ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity

Abstract

Background/aims: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state.

Methods: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents.

Results: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity.

Conclusion: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase.

Fig. 1

DTT pretreatment potentiates acid-induced currents. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before DTT pretreatment (control), and after 5-6 min of 0.5 and 2 mM DTT pretreatment. Group data of the effect of DTT (0.5 or 2 mM) pretreatment on the normalized peak amplitude (B) and accumulated currents (C, area under curve, AUC) induced by extracellular [H⁺], pH 6.0 (n=8 and 7, respectively). Reproducibility of the potentiation effect of DTT (2 mM) on the same cell after wash-off (10-15 min). Peak current (D) and AUC (E) are not different between first and second DTT exposure (n=5). Effect of DTT (2 mM) on pH dose response for peak current (F), AUC (G), and percent responding VSMCs (H) (n=4-8). ^∗Significantly different, p<0.05.

Fig. 2

DTT potentiation is reversed by oxidizing agent DTNB. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before and after DTT pretreatment and following sequential treatment with 1 mM DTT and 1 mM DTNB (time bar not drawn to scale). Group data of the effect of DTT followed by DTNB pretreatment on the normalized peak amplitude (B) and accumulated currents (C, area under curve, AUC) induced by extracellular [H⁺], (n= 6, 7 and 7, respectively). ^∗Significantly different, p<0.05.

Fig. 3

The ASIC channel blocker amiloride inhibits extracellular [H⁺] evoked currents potentiated by DTT. A. Representative traces. VSMCs pretreated with 1mM DTT for 4 min were exposed to normal external solution ± 100 μM amiloride for 45 sec before exposure to extracellular [H⁺] (pH 6.0). Group data of the effect of amiloride on DTT potentiation of extracellular [H⁺] evoked currents on the normalized peak amplitude (B) and accumulated currents (C, area under curve AUC), (n= 7-9 cells). Note that time references are not drawn to scale. ∗Significantly different, p<0.05.

Fig. 4

GSH pretreatment potentiates acid-induced currents. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before GSH pretreatment (control), and after 5-6 min GSH 0.5 and 2 mM pretreatment (n=9 and 10, respectively). Group data of the peak amplitude (B), and normalized accumulated current (C, area under curve, AUC) induced by extracellular [H⁺]. ^∗Significantly different, p<0.05.

Fig. 5

Inhibition of NOX with apocynin (Apo) potentiates extracellular [H⁺] gated currents. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before Apo pretreatment (control, con), and after 5-6 min of 50 or 200 mM Apo pretreatment. Group data of the effect of Apo (50, or 200 mM) pretreatment on normalized peak current amplitude (B) and accumulated currents (C, area under curve, AUC) induced by extracellular [H⁺] (n=5-14). ^∗Significantly different, p<0.05.

Fig. 6

Rac 1 inhibitor NSC23766 (100 μM) potentiates the effect of DTT (1 mM) on extracellular [H⁺] gated currents. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before DTT pretreatment (control, con), right after 1 min of DTT pretreatment, and after 5 min wash-off of DTT, with (+) or without (-) NSC23766 in the pipette solution. Group data of the effect of 1 mM DTT pretreatment for 1 min on normalized peak amplitude (B) and accumulated currents (C, area under curve, AUC) induced by extracellular [H⁺] without NSC23766, as compared to those with (+) NSC23766 in the pipette solution. (n=11 − 12). ^∗Significantly different, p<0.05.

Fig. 7

Inhibition of xanthine oxidase using allopurinol (Alio) potentiates extracellular [H⁺] gated currents. A. Representative traces of currents evoked by extracellular [H⁺] (pH 6.0) in VSMCs before Alio pretreatment (control, con), and after 5-6 min of 50 or 200 mM Alio pretreatment. Group data of the effect of 5 min Alio (50, 200 mM) pretreatment on normalized peak amplitude (B) and normalized accumulated currents (C, area under curve, AUC) induced by extracellular [H⁺] (n=5-6). ∗ Significantly different, p<0.05.