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The development of effective scaffolds for bone regeneration is crucial given the increasing demand for innovative solutions to address bone defects and enhance healing process. In this study, a polycaprolactone/fish collagen/alginate (P/FC/A) 3D scaffold incorporating phlorotannin was developed to promote bone tissue regeneration. While the efficacy of the P/FC/A scaffold has been demonstrated through in vitro and in vivo experiments, its sub-chronic toxicity in animal models remains understudied, raising concerns regarding its safety in clinical application. Therefore, this study assessed the sub-chronic toxicity of the P/FC/A scaffold over 12 week using a New Zealand White rabbit model. Our results indicate no significant adverse effects in the group exposed to the P/FC/A scaffold compared with the negative control group implanted with a high-density polyethylene scaffold. These findings underscore the non-toxicity and safety profile of the P/FC/A scaffold, further supporting its potential suitability for clinical use in bone regeneration., Competing Interests: Declaration of competing interest Won-Kyo Jung reports financial support was provided by National Research Foundation of Korea. Won-Kyo Jung reports financial support was provided by Korea Ministry of Oceans and Fisheries. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background and Aims: Oxidative stress and abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) influence atherosclerosis formation and development. Oxidative stress significantly influences the abnormal proliferation and migration of VSMCs, and nuclear factor erythroid 2-related factor 2 (Nrf2) is a major antioxidant factor. However, the precise function of Nrf2 in the regulation of abnormal proliferation and migration of VSMCs and atherosclerosis is unclear., Methods: We investigated the proliferation and migration of VSMCs in atherosclerosis in male Apoe -/- and Apoe -/- Nrf2 -/- mice fed a high-fat diet for 12 weeks. In cultured mouse VSMCs, we studied the effect of Nrf2 on ox-LDL-stimulated proliferation and migration by using siRNA treatment to silence Nrf2. We then performed dual luciferase reporter and immunoprecipitation assays to study the interaction between Nrf2 and the promoter sequence of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)., Results: Our results demonstrate that Nrf2 expression levels were increased in the aorta and VSMCs of mice in the atherosclerosis model group compared with the control group. We also provide evidence that Nrf2 deficiency attenuated atherosclerotic plaque burden, diminished proliferation, and migration of VSMCs but enhanced VSMC-specific marker gene expression in vitro and in vivo. This is related to Nrf2 binding to the promoter sequence of LOX-1. Furthermore, Nrf2 downregulation contributes to restrain both transcriptional and translational activities of LOX-1., Conclusions: Together, our data indicate that Nrf2 insufficiency is linked to attenuation of atherosclerosis, and could diminish the pathological process by blunting LOX-1-mediated proliferation and migration of VSMCs., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aims: We investigated the changes in large-conductance Ca 2+ -activated K + (BK Ca ) channels from human umbilical arterial smooth muscle cells experiencing gestational diabetes mellitus (GDM)., Main Methods: Whole-cell patch-clamp technique, arterial tone measurement, RT-PCR, Quantitative real-time PCR, western blot were performed in human umbilical arterial smooth muscle cells., Key Findings: Whole-cell BK Ca current density was decreased in the GDM group compared with the normal group. The vasorelaxant effects of the synthetic BK Ca channel activator NS-1619 (10 μM) were impaired in the GDM group compared with the normal group. Reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analyses suggested that the mRNA, total RNA, and protein expression levels of the BK Ca channel were decreased in the GDM group relative to the normal group. In addition, the expression levels of protein kinase A and protein kinase G, which regulate BK Ca channel activity, remained unchanged between the groups. Applying the BK Ca channel inhibitor paxilline (10 μM) induced vasoconstriction and membrane depolarization of isolated umbilical arteries in the normal group but showed less of an effect on umbilical arteries in the GDM group., Significance: Our results demonstrate for the first time impaired BK Ca current and BK Ca channel-induced vasorelaxation activities that were not caused by impaired BK Ca channel-regulated protein kinases, but by decreased expression of the BK Ca channels, in the umbilical arteries of GDM patients., (Copyright © 2021. Published by Elsevier Inc.)

Aims: Canagliflozin is an anti-diabetic agent and sodium glucose co-transporter-2 inhibitor. Despite numerous clinical trials demonstrating its beneficial effects on blood pressure, the cellular mechanisms underlying the effects of canagliflozin on vascular reactivity have yet to be clarified. We investigated the vasodilatory effect of canagliflozin on aortic rings isolated from rabbits., Main Methods: We used rabbit thoracic aortic rings and its arterial tone was tested by using wire myography system., Key Findings: Canagliflozin caused concentration-dependent vasodilation in aortic rings pre-constricted with phenylephrine or high K + . However, the degree of canagliflozin-induced vasodilation of the aortic rings pre-constricted with high K + was less than that of rings pre-constricted with phenylephrine. Application of 4-aminopyridine, a voltage-dependent K + (Kv) channel inhibitor, reduced canagliflozin-induced vasodilation. However, pre-incubation of an inwardly rectifying K + channel inhibitor, a large-conductance Ca 2+ -activated K + channel inhibitor, and an ATP-sensitive K + inhibitor did not modulate the vasodilatory effects of canagliflozin. Indeed, canagliflozin increased Kv currents in aortic smooth muscle cells. Pre-treatment with thapsigargin or cyclopiazonic acid, a sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitors, reduced the vasodilatory effects of canagliflozin. Conversely, pre-treatment with a Ca 2+ channel inhibitor, adenylyl cyclase/PKA inhibitors, and guanylyl cyclase/PKG inhibitors did not modulate the vasodilatory effects of canagliflozin. Endothelium removal, and pre-treatment with the nitric oxide synthase inhibitor L-NAME, and small- and intermediate-conductance Ca 2+ -activated K + channel inhibitor apamin and TRAM-34, did not diminish the vasodilatory effects of canagliflozin., Significance: Our results indicate that canagliflozin induces vasodilation, which is dependent on the robust SERCA activity and Kv channel activation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: In this study, we investigated the vasodilatory effects of trelagliptin (a dipeptidyl peptidase-4 inhibitor) and its related mechanisms using rabbit aortic rings., Main Methods: Arterial tone measurement was performed in rabbit thoracic aortic rings., Key Findings: Trelagliptin induced vasodilation in a dose-dependent manner. Pretreatment with the ATP-sensitive K + channel inhibitor glibenclamide, large-conductance Ca 2+ -activated K + channel inhibitor paxilline, and inwardly rectifying K + channel inhibitor Ba 2+ did not affect the vasodilatory effect of trelagliptin. However, pretreatment with the voltage-dependent K + (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly attenuated the vasodilatory effect of trelagliptin, suggesting that the vasodilatory effect of trelagliptin is associated with Kv channel activation. Although pretreatment with Kv1.5 and Kv2.1 subtype inhibitors did not affect the response to trelagliptin, pretreatment with a Kv7.X subtype inhibitor effectively reduced the vasodilatory effect of trelagliptin. Furthermore, sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitors also significantly attenuated the vasodilatory effect of trelagliptin. These effects, however, were not affected by pretreatment with Ca 2+ channel inhibitors, adenylyl cyclase/PKA inhibitors, guanylyl cyclase/PKG inhibitors, or removal of the endothelium., Significance: From these results, we concluded that the vasodilatory effect of trelagliptin was associated with the activation of Kv channels (primary the Kv7.X subtype) and SERCA pump regardless of other K + channels, Ca 2+ channels, cAMP/PKA-related or cGMP/PKG-related signaling pathways, and the endothelium. Therefore, caution is required when prescribing trelagliptin to the patients with hypotension and diabetes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Due to their short half-lives, repeated administration of anti-hyperglycemic drugs can cause pain, discomfort, tissue damage, and infection in diabetic patients. Therefore, there is a need to develop long-term drug delivery systems to treat diabetes and its complications. C-peptide can prevent diabetic complications, including diabetic vasculopathy, but its clinical application is limited by its short half-life. Here, we developed K9-C-peptide (human C-peptide conjugated to an elastin-like biopolymer) and investigated its long-term influence on hyperglycemia-induced vascular dysfunction using an aortic endothelium model in diabetic mice. Using pharmacokinetics and in vivo imaging, we found that subcutaneously injected K9-C-peptide formed a hydrogel depot that slowly released human C-peptide into the blood circulation for 19 days. Administration of K9-C-peptide, human C-peptide, or K8 polypeptide had no effect on body weight or blood glucose levels. The slow release of C-peptide from K9-C-peptide hydrogels provided prolonged prevention of oxidative stress, inflammatory responses, and endothelial apoptosis in a hyperglycemia-induced vascular dysfunction model using the diabetic mouse aorta. Subcutaneous administration of unbound human C-peptide and K8 polypeptide were used as negative controls and had no effects. These results suggest that K9-C-peptide is suitable for the long-term delivery of human C-peptide for treating vascular dysfunction in diabetic patients., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Acylated chitosan sulfate (ChS1), a sulfated polysaccharide with high anticoagulant activity, was chemically synthesized and structurally characterized using FT-IR analysis. The beneficial structural properties and high availability of the sulfate group in ChS1 led to greater anticoagulant activity through both the intrinsic and common pathways with antithrombin III (AT III)-mediated inhibition, particularly involving coagulation factors FXa and FIIa. The analysis of the binding affinities using surface plasma resonance found that the equilibrium dissociation constant (K D ) of ChS1 for FXa and FIIa in the presence of AT III was 67.4 nM and 112.6 nM, respectively, indicating the stronger interaction of the AT III/ChS1 complex with the ligands and the inhibition of activated FX and FII. The results of amidolytic assays further demonstrated the stronger inhibition of the proteolytic conversion of factor X by the intrinsic FXase complex and of FII by the prothrombinase complex. Molecular docking analysis further validated the protein-ligand interactions of ChS1 with AT III and their binding affinity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We investigated the effect of ziprasidone, a widely used treatment for schizophrenia, on voltage-dependent K + (Kv) channels of coronary arterial smooth muscle cells using the patch-clamp technique. Ziprasidone dose-dependently inhibited Kv channels with an IC 50 value of 0.39 ± 0.06 μM and a Hill coefficient of 0.62 ± 0.03. Although ziprasidone had no effect on the steady-state inactivation kinetics of the Kv channels, the steady-state activation curve shifted towards a more positive potential. These results suggest that ziprasidone inhibits Kv channels by targeting their voltage sensors. The recovery time constant of Kv channel inactivation was increased in the presence of ziprasidone. Furthermore, application of train steps (of 1 and 2 Hz) in the presence of ziprasidone led to a progressive increase in the blockade of Kv currents, suggesting that ziprasidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors partially suppressed the ziprasidone-induced inhibition of Kv currents. These results suggest that ziprasidone inhibits vascular Kv channels through its effect on gating properties. The Kv channel-inhibiting action of ziprasidone is concentration- and use (state)-depedent., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this study, we fabricated polyvinyl alcohol hydrogels containing diphlorethohydroxycarmalol (DPHC) from Ishige okamurae for its anti-bacterial effect in wound-dressing applications. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of DPHC against Staphylococcus aureus and Pseudomonas aeruginosa were investigated, and these were found to be about 128 μg/mL and 512 μg/mL, respectively. Polyvinyl alcohol hydrogels loaded with different concentrations of DPHC were then produced for the dressing of wounds to assist in the healing process and to provide an antibacterial effect. To investigate the characteristics of the proposed PVA/DPHC hydrogels, we conducted SEM analysis, rheological analysis, thermogravimetric analysis, water swelling analysis, drug release testing, and gel fraction assessment. The antibacterial activity of the PVA/DPHC hydrogels was also tested against the gram-positive bacterium S. aureus and the gram-negative bacterium P. aeruginosa using ASTM E2149 tests. The biocompatibility of the PVA/DPHC hydrogels was assessed using in vitro indirect and direct contact tests and in vivo tests on ICR mice. The PVA/DPHC hydrogels exhibited the ability to reduce the viability of S. aureus and P. aeruginosa by about 99% in ASTM E2149 testing, while not producing any toxic effect on NHDF-Neo or HaCaT cells as shown in MTT assays and in vitro FDA fluorescence analysis. In addition, the PVA/DPHC hydrogels had a strong wound healing effect when compared to non-treated groups of ICR mice in vivo. Based on the characterization of the PVA/DPHC hydrogels in vitro and in vivo, this study suggests that the proposed hydrogel has significant potential for use in wound dressing., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aims: Insulin is a central peptide hormone required for carbohydrate metabolism; however, its role in diabetes-associated pulmonary disease is unknown. Here, we investigated the preventative effect of insulin against hyperglycemia-induced pulmonary vascular leakage and its molecular mechanism of action in the lungs of diabetic mice., Main Methods: Vascular endothelial growth factor (VEGF) activated transglutaminase 2 (TGase2) by sequentially elevating intracellular Ca 2+ and reactive oxygen species (ROS) levels in primary human pulmonary microvascular endothelial cells (HPMVECs)., Key Findings: Insulin inhibited VEGF-induced TGase2 activation, but did not affect intracellular Ca 2+ elevation and ROS generation. Insulin prevented VEGF-induced vascular leakage by inhibiting TGase2-mediated c-Src phosphorylation, disassembly of VE-cadherin and β-catenin, and stress fiber formation. Insulin replacement therapy prevented hyperglycemia-induced TGase2 activation, but not ROS generation, in the lungs of diabetic mice. Insulin also prevented vascular leakage and cancer metastasis in the diabetic lung. Notably, vascular leakage was not detectable in the lungs of TGase2-null (Tgm2 -/- ) diabetic mice., Significance: These findings demonstrate that insulin prevents hyperglycemia-induced pulmonary vascular leakage in diabetic mice by inhibiting VEGF-induced TGase2 activation rather than ROS generation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: The vasodilatory effects of the anti-diabetic drug, linagliptin in phenylephrine-precontracted aortic rings were investigated., Materials and Methods: Male New Zealand White rabbits were used in the experiment and its arterial tone was measured by using myogragh system., Key Findings: Linagliptin induced vasodilation in a concentration-dependent manner. The vasodilatory effect of linagliptin was not affected by the absence of the endothelium, or by pretreatment with a nitric oxide synthase inhibitor (L-NAME) or a small-conductance Ca 2+ -activated K + channel inhibitor (apamin). Moreover, application of the adenylyl cyclase inhibitor SQ22536, protein kinase A (PKA) inhibitor KT5720, guanylyl cyclase inhibitor ODQ, or protein kinase G (PKG) inhibitor KT5823 did not alter the vasodilatory effect of linagliptin. However, inhibition of Rho-associated protein kinase by Y-27632 significantly attenuated linagliptin-induced vasodilation. Ion channel involvement in the vasodilatory effect of linagliptin was also investigated. Pretreatment with the vascular K + channel inhibitors glibenclamide (ATP-sensitive K + channels), Ba 2+ (inwardly rectifying K + channels), 4-AP (voltage-dependent K + channels), and paxilline (large conductance Ca 2+ -activated K + channels) did not affect linagliptin-induced vasodilation. Furthermore, the L-type Ca 2+ channel inhibitor, nifedipine, and the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitor, thapsigargin, did not change the vasodilatory effect of linagliptin., Significance: We suggests that linagliptin-induced vasodilation was mediated by the inhibition of Rho-associated kinase, but not with the endothelium, cAMP-PKA or cGMP-PKG-dependent signaling pathways, K + channels, Ca 2+ influx, or SERCA pump., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg 2+ , and Ca 2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 μg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC 50 ). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC 50 values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC 50 values ranging from 0.005 to 1.937 mM; however, Ca 2+ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 BK Ca channel inhibitor paxilline, the K ATP channel inhibitor glibenclamide, and the Kir channel inhibitor Ba 2+ did not change dapagliflozin-induced vasodilation. However, application of the Kv channels inhibitor 4-AP effectively inhibited dapagliflozin-induced vasodilation. Application of the Ca 2+ channel inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca 2+ -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 Ca 2+ -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, Ca 2+ channels, intracellular Ca 2+ , and the endothelium., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix. Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA). Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified., Methods: The immortalized human HSCs, LX-2 cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were fed with a methionine-choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine-choline-sufficient diet (MCS diet group)., Results: In our study, metformin and 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2 cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2 cells were treated with palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-β1 production in succinate-treated LX-2 cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration. Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis, liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet-fed mice., Conclusion: This study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway. Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: The vasorelaxant effects of the anti-diabetic drug, mitiglinide in phenylephrine (Phe)-pre-contracted aortic rings were examined., Materials and Methods: Arterial tone measurement was performed in aortic smooth muscle cells., Key Findings: Mitiglinide dose-dependently induced vasorelaxation. Application of the large-conductance Ca 2+ -activated K + (BK Ca ) channel blocker paxilline, inwardly rectifying K + (Kir) channel blocker Ba 2+ , and ATP-sensitive K + (K ATP ) channel blocker glibenclamide did not affect the vasorelaxant effect of mitiglinide. However, application of the voltage-dependent K + (Kv) channel blocker 4-AP, effectively inhibited mitiglinide-induced vasorelaxation. Although pretreatment with the Ca 2+ channel blocker nifedipine did not alter the mitiglinide-induced vasorelaxation, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitor thapsigargin and cyclopiazonic acid reduced the vasorelaxant effect of mitiglinide. In addition, the vasorelaxant effect of mitiglinide was not affected by the inhibitors of adenylyl cyclase, protein kinase A, guanylyl cyclase, or protein kinase G. Elimination of the endothelium and inhibition of endothelium-dependent vasorelaxant mechanisms also did not change the vasorelaxant effect of mitiglinide., Significance: We proposed that mitiglinide induces vasorelaxation via activation of Kv channels and SERCA pump. However, the vasorelaxant effects of mitiglinide did not involve other K + channels, Ca 2+ channels, PKA/PKG signaling pathways, or the endothelium., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The variable number of tandem repeats (VNTRs) provides valuable information about both the functional and evolutionary aspects of genetic diversity. Comparative analysis of 3 Plasmodium falciparum genomes has shown that more than 9% of its open reading frames (ORFs) harbor VNTRs. Although microsatellites and VNTR genes of P. vivax were reported, the VNTR polymorphism of genes has not been examined widely. In this study, 230 P. vivax genes were analyzed for VNTRs by SERV, and 33 kinds of TR deletions or insertions from 29 P. vivax genes (12.6%) were found. Of these, 9 VNTR fragments from 8 P. vivax genes were used for PCR amplification and sequence analysis to examine the genetic diversity among 134 isolates from four Southeast Asian countries (China, Republic of Korea, Thailand, and Myanmar) with different malaria endemicity. We confirmed the existence of extensive polymorphism of VNTR fragments in field isolates. This detection provides several suitable markers for analysis of the molecular epidemiology of P. vivax field isolates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Analysis of the signaling mechanism triggered by endotoxin-mediated toll-like receptor-4 activation using immune cell systems or rodent models may help identify potential agents for the prevention of Gram-negative bacteria infection. β-agarase cleaves the β-1,4-linkages of agar to produce neoagarooligosaccharides (NAOs), which have various physiological functions. The aim of this study was to investigate the efficacy of NAOs in preventing experimental sepsis caused by the administration of endotoxin or Gram-negative bacteria. Organ damage and neutrophil infiltration in an endotoxemia and septic-shock mouse model were suppressed by NAOs. Pro-inflammatory cytokine level was decreased, but IL-10 level was increased by NAO-treatment. Further induction by NAOs in the presence of endotoxin was associated with a significant induction of A20 and cyclooxygenase (COX)-2 expressions. Our data suggest that NAOs have a beneficial preventive effect in septic shock correlated with the enhancement of IL-10 via the induction of A20 and COX-2., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microglia are the immune cells of the central nervous system (CNS). Overexpression of inflammatory mediators by microglia can induce several neurological diseases. Thus, the underlying basic requirement for neural tissue engineering is to develop materials that exhibit little or no neuro-inflammatory effects. In this study, we have developed a method to create porous scaffolds by adding fucoidan (Fu) into porous sodium alginate (Sa)/gelatine (G) (SaGFu). For mechanical characterization, in vitro degradation, stress/strain, swelling, and pore size were measured. Furthermore, the biocompatibility was evaluated by assessing the adhesion and proliferation of BV2 microglial cells on the SaGFu porous scaffolds using scanning electron microscopy (SEM) and lactate dehydrogenase (LDH) assay, respectively. Moreover, we studied the neuro-inflammatory effects of SaGFu on BV2 microglial cells. The effect of gelatine and fucoidan content on the various properties of the scaffold was investigated and the results showed that mechanical properties increased porosity and swelling ratio with an increase in the gelatine and fucoidan, while the in vitro biodegradability decreased. The average SaGFu diameter attained by fabrication of SaGFu ranged from 60 to 120μm with high porosity (74.44%-88.30%). Cell culture using gelatine 2.0% (SaG2Fu) and 4.0% (SaG4Fu), showed good cell proliferation; more than 60-80% that with Sa alone. Following stimulation with 0.5μg/mL LPS, microglia cultured in porous SaGFu decreased their expression of nitric oxide (NO), prostaglandin E2 (PGE2), and reactive oxygen species (ROS). SaG2Fu and SaG4Fu also inhibited the activation and translocation of p65 NF-κB protein levels, resulting in reduction of NO, ROS, and PGE2 production. These results provide insights into the diverse biological effects and opens new avenues for the applications of SaGFu in neuroscience., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We investigated the effect of cisapride, a selective serotonin 5-HT4-receptor agonist, on voltage-dependent K(+) (Kv) channels using freshly isolated smooth muscle cells from the coronary arteries of rabbits. The amplitude of Kv currents was reduced by cisapride in a concentration-dependent manner, with an IC50 value of 6.77 ± 6.01 μM and a Hill coefficient of 0.51 ± 0.18. The application of cisapride shifted the steady-state inactivation curve toward a more negative potential, but had no significant effect on the steady-state activation curve. This suggested that cisapride inhibited the Kv channel in a closed state by changing the voltage sensitivity of Kv channels. The application of another selective serotonin 5-HT4-receptor agonist, prucalopride, did not affect the basal Kv current and did not alter the inhibitory effect of cisapride on Kv channels. From these results, we concluded that cisapride inhibited vascular Kv current in a concentration-dependent manner by shifting the steady-state inactivation curve, independent of its own function as a selective serotonin 5-HT4-receptor agonist., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An emerging paradigm in wound healing techniques is that a tissue-engineered skin substitute offers an alternative approach to create functional skin tissue. Here we developed a fish collagen/alginate (FCA) sponge scaffold that was functionalized by different molecular weights of chitooligosaccharides (COSs) with the use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as a cross-linking agent. The effects of cross-linking were analyzed by Fourier transform infrared spectroscopy. The results indicate that the homogeneous materials blending and cross-linking intensity were dependent on the molecular weights of COSs. The highly interconnected porous architecture with 160-260μm pore size and over 90% porosity and COS's MW driven swelling and retention capacity, tensile property and in vitro biodegradation behavior guaranteed the FCA/COS scaffolds for skin tissue engineering application. Further improvement of these properties enhanced the cytocompatibility of all the scaffolds, especially the scaffolds containing COSs with MW in the range of 1-3kDa (FCA/COS1) showed the best cytocompatibility. These physicochemical, mechanical, and biological properties suggest that the FCA/COS1 scaffold is a superior candidate that can be used for skin tissue regeneration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aim: B7-H1, which belongs to the B7 family of costimulatory molecules, is implicated in the ability of tumors to evade the host immune response. The development of evasion mechanisms within the tumor microenvironment may be responsible for poor therapeutic responses. In this manuscript, we report that the 15-deoxy-δ(12,14)-prostaglandin J2 (15d-PGJ2), peroxisome proliferator-activated receptor gamma (PPARγ) activator leads to the downregulation of the cancer-associated expression of B7-H1 in response to interferon-gamma (IFN-γ) and the associated signaling cascades., Main Methods: The expression of B7-H1 from IFN-γ-induced B16F10 melanoma cells was measured with flow cytometric analysis. The regulatory mechanisms of 15d-PGJ2 on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays., Key Findings: The flow cytometric analysis revealed that the B7-H1 costimulatory molecule is significantly upregulated in B16F10 melanoma cells by stimulation with IFN-γ. However, 15d-PGJ2 strongly downregulates B7-H1 expression in IFN-γ-stimulated B16F10 melanoma cells. Furthermore, the significant damping effect of 15d-PGJ2 on B7-H1 expression involves the inhibition of the tyrosine phosphorylation of Janus kinase (Jak) and signal transducer(s) and activator(s) of transcription (STAT) and, thereby, the interferon regulatory factor-1 (IRF-1) trans-activation of STAT. These effects of 15d-PGJ2 were not abrogated by the PPARγ antagonist GW9662, indicating that they occur through a PPARγ-independent mechanism., Significance: In this study, we demonstrate that 15d-PGJ2 suppresses the IFN-γ-elicited expression of B7-H1 by the inhibition of IRF-1 transcription via the Jak/STAT signaling pathway through a PPARγ-independent mechanism in mouse melanoma cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We investigated the effect of the calmodulin inhibitor and antipsychotic drug trifluoperazine on voltage-dependent K(+) (Kv) channels. Kv currents were recorded by whole-cell configuration of patch clamp in freshly isolated rabbit coronary arterial smooth muscle cells. The amplitudes of Kv currents were reduced by trifluoperazine in a concentration-dependent manner, with an apparent IC50 value of 1.58±0.48 μM. The rate constants of association and dissociation by trifluoperazine were 3.73±0.33 μM(-1) s(-1) and 5.84±1.41 s(-1), respectively. Application of trifluoperazine caused a positive shift in the activation curve but had no significant effect on the inactivation curve. Furthermore, trifluoperazine provoked use-dependent inhibition of the Kv current under train pulses (1 or 2 Hz). These findings suggest that trifluoperazine interacts with Kv current in a closed state and inhibits Kv current in the open state in a time- and use-dependent manner, regardless of its function as a calmodulin inhibitor and antipsychotic drug., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: Cilostazol is a selective inhibitor of type III phosphodiesterase that inhibits platelet aggregation. Cilostazol is a useful vasodilator, antithrombotic, and cardiotonic agent. Ultraviolet B (UVB) irradiation increases the production of matrix metalloproteinase-1 (MMP-1) during skin photoaging. The UVB-induced increase of MMP-1 results in connective tissue damage, and the skin becomes wrinkled and aged. Here, we investigated the capacity of cilostazol to inhibit MMP-1 expression in UVB-irradiated human dermal fibroblasts., Main Methods: Cultured human dermal fibroblasts were irradiated with UVB, followed by the addition of cilostazol to the culture medium., Key Findings: Post-treatment with cilostazol attenuated UVB-induced production of MMP-1 and prevented the reduction of type I procollagen. Cilostazol inhibited UVB irradiation-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling molecules Jun-N-terminal kinase (JNK) and p38 kinase, as well as activator protein-1 (AP-1) in dermal fibroblasts., Significance: Overall, these results demonstrate that cilostazol regulates UVB-induced MMP-1 expression and type I procollagen synthesis by inhibiting MAPK signaling and AP-1 activity. Therefore, we suggest that cilostazol may be useful for the prevention and treatment of skin photodamage caused by UVB-irradiation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K(+) (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K(d) value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM(-1)s(-1) and 1.47 ± 0.17s(-1), respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Ischemic preconditioning (IPC) is a powerful mechanism for limiting myocardial infarction during or after ischemia-reperfusion (IR) injury. However, effective target genes and proteins for IPC are unknown. We characterized global changes in gene expression in the heart during IR, and identified effective target genes for IPC., Methods: Hearts were isolated from Sprague-Dawley rats under control, IR, and IPC conditions. We generated expressed-sequence-tags (ESTs) for each group and investigated their functions and the major biological processes in which they are involved using the eukaryotic clusters of orthologous groups (KOG) database and bioinformatics analysis tools., Results: IR modified the expression of 126 genes. Of these, 62 were upregulated, 64 were downregulated, and 77 were found to be effective target genes for IPC. In KOG analysis, most of the genes whose expression was modified were involved in energy production and conversion and the cytoskeleton. A gene-to-gene interaction map revealed that IR modified the expression of genes in four major functional modules: electron transport chain/oxidative phosphorylation; tricarboxylic acid cycle/glucose metabolism/amino acid metabolism; cellular structure and contraction; and gene transcription, translation, and protein folding. At the individual gene level, the genes encoding mitochondrial cytochrome c oxidase subunits 2 and 3 were downregulated, and those encoding the major cytoskeleton components tropomyosin, myosin light chain, myomesin 2, and myosin regulatory light chain 2, as well as the gene encoding the iron-storage protein ferritin, were upregulated, and thus were identified as potential target genes. Real time PCR evaluated expression patterns of three mitochondrial IPC effective genes. Two-dimensional electrophoresis proteomic analyses revealed altered expression of 14 target proteins. The expression patterns of six proteins matched the corresponding EST expression patterns., Conclusion: The global profiling of cardiac ischemia-related genes provides the possible mechanisms of IR and IPC and ways of treating IR injury., (Copyright © 2012 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

We investigated the effect of a specific protein kinase C (PKC) inhibitor, bisindolylmaleimide I [BIM (I)], on L-type Ca(2+) channels in rat ventricular myocytes. BIM (I) alone inhibited the L-type Ca(2+) current in a concentration-dependent manner, with a K(d) value of 3.31 ± 0.25 μM, and a Hill coefficient of 2.34 ± 0.23. Inhibition was immediate after applying BIM (I) in the bath solution and then it partially washed out. The steady-state activation curve was not altered by applying 3μ M BIM (I), but the steady-state inactivation curve shifted to a more negative potential with a change in the slope factor. Other PKC inhibitors, PKC-IP and chelerythrine, showed no significant effects either on the L-type Ca(2+) current or on the inhibitory effect of BIM (I) on the L-type Ca(2+) current. The results suggest that the inhibitory effect of BIM (I) on the L-type Ca(2+) current is independent of the PKC pathway. Thus, our results should be considered in studies using BIM (I) to inhibit PKC activity and ion channel modulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: We investigated the effects of BMS-204352 (BMS), a big-conductance calcium-activated potassium (BK(Ca)) channel activator, on L-type Ca(2+) channels., Main Methods: Electrophysiological recordings were performed in isolated rat ventricular myocytes. Whole-cell configuration was used., Key Findings: BMS caused inhibition of the Ca(2+) current in a dose-dependent manner, with K(d) of 6.00 ± 0.67 μM and a Hill coefficient of 1.33 ± 0.18. BMS did not affect the steady-state activation of L-type Ca(2+) channels. However, for those in steady-state inactivation, BMS shifted the half-maximal potential (V(1/2)) by -11 mV, but the slope value (k) was not altered. Iberiotoxin, inhibitor of membrane BK(Ca) channels and paxilline, inhibitor of mitochondrial BK(Ca) channel did not affect the inhibitory effect of BMS on L-type Ca(2+) channels. Pretreatment with inhibitors of protein kinase A (PKA), protein kinase C (PKC), and protein kinase G (PKG) did not significantly alter the inhibitory effect of BMS on L-type Ca(2+) current. The presence of a selective β-adrenergic receptor agonist, isoproterenol did not affect the inhibitory effect of BMS on L-type Ca(2+) current. Based on these results, we concluded that the inhibition of L-type Ca(2+) channels by BMS is independent of the inhibition of BK(Ca) channels or intracellular signaling pathways., Significance: It is important to take BMS-204352 (BMS) effects on L-type Ca(2+) channels into consideration when using BMS as a BK(Ca) channel activator or therapeutic target in ventricular myocytes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Galectin-9 exhibited potent and selective eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by the marked presence of inflammatory cells, particularly eosinophils. Thus, galectin-9 may be implicated in the pathogenesis of nasal polyposis. The study was designed to investigate whether interferon-gamma (IFN-γ) can induce the augmentation of galectin-9 expression and induce the expression of galectin-9 in nasal polyps. We examined the correlation between galectin-9 expression and eosinophil infiltration in nasal polyps. In addition, we identified the signaling pathways involved in the elevation of galectin-9 expression in response to IFN-γ. Our data demonstrate that the involvement of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 phosphate kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) may play important roles in the selective recruitment of eosinophils in nasal polyp tissues through the production of galectin-9. These findings suggest that galectin-9 expression is associated with eosinophil infiltration in polyps of patients with nasal polyposis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A 63 year-old man developed sudden pulmonary edema after uneventful robot-assisted laparoscopic radical prostatectomy (RALP) for prostate cancer despite normal preoperative laboratory findings and appropriate anesthetic management. The pulmonary edema was attributed to prolonged (4 hrs) pneumoperitoneum with concomitant high intraabdominal pressure (15-20 mmHg)., (Copyright 2010 Elsevier Inc. All rights reserved.)

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-gamma by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-kappaB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses., (2010 Elsevier Inc. All rights reserved.)

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK(Ca) channel alpha-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK(Ca) channel alpha-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK(Ca) channel subfamily M alpha member 1. These findings suggest that a unique BK(Ca) channel alpha-subunit is expressed in mouse cardiomyocytes.

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.

Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.

We examined the effects of the protein kinase A (PKA) inhibitor H-89 on voltage-dependent K(+) (K(V)) currents in freshly isolated rabbit coronary arterial smooth muscle cells, using a whole-cell patch clamp technique. H-89 inhibited the K(V) current in a concentration-dependent manner, with a K(d) value of 1.02 microM. However, the PKA inhibitors KT 5720 and Rp-8-CPT-cAMPS did not significantly alter the K(V) current or the inhibitory effects of H-89 on the K(V) current. Moreover, H-85, a structurally similar but inactive analog of H-89, showed similar inhibitory effects on the K(V) channel. H-89 had no effect on the voltage-dependency of activation or inactivation, or on recovery kinetics. These results suggest that in rabbit coronary arterial smooth muscle cells, H-89 inhibits the K(V) current directly by blocking the pore cavity, an effect independent of PKA inhibition.

We investigated the effects of the vasoconstrictor angiotensin (Ang) II on the whole cell inward rectifier K(+) (Kir) current enzymatically isolated from small-diameter (<100 microm) coronary arterial smooth muscle cells (CASMCs). Ang II inhibited the Kir current in a dose-dependent manner (half inhibition value: 154 nM). Pretreatment with phospholipase C inhibitor and protein kinase C (PKC) inhibitors prevented the Ang II-induced inhibition of the Kir current. The PKC activator reduced the Kir currents. The inhibitory effect of Ang II was reduced by intracellular and extracellular Ca(2+) free condition and by Gö6976, which inhibits Ca(2+)-dependent PKC isoforms alpha and beta. However, the inhibitory effect of Ang II was unaffected by a peptide that selectively inhibits the translocation of the epsilon isoform of PKC. Western blot analysis confirmed that PKCalpha, and not PKCbeta, was expressed in small-diameter CASMCs. The Ang II type 1 (AT(1))-receptor antagonist CV-11974 prevented the Ang II-induced inhibition of the Kir current. From these results, we conclude that Ang II inhibits Kir channels through AT(1) receptors by the activation of PKCalpha.

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (<100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.

We examined the effects of the protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM) (I), on voltage-dependent K+ (K(V)) channels in rabbit coronary arterial smooth muscle cells using whole-cell patch clamp technique. BIM (I) reversibly and dose-dependently inhibited the K(V) currents with an apparent Kd value of 0.27 microM. The inhibition of the K(V) current by BIM (I) was highly voltage-dependent between -30 and +10 mV (voltage range of channel activation), and the additive inhibition of the K(V) current by BIM (I) was voltage-dependence in the full activation voltage range. The rate constants of association and dissociation for BIM (I) were 18.4 microM(-1) s(-1) and 4.7 s(-1), respectively. BIM (I) had no effect on the steady-state activation and inactivation of K(V) channels. BIM (I) caused use-dependent inhibition of K(V) current, which was consistent with the slow recovery from inactivation in the presence of BIM (I) (recovery time constants were 856.95 +/- 282.6 ms for control, and 1806.38 +/- 110.0 ms for 300 nM BIM (I)). ATP-sensitive K+ (K(ATP)), inward rectifier K+ (K(IR)), Ca2+-activated K+ (BK(Ca)) channels, which regulate the membrane potential and arterial tone, were not affected by BIM (I). The PKC inhibitor, chelerythrine, and protein kinase A (PKA) inhibitor, PKA-IP, had little effect on the K(V) current and did not significantly alter the inhibitory effects of BIM (I) on the K(V) current. These results suggest that BIM (I) inhibits K(V) channels in a phosphorylation-independent, and voltage-, time- and use-dependent manner.

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.