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Tegaserod, a gastroprokinetic agent, is used to treat irritable bowel syndrome. Despite its extensive clinical use, little is known about the effects of tegaserod on vascular ion channels, especially K+ channels. Therefore, we examined the effects of tegaserod on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Tegaserod inhibited Kv channels in a concentration-dependent manner with an IC50 value of 1.26 ± 0.31 µmol/L and Hill coefficient of 0.81 ± 0.10. Although tegaserod had no effect on the steady-state activation curves of the Kv channels, the steady-state inactivation curve was shifted toward a more negative potential. These results suggest that tegaserod inhibits Kv channels by influencing their voltage sensors. The recovery time constant of channel inactivation was extended in the presence of tegaserod. Furthermore, application of train steps (1 and 2 Hz) in the presence of tegaserod progressively increased the inhibition of Kv currents suggesting that tegaserod-induced Kv channel inhibition is use (state)-dependent. Pretreatment with a Kv1.5 subtype inhibitor suppressed the Kv current. However, additional application of tegaserod did not induce further inhibition. Pretreatment with a Kv2.1 or Kv7 inhibitor did not affect the inhibitory effect of tegaserod on Kv channels. Based on these results, we conclude that tegaserod inhibits vascular Kv channels in a concentration- and use (state)-dependent manner independent of its own functions. Furthermore, the major Kv channel target of tegaserod is the Kv1.5 subtype.

Iloperidone, a second-generation atypical antipsychotic drug, is widely used in the treatment of schizophrenia. However, the side-effects of iloperidone on vascular K+ channels remain to be determined. Therefore, we explored the effect of iloperidone on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Iloperidone inhibited vascular Kv channels in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 2.11 ± 0.5 μM and a Hill coefficient of 0.68 ± 0.03. Iloperidone had no effect on the steady-state inactivation kinetics. However, it shifted the steady-state activation curve to the right, indicating that iloperidone inhibited Kv channels by influencing the voltage sensors. Application of 20 repetitive depolarizing pulses (1 and 2 Hz) progressively increased the inhibition of the Kv current in the presence of iloperidone. Furthermore, iloperidone increased the recovery time constant from Kv channel inactivation, suggesting that iloperidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with a Kv1.5 inhibitor (diphenyl phosphine oxide 1 [DPO-1]) inhibited the Kv current to a level similar to that with iloperidone alone. However, pretreatment with a Kv2.1 or Kv7.X inhibitor (guangxitoxin or linopirdine) did not affect the inhibitory effect of iloperidone on Kv channels. Therefore, iloperidone directly inhibits Kv channels in a concentration- and use (state)-dependent manner independently of its antagonism of serotonin and dopamine receptors. Furthermore, the primary target of iloperidone is the Kv1.5 subtype.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.—Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory–induced endothelial dysfunction.

This study demonstrates the inhibitory effect of anticholinergic drug oxybutynin on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Oxybutynin inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 11.51 ± 0.38 μmol/L and a Hill coefficient (n) of 2.25 ± 0.12. Application of oxybutynin shifted the activation curve to the right and the inactivation curve to the left. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 and the Kv2.1 subtype inhibitor guangxitoxin suppressed the oxybutynin-induced inhibition of the Kv current. However, application of the Kv7 subtype inhibitor linopirdine did not affect the inhibition by oxybutynin of the Kv current. The anticholinergic drug atropine did not inhibit the Kv current nor influence oxybutynin-induced inhibition of the Kv current. From these results, we concluded that oxybutynin inhibited the vascular Kv current in a concentration-dependent manner by influencing the steady-state activation and inactivation curves independent of its anticholinergic effect.

Prolonged endotracheal intubation is the most common cause of tracheal stenosis, which may lead to serious airway obstruction. Development of an endotracheal tube coated with biomaterials that exhibit anti-inflammatory or anti-fibrogenic effects may prevent tracheal stenosis. This study demonstrates that an endotracheal tube coated with phlorotannin, which is present in extracts of the brown alga Ecklonia cava, can prevent tracheal stenosis in a rabbit model. An in vitro study shows that phlorotannin inhibits proliferation of human tracheal fibroblasts treated with transforming growth factor β1. Phlorotannin-coated endotracheal tubes show steady release of phlorotannin for up to 7 days, and removal of the tube 1 week after insertion reveals a reduction in both fibrogenesis and thickening of tracheal submucosa. Western blot analysis of tracheal tissues after removal of the phlorotannin-coated tube shows decreased protein expression levels of phenotypic markers of fibrosis such as collagen type I and α-smooth muscle actin. The ability of phlorotannin-coated endotracheal tube to prevent tracheal stenosis caused by endotracheal intubation indicates that phlorotannin may be considered as a candidate biomaterial for coating the cuff of endotracheal tubes to prevent tracheal stenosis.

This study examined the inhibitory effect of flecainide, a class 1c antiarrhythmic agent (Na+ channel blocker), on voltage-dependent K+ (Kv) channels in smooth muscle cells isolated from coronary arteries. Flecainide decreased the vascular Kv channel current in a dose-dependent manner with an IC50 value of 5.90 ± 0.87 μmol/L and a Hill coefficient of 0.77 ± 0.06. Although the steady-state activation curve was not affected by flecainide, it shifted the steady-state inactivation curves toward a more negative potential. Application of train pulses such as 1 or 2 Hz did not change the flecainide-induced inhibition of Kv channels, indicating that the inhibitory effect of flecainide was not use-dependent. Using perforated-patch clamp experiments, we found that inhibition of Kv channels by flecainide caused membrane depolarization. Together, these results suggest that flecainide inhibits Kv channels in a concentration-dependent, but not use-dependent manner by changing the inactivation gating properties. Furthermore, Kv channel inhibition by flecainide occurs regardless of Na+ channel inhibition.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

Amitriptyline, a tricyclic antidepressant (TCA) drug, is widely used in treatment of psychiatric disorders. However, the side effects of amitriptyline on vascular K+ channels remain to be determined. Therefore, we investigated the effect of the tricyclic antidepressant and serotonin reuptake inhibitor amitriptyline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells, using the whole-cell patch clamp technique. The Kv current amplitudes were inhibited by amitriptyline in a concentration-dependent manner, with an apparent IC50 value of 2.2 ± 0.14 μmol/L and a Hill coefficient of 0.87 ± 0.03. Amitriptyline shifted the activation curve to a more positive potential, but had no significant effect on the inactivation curve, suggesting that amitriptyline altered the voltage sensitivity of Kv channels. Pretreatment with Kv1.5 and Kv1.2 channel inhibitors did not alter the inhibitory effect of amitriptyline on Kv channels. Additionally, application of train pulses (1 and 2 Hz) did not affect amitriptyline-induced inhibition of Kv currents, which suggested that the action of amitriptyline on Kv channels was not use (state)-dependent. From these results, we concluded that amitriptyline inhibited the channels in a concentration-dependent, but state-independent manner.

Summary We investigated the inhibitory effect of dapoxetine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K+ (Kv) channels using native smooth muscle cells from rabbit coronary arteries. Dapoxetine inhibited Kv channel currents in a concentration-dependent manner, with an IC50 value of 2.68 ± 0.94 μM and a slope value (Hill coefficient) of 0.63 ± 0.11. Application of 10 μM dapoxetine accelerated the rate of inactivation of Kv currents. Although dapoxetine did not modify current activation kinetics, it caused a significant negative shift in the inactivation curves. Application of train step (1 or 2 Hz) progressively increased the inhibitory effect of dapoxetine on Kv channels. In addition, the recovery time constant was extended in its presence, suggesting that the longer recovery time constant from inactivation underlies a use-dependent inhibition of the channel. From these results, we conclude that dapoxetine inhibits Kv channels in a dose-, time-, use-, and state (open)-dependent manner, independent of serotonin reuptake inhibition. This article is protected by copyright. All rights reserved.

This study investigated the alteration of voltage-dependent K(+) (Kv) channels in mesenteric arterial smooth muscle cells from control (Long-Evans Tokushima Otsuka [LETO]) and diabetic (Otsuka Long-Evans Tokushima Fatty [OLETF]) rats during the early and chronic phases of diabetes. We demonstrated alterations in the mesenteric Kv channels during the early and chronic phase of diabetes using the patch-clamp technique, the arterial tone measurement system, and RT-PCR in Long-Evans Tokushima (LETO; for control) and Otsuka Long-Evans Tokushima Fatty (OLETF; for diabetes) type 2 diabetic model rats. In the early phase of diabetes, the amplitude of mesenteric Kv currents induced by depolarizing pulses was greater in OLETF rats than in LETO rats. The contractile response of the mesenteric artery induced by the Kv inhibitor, 4-aminopyridine (4-AP), was also greater in OLETF rats. The expression of most Kv subtypes- including Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.2, Kv4.1, Kv4.3, Kv5.1, Kv6.2, Kv8.1, Kv9.3, and Kv10.1-were increased in mesenteric arterial smooth muscle from OLETF rats compared with LETO rats. However, in the chronic phase of diabetes, the Kv current amplitude did not differ between LETO and OLETF rats. In addition, the 4-AP-induced contractile response of the mesenteric artery and the expression of Kv subtypes did not differ between the two groups. The increased Kv current amplitude and Kv channel-related contractile response were attributable to the increase in Kv channel expression during the early phase of diabetes. The increased Kv current amplitude and Kv channel-related contractile response were reversed during the chronic phase of diabetes.

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free-radical-scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free-radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC-5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.

Background and purpose: Cilostazol is a specific inhibitor of 3′-5′-cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti-inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti-inflammatory effect of cilostazol on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. Experimental approach: We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs). Key results: Cilostazol suppressed production of nitric oxide (NO), prostaglandin E2 (PGE2) and the proinflammatory cytokines, interleukin-1 (IL-1), tumour necrosis factor-α, and monocyte chemoattractant protein-1 (MCP-1), in a concentration-dependent manner. Inhibitory effects of cilostazol were not affected by treatment with an adenylate cyclase inhibitor, SQ 22536, indicating that these actions of cilostazol were cAMP-independent. Cilostazol significantly inhibited the DNA binding and transcriptional activity of NF-κB. Moreover, cilostazol blocked signalling upstream of NF-κB activation by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but without affecting the activity of p38 MAPK. Conclusion and implications: Our results demonstrate that suppression of the NF-κB, ERK, JNK signalling pathways may inhibit LPS-induced NO and PGE2 production. Therefore, cilostazol may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators and cytokine production in activated microglia.

Diabetic hyperglycemia can lead to stress-related cellular apoptosis of cardiac tissue. However, the mechanism by which hyperglycemia inflicts this damage on the structure and function of the heart is unclear. In this study, we examined the relationship between proteome alterations, mitochondrial function, and major biochemical and electrophysiological changes affecting cardiac performance during simulated short-term hyperglycemia. Two-dimensional comparative proteomics analysis of rat hearts perfused with glucose at high (30 mM) or control (5.5 mM) levels revealed that glucose loading alters cardiomyocyte proteomes. It increased expression levels of initial enzymes of the tricarboxylic acid cycle, and of enzymes of fatty acid beta-oxidation, with consequent up-regulation of enzymes of mitochondrial electron transport. It also markedly decreased expression of enzymes of glycolysis and the final steps of the tricarboxylic acid cycle. Glucose loading increased the rate of Bax-independent apoptosis. High glucose increased the duration of the action potential and elevated level of intracellular cytoplasmic calcium. Surprisingly, glucose loading did not influence levels of nitric oxide or mitochondrial superoxide in isolated cardiomyocytes. In summary, short-term simulated hyperglycemia attenuated expression of many anti-apoptotic proteins. This effect was apparently mediated via alterations in multiple biochemical pathways that collectively increased apoptotic susceptibility.

SummaryAims We investigated the vasorelaxant effect of nateglinide and its related mechanisms using phenylephrine (Phe)-induced pre-contracted 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. This article is protected by copyright. All rights reserved.

BACKGROUND: This study was conducted to evaluate the occurrence and fate of fetal lumbar rib induced by Scutellariae radix (SR) in rats. METHODS: Water extracts of SR were orally administered to pregnant rats from day 7 to day 17 of gestation at a dose of 186 mg/kg/day, equivalent to 25 g/kg of starting material, representing a 100-fold increase over typical human intake level. RESULTS: The incidence of fetal lumbar rib in the SR-treated group was increased on gestational day 20 and then decreased on postnatal day 50. The weight of fetuses in the SR-treated group tended to be less than that in the control group. Alkaline phosphatase in SR-treated dams was increased on gestational day 20, but was decreased on postnatal day 50. There were no significant differences between the vehicle control and SR-treated groups in maternal body weight, embryological, histopathological, hematological, and serum biochemical changes. CONCLUSIONS: The present data suggest that the appearance of lumbar rib induced by SR is a transient fetal variation rather than teratogenicity or maternal toxicity. Birth Defects Res (Part B) 89:201–206, 2010. © 2010 Wiley-Liss, Inc.