Inhibition of ion channels and heart beat in Drosophila by selective COX-2 inhibitor SC-791

Abstract

Recent findings suggest that modulation of ion channels might be implicated in some of the clinical effects of coxibs, selective inhibitors of cyclooxygenase-2 (COX-2). Celecoxib and its inactive analog 2,5-dimethyl-celecoxib, but not rofecoxib, can suppress or augment ionic currents and alter functioning of neurons and myocytes. To better understand these unexpected effects, we have recently investigated the mechanism of inhibition of human K(v)2.1 channels by a highly selective COX-2 inhibitor SC-791. In this study we have further explored the SC-791 action on ion channels and heartbeat in Drosophila, which lacks cyclooxygenases and thus can serve as a convenient model to study COX-2-independent mechanisms of coxibs. Using intracellular recordings in combination with a pharmacological approach and utilizing available Drosophila mutants, we found that SC-791 inhibited voltage-activated K(+) and L-type Ca(2+) channels in larval body-wall muscles and reduced heart rate in a concentration-dependent manner. Unlike celecoxib and several other K(+) channel blockers, SC-791 did not induce arrhythmia. Instead, application of SC-791 resulted in a dramatic slowing of contractions and, at higher concentrations, in progressively weaker contractions with gradual cessation of heartbeat. Isradipine, a selective blocker of L-type Ca(2+) channels, showed a similar pattern of heart arrest, though no prolongation of contractions was observed. Ryanodine was the only channel modulating compound of those tested additionally that was capable of slowing contractions. Like SC-791, ryanodine reduced heart rate without arrhythmia. However, it could not stop heartbeat completely even at 500 µM, the highest concentration used. The magnitude of heart rate reduction, when SC-791 and ryanodine were applied together, was smaller than expected for independent mechanisms, raising the possibility that SC-791 might be interfering with excitation-contraction coupling in Drosophila heart.

Figure 1. Inhibition of L-type Ca²⁺ channels by SC-791.

L-type currents in larval body-wall muscles in control (A) and in the presence of 30 μM SC-791 (B) were elicited by 500 ms voltage pulses between −60 and +40 mV in 10 mV increments from a holding potential (HP) of −40 mV; averaged traces are shown. (C) SC-791 slowed activation of L-type Ca²⁺ channels. Activation time constants were determined by fitting the rising part of the current with a single exponential function; (n), number of experiments. (D) Dose-response relation for inhibition of the peak L-type current by SC-791 at −10 mV; the curves in this and following dose-response relations represent fits to Hill equation.

Figure 2. Inhibition of K⁺ currents by SC-791.

(A) Schematic representation of three voltage-activated K⁺ currents. The average I_KS in control (B) and in the presence of 30 μM SC-791 (C) in the wild-type larvae; currents were evoked by 500 ms voltage pulses between −40 and +40 mV from a HP of −80 mV in 10 mV increments in the presence of 200 μM CdCl₂ in bath solution to block the I_KF; the I_A can be seen in panels B, C, E, F in the beginning of each trace; it was not removed as it did not obscure the effects of SC-791 on other currents. (D) Dose-response relations for inhibition of the I_KS by SC-791 at 50 ms after the onset (open circles) and at the end of a voltage pulse to +40 mV (closed circles); the number of experiments varied from 5 to 12. The average I_KF in control (E) and in the presence of 100 μM SC-791(F) in Shab mutant larvae; the currents were elicited by 500 ms voltage pulses between −40 and +40 mV from a HP of −80 mV in 10 mV increments; the number of experiments for each condition was 8. (G) SC-791 did not affect the I_A; the currents were recorded from Shab larvae as in the panels (B–C) in the presence of 200 μM CdCl₂ to block the I_KF; the number of experiments for each condition was 9.

Figure 3. Effects of SC-791 on heartbeat.

Traces represent the relative displacement of an edge of the larval heart in the experiments with SC-791 (A) or celecoxib (C); heartbeat was video recorded immediately before drug application, at 18–20 min after wash-in of 30 µM SC-791 or at 8–10 min after application of 10 μM celecoxib, and at 8–10 min after washing the drug out; (B) depicts heartbeat irregularity in the presence of 30 μM SC-791; the coefficient of variation (CV) was calculated as described previously ; the number of experiments was 8. (D) Average heart rate is shown for 6–10 larvae before drug application, after rapid wash-in of 3 μM, 10 μM, 30 μM, or 100 μM SC-791 (horizontal bar), and after the wash-out; treatment with 0.5% Me₂SO, the maximum concentration of Me₂SO used with SC-791, did not change the heart rate significantly. (E) A dose-response curve for the effect of SC-791 on heart rate is shown.

Figure 4. Heartbeat disruption by SC-791 and isradipine.

Development of heartbeat stoppage in the presence of 50 μM SC-791 (A) or 3 μM isradipine (B). The corresponding averaged contraction waveforms demonstrate similarities (decrease in amplitude) and differences (prolongation of contraction) between the effects of SC-791 (C) and isradipine (D); each waveform is an average of 10 to 20 individual contraction waveforms.

Figure 5. Effects of ryanodine on heartbeat.

Typical examples of heartbeat in control, after 20 min application of either 100 µM ryanodine (A) or 30 µM SC-791 (B) alone followed by 20 min exposure to a solution containing both 100 µM ryanodine and 30 µM SC-791, and after the wash-out. (C, D) Time course of the average heart rate in the experiments described in the panels (A) and (B), respectively; dotted lines indicate the value of BIC (82% heart rate inhibition) (see Results); panels (C) and (D), averages of 7 and 6 experiments, respectively. (E) Bar plot illustrates differences between the BIC of 0.82 and the experimentally determined total fractional reduction in heart rate for three experimental conditions: when SC-791/ryanodine was applied after (1) exposure to ryanodine alone, (2) exposure to SC-791 alone, and (3) without pre-exposure of the preparation to any compound. (F) Bar plot summarizes effects of ryanodine and SC-791 on the duration of heart contraction when the chemicals were applied in different order as in the panels (A) and (B).