Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels.

Christopher Katnik,Javier Cuevas

doi:10.3389/fphar.2021.732922

Abstract

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Highlights

Preservation of neuronal [Ca2+]i and [Na+]i homeostasis is dependent on ATPases, ion exchangers and cotransporters, and disruption of these during ischemia has a major impact on cell survival
Our laboratory has shown that the concurrence of ischemia and acidosis, which occurs during stroke, results in a synergistic [Ca2+]i overload in neurons and concomitant cell death (Mari et al, 2010)
Na+-K+-Cl- cotransporter 1 (NKCC1) inhibitors, bumetanide and ethacrynic acid, on the [Ca2+]i burden produced by simultaneous ischemia and acidosis in neurons

Summary

Introduction

Preservation of neuronal [Ca2+]i and [Na+]i homeostasis is dependent on ATPases, ion exchangers and cotransporters, and disruption of these during ischemia has a major impact on cell survival. Our laboratory has shown that acidosis synergistically potentiates the intracellular Ca2+ dysregulation evoked by ischemia in cortical neurons, enhancing neuronal death (Mari et al, 2010). The acidsensing ion channel, ASIC1a, contributes to this potentiation, but these channels alone cannot account for the long-lived synergy (Mari et al, 2010), since ASIC1a rapidly inactivates and release of Ca2+ from intracellular stores was observed following ASIC1a activation (Herrera et al, 2008; Mari, Katnik, and Cuevas, 2010). It is of significant interest to identify other contributors to this synergistic potentiation of [Ca2+]i dysregulation during ischemia and acidosis since these may, in part, account for the expansion of the ischemic lesion following stroke

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