Na+/H+ exchanger inhibitor prevented endothelial dysfunction induced by high glucose

The aims of this study were to examine whether cariporide, a selective Na+/H+ exchanger inhibitor, has protective effects against endothelial dysfunction induced by high glucose in vitro and to investigate the potential mechanisms. Exposure of rat aorta rings to high glucose (44 mmol/L) for 6 hours caused an inhibition of acetylcholine-induced endothelium-dependent relaxation but had no effect on sodium nitroprusside-induced endothelium-independent relaxation. Treatment with cariporide (0.01, 0.1, 1 micromol/L) of aortic rings incubated with high-glucose medium attenuated the impaired endothelium-dependent relaxation in a dose-dependent manner. Furthermore, high glucose resulted in an increase of malondialdehyde and a decrease of superoxide dismutase activity in rat aorta rings, and these effects were reversed by cariporide. In addition, cariporide was able to inhibit the activation of Na+/H+ exchanger induced by high glucose in cultured endothelial cells. These findings suggest that the endothelial dysfunction induced by high glucose in vitro is caused by the activation of Na+/H+ exchanger.     

Topics on the Na+/Ca2+ exchanger: responses of Na+/Ca2+ exchanger to interferon-gamma and nitric oxide in cultured microglia

The Na(+)/Ca(2+) exchanger (NCX) plays a role in regulation of intracellular Ca(2+) levels, but little is known about the functional role of NCX in microglia. To clarify the role of NCX in microglia, we studied the responses of NCX to pathological conditions such as interferon-gamma or nitric oxide (NO) exposure. Treatment with interferon-gamma caused a biphasic increase in NCX activity. The delayed increase in NCX activity was accompanied by increases in the mRNA and protein levels. Pharmacological studies show that protein kinase C and tyrosine kinase are involved in the transient and delayed increases in NCX activity, and the extracellular signal-regulated protein kinase is involved in the delayed increase in NCX activity. On the other hand, NO causes apoptotic cell death in cultured microglia. We observed, using the specific NCX inhibitor SEA0400, that NO activates NCX activity and NCX is involved in NO-induced depletion of Ca(2+) in the endoplasmic reticulum (ER), leading to ER stress. These results suggest that NCX is involved in the regulation of Ca(2+) levels in the ER. The responses of NCX to interferon-gamma and NO implies that NCX plays a key role in microglial function.     

Effects of volume depletion and NaHCO3 loading on the expression of Na+/H+ exchanger isoform 3, Na+: HCO3- cotransporter type 1 and nitric oxide synthase in rat kidney

1. The effects of volume depletion and NaHCO(3) loading on the expression of Na(+)/H(+) exchanger isoform 3 (NHE3), Na(+) : HCO(3)(-) cotransporter type 1 (NBC1) and neuronal (n) and inducible (i) isoforms of nitric oxide synthase (NOS) were determined in rat kidney. 2. Adult male Sprague-Dawley rats were used. Rats were divided into four groups: (i) euvolaemic (EC); (ii) hypovolaemic (HC); (iii) euvolaemia with NaHCO(3) loading (EB); and (iv) hypovolaemia with NaHCO(3) loading (HB). The expression of NHE3, NBC1, nNOS and iNOS proteins was determined in the cortex of the kidney by immunoblotting and immunohistochemistry. Tissue content of nitric oxide (NO) metabolites (NO(x)) were also determined in the cortex using a colourimetric assay. 3. Compared with the EC group, the expression of NHE3 and NBC1 was increased in the HC group and decreased in the EB group. Comparing the EB and HB groups, the expression of NHE3 and NBC1 was higher in the latter group. The expression of NHE3 was decreased and that of NBC1 was increased in the HB group compared with the HC group. The NO(x) content and nNOS expression were decreased in the hypovolaemic (HC) and NaHCO(3)-loaded (EB and HB) rats. Moreover, the expression of iNOS was decreased in the HB group compared with the other groups. 4. An altered volume status and NaHCO(3) loading may affect the regulation of NHE3 and NBC1 in the kidney and the endogenous NO system may play a role in the observed effects.     

高糖抑制牛主动脉内皮细胞诱导型和结构型一氧化氮合酶的表达

目的:研究高糖对新生小牛主动脉内皮细胞(BAEC)一氧化氮合酶(NOS)表达的影响。方法:BAEC培养并传代于含正常葡萄糖(5.5mmol·L~(-1),NGBAEC),高糖(25 mmol·L~(-1),HG-BAEC)或高渗(葡萄糖5.5 甘露醇19.5 mmol·L~(-1),Mann-BAEC)的无酚红M1640培养基.Griess反应检测脂多糖(LPS)诱导的一氧化氮(NO)产生.Westem blot法检测结构型NOS(ecNOS)及诱导型NOS(iNOS)表达。结果:LPS(0.25-2 mg·L~(-1))剂量依赖性刺激BAEC产生NO,并在LPS 1 mg·L~(-1)达峰值。高糖显著抑制LPS诱导的NO产生(亚硝酸μmol·L~(-1):HG-BAEC 43±8,vs NG-BAEC 71±11,Mann-BAEC 70±9,n=4,P<0.01)。同样,与NG-和Mann-BAEC相比,HG-BAEC iNOS表达下降39.9％和39.3％,ecNOS表达下降28％和24％,而NG-与Mann-BAEC之间,LPS诱导的NO产量和iNOS和ecNOS的表达无差别。结论:高糖抑制BAEC NO的释放,与NOS的低表达有关。     

Regulation of endothelial nitric oxide synthase and endothelin-1 expression by fluvastatin in human vascular endothelial cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on endothelial vasoactive substances using human umbilical vein endothelial cells (HUVECs). Incubation of HUVECs with fluvastatin for 12 h increased endothelial nitric oxide synthase (eNOS) mRNA expression in a concentration-dependent manner (peak, 276 +/- 38%, mean +/- S.D., of the control, at 1.0 microM fluvastatin, P<0.01). In addition, fluvastatin increased eNOS protein production (245 +/- 51% of the control level, P<0.05) as well as nitrite production (165 +/- 35% of the control level, P<0.01). In contrast, incubation of HUVECs with 1.0 microM fluvastatin for 12 h significantly reduced the production of endothelin-1 (ET-1) and preproET-1 mRNA expression in HUVECs (28 +/- 1% and 39 +/- 1% of the control level, respectively, P<0.01). Our results suggest that fluvastatin might be involved in improvement of endothelial function and prevention of the progression of atherosclerosis.     

Na +/H + exchanger inhibitor induces vasorelaxation through nitric oxide production in endothelial cells via intracellular acidification-associated Ca2 + mobilization

The objective of this study was to determine the mechanism by which Na⁺/H⁺ exchanger (NHE) inhibitors induce vasodilatation. The NHE inhibitors, 5-(N,N-dimethyl)-amiloride (DMA), cariporide, and amiloride, evoked endothelium-dependent relaxation in rat aortas with ED₅₀ values of 16, 89, and 148 μM, respectively, and these effects were abolished by treatment with N^G-nitro-l-arginine methyl ester (L-NAME). The relaxation effects induced by DMA and cariporide were strongly attenuated in aortas of the endothelial NO synthase (eNOS)-deficient mice, as compared to the effects in wild-type mice. The DMA-induced relaxation in rat aorta was attenuated by a calmodulin (CaM) inhibitor, calmidazolium, and a soluble guanylyl cyclase inhibitor, [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but was not affected by a phosphoinositide 3-kinase inhibitor, wortmannin. Immunoblots for endothelial eNOS on immunoprecipitated CaM complexes showed that DMA enhanced the association of eNOS with CaM in rat aortas. Both DMA and cariporide induced the reduction of intracellular pH (pHi) in bovine aortic endothelial cells (BAECs), which was accompanied by a sustained elevation of cytosolic Ca^2 + ([Ca^2 +]i). This DMA-induced rise of [Ca^2 +]i was not affected by removing external Ca^2 + from the buffer, but was abolished in thapsigargin-pretreated BAECs. These results suggest that lowering of pHi by NHE inhibitors in endothelial cells induces the mobilization of Ca^2 + from the thapsigargin-sensitive stores of endoplasmic reticulum, which in turn stimulates NO production via the CaM-dependent activation of eNOS.     

Celecoxib modulates adhesion of HT29 colon cancer cells to vascular endothelial cells by inhibiting ICAM-1 and VCAM-1 expression

BACKGROUND AND PURPOSE: Cyclooxygenase-2 (COX-2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX-2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM-1 and VCAM-1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS-coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM-1 and VCAM-1, to assess further the potential protective effects of celecoxib on cancer development. EXPERIMENTAL APPROACH: Celecoxib was incubated for 4 h with HT29 cells and HUVEC and adhesion was quantified by a computerized micro-imaging system. Expression analysis of ICAM-1 and VCAM-1 cell adhesion molecules was performed by western blot. KEY RESULTS: Celecoxib (1 nM-10 microM) inhibited, with the same potency, adhesion of HT29 cells to resting HUVEC or to HUVEC stimulated by tumour necrosis factor-alpha (TNF-alpha), mimicking inflammatory conditions. Analysis of ICAM-1 and VCAM-1 expression showed that celecoxib inhibited expression of both molecules in TNF-alpha-stimulated HUVEC, but not in resting HUVEC; inhibition was concentration-dependent and maximal (about 50%) at 10 microM celecoxib. CONCLUSIONS AND IMPLICATIONS: In conclusion, our data show that celecoxib inhibits HT29 cell adhesion to HUVEC and expression of ICAM-1 and VCAM-1, in stimulated endothelial cells. These effects may contribute to the chemopreventive activity of celecoxib in the development of colorectal cancer.     

Aspirin and PPAR-alpha activators inhibit monocyte chemoattractant protein-1 expression induced by high glucose concentration in human endothelial cells

Activated endothelial cells express monocyte chemoattractant protein-1 (MCP-1), a chemokine which is reportedly involved in the recruitment of plasma monocytes in the early stages of atherosclerosis. Since accelerated atherosclerosis is the main complication of diabetes and both diseases encompass an inflammatory reaction, we hypothesized that the anti-inflammatory drugs, aspirin and peroxisome proliferator-activated receptor (PPAR-alpha) activators (fenofibrate and clofibrate), could have an effect on the high glucose-induced MCP-1 expression in endothelial cells. To test this assumption, as well as the possible mechanisms involved, the MCP-1 expression and secretion, the reactive oxygen species levels, nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) expression were determined in human endothelial cells exposed to high glucose concentrations in the presence of aspirin, fenofibrate and clofibrate. Human endothelial cells kept in normal glucose concentration in the absence of drugs were used as control. The results showed that (i) aspirin, fenofibrate and clofibrate decrease significantly the MCP-1 expression and secretion in human endothelial cells; (ii) the high glucose up-regulated expression of MCP-1 in endothelial cells was significantly reduced by inhibitors of NF-kB and reactive oxygen species; (iii) all drugs notably decrease the level of the reactive oxygen species and activation of NF-kB and AP-1. Together, the findings indicate that in endothelial cells aspirin and PPAR-alpha activators reduce the high glucose-increased expression of MCP-1 by a mechanism that includes the inhibition of reactive oxygen species, and decrease of AP-1 and NF-kB activation.     

Kinin B1 receptors stimulate nitric oxide production in endothelial cells: signaling pathways activated by angiotensin I-converting enzyme inhibitors and peptide ligands

We reported previously a novel mode of action of angiotensin I-converting enzyme (kininase II; ACE) inhibitors mediated through the direct activation of bradykinin B(1) receptor, independent of endogenous kinins or ACE (J Biol Chem 277:16847-16852, 2002). We aimed to further clarify the mechanism of activation of B(1) receptor, which leads to prolonged nitric oxide (NO) release. The ACE inhibitor enalaprilat and the peptide ligand desArg(10)-kallidin (in nanomolar concentrations) release NO by activating endothelial NO synthase (eNOS) in bovine and inducible NO synthase (iNOS) in stimulated human endothelial cells. The peptide and the ACE inhibitor ligands activate eNOS by facilitating different signaling pathways. DesArg(10)-kallidin enhances inositol-phosphate generation and elevates [Ca(2+)](i) by first augmenting intracellular release and then the influx of extracellular Ca(2+). In contrast, enalaprilat stimulates only the influx of extracellular Ca(2+) through rare earth-sensitive channels, and its effect is blocked by cholera toxin or protein kinase C inhibitors. In addition, unlike desArg(10)-kallidin, enalaprilat can also release NO independent of Ca(2+) in bovine endothelial cells. The inflammatory cytokines interleukin-1beta and interferon-gamma induce both B(1) receptor and iNOS in human endothelial cells. In contrast to eNOS, B(1) ligands activate iNOS similarly. Both desArg(10)-kallidin and ACE inhibitors enhance arginine uptake and release NO independent of [Ca(2+)](i) elevation. This is the first report on the direct activation of B(1) receptor by ACE inhibitors in human endothelial cells. This interaction leads to prolonged NO release and possibly contributes to the documented benefits of the use of ACE inhibitors.     

野百合碱对培养的肺动脉内皮细胞NO含量、eNOS蛋白表达及肺动脉平滑肌细胞收缩性的影响

目的研究野百合碱(monocrotaline pyrrole,MCTP)对培养的牛肺动脉内皮细胞(calf pulmonary artery endothelial cells,CPAEs)一氧化氮(NO)含量、内皮型一氧化氮合酶(eNOS)蛋白表达及肺动脉平滑肌细胞收缩的影响。方法采用荧光共聚焦激光显微镜检测培养的牛肺动脉内皮细胞的NO含量;Western blot检测eNOS蛋白表达;胶原凝胶实验分析肺动脉平滑肌细胞收缩性。结果经野百合碱处理的牛肺动脉内皮细胞NO含量和eNOS蛋白表达较对照组明显减少、肺动脉平滑肌细胞收缩性增强。结论MCTP可抑制eNOS蛋白表达,肺动脉内皮细胞NO产生受到抑制,使肺动脉平滑肌细胞收缩性增强,这些改变与肺动脉高压的形成可能存在一定关系。     

Lipophilic fractions from the marine sponge Halichondria sitiens decrease secretion of pro-inflammatory cytokines by dendritic cells and decrease their ability to induce a Th1 type response by allogeneic CD4+ T cells

Context: Halichondria (Halichondriidae) marine sponges contain components possessing various biological activities, but immunomodulation is not among the ones reported.

Objective: This study evaluated the immunomodulatory effects of fractions/compounds from Halichondria sitiens Schmidt.

Materials and methods: Crude dichloromethane/methanol extracts of H. sitiens were subjected to various chromatographic techniques to obtain fractions/compounds with immunomodulatory activity, using bioassay-guided isolation. The effects of the fractions/compounds were determined by measuring secretion of cytokines and expression of surface molecules by dendritic cells (DCs) and their ability to stimulate and modify cytokine secretion by allogeneic CD4⁺ T cells. The bioactive fractions were chemically analyzed to identify the immunomodulatory constituents by 1D, 2D NMR, and HRMS data.

Results: Several lipophilic fractions from H. sitiens at 10?μg/mL decreased secretion of the pro-inflammatory cytokines IL-12p40 and IL-6 by the DCs, with maximum inhibition being 64% and 25%, respectively. In addition, fractions B3b3F and B3b3J decreased the ability of DCs to induce T cell secretion of IFN-γ. Fraction B3b3 induced morphological changes in DCs, characterized by extreme elongation of dendrites and cell clustering. Chemical screening revealed the presence of glycerides and some minor unknown constituents in the biologically active fractions. One new glyceride, 2,3-dihydroxypropyl 2-methylhexadecanoate (1), was isolated from one fraction and two known compounds, 3-[(1-methoxyhexadecyl)oxy]propane-1,2-diol (2) and monoheptadecanoin (3), were identified in another, but none of them had immunomodulatory activity.

Discussion and conclusions: These results demonstrate that several lipophilic fractions from H. sitiens have anti-inflammatory effects on DCs and decrease their ability to induce a Th1 type immune response.     

17Beta-oestradiol regulates the expression of Na+/K+-ATPase beta1-subunit, sarcoplasmic reticulum Ca2+-ATPase and carbonic anhydrase iv in H9C2 cells

1. It is necessary to improve our understanding of the effect of 17beta-oestradiol (E2) on the heart at a molecular and cellular level. In the present study, the effects of E2 on Na(+)/K(+)-ATPase, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and carbonic anhydrase IV (CAIV) in H9C2 cells were investigated. To identify the mechanism of action of E2 on these proteins, the oestrogen receptor (ER) antagonist tamoxifen was used. 2. The results indicated that 1 and 100 nmol/L E2 can enhance the activity of Na(+)/K(+)-ATPase and SERCA and upregulate the expression of the Na(+)/K(+)-ATPase beta1-subunit, SERCA2a and CAIV at both the mRNA and protein level compared with 0 and 0.01 nmol/L E2. 17beta-Oestradiol had the greatest effect at 100 nmol/L; 1 micromol/L E2 did not further protein expression compared with 100 nmol/L E2. 3. Tamoxifen (10 nmol/L) significantly decreased the activity of SERCA, as well as the expression of the Na(+)/K(+)-ATPase beta1-subunit and SERCA at the mRNA and protein level, in H9C2 cells cultured with 1 nmol/L E2. Tamoxifen alone had no significant effect on these proteins in H9C2 cells. 4. It may be hypothesized that a suitable E2 concentration has a protective effect on the heart and that the actual dose of E2 used in hormone-replacement therapy is important in menopausal women.     

Tanshinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxide synthase expression and production of TNF-alpha, IL-1beta and IL-6 in activated RAW 264.7 cells

The inhibitory effects of tanshinone IIA, a diterpene isolated from Salvia miltiorrhiza root, on the production of nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and the expression of inducible nitric oxide synthase (iNOS) were investigated in activated RAW 264.7 cells. This compound markedly inhibited the production of NO, IL-1beta and TNF-alpha, and suppressed the expression of iNOS in a dose-dependent manner. These results suggest that the traditional use of S. miltiorrhiza as an anti-inflammatory herbal medicine may be explained, in part, by the inhibition of NO, IL-1beta, IL-6 and TNF-alpha production, and expression of iNOS.     

下丘脑-垂体条件培养基对同型半胱氨酸作用的内皮细胞NOS和ICAM-1表达的影响

目的：探讨下丘脑－垂体条件培养基对同型半胱氨酸作用过的内皮细胞（EC）一氧化氮合酶（NOS）和血管细胞间粘附分子（ICAM－1）表达的影响。方法：取5个月龄水囊引产胚胎的下丘脑和垂体组织进行培养,并收集其条件培养基。用其条件培养基培养同型半胱氨酸（Hcy)作用的EC,用免疫细胞化学和图像分析方法检测其内皮细胞ICAM－1和NOS表达的变化。观察下丘脑－垂体分泌的神经因子对同型半胱氨酸作用后的内皮细胞ICAM－1和NOS表达是否有调控作用。结果：单纯同型半胱氨酸作用EC后,ICAM－1呈高表达,NOS呈略高表达,而用下丘脑－垂体条件培养基培养同型半胱氨酸作用过的内皮细胞后,NOS的表达明显上调（P＜0．05）,而对ICAM－1的高表达有明显下调作用（P＜0．05）。结论：EC受到同型半胱氨酸作用后,下丘脑－垂体水平可能通过上调内皮细胞NOS的表达和下调ICAM－1的表达来起保护作用,其条件培养基中可能存在对内皮细胞起调控作用的物质。     

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles induce CYP1A1 expression, become metabolized, and bind to macromolecules in sensitive human cancer cells.

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any "resistant" cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.     

Evidence for direct regulation of myocardial Na+/H+ exchanger isoform 1 phosphorylation and activity by 90-kDa ribosomal S6 kinase (RSK): effects of the novel and specific RSK inhibitor fmk on responses to alpha1-adrenergic stimulation

Multiple stimuli of physiological and pathophysiological significance, including alpha1-adrenoceptor agonists, stimulate the cardiac sarcolemmal Na+/H+ exchanger isoform 1 (NHE1) through activation of the mitogen-activated or extracellular signal-regulated kinase (ERK) kinase (MEK) ERK-90-kDa ribosomal S6 kinase (RSK) signaling cascade. However, the individual contributions of ERK and RSK, which can each phosphorylate the NHE1 regulatory domain, to such stimulation are unknown. In the present study, we have used the novel RSK inhibitor fmk to determine the role of RSK as a direct regulator of NHE1 phosphorylation and activity in response to alpha1-adrenergic stimulation, in ventricular myocytes isolated from the adult rat heart. Initial experiments confirmed that pretreatment of myocytes with fmk before exposure to the alpha1-adrenoceptor agonist phenylephrine inhibited RSK C-terminal kinase activity and thereby RSK N-terminal kinase activation, without affecting MEK or ERK activation. Pretreatment of myocytes with fmk also inhibited phenylephrine-induced increases in NHE1 phosphorylation and the rate of NHE1-mediated H+ efflux under conditions of intracellular acidosis. These findings reveal, for the first time to our knowledge, that RSK is the principal regulator of NHE1 phosphorylation and activity after alpha1-adrenergic stimulation in adult myocardium.