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Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

ABSTRACT: An important contributor to brain ischemia is known to be extracellular acidosis, which activates acid-sensing ion channels (ASICs), a family of proton-gated sodium channels. Lines of evidence suggest that targeting ASICs may lead to novel therapeutic strategies for stroke. Investigations of the role of ASICs in ischemic brain injury have naturally focused on the role of extracellular pH in ASIC activation. By contrast, intracellular pH (pHi) has received little attention. This is a significant gap in our understanding because the ASIC response to extracellular pH is modulated by pHi, and activation of ASICs by extracellular protons is paradoxically enhanced by intracellular alkalosis. Our previous studies show that acidosis-induced cell injury in in vitro models is attenuated by intracellular acidification. However, whether pHi affects ischemic brain injury in vivo is completely unknown. Furthermore, whereas ASICs in native neurons are composed of different subunits characterized by distinct electrophysiological/pharmacological properties, the subunit-dependent modulation of ASIC activity by pHi has not been investigated. Using a combination of in vitro and in vivo ischemic brain injury models, electrophysiological, biochemical, and molecular biological approaches, we show that the intracellular alkalizing agent quinine potentiates, whereas the intracellular acidifying agent propionate inhibits, oxygen-glucose deprivation-induced cell injury in vitro and brain ischemia-induced infarct volume in vivo Moreover, we find that the potentiation of ASICs by quinine depends on the presence of the ASIC1a, ASIC2a subunits, but not ASIC1b, ASIC3 subunits. Furthermore, we have determined the amino acids in ASIC1a that are involved in the modulation of ASICs by pHi.

SUBMITTER: Li MH

PROVIDER: S-EPMC5000083 | biostudies-other | 2016 Aug

REPOSITORIES: biostudies-other

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Project description:Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of β-estradiol. However, the detailed mechanisms underlying the neuroprotection by β-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of β-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with β-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca2+ were all attenuated by β-estradiol treatment. In addition, we showed that β-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- β-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.

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Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

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