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(Stroke. 2007;38:670.)
© 2007 American Heart Association, Inc.

Glutamate-Independent Calcium Toxicity: Introduction

Extracellular Zinc Protects Against Acidosis-Induced Injury of Cells Expressing Ca²⁺-Permeable Acid-Sensing Ion Channels

Jessica G. Hey, BS; Xiang-Ping Chu, MD, PhD; Joshua Seeds, MSc; Roger P. Simon, MD Zhi-Gang Xiong, MD, PhD

From Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Ore.

Correspondence to Zhigang Xiong, MD, PhD, Robert S. Dow, Neurobiology Laboratories, Legacy Clinical Research Center, 1225 NE 2nd Ave, Portland, OR 97232. E-mail zxiong{at}Downeurobiology.org

Abstract

Acidosis is a common feature of neurological conditions including brain ischemia, epileptic seizures, and neurotrauma. Activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) is involved in acidosis-mediated ischemic brain injury. Zn²⁺ is a divalent cation concentrated in nerve terminals in various brain regions, and is released into the extracellular space during excitatory stimulation. Our previous studies have demonstrated that the activities of ASIC1a containing channels and acid-induced increased intracellular Ca²⁺ concentrations are inhibited dramatically by the physiological concentration of extracellular Zn²⁺. In this report, we demonstrate that decreasing the concentration of the extracellular Zn²⁺ significantly enhances acid-induced injury of HEK 293 cells, a cell line expressing homomeric ASIC1a-like channels, whereas increasing the concentration of extracellular Zn²⁺ appears to be protective. Although increased concentrations of intracellular Zn²⁺ have been shown to be detrimental to neurons, our findings may suggest that the physiological concentration of extracellular Zn²⁺ might play a protective role in acidosis-induced, ASIC1a-mediated neuronal injury.

Key Words: acid • cell injury • HEK293 • zinc

Acidosis, or significant decrease of tissue pH, is common in brain in acute neurological disorders, particularly in stroke.^1,2 For several decades, acidosis has been known to be associated with neuronal injury.¹ However, the detailed mechanism underlying acid-mediated neuronal injury remained elusive. Previously, we demonstrated that activation of Ca²⁺-permeable acid-sensing ion channels (ASIC1a) and subsequent intracellular Ca²⁺ accumulation is largely responsible for acidosis-mediated neuronal injury,^3,4 disclosing a novel potential therapeutic target for stroke intervention.^5,6 In addition, we have shown that the activities of ASICs are modulated by endogenous signaling molecules.^7,8 For example, the activities of ASIC1a containing channels are substantially inhibited by physiological concentrations of Zn²⁺, a divalent cation concentrated in excitatory nerve terminals and released into the extracellular space during stimulation.^7,9 Because activation of ASIC1a channels is involved in acidosis-mediated neuronal injury, this finding suggests that extracellular Zn²⁺ may reduce acidosis-mediated injury of cell expressing ASIC1a channels. Because native neurons in the brain contain not only homomeric ASIC1a but also heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels,^3,10 and the fact that Zn²⁺ has dramatically different effect on different configurations of ASICs,^7,11 we decide to use HEK 293 cells, a cell line containing only ASIC1a-like channels¹² to test the effect of Zn²⁺ on acid-induced injury.

Methods

Cell Culture
HEK 293 cells were plated in 24-well culture plates and 35-mm culture dishes with minimal essential medium supplemented with 10% fetal bovine serum. Cells were used for electrophysiological recording and cell injury assays 3 to 5 days after plating.

Electrophysiology
Whole-cell voltage-clamp recordings were performed using Axopatch-1D amplifier (Molecular Device). Extracellular solution contained (in mM): 140 NaCl, 5 KCl, 20 HEPES, 10 glucose, 2 CaCl₂, 1 MgCl₂. The pH was adjusted with NaOH or HCl. Osmolarity was adjusted to 320 to 330 mOsm. Patch electrodes contained (in mM): 140 CsF, 2.0 MgCl₂, 1.0 CaCl₂, 10 HEPES, 11 EGTA, 4 MgATP, with pH adjusted to 7.3 and osmolarity to 300 mOsm.

Cell Injury Assay
Lactate dehydrogenase (LDH) release was measured using cytotoxicity detection kit (Roche). Optic density was measured at 490 nm with subtraction of the reference value at 620 nm. Propidium iodide and fluorescein diacetate were used for the staining of alive and dead cells, respectively, and were detected by a fluorescent microscope with excitation/emission wavelength at 530/612 nm for propidium iodide and 500/550 nm for fluorescein diacetate.

Results

Extracellular Acidosis Activates ASIC1a-Like Channels in HEK293 Cells
We first studied the existence of ASIC current in HEK293 cells using patch-clamp recording and fast-perfusion techniques. Decreasing the extracellular pH (pH_o) from 7.4 to 6.0 activates transient inward current in all cells recorded (Figure 1), as demonstrated previously.¹² The current is sensitive to the blockade by amiloride, a nonspecific ASIC blocker (not shown), indicating activation of ASICs in these cells. The acid-activated current in HEK293 cells is largely blocked by PcTX venom (150 ng/mL), a specific blocker for homomeric ASIC1a channels,¹ suggesting that the acid-activated current in these cells is mediated by homomeric ASIC1a channels (Figure 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Lowering extracellular pH activates ASIC1a currents in HEK293 cells and these currents are potentiated by reducing Zn2+ concentrations. A, Representative current traces demonstrating the activation of ASIC1a-like current in HEK293 cells. The current is largely inhibited by PcTX venom (150 ng/mL), a specific blocker for homomeric ASIC1a channels. Reducing the concentration of Zn2+ by perfusion with 10 µmol/L TPEN dramatically increased the amplitude of the ASIC current. In contrast, perfusion of the cells with 10 µmol/L ZnCl2 slightly inhibited the ASIC current. B, Summary bar graph demonstrating the effect of changing the concentration of Zn2+ on ASIC current in HEK293 cells. n=5. **P<0.01 compared with control group.

Zinc Chelator Potentiates Acid-Activated Current in HEK293 Cells
We then determined whether changing the concentration of the extracellular Zn²⁺ affects the amplitude of ASIC current in HEK293 cells. Bath application of TPEN, a high-affinity Zn²⁺ chelator,¹³ dramatically potentiated the ASIC current by 50% (n=5, Figure 1). In contrast, addition of 10 µmol/L Zn²⁺ slightly inhibited the ASIC current (Figure 1). The lack of dramatic inhibition of the current by addition of 10 µmol/L Zn²⁺ is likely attributable to the fact that the ambient concentration of Zn²⁺ present in most physiological solutions (eg, 50 nM) is already near the saturating concentration for high affinity Zn²⁺ inhibition of the ASIC1a current.⁷

Acid Injury of HEK293 Cells
Next, we studied acid injury of HEK293 cells. HEK293 cells grown in 24-well plates were randomly divided into different treatment groups and incubated with either normal (pH 7.4) or acidic (pH 5.5) solutions. Cell injury was determined by LDH release measured at different time points following 3 hour of acid incubation. As shown in Figure 2, acid incubation induced a time-dependent increase in LDH release compared with cells treated with normal extracellular solutions, indicating injury of HEK293 cells by acidosis. Incubation of cells with pH 6.0 solution also induced significant increase of LDH release (n=4, not shown).

View larger version (81K): [in this window] [in a new window]   Figure 2. Acid incubation induces injury of HEK293 cells. A, Representative black and white images showing the acid-induced injury of HEK293 cells. Cells were incubated with extracellular solutions with pH buffered at either 7.4 or 5.5 for 3 hours before switching back to neurobasal medium. Photographs were taken 24 hours after the acid incubation. B, Bar graph summarizing time-dependent injury of HEK293 cells as indicated by increase in LDH release, after the acid incubation. LDH release at various time points has been normalized to the maximal releasable amount obtained by incubating cells with 1% Triton X-100 for 15 minutes. *P<0.05 and **P<0.01 compared with pH 7.4 treated group at the same time points.

Acid Injury of HEK293 Cells Is Inhibited by PcTX Venom
We then determined whether acid-induced injury of HEK293 cells can be inhibited by PcTX venom; 150 ng/mL PcTX venom was included in both normal and acidic solutions. Addition of 150 ng/mL PcTX venom slightly increased the baseline LDH release at pH 7.4; however, it significantly reduced the LDH release induced by incubation with acidic solutions (Figure 3), suggesting that acid-induced injury of HEK293 cells is largely caused by activation of homomeric ASIC1a channels.

View larger version (19K): [in this window] [in a new window]   Figure 3. Acid-induced injury of HEK293 cells is inhibited by specific ASIC1a blockade. Summary bar graph showing the inhibition of acidosis induced injury of HEK293 cells by PcTX venom (150 ng/mL), a specific blocker for homomeric ASIC1a channels. n=4. **P<0.01 between control (pH 7.4) and pH 5.5, and *P<0.05 between pH 5.5 and pH 5.5+PcTX venom.

Acid-Induced Injury of HEK293 Cells Is Potentiated by Zn²⁺ Chelation
We next determined whether changing the concentration of extracellular Zn²⁺ has an effect on acid-induced injury of HEK293 cells. The Zn²⁺ chelator TPEN, at 10 µmol/L, was added to the treatment solutions. Addition of 10 µmol/L TPEN did not significantly affect the baseline LDH release at 7.4; however, it dramatically potentiated the LDH release induced by incubating cells with acidic solutions (Figure 4). In contrast, addition of 10 µmol/L ZnCl₂ slightly increased acid-induced LDH release.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of Zn2+ on acid-induced injury of HEK293 cells. A, Fluorescent images (upper panel) and summary bar graph (lower panel) showing the potentiation of acidosis-induced injury of HEK293 cells by Zn2+ chelation with TPEN (10 µmol/L). n=4. **P<0.01 between control (pH 7.4) and pH 5.5, and between pH 5.5 and pH 5.5+TPEN. B. Summary bar graph showing inhibition of acid-induced injury of HEK293 cells by addition of 10 µmol/L Zn2+. n=4. **P<0.01 between control and pH 5.5, and *P<0.05 between pH 5.5 and pH 5.5+10 µmol/L Zn2+.

Discussion

Brain acidosis, characterized by an increase in proton concentration or decrease in tissue pH, is an important component of the pathogenic events associated with various neurological conditions including brain ischemia.¹⁴ The decrease of brain pH, even moderately, is expected to activate ASICs, especially the Ca²⁺-permeable ASIC1a channels that are highly expressed in neurons of the central nervous system. Activation of these channels results in membrane depolarization and intracellular Ca²⁺ accumulation, which contributes substantially to acidosis-mediated ischemic brain injury.^3,4

Zn²⁺, an endogenous divalent cation and trace element, is stored at presynaptic terminals in various brain regions and released during intense neuronal excitation.^9,15 The role of these Zn²⁺ stores in neuronal injury is not clear. Although abnormal increases of extracellular Zn²⁺ are associated with neurotoxicity^16,17 likely caused by entry of Zn²⁺ into neurons,¹⁸ high-affinity inhibition of ASIC1a channels, as shown by our studies,⁷ along with Zn²⁺ inhibition of NMDA channels,^19,20 strongly suggest that moderate increase of the extracellular Zn²⁺ may serve as a negative feedback mechanism to maintain neuronal excitation at a moderate level thus protecting neurons from excitatory and acidosis-mediated injury.

This notion is consistent with a previous report that disrupting Zn²⁺ storage in synaptic vesicles, by knockout of the Zn²⁺ transporter, rendered mice more susceptible to kainic acid-induced seizures.²¹ Similarly, studies by Blasco-Ibanez et al demonstrated that chelation of synaptic zinc induced over-excitations of hilar mossy cells in rat hippocampus.²²

In summary, our present studies strongly suggest that in pathological conditions in which severe acidosis takes place, a moderate increase of Zn²⁺ in the extracellular space may serve as a neuroprotective measure against acid injury through inhibition of Ca²⁺-permeable ASIC1a channels. It is, however, worth mentioning that the current study using HEK 293 cells is largely for "proof of principle," which cannot be directly translated to the stroke brain. The HEK cells are generally tolerant to various injury paradigms and the pH 5.5 used in this study is unlikely to be achieved in the ischemic brain. Future studies will need to use primary neurons, or at least neuronal cell lines to confirm these findings.

Acknowledgments

Sources of Funding

This work was supported by NIH R01NS47506 and R01NS049470 (to Z.G.X.).

Disclosures

None.

Received October 13, 2006; accepted October 19, 2006.

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