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

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.

Keywords: acidosis; bumetanide; calcium; ethacrynic acid; ischemia; neurons; sodium; voltage-gated channels.

FIGURE 1

Ischemia-acidosis evoked [Ca²⁺]_i increases in cultured rat cortical neurons are inhibited by bumetanide and ethacrynic acid. A, Representative traces of [Ca²⁺]_i as a function of time recorded from a single neuron in response to an initial 2-min ischemia-acidosis insult (Isc. pH 6; Control 1, black trace) and a second application of the Isc. pH 6 solution following a 20-min recovery period (Control 2, gray trace). Representative traces of [Ca²⁺]_i recorded from two neurons using the same protocol as in (A), in the absence and presence of 100 μM bumetanide [(B); Control, black trace; 100 μM BMN, blue trace] or 100 μM ethacrynic acid [(C); Control, black trace, 100 μM EA, red trace]. (D), Relative mean [Ca²⁺]_i (±SEM) responses obtained using the paired protocol with the second response, in the absence (Control, n = 59) and presence of 100 μM bumetanide (BMN, n = 156) or 100 μM Ethacrynic Acid (EA, n = 140), normalized to the first control response. Peak [Ca²⁺]_i increase was measured immediately after application of the ischemia-acidosis solution. Sustained [Ca²⁺]_i was measured just prior to washout of the Isc. pH 6 solution. Rebound [Ca²⁺]_i was the maximum [Ca²⁺]_i measured following washout of Isc. pH 6 solution. The percent recovery (%Rec) was calculated as the final [Ca²⁺]_i normalized to the maximum [Ca²⁺]_i after the initial transient peak ([final-baseline]/[rebound peak-baseline]). Total [Ca²⁺]_i increases were calculated by integrating beneath the traces of [Ca²⁺]_i vs. time. (E), Same data in (D) with ratios normalized to Control/Control ratios. Asterisks denote significant difference from Control (p < 0.05) and pound symbols indicate significant difference between BMN and EA (p < 0.05). Bars above (A), (B) and (C) indicate duration of ischemia-acidosis (Isc. pH 6.0).

FIGURE 2

Increases in [Na⁺]_i, depicted by R_SBFI, evoked by concurrent ischemia and acidosis are not inhibited by NKCC1 inhibitors. (A), Representative traces of R_SBFI as a function of time recorded from a single neuron during two, 2-min ischemia-acidosis insults with a 20 min recovery period in between. (B), Representative traces of R_SBFI as a function of time recorded from a single neuron in response to two, 2-min ischemia-acidosis applications, first in the absence (Control, black trace) and 20 min later in the presence of 100 μM bumetanide (100 μM BMN, blue trace). (C), Representative traces of R_SBFI as a function of time recorded from a single neuron in response to two, 2-min ischemia-acidosis applications, first in the absence (Control, black trace) and 20 min later in the presence of 100 μM ethacrynic acid (100 μM EA, red trace). (D), Relative mean R_SBFI (±SEM) measured in the absence (Control) and presence of 100 μM bumetanide (100 μM BMN, n = 108) or 100 μM ethacrynic acid (100 μM EA, n = 49). Responses were normalized to control responses in the same cells. Baseline and Initial Peak represent R_SBFI values measured immediately prior to and 15 s after initiation of ischemia-acidosis. Max Peak represents highest R_SBFI measured during ischemia + acidosis. Recovery was calculated as (final-baseline)/(maximum-baseline). (E), Same data presented in (D) but normalized to the Response2/Response1 ratio for Control. Asterisks denote significant difference from Control (p < 0.05) and pound symbol indicates significant difference between BMN and EA (p < 0.05). Bars above (A) and (B) indicate duration of ischemia + acidosis (Isc. pH 6.0).

FIGURE 3

Peak inward ASIC-mediated currents are not inhibited by bumetanide or ethacrynic acid. (A), Representative traces of inward currents activated by two 10 s applications of acidic solution (pH 6.0) onto a single cell voltage clamped at −70 mV. (B), Representative traces of inward currents activated by 10 s applications of PSS at pH 6.0 onto a single cell voltage-clamped at −70 mV in the absence (Control, black trace) and presence of 100 μM bumetanide (100 μM BMN, blue trace). (C), Representative traces of inward currents activated by 10 s applications of PSS at pH 6.0 onto a single cell voltage-clamped at −70 mV in the absence (Control, black trace) and presence of 100 μM ethacrynic acid (100 μM EA, red trace). (D), Mean peak inward currents, rates of current decay (τ) and net current (area under current trace) (±SEM) measured in the absence (Control, n = 16) and presence of 100 μM bumetanide (BMN, n = 6) and 100 μM ethacrynic acid (EA, n = 10) normalized to initial control responses (absence of drugs). (E), Data in (D) normalized to Response2/Response1 ratio for Control. Asterisks denote significant difference from Control (p < 0.05) and pound symbols indicate significant difference between BMN and EA (p < 0.05). Bars above (A), (B), and (C) indicate duration of low pH application (pH 6.0). In all experiments, second low pH solution application was carried out after a 5 min washout of the initial acid stimulation.

FIGURE 4

Glutamate activated inward currents are not inhibited by bumetanide or ethacrynic acid. (A), Representative traces of whole-cell currents recorded from a single neuron voltage clamped at −70 mV in response to two 10 s applications of 20 μM glutamate. (B), Representative traces of whole-cell currents recorded from a single neuron voltage clamped at −70 mV in response to a 10 s application of 20 μM glutamate in the absence (Control, black trace) and presence of 100 μM bumetanide (100 μM BMN, blue trace). (C), Representative traces of whole cell currents recorded from a single neuron voltage clamped at −70 mV in response to a 10 s application of 20 μM glutamate in the absence (Control, black trace) and presence of 100 μM ethacrynic acid (100 μM EA, red trace). (D), Bar graph of compiled data from experiments identical to those in A (n = 5), B (n = 4) and C (n = 4). Data are shown as the ratio of second responses to first responses in the absence (Control) and presence of drug (BMN or EA). Data shown are mean response ratios (±SEM) of holding current, initial peak and steady state (measured at the end of the 10 s application) inward currents, and charge influx during glutamate application (Glut Dependent) and throughout the recording (Total). No significant differences were noted for any of the parameters measured (p > 0.12 for all). Bars above traces in A-C denote times of glutamate application.

FIGURE 5

Bumetanide and ethacrynic acid inhibit voltage-gated calcium channels in a dose-dependent manner. (A), Representative traces of whole-cell VGCC currents recorded from a single neuron in response to a 500 msec step from a holding potential of −70 mV to −10 mV in the absence (Control, black trace) and presence of 10 μM bumetanide (10 μM BMN, blue trace). (B), Representative traces of whole-cell VGCC currents recorded from a single neuron in response to a 500 msec step from a holding potential of -70 mV to −10 mV in the absence (Control, black trace) and presence of 56 μM ethacrynic acid (56 μM EA, red trace). (C), Concentration-response relationships of relative peak inward VGCC currents (mean ± SEM) activated by voltage steps from −70 mV to −10 mV. Currents were normalized to control peak currents measured in the absence of bumetanide or ethacrynic acid. Data points (bumetanide blue circles; ethacrynic acid red circles) were best fit (bumetanide blue line, ethacrynic acid red line) using a single-site Langmuir-Hill equation with an asymptotic minimum (dashed line for BMN). n > 7 for each data point. Asterisks denote significant difference from Control (p < 0.05).

FIGURE 6

Bumetanide and ethacrynic acid inhibit voltage-gated sodium channels in a dose-dependent manner. (A), Representative traces of whole-cell VGSC currents recorded from a single neuron in response to 50 msec steps from a holding potential of −70 mV to −30 mV in the absence (Control, black trace) and presence of 30 μM Bumetanide (30 μM BMN, blue trace). (B), Representative traces of whole-cell VGSC currents recorded from a single neuron in response to 50 msec steps from a holding potential of −70 mV to −30 mV in the absence (Control, black trace) and presence of 100 μM ethacrynic acid (100 μM EA, red trace). (C), Concentration-response relationship of relative peak inward VGSC currents (mean ± SEM) elicited by voltage steps from −70 to −30 mV. Currents were normalized to control peak currents measured in the absence of bumetanide or ethacrynic acid. Data points (bumetanide blue circles; ethacrynic acid red circles) were best fit (bumetanide blue line, ethacrynic acid red line) to a single-site Langmuir-Hill equation with an asymptotic minimum (dashed line). n > 15 for all bumetanide data points and n > 6 for all ethacrynic acid data points. Asterisks and pound symbols denote significant difference from Control for BMN and EA, respectively (p < 0.05). Neurons for these experiments were electrically accessed using the conventional (dialyzing) whole-cell patch clamp recording configuration.

FIGURE 7

Sensitivity to inhibition by bumetanide distinguishes two populations of voltage-gated sodium channels in cultured neurons. (A), Normalized peak inward VGSC currents (mean ± SEM) as a function of bumetanide concentration recorded from neurons with VGSC resistant to bumetanide blockade (n > 7). Currents were activated by voltage steps from a holding potential of −70 mV to −30 mV and normalized to control peak currents measured in the absence of bumetanide at the indicated concentrations. Data points were best fit (solid line) to a linear equation with a slope of −0.2 nM⁻¹. Dotted line is the single-site Langmuir-Hill equation fit to the data from the bumetanide-sensitive neurons shown in Figure 6. (B), Representative traces of whole-cell currents evoked by 50 msec voltage steps from a holding potential of −70 mV to −30 mV recorded from two different neurons, one with primarily VGSC currents resistant to bumetanide (i, Resistant) and one with VGSC sensitive to inhibition by bumetanide (ii, Sensitive). Currents were elicited from the neurons in the absence of drug (Control, black traces), and in the presence of 1 mM Bumetanide (BMN, blue traces), 100 nM TTX (TTX, green traces) and 1 mM Bumetanide +100 nM TTX (BMN + TTX, red traces). (C), Peak inward VGSC currents (mean ± SEM) measured from 4 bumetanide-resistant (Resistant, left panel) and 6 bumetanide-sensitive (Sensitive, right panel) neurons using the same protocol as (B). A two-way ANOVA followed by a post-hoc Tukey Test indicates no significant interaction between BMN and TTX in the Resistant group (p = 0.336), but a significant interaction between TTX and BMN in the Sensitive group (p < 0.02). A one-way ANOVA followed by a post-hoc Tukey Test on the Resistant group indicates TTX and BMN + TTX are both significantly different from Control (p < 0.001) and BMN (p < 0.05 and 0.001, respectively) but not each other and BMN was not significantly different from Control (p = 0.079). (D), Percent block of normalized peak VGSC currents (Percent block = (1-I/I_Control)*100) calculated from the currents measured in (B) for cells Resistant (left panel) or Sensitive (right panel) to BMN. A one-way ANOVA indicates BMN produces a statistically significant lower block than TTX or TTX + BMN in resistant cells (p < 0.001). No significant differences were noted for block in the BMN-sensitive cells (p = 0.10). Asterisks indicate significant difference from Control, pound sign indicates significant difference from BMN determined by Two-Way ANOVA (*^,# p < 0.05: **^,## p < 0.001).