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In the present study, we investigated the inhibitory effect of tacrolimus, a macrolide immunosuppressive drug that acts through calcineurin inhibition, on voltage-gated K+ (Kv) channels expressed in native smooth muscle cells isolated from the coronary arteries of rabbits. Tacrolimus reduced the amplitude of Kv currents in a dose-dependent manner with an IC50 value and Hill coefficient of 7.80 ± 3.01 μM and 1.07 ± 0.25, respectively. Tacrolimus caused a shift in the activation curve toward a more positive potential and in the inactivation curve toward a more negative potential. Tacrolimus-induced inhibition of Kv current was increased by the application of train pulses (1 or 2 Hz). Furthermore, the recovery time constant of inactivation was extended in the presence of tacrolimus, suggesting that tacrolimus inhibited Kv channels in a use-dependent manner. Two kinds of Kv subtype inhibitors, DPO-1 and guangxitoxin did not affect the degree of tacrolimus-induced inhibition of Kv current. Furthermore, pretreatment with another calcineurin inhibitor, cyclosporine A, did not affect the Kv current, and did not alter the inhibitory effect of tacrolimus. Using perforated-patch clamp experiments, inhibition of Kv channels by tacrolimus caused membrane depolarization. From these results, we concluded that tacrolimus inhibited the vascular Kv currents in a dose, state (open and closed)-dependent manner.

We examined the effects of the PPARα activator fenofibrate on voltage-dependent K+ (Kv) channels using a patch clamp technique in native rabbit coronary arterial smooth muscle cells. Kv current was inhibited by application of fenofibrate in a concentration-dependent manner, with an apparent IC50 value of 6.39 ± 0.53μM and a slope value (Hill coefficient) of 1.63 ± 0.10. Fenofibrate accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for fenofibrate were 0.81± 0.05μM-1s-1 and 4.70 ± 0.47s-1, respectively. Although fenofibrate did not affect the steady-state activation curves, fenofibrate shifted the inactivation curves toward a more negative potential. Application of train pulses (1 or 2Hz) progressively increased the fenofibrate-induced inhibition of the Kv channel, and the recovery time constant from inactivation was increased in the presence of fenofibrate, which suggested that the inhibitory effect of fenofibrate is use-dependent. Another PPARα activator, bezafibrate and PPARα inhibitor, GW 6471, did not affect the Kv current and also did not change the inhibitory effect of fenofibrate on the Kv current. From these results, we suggest that fenofibrate inhibited Kv current in a state-, time-, and use-dependent manner, completely independent of PPARα activation.

We evaluated the inhibitory effects of the atypical antipsychotic drug risperidone on voltage-dependent K

We evaluated the inhibitory effects of the atypical antipsychotic drug risperidone on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Risperidone suppressed Kv currents in reversible and concentration-dependent manners with an apparent half-maximal effective concentration (IC50 value) of 5.54 ± 0.66 μM and a slope factor of 1.22 ± 0.07. The inactivation of Kv currents was significantly accelerated by risperidone. The rate constants of association and dissociation for risperidone were 0.25 ± 0.01 μM−1s−1 and 1.36 ± 0.14 s−1, respectively. Application of risperidone shifted the steady-state activation curve in the positive direction and the inactivation curve in the negative direction, suggesting that the risperidone-induced inhibition of Kv channels was mediated by effects on the voltage sensors of the channels. Application of train pulses at 1 and 2 Hz led to a progressive increase in the blockage of Kv currents by risperidone. In addition, the recovery time constants from inactivation were extended in the presence of risperidone, indicating that risperidone inhibited Kv channels in a use (state)-dependent manner. Pretreatment with the Kv1.5 subtype inhibitor reduced the inhibitory effects of risperidone on Kv channels. However, pretreatment with a Kv2.1 or Kv7.X subtype inhibitor did not affect the inhibitory effects of risperidone. Risperidone induced vasoconstriction and membrane depolarization. Based on these results, we conclude that risperidone inhibits Kv channels in a concentration-, time-, and state-dependent manners. Our results should be taken into consideration when using risperidone to study the kinetics of K+ channels in vascular smooth muscle.

In the present study, we investigated the inhibitory effect of tacrolimus, a macrolide immunosuppressive drug that acts through calcineurin inhibition, on voltage-gated K

We examined the effects of the PPARα activator fenofibrate on voltage-dependent K

article i nfo Taurine, a non essential sulfur-containing amino acid, plays a critical role in cardiovascular functions. We here examined the effect of taurine on angiogenesis and its underlying signal pathway. Taurine treatment in- creased angiogenesis in vitro and in vivo, which was followed by activation of the phosphatidylinositol 3- kinase (PI3K)/Akt, MEK/ERK, and Src/FAK signaling pathways. Further, taurine promoted endothelial cell cycle progression to the S and G2/M phases by up-regulating the positive cell cycle proteins, particularly cyclins D1 and B, as well as down-regulating the negative cell cycle proteins, p53 and p21 WAF1/CIP1 , resulting in Rb phosphorylation. This angiogenic event was inhibited by inhibitors of PI3K and MEK. In addition, a PI3K inhibitor blocked the activation of Akt and ERK, while Akt knockdown did not affect taurine-induced ERK ac- tivation, indicating that PI3K is an upstream mediator of both MEK and Akt. Taurine-induced endothelial cell migration was suppressed by Src inhibitor, but not by other inhibitors, suggesting that the increase in cell mi- gration is regulated by Src-dependent pathway. Moreover, inhibition of cellular taurine uptake by β-alanine and taurine transporter knockdown promoted taurine-induced cell proliferation, ERK and Akt activation, and in vivo angiogenesis, suggesting that extracellular taurine induces angiogenesis. However, taurine did not in- duce vascular inflammation and permeability in vitro and in vivo. These data demonstrate that extracellular taurine promotes angiogenesis by Akt- and ERK-dependent cell cycle progression and Src/FAK-mediated cell migration without inducing vascular inflammation, indicating that it is potential use for the treatment of vascular dysfunction-associated human diseases.

We here investigated the functional effect of 4-O-methylgallic acid (4-OMGA), a major metabolite of gallic acid abundant in red wine, on vascular inflammation and its action mechanism. 4-OMGA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to HUVECs. In addition, 4-OMGA inhibited the promoter activities of ICAM-1 and VCAM-1 and the activity of nuclear factor-kappaB (NF-kappaB) without affecting cytosolic IkappaB kinase (IKK) activation, inhibitor of kappaB (IkappaB) phosphorylation and degradation, and nuclear translocation of NF-kappaB. This compound did not alter nitric oxide (NO) generation, but inhibited reactive oxygen species (ROS) production in TNF-alpha-stimulated HUVECs, suggesting that NO and ROS are not involved in 4-OMGA-mediated inhibition of NF-kappaB activity. Moreover, 4-OMGA directly blocked the binding activity of NF-kappaB to its consensus DNA oligonucleotide, when pre-incubated with the nuclear extract from TNF-alpha-stimulated HUVECs, but not with the oligonucleotide alone. This inhibition was completely abolished by the addition of dithiothreitol. 4-OMGA exhibits an anti-inflammatory property by interfering with the formation of the NF-kappaB-DNA complex in the nuclei through direct and redox-sensitive interactions and may play an important role in the prevention of inflammatory responses such as the atherosclerotic process.

We investigated the effect of desmethylanhydroicaritin (DMAI), a major compound of the Chinese herbal medicine Epimedium, on inflammatory gene expression and the NF-kappaB signaling pathway. We found that DMAI suppressed the expression of NF-kappaB-responsive genes, such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-1beta, and tumor necrosis factor-alpha, in lipopolysaccharide (LPS)-stimulated macrophages and endotoxemic mice as well as protected mice against LPS-induced lethality. DMAI inhibited NF-kappaB activation through the inhibition of IkappaB kinase (IKK) activation, IkappaB phosphorylation and degradation, and NF-kappaB nuclear translocation in LPS-stimulated macrophages. This compound inhibited in vitro and in vivo LPS-induced phosphatidylinositol 3-kinase (PI3K) activation, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) oxidation, and Akt phosphorylation, which are upstream modulators of IKK activation. Moreover, treatment with DMAI was not observed to affect the interaction between the Toll-like receptor 4, MyD88, and TRAF6 as well as mitogen-activated protein kinase activation. DMAI also suppressed intracellular H(2)O(2) accumulation, hydroxyl radical production, and glutathione oxidation without affecting superoxide generation and accumulation by NADPH oxidase. Moreover, DMAI inhibited redox-sensitive activation of the PI3K/PTEN/Akt pathway and NF-kappaB activation in macrophages treated with H(2)O(2). These results indicate that DMAI negatively regulates canonical NF-kappaB-regulated inflammatory gene expression by functioning as an inhibitor of the NF-kappaB pathway through the suppression of redox-based PI3K activation and PTEN inactivation and therefore can be considered as a potential drug for inflammatory diseases.