Mild acidosis enhances AMPA receptor-mediated intracellular zinc mobilization in cortical neurons

Abstract

Overactivation of glutamate receptors and subsequent deregulation of the intraneuronal calcium ([Ca2+]i) levels are critical components of the injurious pathways initiated by cerebral ischemia. Another hallmark of stroke is parenchymal acidosis, and we have previously shown that mild acidosis can act as a switch to decrease NMDAR-dependent neuronal loss while potentiating the neuronal loss mediated by AMPARs. Potentiation of AMPAR-mediated neuronal death in an acidotic environment was originally associated only with [Ca2+]i dyshomeostasis, as assessed by Ca2+ imaging; however, intracellular dyshomeostasis of another divalent cation, Zn2+, has recently emerged as another important co-factor in ischemic neuronal injury. Rises in [Zn2+]i greatly contribute to the fluorescent changes of Ca2+-sensitive fluorescent probes, which also have great affinity for Zn2+. We therefore revisited our original findings (Mcdonald et al., 1998) and investigated if AMPAR-mediated fura-2 signals we observed could also be partially due to [Zn2+]i increases. Fura-2 loaded neuronal cultures were exposed to the AMPAR agonist, kainate, in a physiological buffer at pH 7.4 and then washed either at pH 7.4 or pH 6.2. A delayed recovery of fura-2 signals was observed at both pHs. Interestingly this impaired recovery phase was found to be sensitive to chelation of intracellular Zn2+. Experiments with the Zn2+ sensitive (and Ca2+-insensitive) fluorescent probe FluoZin-3 confirmed the idea that AMPAR activation increases [Zn2+]i, a phenomenon that is potentiated by mild acidosis. Additionally, our results show that selective Ca2+ imaging mandates the use of intracellular heavy metal chelators to avoid confounding effects of endogenous metals such as Zn2+.

Figure 1

AMPAR activation increases both [Ca²⁺]_i and [Zn²⁺]_i levels. A, B. Time course of Ca²⁺ and Zn²⁺-dependent changes in fura-2 fluorescence upon activation of AMPARs: Neuronal cultures loaded with fura-2 were imaged before, during, and after a 1 min exposure to 300 μM kainate. After the kainate challenge, neurons were washed for 30 min in a physiological buffer either at pH 7.4 (A) or pH 6.2 (B). At the end of the washout period, neurons were exposed for 5 min to the cell permeant Zn²⁺ chelator TPEN (20 μM). Traces show mean fura-2 ΔF (expressed as % over baseline of 340/380 nm ratios) (±SEM) of 13–24 neurons from one experiment representative of 5–6, respectively.

Figure 2

Mild acidosis enhances AMPAR-mediated [Zn²⁺]_i rises. A, B. FluoZin-3 loaded cultures were exposed to kainate (300 μM) for 1 min and washed out in a physiological buffer either at pH 7.4 (A) or pH 6.2 (B) for 20 min. Traces show mean FluoZin-3 ΔF (±SEM) of > 50 neurons from 4–7 experiments. C. Bar graph depicts the area under the curve during the recovery phase in the two conditions, * indicates differences between washout at pH 7.4 versus washout at pH 6.2 (P < 0.01). Note that AMPAR-mediated [Zn²⁺]_i rises are greatly enhanced upon exposure to an acidotic environment.

Figure 3

AMPAR-mediated [Zn²⁺]_i rises are mainly dependent on intracellular Zn²⁺ mobilization. A. AMPAR-mediated [Zn²⁺]_i rises are not modulated by extracellular Zn²⁺ chelation. FluoZin-3 loaded neurons were exposed, as in Figure 1, to kainate in the presence of the extracellular Zn²⁺ chelator Ca²⁺ EDTA (20 μM). Note that the [Zn²⁺]_i rises occurring both during and after kainate exposure are not affected by extracellular chelation of contaminant Zn²⁺, indicating that the source of these rises is intracellular. B. AMPAR-mediated release of intra-mitochondrial Zn²⁺. FluoZin-3 loaded cultures were exposed for 5 min to the mitochondrial protonophore, CCCP (1 μM), challenged with 300 μM kainate for 5 min and washed at pH 7.4, in the presence of CCCP. Note that prior exposure to CCCP greatly inhibits subsequent kainate-triggered [Zn²⁺]_i rises, indicating that these two manipulations target a common intracellular Zn²⁺ pool. Traces show time course of FluoZin-3 Delta;F (±SEM), and are derived from > 15 neurons from 3–5 experiments.

Figure 4

AMPAR-mediated ROS generation is enhanced by low extracellular pH. A, B. Cortical cultures were loaded with HEt, and exposed for 5 min to 300 μM kainate and washed for 50 min with a buffer at pH 7.4 (A) or pH 6.2 (B). HEt fluorescence changes for each neuron are expressed as the ratio of fluorescence at each time point (F_x) to its own baseline fluorescence (F₀). Traces show mean (± SEM) of 29 (A) or 40 (B) neurons from 3 experiments. C. Bar graph depicts the cumulative ROS production as area under the curve during the recovery phase in the two conditions, * indicates differences between washout at pH 7.4 versus washout at pH 6.2 (P < 0.01) Note the marked increase in HEt fluorescence only in the case of kainate exposures followed by an acidotic wash out.