Hydrogen sulfide induced disruption of Na+ homeostasis in the cortex

Abstract

Maintenance of ionic balance is essential for neuronal functioning. Hydrogen sulfide (H(2)S), a known toxic environmental gaseous pollutant, has been recently recognized as a gasotransmitter involved in numerous biological processes and is believed to play an important role in the neural activities under both physiological and pathological conditions. However, it is unclear if it plays any role in maintenance of ionic homeostasis in the brain under physiological/pathophysiological conditions. Here, we report by directly measuring Na(+) activity using Na(+) selective electrodes in mouse cortical slices that H(2)S donor sodium hydrosulfide (NaHS) increased Na(+) influx in a concentration-dependent manner. This effect could be partially blocked by either Na(+) channel blocker or N-methyl-D-aspartate receptor (NMDAR) blocker alone or almost completely abolished by coapplication of both blockers but not by non-NMDAR blocker. These data suggest that increased H(2)S in pathophysiological conditions, e.g., hypoxia/ischemia, potentially causes a disruption of ionic homeostasis by massive Na(+) influx through Na(+) channels and NMDARs, thus injuring neural functions. Activation of delta-opioid receptors (DOR), which reduces Na(+) currents/influx in normoxia, had no effect on H(2)S-induced Na(+) influx, suggesting that H(2)S-induced disruption of Na(+) homeostasis is resistant to DOR regulation and may play a major role in neuronal injury in pathophysiological conditions, e.g., hypoxia/ischemia.

FIG. 1.

Extracellular Na⁺ response to NaHS at different concentrations. The bars indicate the response rate of the examined cortical slices. Note that NaHS at concentration less than 100μM has very little effect on extracellular Na⁺ activity in all slices. With the increase in its concentration (≥ 150μM), NaHS evokes a large drop in [Na⁺]o (a direct index of Na⁺ influx) in a concentration-dependent fashion in almost all the slices that were investigated. Chi-square test showed a statistically significant difference in comparison with control when the concentration of NaHS increased to 150 (p = 0.0013), 300 (p < 0.0001), 600 (p = 0.0005), and 1200μM (p = 0.0001).

FIG. 2.

NaHS-evoked Na⁺ influx in the cortical slices. Trace recordings of (A) control, (B–F) 100, 150, 300, 600, 1200μM of NaHS respectively. (G–I) Statistical results of each recording parameter. ***p < 0.001 as compared with the control; ###p < 0.001 as compared with 100μM of NaHS; &p < 0.05, &&p < 0.01, &&&p < 0.001 as compared with 150μM of NaHS; +p < 0.05, +++p < 0.001 as compared with 300μM of NaHS. Note that NaHS, at the concentrations ≥ 150μM, evoked a large concentration-dependent fall in [Na⁺]o in almost all the slices investigated with a significantly shortened interval to maximal fall in [Na⁺]o and prolonged the recovery time from Na⁺ drop.

FIG. 3.

Differential effects of TTX, MK 801, CNQX, and UFP 512 on extracellular Na⁺ response to 150 (A) and 300 (B) μM NaHS. The bars indicate the response rate of the cortical slices investigated. Na⁺ channel blocker TTX (1μM) completely blocked extracellular Na⁺ response to 150μM of NaHS in all the seven slices investigated (100%) (p = 0.0004, chi-square test). In the presence of NMDAR antagonist MK 801 (10μM) and non-NMDAR antagonist CNQX (10μM), respectively, 67 and 73% slices no longer showed responses to NaHS (150μM) (p = 0.011 and 0.006, respectively, chi-square test). Activation of DOR with UFP 512 (5μM) did not increase the number of slices that showed a lack of response to 150μM of NaHS (p = 0.881, chi-square test). In contrast, in the presence of TTX (1μM) and MK 801 (10μM), respectively, there were 21 and 18% of the slices showing lack of response to 300μM of NaHS (p = 0.088 and 0.124, respectively, chi-square test). In the presence of CNQX (10μM) or UFP 512 (5μM), all slices still responded to 300μM NaHS, as seen in NaHS alone. However, NaHS (300μM)-evoked drop in [Na⁺]o was completely blocked by coperfusion of TTX (1μM) and MK 801 (10μM) in eight of nine slices investigated (89%) (p < 0.0001, chi-square test).

FIG. 4.

Different roles of Na⁺ channels, NMDAR, and non-NMDAR in NaHS-evoked Na⁺ influx. Trace recordings of (A) control, (B) TTX (1μM), (C) MK 801 (10μM), (D) CNQX (10μM), and (E)TTX + MK 801. (F–H) Statistical results of each recording parameter. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the control. Note that 300μM NaHS-evoked Na⁺ influx could be partially attenuated by Na⁺ channel blocker TTX or NMDAR antagonist MK 801 but could not be decreased by non-NMDAR antagonist CNQX.

FIG. 5.

Effect of UFP 512 on NaHS-evoked Na⁺ influx in the cortex. Trace recordings of (A) 150μM of NaHS (NaHS-150), (B) NaHS-150 + UFP 512 (5μM), (C) 300μM of NaHS (NaHS-300), and (D) NaHS-300 + UFP 512 (5μM). (E–G) Statistical results of each recording parameter. Note that activation of DOR with UFP 512 did not affect the NaHS-evoked Na⁺ influx in the cortex.