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.