Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

Abstract

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 °C and the Q(10) value of 5.6. After 1 h exposure to acidic solution at 37 °C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 °C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.

Figure 1. Whole-cell anionic currents activated by extracellular acidification in cortical neurons at 25 °C. (A) Top: Representative record of whole-cell currents before, during (at bar), and after exposure to acidic solution (pH 4.5). Alternating pulses (0.25 s duration, every 5 s) of ± 60 mV or step pulses (0.5 s duration, every 3 s) of ± 100 mV in 20-mV increments (at arrows) were applied. Bottom: Expanded traces of current responses to step pulses (applied at a, b, and c). (B) I-V relationships for the mean currents (with the SEM bars) of steady-state peak responses to step pulses before (pH 7.4) and during (pH 4.5) exposure to acidic solution. Asterisks indicate significant differences between data recorded at pH 7.4 and pH 4.5 (P < 0.05). (C) Relation between the mean reversal potential (E_rev) for the acid-activated current components and the logarithm of [Cl]_i/[Cl]_o. The slope determined by a linear regression analysis is −56.5 mV/decade.

Figure 2. Sensitivity of ASOR anion currents recorded at 25 °C in cortical neurons to DIDS (A) and phloretin (B). Top: Representative records of ASOR currents before and during application of 100 μM DIDS and 300 μM phloretin. Alternating pulses were applied as in Figure 1A. Bottom: Concentration-dependent inhibition of ASOR currents (acid-activated components) recorded at +60 mV and 25 °C by DIDS and phloretin. The data are fitted by the Hill equation with the indicated IC₅₀ values.

Figure 3. Effects of temperature changes on the ASOR anion channel currents recorded in cortical neurons. (A) Representative trace (top) of ASOR current activation during application of alternating pulses from 0 to ± 60 mV at pH 5.25 in response to a ramp increase in temperature (bottom) from 22 to 38 °C. (B) Arrhenius plot for the mean ASOR currents (with the SEM bars) recorded at +60 mV and pH 5.25. The whole-cell current (I) and temperature were simultaneously recorded as done in A. (Q₁₀ values and two lines, see in the text). (C) Representative current responses to step pulses (left) and mean peak currents recorded at +100 mV (right) at 37 and 25 °C (at pH 5.25). Asterisk indicates a significant difference (P < 0.01) between the mean peak values (with the SEM bars) recorded at 37 and 25 °C. (D) Shift of pH dependence of relative mean ASOR currents (recorded at +60 mV) by a temperature increase from 25 to 37 °C. The data are fitted by the Hill equation with the EC₅₀ values given on the figures.

Figure 4. Acidotoxic necrotic death of cortical neurons and its sensitivity to hypothermia and anion channel blockers. (A) Representative fluorescence micrographs of cortical neurons stained with Hoechst 33342 (blue) for nuclei of all neurons and with PI for nuclei of injured neurons 1 h after exposure to control (pH 7.4) or acidic (pH 5.25) solution at 25 or 37 °C. (B) Summarized data showing the fraction of PI-positive neurons at 25 °C (top) and 37 °C (bottom) in neutral pH or acidic solution in the absence or presence of 10 μM DIDS or 50 μM phloretin. (C) Caspase-3 activity after exposure to control (pH 7.4) or acidic (pH 5.25) solution for 1 h at 37 °C in the absence or presence of 0.5 μM STS. Each column represents the mean (with the SEM bar) of 28−96 (B) or 5−10 (C) samples. Daggers indicate significant differences (P < 0.01) between the data obtained at 25 and 37 °C (B). Single asterisks indicate significant differences (P < 0.01) between the data obtained at 37 °C without and with anion channel blockers (B) or between those in the presence and absence of STS (C).

Figure 5. Acidotoxic cell swelling (NVI) and its sensitivity to hypothermia and anion channel blockers in cortical neurons. (A) Time courses of cell size (CSA) changes before (pH 7.4) and after application (started at arrows) of acidic (pH 5.25) solution at 25 °C (top) or 37 °C (bottom) in the absence (open symbols) or presence (filled symbols) of 10 μM DIDS or 50 μM phloretin. Insets: Representative microscopic images of a neuron before (0 min) and 2.5 min after the beginning of exposure to acidic solution at 25 °C (top photos) and 37 °C (bottom photos). (B) Summarized data showing the peak cell swelling (CSA recorded at 2.5 min) relative to the control cell size (CSA recorded at 0 min) at 25 °C (top) or 37 °C (bottom) in the absence (open column) or presence of 10 μM DIDS or 50 μM phloretin. Asterisks indicate significant difference (P < 0.01) between the data obtained at pH 5.25 without anion channel blockers and those obtained at pH 7.4 without anion channel blockers or at pH 5.25 with anion channel blockers. Daggers indicate significant difference (P < 0.01) between the data obtained at 25 and 37 °C.