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

Frolov RV, Singh S - PLoS ONE (2012)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York, United States of America. rvfrolov@gmail.com

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.

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Inhibition of K+ currents by SC-791.(A) Schematic representation of three voltage-activated K+ currents. The average IKS 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 IKF; the IA 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 IKS 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 IKF 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 IA; the currents were recorded from Shab larvae as in the panels (B–C) in the presence of 200 μM CdCl2 to block the IKF; the number of experiments for each condition was 9.
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pone-0038759-g002: Inhibition of K+ currents by SC-791.(A) Schematic representation of three voltage-activated K+ currents. The average IKS 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 IKF; the IA 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 IKS 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 IKF 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 IA; the currents were recorded from Shab larvae as in the panels (B–C) in the presence of 200 μM CdCl2 to block the IKF; the number of experiments for each condition was 9.

Mentions: Several ionic currents have been previously identified in Drosophila larval body-wall muscles: T-type and L-type Ca2+ currents [19], [20], [21], two calcium-activated K+ currents, and three voltage-activated K+ currents [22], [23]. The latter include Shaker channels, conducting fast activating and inactivating IA; Shab channels, mediating relatively slow delayed rectifier IKS; and rapidly activating IKF with unknown molecular basis [24]. In this study we have assessed effects of SC-791 on four major currents: L-type current and three K+ currents, IA, IKS, and IKF (Fig. 1A, 2A). These currents can be separated by electrophysiological, pharmacological and genetic means. The L-type current can be isolated by recording in the presence of K+ channel blockers (TEA, 4-AP and quinidine) from a holding potential (HP) of −40 mV (to inactivate T-type Ca2+ channels) with Ba2+ ions as charge carrier (to prevent muscle contractions and to additionally block K+ channels) [20], [24]. The IKF can be recorded from the Shab strain in the presence of 4-AP to block the IA; the IKF can be completely abolished by 200 μM Cd2+ in the external solution [8], allowing recording of IKS from the wild-type larvae. The IA can be recorded from wild-type animals with Cd2+ (to inhibit IKF) and 100 μM quinidine (to selectively inhibit the IKS) in the bath or from the Shab in the presence of Cd2+ only.


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

Frolov RV, Singh S - PLoS ONE (2012)

Inhibition of K+ currents by SC-791.(A) Schematic representation of three voltage-activated K+ currents. The average IKS 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 IKF; the IA 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 IKS 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 IKF 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 IA; the currents were recorded from Shab larvae as in the panels (B–C) in the presence of 200 μM CdCl2 to block the IKF; the number of experiments for each condition was 9.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368905&req=5

pone-0038759-g002: Inhibition of K+ currents by SC-791.(A) Schematic representation of three voltage-activated K+ currents. The average IKS 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 IKF; the IA 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 IKS 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 IKF 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 IA; the currents were recorded from Shab larvae as in the panels (B–C) in the presence of 200 μM CdCl2 to block the IKF; the number of experiments for each condition was 9.
Mentions: Several ionic currents have been previously identified in Drosophila larval body-wall muscles: T-type and L-type Ca2+ currents [19], [20], [21], two calcium-activated K+ currents, and three voltage-activated K+ currents [22], [23]. The latter include Shaker channels, conducting fast activating and inactivating IA; Shab channels, mediating relatively slow delayed rectifier IKS; and rapidly activating IKF with unknown molecular basis [24]. In this study we have assessed effects of SC-791 on four major currents: L-type current and three K+ currents, IA, IKS, and IKF (Fig. 1A, 2A). These currents can be separated by electrophysiological, pharmacological and genetic means. The L-type current can be isolated by recording in the presence of K+ channel blockers (TEA, 4-AP and quinidine) from a holding potential (HP) of −40 mV (to inactivate T-type Ca2+ channels) with Ba2+ ions as charge carrier (to prevent muscle contractions and to additionally block K+ channels) [20], [24]. The IKF can be recorded from the Shab strain in the presence of 4-AP to block the IA; the IKF can be completely abolished by 200 μM Cd2+ in the external solution [8], allowing recording of IKS from the wild-type larvae. The IA can be recorded from wild-type animals with Cd2+ (to inhibit IKF) and 100 μM quinidine (to selectively inhibit the IKS) in the bath or from the Shab in the presence of Cd2+ only.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York, United States of America. rvfrolov@gmail.com

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.

Show MeSH
Related in: MedlinePlus