Modafinil exerts anti-inflammatory and anti-fibrotic effects by upregulating adenosine A2A and A2B receptors.

Adenosine receptor (AR) suppresses inflammation and fibrosis by activating cyclic adenosine monophosphate (cAMP) signaling. We investigated whether altered AR expression contributes to the development of fibrotic diseases and whether A2AAR and A2BAR upregulation inhibits fibrotic responses. Primary human lung fibroblasts (HLFs) from normal (NHLFs) or patients with idiopathic pulmonary fibrosis (DHLF) were used for in vitro testing. Murine models of fibrotic liver or pulmonary disease were developed by injecting thioacetamide intraperitoneally, by feeding a high-fat diet, or by intratracheal instillation of bleomycin. Modafinil, which activates cAMP signaling via A2AAR and A2BAR, was administered orally. The protein amounts of A2AAR, A2BAR, and exchange protein directly activated by cAMP (Epac) were reduced, while collagen and α-smooth muscle actin (α-SMA) were elevated in DHLFs compared to NHLFs. In liver or lung tissue from murine models of fibrotic diseases, A2AAR and A2BAR were downregulated, but A1AR and A3AR were not. Epac amounts decreased, and amounts of collagen, α-SMA, KCa2.3, and KCa3.1 increased compared to the control. Modafinil restored the amounts of A2AAR, A2BAR, and Epac, and reduced collagen, α-SMA, KCa2.3, and KCa3.1 in murine models of fibrotic diseases. Transforming growth factor-ß reduced the amounts of A2AAR, A2BAR, and Epac, and elevated collagen, α-SMA, KCa2.3, and KCa3.1 in NHLFs; however, these alterations were inhibited by modafinil. Our investigation revealed that A2AAR and A2BAR downregulation induced liver and lung fibrotic diseases while upregulation attenuated fibrotic responses, suggesting that A2AAR and A2BAR-upregulating agents, such as modafinil, may serve as novel therapies for fibrotic diseases.

Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease.

Small- and intermediate-conductance Ca2+-activated K+ channels, KCa2.3 and KCa3.1, are involved in cellular signaling processes associated with inflammation and fibrosis. KCa2.3 and KCa3.1 are upregulated by proinflammatory cytokines and profibrotic growth factors. Cyclic AMP, which downregulates KCa2.3 and KCa3.1, is elevated by modafinil in cells; accordingly, we investigated whether modafinil exerts anti-inflammatory and anti-fibrotic responses via KCa2.3- and KCa3.1-mediated pathways in high-fat diet (HFD)- or thioacetamide-induced liver disease models in mice. Modafinil was administered orally in the form of a racemate, (R)-isomer, or (S)-isomer. We also determined whether the treatment targeted the profibrotic activity of hepatic stellate cells using immortalized human hepatic stellate cells (LX-2 cells). Modafinil improved HFD- or thioacetamide-induced changes compared to the control, leading to a reduced inflammatory response, collagen deposition, and α-smooth muscle actin expression both in vivo and in vitro. However, modafinil did not relieve HFD-induced steatosis. There were no significant differences in the effects of the (R)- and (S)-isomers of modafinil. KCa2.3 and KCa3.1 were upregulated and catalase was downregulated in liver tissues from thioacetamide- or HFD-induced liver disease models or in TGF-ß-treated LX-2 cells. TGF-ß-induced upregulation of KCa2.3, KCa3.1, collagen, and α-smooth muscle actin and downregulation of catalase were reversed by modafinil, polyethylene glycol catalase, N-acetylcysteine, siRNA against KCa2.3 or KCa3.1, and Epac inhibitors. Our investigation revealed that modafinil attenuated inflammatory and fibrotic progression via KCa2.3- and KCa3.1-mediated pathways in nonalcoholic hepatitis, suggesting that inhibiting KCa2.3- and KCa3.1-mediated signaling may serve as a novel therapeutic approach for inflammatory and fibrotic liver diseases.

Internalization and Transportation of Endothelial Cell Surface KCa2.3 and KCa3.1 in Normal Pregnancy and Preeclampsia.

Altered redox state modulates the expression levels of endothelial KCa2.3 and KCa3.1 (KCas) in normal pregnancy (NP) and preeclampsia (PE), thereby regulating vascular contractility. The mechanisms underlying KCas endocytosis and transportation remain unknown. We investigated the regulation of KCas expression in plasma membrane (PM) during NP and PE. Cultured human uterine artery endothelial cells were incubated in serum from normal nonpregnant women and women with NP or PE, or in oxidized LDL-, or lysophosphatidylcholine- (LPC-) containing a medium for 24 hours. NP serum elevated PM levels of KCas and reduced caveolin-1 and clathrin levels. PE serum, oxidized LDL, or LPC reduced PM levels of KCas and elevated caveolin-1, clathrin, Rab5c, and early endosome antigen-1 (EEA1) levels. Reduced KCas levels by PE serum or LPC were reversed by inhibition of caveolin-1, clathrin, or EEA1. Catalase and glutathione peroxidase 1 (GPX1) knockdown elevated PM-localized KCas levels and reduced caveolin-1 and clathrin levels. Elevated KCa2.3 levels upon catalase and GPX1 knockdown were reversed by PEG-catalase treatment. An H2O2 donor reduced clathrin and Rab5c. In contrast, elevated clathrin, caveolin-1, or colocalization of caveolin-1 with KCa3.1 by PE serum or LPC was reversed by NADPH oxidase inhibitors or antioxidants. A superoxide donor xanthine+xanthine oxidase elevated caveolin-1 or Rab5c levels. We concluded that KCas are endocytosed in a caveola- or a clathrin-dependent manner and transported in a Rab5c- and EEA1-dependent manner during pregnancy. The endocytosis and transportation processes may slow down via H2O2-mediated pathways in NP and may be accelerated via superoxide-mediated pathways in PE.

Altered Redox State Modulates Endothelial KCa2.3 and KCa3.1 Levels in Normal Pregnancy and Preeclampsia.

AIMS: Altered redox state has been related to the development of normal pregnancy (NP) and preeclampsia (PE). Endothelial KCa2.3 and KCa3.1 (KCas) play an important role in vasodilation, and KCas levels are affected by oxidative stress. We investigated the mechanisms of oxidative stress-mediated KCas expression modulation during NP and PE. RESULTS: Human uterine microvascular endothelial cells were incubated in serum from normal nonpregnant women (n = 13) and women with NP (n = 24) or PE (n = 15), or in vascular endothelial growth factor (VEGF), oxidized low-density lipoprotein (ox-LDL), progesterone, or estradiol-17ß (E2)-containing medium for 24 h. NP serum elevated H2O2 levels via reducing catalase and glutathione peroxidase 1 levels, thereby enhancing KCas levels via a H2O2/fyn/extracellular signal-regulated kinase (ERK)-mediated pathway. VEGF enhanced H2O2 and KCas levels and KCa3.1 currents. KCas were upregulated and KCas activation-induced endothelium-dependent relaxation (EDR) was augmented in vessels from pregnant mice and rats. Whereas PE serum, ox-LDL, progesterone, or soluble fms-like tyrosine kinase 1 (sFlt-1) elevated superoxide levels via elevating NADPH oxidase 2 (NOX2) and NOX4 levels and reducing superoxide dismutase (SOD) 1 levels, thereby downregulating KCas. sFlt-1 inhibited EDR. PE serum- or progesterone-induced alterations in levels of KCas were reversed by polyethylene glycol-SOD, NOX inhibition, or E2. Innovation and Conclusions: This is the first study of how endothelial KCas levels are modulated during NP and PE. KCas were upregulated by soluble serum factors such as VEGF via H2O2 generation in NP, and were downregulated by serum factors such as progesterone and ox-LDL via superoxide generation in PE, which may contribute to hemodynamic adaptations in NP or to the development of PE.

KCa 3.1 upregulation preserves endothelium-dependent vasorelaxation during aging and oxidative stress.

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium-dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial KCa 3.1, which contributes to EDR, is upregulated by H2 O2 . We investigated whether KCa 3.1 upregulation compensates for diminished EDR to NO during aging-related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1-phosphate were increased in aged wild-type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild-type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age-matched wild-type mice. Increased H2 O2 levels induced Fyn and extracellular signal-regulated kinases (ERKs) phosphorylation and KCa 3.1 upregulation. Catalase/GPX1 double knockout (catalase(-/-) /GPX1(-/-) ) upregulated KCa 3.1 in MAECs. NO production was decreased in aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) MAECs. However, KCa 3.1 activation-induced, N(G) -nitro-l-arginine-, and indomethacin-resistant EDR was increased without a change in acetylcholine-induced EDR in aortic rings from aged wild-type, CerS2 null, and catalase(-/-) /GPX1(-/-) mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1-phosphate induced similar changes in levels of the antioxidant enzymes and upregulated KCa 3.1. Our findings suggest that, during aging-related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H2 O2 and thereby upregulate KCa 3.1 expression and function via a H2 O2 /Fyn-mediated pathway. Altogether, enhanced KCa 3.1 activity may compensate for decreased NO signaling during vascular aging.

Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via K(Ca) 1.1 upregulation.

K(Ca) 1.1 regulates smooth muscle contractility by modulating membrane potential, and age-associated changes in K(Ca) 1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K(Ca) 1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2-null mice. The levels of C16- and C18-ceramides, sphinganine, sphingosine, and sphingosine 1-phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild-type and young CerS2-null mice. The altered SL composition upregulated K(Ca) 1.1 and increased K(Ca) 1.1 currents, while no change was observed in K(Ca) 1.1 channel activity. The upregulation of KC a 1.1 impaired intracellular Ca²âºmobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3-kinase, protein kinase Cζ , c-Jun N-terminal kinases, and nuclear factor kappa-B were found to be involved in K(Ca) 1.1 upregulation. Our findings suggest that age-associated changes in SL composition or CerS2 ablation upregulate K(Ca) 1.1 via the phosphoinositide 3-kinase/protein kinase Cζ /c-Jun N-terminal kinases/nuclear factor kappa-B-mediated pathway and impair Ca²âº mobilization, which thereby induces the contractile dysfunction of GSM. CerS2-null mice exhibited similar effects to aged wild-type mice; therefore, CerS2-null mouse models may be utilized for investigating the pathogenesis of aging-associated motility disorders.

Isolation and in vitro culture of vascular endothelial cells from mice.

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidate the disease mechanism. Although the mouse model has many advantages for in vivo and in vitro research, efficient procedures for the isolation and propagation of primary mouse EC have been problematic. We describe a high yield process for isolation and in vitro culture of primary EC from mouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle of Willis). Mouse arteries were carefully dissected without damage under a light microscope, and small pieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growth supplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetal calf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity of the cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized by a monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers for von-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Our simple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

Globotriaosylceramide induces lysosomal degradation of endothelial KCa3.1 in fabry disease.

OBJECTIVE: Globotriaosylceramide (Gb3) induces KCa3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces KCa3.1 endocytosis and degradation. APPROACH AND RESULTS: KCa3.1, especially plasma membrane-localized KCa3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced KCa3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress-inducing agents did not induce KCa3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated KCa3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered KCa3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored KCa3.1 expression, the current, and endothelium-dependent relaxation. CONCLUSIONS: -Gb3 accelerates the endocytosis and lysosomal degradation of endothelial KCa3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells.

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K(+) current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 µM Ca(2+) and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K(+) current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K(+) current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia.

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1-NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia.

Modafinil inhibits K(Ca)3.1 currents and muscle contraction via a cAMP-dependent mechanism.

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator.

Globotriaosylceramide leads to K(Ca)3.1 channel dysfunction: a new insight into endothelial dysfunction in Fabry disease.

AIMS: Excessive endothelial globotriaosylceramide (Gb3) accumulation is associated with endothelial dysfunction and impaired endothelium-dependent relaxation in Fabry disease. In endothelial cells, K(Ca)3.1 channels contribute to endothelium-dependent relaxation. However, the effect of Gb3 on K(Ca)3.1 channels and the underlying mechanisms of Gb3-induced dysfunction are unknown. Herein, we hypothesized that Gb3 accumulation induces K(Ca)3.1 channel dysfunction and aimed to clarify the underlying mechanisms. METHODS AND RESULTS: The animal model of Fabry disease, α-galactosidase A (Gla) knockout mice, displayed age-dependent K(Ca)3.1 channel dysfunction. K(Ca)3.1 current and the channel expression were significantly reduced in mouse aortic endothelial cells (MAECs) of aged Gla knockout mice, whereas they were not changed in MAECs of wild-type and young Gla knockout mice. In addition, K(Ca)3.1 current and the channel expression were concentration-dependently reduced in Gb3-treated MAECs. In both Gb3-treated and aged Gla knockout MAECs, extracellular signal-regulated kinase (ERK) and activator protein-1 (AP-1) were down-regulated and repressor element-1 silencing transcription factor (REST) was up-regulated. Gb3 inhibited class III phosphoinositide 3-kinase and decreased intracellular levels of phosphatidylinositol 3-phosphate [PI(3)P]. In addition, endothelium-dependent relaxation was significantly attenuated in Gb3-treated mouse aortic rings. CONCLUSION: Gb3 accumulation reduces K(Ca)3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes K(Ca)3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

Modulation of nonselective cation current by oxidized LDL and lysophosphatidylcholine and its inhibitory contribution to endothelial damage.

AIMS: This study examined the effects of oxidized low-density lipoprotein (LDL) and its major lipid constituent lysophosphatidylcholine (LPC) on nonselective cation (NSC) current and its inhibitory contribution to LPC-induced cytotoxicity in cultured human umbilical endothelial cells (HUVECs). MAIN METHODS: Patch-clamp technique and the resazurin-based cell viability assay were used. KEY FINDINGS: In voltage-clamped cells, oxidized LDL or LPC slowly activated NSC current. NSC current was also activated by loading cells with Ca(2+) solution buffered at various concentrations using a patch pipette or by applying the sarcoplasmic reticulum Ca(2+) pump blocker 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), the metabolic inhibitor CN(-) or the hydroperoxide donor tert-butyl hydroperoxide (TBHP). On the contrary, when intracellular Ca(2+) was strongly buffered with 12mM BAPTA or cells were loaded with superoxide dismutase using a patch pipette, LPC or BHQ did not activate NSC current. Furthermore, NSC current activated by LPC, TBHP or CN(-) was inhibited by the antioxidant tempol or extracellular Ca(2+) depletion and NSC current activated by intracellular Ca(2+) was further augmented by oxidized LDL or LPC. LPC or oxidized LDL released Ca(2+) from intracellular stores and further enhanced store-operated Ca(2+) entry. LPC-induced cytotoxicity was augmented by inhibiting Ca(2+) influx and NO synthesis. SIGNIFICANCE: Oxidized LDL or its main component LPC activated Ca(2+)-permeable NSC current via releasing Ca(2+) from intracellular stores and producing ROS and thereby increased Ca(2+) influx. Ca(2+) influx through NSC channel might protect endothelial cells by producing NO.

Superoxide generated by lysophosphatidylcholine induces endothelial nitric oxide synthase downregulation in human endothelial cells.

We examined the mechanism through which lysophosphatidylcholine (LPC) induces endothelial nitric oxide (eNOS) downregulation. Human umbilical vein endothelial cells (HUVECs) were treated with LPC (50-150 microM) for 0.5-2 h or the reactive oxygen species (ROS) donors, xanthine/xanthine oxidase (X/XO), 1,4-hydroquinone (HQ) or tert-butylhydroperoxide (TBHP) for 2 h. Protein levels of eNOS, superoxide dismutase1 (SOD1), catalase, and phospho-extracellular signal regulated kinase 1/2 (pERK 1/2) were assessed using immunoblotting. LPC treatment reduced SOD1 levels but increased catalase levels. The superoxide donors X/XO and HQ showed similar effects. The hydroperoxide donor TBHP increased SOD1 levels but did not change catalase levels. LPC concentration- and time-dependently decreased eNOS levels, but this effect was blocked by antioxidants and SOD and potentiated by the SOD1 inhibitor, ammonium tetrathiomolybdate. LPC and X/XO inhibited ERK1/2 phosphorylation, whereas TBHP stimulated phosphorylation. Taken together, these data indicate that LPC induces superoxide overload in HUVECs via SOD1 inhibition and downregulates phospho-ERK1/2 and eNOS levels.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca Mobilization in Human Endothelial Cells.

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca(2+) entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca(2+) concentration ([Ca(2+)](i)), and the increase of [Ca(2+)](i) by OxLDL or by LPC was inhibited by La(3+) or heparin. LPC failed to increase [Ca(2+)](i) in the presence of an antioxidant tempol. In addition, store-operated Ca(2+) entry (SOC), which was evoked by intracellular Ca(2+) store depletion in Ca(2+)-free solution using the sarcoplasmic reticulum Ca(2+) pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca(2+)](i) and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La(3+) or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca(2+) to 1 microM activated large-conductance Ca(2+)-activated K(+) (BK(Ca)) current spontaneously, and this activated BK(Ca) current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca(2+)-permeable Ca(2+)-activated NSC current and BK(Ca) current simultaneously, thereby increasing SOC.

Intracellular Na(+) modulates large conductance Ca(2+)-activated K (+) currents in human umbilical vein endothelial cells.

We studied the effects of Na(+) influx on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na(+) replacement by NMDG(+) or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na(+) concentration ([Na(+)](i)) and vice versa. In addition, extracellular Na(+) depletion decreased [Na(+)](i). In voltage-clamped cells, BK(Ca) currents were markedly increased by extracellular Na(+) depletion. In inside-out patches, increasing [Na(+)](i) from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK(Ca) channels and decreasing intracellular K(+) concentration ([K(+)](i)) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na(+)](i) gradually from 0 to 70 mM, by replacing K(+), markedly reduced single channel conductance and NPo. The Na(+)-Ca(2+) exchange blocker Ni(2+) or KB-R7943 decreased [Na(+)](i) and increased BK(Ca) currents simultaneously, and the Na(+) ionophore monensin completely inhibited BK(Ca) currents. BK(Ca) currents were significantly augmented by increasing extracellular K(+) concentration ([K(+)](o)) from 6 to 12 mM and significantly reduced by decreasing [K(+)](o) from 12 or 6 to 0 mM or applying the Na(+)-K(+) pump inhibitor ouabain. These results suggest that intracellular Na(+) inhibit single channel conductance of BK(Ca) channels and that intracellular K(+) increases single channel conductance and NPo.

The Na(+)/Ca(2+) exchanger inhibitor KB-R7943 activates large-conductance Ca(2+)-activated K(+) channels in endothelial and vascular smooth muscle cells.

The effect of the selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), KB-R7943, on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK(Ca) currents in HUVECs and MASMCs. The EC(50) of KB-R7943 for BK(Ca) current activation in HUVECs was determined to be 6.78+/-0.7 microM. In inside-out and outside-out patches, KB-R7943 markedly increased BK(Ca) channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca(2+)](i) and open probability (P(o)) to the left; the [Ca(2+)](i) required to evoke half-maximal activation changed from 1220+/-68 nM (in the absence of KB-R7943) to 620+/-199 nM (in the presence of 10 microM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P(o) to the left; the membrane potential to evoke half-maximal activation changed from 76.86+/-1.09 mV (in the absence of KB-R7943) to 49.62+/-2.55 mV (in the presence of 10 microM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK(Ca) channel activator, which increases the sensitivity of BK(Ca) channels to cytosolic free Ca(2+) and membrane potential, and thereby BK(Ca) channel activity. These results should be considered when KB-R7943 is used as NCX blocker.

Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells.

The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca(2+)](i). [Ca(2+)](i) and membrane potentials were strongly correlated. In whole cell clamped cells, BK(Ca) currents were activated by increasing [Ca(2+)](i) via cell dialysis with pipette solution, and the activated BK(Ca) currents were further enhanced by S1P. When [Ca(2+)](i) was buffered at 1 microM, the S1P concentration required to evoke half-maximal activation was 403 +/- 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK(Ca) channel activity in a reversible manner and shifted the relationship between Ca(2+) concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; 1 mM) using a patch pipette, GDPbetaS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK(Ca) current and channel activation. These results suggest that S1P enhances BK(Ca) channel activity by increasing Ca(2+) sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca(2+) influx through Ca(2+) entry channels. Inasmuch as S1P activates BK(Ca) channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca(2+) mobilization in human endothelial cells.

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta.

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.

Contribution of Na+-K+ pump and KIR currents to extracellular pH-dependent changes of contractility in rat superior mesenteric artery.

We compared the branches and trunk of rat superior mesenteric artery (SMA) with respect to extracellular pH (pHo)-dependent changes in vascular contractility. Decreases in pHo from 7.8 to 6.4 significantly reduced apparent affinity (pD2) to norepinephrine (NE) and maximal contraction by NE, which were more prominent in larger-diameter arteries. On the other hand, decreases in pHo significantly reduced Ba2+-sensitive K+-induced relaxation (which was evoked by elevation of extracellular K+ concentration from 6 to 12 mM) in the first branch and inhibited inwardly rectifying K+ (K(IR)) currents in cultured smooth muscle cells (SMCs) of SMA. RT-PCR revealed transcripts for Kir2.1 in the SMCs. Real-time PCR analysis revealed 6.1-, 3.3-, and 2.2-fold increases in the Kir2.1 mRNA-to-beta-actin mRNA ratios of SMCs of the third, second, and first branches, respectively, vs. the corresponding relative levels of trunk SMCs. The magnitudes of K+-induced relaxation were significantly greater in smaller-diameter arteries, and there was a strong correlation between the transcript levels of Kir2.1 and K+-induced relaxation. A decrease in pHo reduced ouabain-sensitive K+-induced relaxation and ouabain-induced contraction. A decrease in pHo from 7.4 to 6.4 depolarized membrane potential of the cultured SMCs. From these results, we conclude that an increase in pHo activates K(IR) currents and the Na+ -K+ pump, which then reduces vascular contractility. Inasmuch as K(IR) channel densities are significantly greater in smaller-diameter arteries, the reduction in vascular contractility on increasing pHo is more pronounced in smaller-diameter arteries.