The loop diuretics bumetanide and ethacrynic acid inhibit voltage‐gated calcium and sodium channels in cultured cortical neurons

Abstract

One of the consequences of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ have 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 a number of plasma membrane transport proteins, such as increased activity of the sodium‐potassium‐chloride cotransporter 1 (NKCC1). Experiments using the NKCC1 inhibitors, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects the Na+ and Ca2+ overload observed following in vitro ischemia‐acidosis. Fluorometric Ca2+ and Na+ measurements were performed on cultured cortical neurons from embryonic (E18) rats. Both BMN and EA depressed ischemia‐acidosis induced [Ca2+]i overload, but did not appreciably alter [Na+]i increases observed under these conditions. Patch‐clamp electrophysiology was used to measure whole‐cell membrane currents to identify target(s) of BMN and EA, other than the electroneutral NKCC‐1, responsible for the effects of these drugs on [Ca2+]i. Voltage‐gated Ca2+ channels (VGCC) were inhibited by both BMN and EA with half‐maximal inhibitory concentration (IC50) values of 4 μM and 36 μM respectively. However, maximal concentrations of BMN failed to block ~40% of VGCC mediated currents, while EA completely abolished these responses. Similarly, voltage‐gated Na+ channels (VGSC) were blocked by BMN and EA with IC50 values of 13 μM and 30 μM, respectively. In contrast to the VGCC responses, both loop diuretics inhibited VGSC currents by ~80%. Experiments using the VGSC inhibitor, tetrodotoxin, showed that BMN was preferentially inhibiting TTX‐sensitive currents in these cells. Neither BMN nor EA affected currents mediated by acid‐sensing ion channels or ionotropic glutamatergic receptors. Taken together, our data suggest that these two loop diuretics are likely to decrease injury following ischemic stroke by effectively mitigating [Ca2+]i overload in neurons via the inhibition of both voltage‐gated Ca2+ and Na+ channels. Moreover, the depression of ischemia‐acidosis evoked [Ca2+]i overload by these compounds does not appear to be dependent on inhibition of NKCC1. Given that the inhibitory concentrations observed here for VGCC and VGSC are similar to those reported for block of NKCC1 and NKCC2 by these compounds, loop diuretics are likely to be inhibiting these channels at therapeutic doses used in humans. Therefore, blockade of these channels by loop diuretics may also contribute to other clinical effects of the compounds observed in animal studies and in humans.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid, 17GRNT33661273 (JC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.