Sung Hun Na

The Ca2+ channel inhibitor efonidipine decreases voltage-dependent K+ channel activity in rabbit coronary arterial smooth muscle cells

The effect of efonidipine, a commercially available antihypertensive drug and Ca(2+) channel inhibitor, on voltage-dependent K(+) (Kv) channels was studied in freshly isolated rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. The amplitude of Kv current was decreased by application of efonidipine in a dose-dependent manner, with IC50 of 0.26μM and a Hill coefficient of 0.91, which suggests 1:1 binding stoichiometry. Efonidipine did not affect voltage-dependent activation of the Kv channel, but shifted the inactivation curve by -8.87mV. The inhibitory effect of efonidipine was not significantly changed by depletion of extracellular Ca(2+) or intracellular ATP, which indicated no involvement of the Ca(2+) channel or intracellular protein kinase-dependent cascades. We conclude that efonidipine dose-dependently inhibits Kv current in a phosphorylation- and Ca(2+) channel-independent manner.

The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K(+) (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 0.81μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77±0.04μM(-1)s(-1) and 2.55±1.50s(-1), respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition.

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K(+) channels in rabbit coronary arterial smooth muscle cells.

AIMS We examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K(+) (Kv) channels. MAIN METHODS Electrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells. KEY FINDINGS The Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a Kd value of 1.48μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83±0.01μM(-1)s(-1) and 2.59±0.14s(-1), respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002. SIGNIFICANCE Based on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.

Cilostazol induces vasodilation through the activation of Ca2 +-activated K+ channels in aortic smooth muscle

We investigated the vasorelaxant effect of cilostazol and related signaling pathways in phenylephrine (Phe)-induced pre-contracted aortic rings. Cilostazol induced vasorelaxation in a concentration-dependent manner when aortic rings were pre-contracted with Phe. Application of the voltage-dependent K(+) (Kv) channel inhibitor 4-AP, the ATP-sensitive K(+) (K(ATP)) channel inhibitor glibenclamide, and the inwardly rectifying K(+) (Kir) channel inhibitor Ba(2+) did not alter the vasorelaxant effect of cilostazol; however, pre- and post-treatment with the big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel inhibitor paxilline inhibited the vasorelaxant effect of cilostazol. This vasorelaxant effect of cilostazol was reduced in the presence of an adenylyl cyclase or a protein kinase A (PKA) inhibitor, but not a protein kinase G inhibitor. Inside-out single channel recordings revealed that cilostazol induced the activation of BK(Ca) channel activity. The vasorelaxant effect of cilostazol was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor and a small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel inhibitor did not affect cilostazol-induced vasorelaxation. We conclude that cilostazol induced vasorelaxation of the aorta through activation of BK(Ca) channel via a PKA-dependent signaling mechanism independent of endothelium.

W-7 inhibits voltage-dependent K(+) channels independent of calmodulin activity in rabbit coronary arterial smooth muscle cells.

We investigated the effect of W-7, a calmodulin inhibitor, on voltage-dependent K(+) (Kv) channels in freshly isolated coronary arterial smooth muscle cells using the whole-cell patch clamp technique. The amplitude of Kv currents was inhibited by W-7 in a concentration-dependent manner, with an IC50 value of 3.38±0.47μM and a Hill coefficient of 0.84±0.10. W-7 shifted the activation curve to a more positive potential but had no significant effect on the inactivation curve, which indicated that W-7 inhibited the Kv current in a closed state of the Kv channel. Another calmodulin inhibitor, W-13, had no significant effect on Kv currents and did not change the inhibitory effect of W-7 on Kv channels. From these results, we conclude that W-7 inhibited the Kv current in a dose-dependent manner, but this inhibition occurred independent of calmodulin activity and in a closed (inactivated) state of the Kv channels.

The Effects of the Selective Serotonin Reuptake Inhibitor Fluvoxamine on Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We demonstrated the inhibitory effect of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K(+) (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Fluvoxamine reduced the amplitude of Kv currents in a concentration-dependent manner with an IC50 value of 3.71±1.09 µM and a Hill coefficient of 0.62±0.14. Although fluvoxamine did not significantly affect the steady-state activation curve, it shifted the steady-state inactivation curve toward a more negative potential. Pretreatment with another SSRI, paroxetine, did not affect the basal Kv current and did not alter the inhibitory effect of fluvoxamine on Kv channels. We concluded that fluvoxamine inhibits the Kv current in a concentration-dependent manner and in a closed (inactivated) state of the Kv channels independent of serotonin reuptake inhibition.

The anti-diabetic drug dapagliflozin induces vasodilation via activation of PKG and Kv channels

Aim: Considering the clinical efficacy of dapagliflozin in patients with type 2 DM and the pathophysiological relevance of Kv channels for vascular reactivity. We investigate the vasodilatory effect of dapagliflozin and related mechanisms using phenylephrine (Phe)‐induced contracted aortic rings. Material and methods: Arterial tone measurement was performed in aortic smooth muscle. Key findings: Application of dapagliflozin induced vasodilation in a concentration‐dependent manner. Pre‐treatment with the BKCa channel inhibitor paxilline, the KATP channel inhibitor glibenclamide, and the Kir channel inhibitor Ba2+ did not change dapagliflozin‐induced vasodilation. However, application of the Kv channels inhibitor 4‐AP effectively inhibited dapagliflozin‐induced vasodilation. Application of the Ca2+ channel inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump inhibitor thapsigargin did not alter the vasodilatory effect of dapagliflozin. Moreover, the adenylyl cyclase inhibitor SQ 22536 and the protein kinase A (PKA) inhibitor KT 5720 had no effect on dapagliflozin‐induced vasodilation. Although guanylyl cyclase inhibitors, NS 2028 and ODQ, did not reduce the vasodilatory effect of dapagliflozin, the protein kinase G (PKG) inhibitor KT 5823 effectively inhibited dapagliflozin‐induced vasodilation. The vasodilatory effect of dapagliflozin was not affected by elimination of the endothelium. Furthermore, pretreatment with the nitric oxide synthase inhibitor L‐NAME or the small‐conductance Ca2+‐activated K (SKCa) channel inhibitor apamin did not change the vasodilatory effect of dapagliflozin. Significance: We concluded that dapagliflozin induced vasodilation via the activation of Kv channels and PKG, and was independent of other K+ channels, Ca2+ channels, intracellular Ca2+, and the endothelium.

The vasorelaxant effect of anti‐diabetic drug nateglinide via activation of voltage‐dependent K+ channels in aortic smooth muscle

AIMS We investigated the vasorelaxant effect of nateglinide and its related mechanisms using phenylephrine (Phe)-induced precontracted aortic rings. METHODS Arterial tone measurement was performed in aortic smooth muscle. RESULTS The application of nateglinide induced vasorelaxation in a concentration-dependent manner. Pretreatment with the large-conductance Ca2+ -activated K+ (BKCa ) channel inhibitor paxilline, the inwardly rectifying K+ (Kir) channel inhibitor Ba2+ , and ATP-sensitive K+ (KATP ) channel inhibitor glibenclamide did not affect the vasorelaxant effect of nateglinide. However, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) effectively reduced the vasorelaxant effect of nateglinide. Pretreatment with the Ca2+ inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin did not change the vasorelaxant effect of nateglinide. Additionally, the vasorelaxant effect of nateglinide was not altered in the presence of an adenylyl cyclase, a protein kinase A, a guanylyl cyclase, or a protein kinase G inhibitor. The vasorelaxant effect of nateglinide was not affected by the elimination of the endothelium. In addition, pretreatment with a nitric oxide synthase inhibitor, L-NAME, and a small-conductance Ca2+ -activated K+ (SKCa ) channel inhibitor, apamin, did not change the vasorelaxant effect of nateglinide. CONCLUSION Nateglinide induced vasorelaxation via the activation of the Kv channel independent of other K+ channels, Ca2+ channels, intracellular Ca2+ ([Ca2+ ]i ), and the endothelium.

The Effects of Heme Oxygenase By-Products on the Proliferation and Invasion of HUVECs, HTR-8/SVneo Cells, 3A(tPA 30-1) Cells, and HESCs Under Varying Oxygen Concentrations

Objective: Abnormal spiral artery remodeling during early pregnancy leads to preeclampsia. The proliferation and invasion of trophoblasts in pregnancy are important for spiral artery remodeling. This study examined whether heme oxygenase (HO) by-products (carbon monoxide biliverdin, and iron) play roles in regulating the restoration of proliferation and invasion of human umbilical vein endothelial cells (HUVECs), HTR-8/SV-neo cells originating from first-trimester trophoblasts, 3A(tPA 30-1) obtained from term trophoblasts, and human endometrial stromal cells (HESCs) inhibited by zinc protoporphyrin IX (Znpp-9). Study Design: We explored whether HO by-products restored the proliferation and invasion of HUVECs, HTR-8/SVneo cells, 3A(tPA 30-1) cells, and HESCs inhibited by Znpp-9 depending on the oxygen concentration. Results: Bilirubin promoted proliferation of HUVECs, HTR-8/SVneo cells, 3A(tPA-30-1) cells, and HESCs under both hypoxic and normoxic conditions. Biliverdin also promoted invasion of HUVECs, HTR-8/SVneo cells, 3A(tPA30-1) cells, and HESCs under both hypoxic and normoxic conditions. Carbon monoxide-releasing molecule 2 promoted the proliferation and invasion of specific cell types depending on the oxygen concentration. Conclusion: Our data suggest that HO by-products differentially stimulate the proliferation and invasion of cells involved in pregnancy maintenance. When HO by-products are considered to be stimulants during the invasion and proliferation of such cells, both target cells and the gestational period should be considered.