.4) has little or no
effect on pHi of cortical neurons (data not shown). The
H+ flux associated with Zn2+ transport under
these conditions may be small and difficult to measure because cortical
neurons have a large intrinsic buffer capacity (20 mmol/pH unit; Ref.
26). Another approach would be to attempt to measure
Zn2+-dependent H+ fluxes after an acid load.
Unfortunately, cortical neurons contain an active
Na+/H+ exchanger (net H+ flux = 3 mmol · l1 · min
1; Ref.
26) that is resistant to inhibition by EIPA (see Fig. 5B), thus making it difficult to measure a small change in
H+ flux. Our laboratory is currently working on
experiments in which pHi is measured in the absence of
Na+ (to inhibit Na+/H+ exchange) in
hopes of uncovering a small Zn2+-dependent H+
flux. Finally, an approach that has been attempted is to load cortical
neurons with Zn2+ using the ionophore pyrithione and
determine changes in pHi. In fact, it has been reported
that such treatments resulted in a prolonged intracellular
acidification of cultured neurons (10a), which can be reversed on
application of TPEN. Unfortunately, Reynolds and coworkers
(10a) were unable to find any direct evidence of Zn2+/H+ antiport in their studies. Thus it is
likely that large elevations in intracellular or extracellular
Zn2+ affect pHi by several different
mechanisms. For example, micromolar Zn2+ has been shown to
inhibit Na+/H+ exchanger activity
(36).
Evidence for extrasynaptic compartmentalization of Zn2+. In the present study, evidence is presented for the extrasynaptic compartmentalization of Zn2+ in preexisting cytoplasmic organelles. These conclusions are based on the pattern of zinquin labeling observed in cortical neurons (see Fig. 8). The uniform, albeit punctate, zinquin labeling pattern and its occurrence in cellular regions apparently devoid of cell-to-cell apposition (putative synaptic contacts) suggested that zinquin labeling was not restricted to presynaptic vesicles.
In light of the physical properties of zinquin (33), several concerns can be raised as to whether the presence of zinquin will itself perturb Zn2+ compartmentalization. 1) This could occur first by cytosolic zinquin acting as a "sink" for intracellular Zn2+. This phenomenon is very unlikely because of zinquin's low affinity for Zn2+. Zinquin will not perturb the status of MT-III-bound Zn2+ in the cytosol (binding affinity of MT-III for Zn2+ is much too high to be affected by zinquin), and free cytosolic Zn2+ concentrations are well below the binding affinity of the zinquin-Zn2+ complex. 2) It was reported that high concentrations of zinquin can act as an ionophore for Zn2+ at acidic pH (33). Could zinquin load cytoplasmic compartments with Zn2+ by virtue of its ionophoric properties? Probably not, because the ionophoric effects of zinquin are concentration dependent and are negligible at the concentrations used in the proposed studies (Biological significance of pH-dependent Zn2+ transport. The results with zinquin demonstrate that, after Zn2+ depletion with TPEN, pH-dependent plasma membrane Zn2+ transport can supply Zn2+ directly to subcellular compartments and effect a rapid refilling of these stores in the presence of an inwardly directed Zn2+ gradient and physiological pH values. This suggests that the plasma membrane pH-dependent pathway may provide the means for released Zn2+ to reenter neurons. Such recycling of histochemically reactive Zn2+ has been observed after electrical stimulation of the mossy fibers innervating the hippocampus (19). The pH-dependent pathway may also provide the primary means for Zn2+ entry into neurons under resting conditions as well. Because the cortical cultures used in the present studies may contain as much as 10% glial cells, a similar pH-dependent Zn2+ transport may occur in glial cells. Eventually, a better understanding of plasma membrane transport mechanisms for Zn2+ and the fate of transported Zn2+ will aid in the clarification of the role that Zn2+ plays in selective neuronal death after transient brain ischemia. Although our knowledge of the mechanisms of plasma membrane Zn2+ transport and homeostasis is growing, there is still much more to be learned.
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ACKNOWLEDGEMENTS |
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This work would not have been possible without the technical assistance of Philip A. Carter, Jason Zaros, and Lynn Bowman. Special thanks go to Mark Berryman, Department of Biomedical Sciences, Ohio University, who supplied the microscope and digital camera setup. Also contributing to this work was Tatsuhiko Kawaguchi, a summer undergraduate research fellow in cellular and molecular biology.
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FOOTNOTES |
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This work was supported by National Institute on Aging Grant AG-17741.
Address for reprint requests and other correspondence: R. A. Colvin, Dept. of Biological Sciences, Ohio Univ., Athens, OH 45701 (E-mail: colvin{at}ohio.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 17, 2001; 10.1152/ajpcell.00143.2001
Received 19 March 2001; accepted in final form 17 October 2001.
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