Choline uptake by mouse brain capillary endothelial cells in culture.

Choline, a precursor of the neurotransmitter acetylcholine, is synthesized in only small amounts in the brain, so the choline concentration in the brain may vary depending on the plasma concentration and the transport rate across the blood-brain barrier. To elucidate the transport mechanism of choline, we carried out uptake experiments with mouse brain capillary endothelial cells in culture (MBEC4). [3H]Choline uptake was linear for up to 5 min. An examination of the concentration dependence of [3H]choline uptake revealed the operation of both saturable (Jmax = 423+/-27 pmol min(-1) (mg protein)(-1) and Kt = 20.0+/-3.1 microM) and non-saturable (kd = 1.23+/-0.045 microL min(-1)(mgprotein)-1) processes. The saturable process was independent of Na+ and pH, but was dependent on membrane potential as a driving force. Various basic drugs and endogenous substances, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [3H]choline uptake. These data suggest that choline was taken up into the endothelial cells via two routes and that a membrane potential-dependent carrier-mediated transport system may participate in choline transport across the blood-brain barrier.     

Limethason as a lipid microsphere preparation: An overview

Limethason is an i.v. injectable lipid emulsion in which dexamethasone-21-palmitate is dissolved as an active ingredient. Limethason exhibited 2 to 5 times as potent anti-inflammatory activity as water-soluble dexamethasone phosphate on chronic inflammatory disease models. The strong anti-inflammatory activity of the drug was primarily based on a high distribution in the inflammatory lesion, a high uptake by macrophages and a suppressive effect on the macrophage function. A multicenter double-blind comparative clinical trial showed a tendency to a significantly higher rate of improvement with lower frequency of side effects in the Limethason group than the dexamethasone phosphate group. These results indicate that Limethason is more useful for rheumatoid arthritis and that the separation of the efficacy and side-effects of steroid could be clinically confirmed to some extent.     

Calcium mobilization in activated mast cells monitored by flow cytometric analysis

Changes in intracellular calcium levels induced in mast cells by either antigen or ionomycin were monitored using flow cytometry and the calcium binding dye indo-1. Both stimuli increased calcium levels in responsive cells by a similar amount, but not all cells examined were responsive to antigen. Antigen-induced increases in intracellular calcium levels could be completely blocked by the calcium antagonist TMB-8. Flow cytometry appears to be a useful method for monitoring mast cell calcium concentrations, as it provides the capability to study large numbers of individual cells.     

Analysis of heterogeneity in daunorubicin uptake by human leukemia cells using laser flow cytometry

Summary Heterogeneity in daunorubicin uptake by leukemic blast cells obtained from the bone marrow of 16 patients with acute leukemia (10 acute nonlymphocytic leukemia and 6 acute lymphocytic leukemia) was determined by laser flow cytometry following drug treatment in vitro for 1 hr. Fluorescence profiles of cellular anthracycline levels in the various samples indicated a marked heterogeneity in daunorubicin uptake and the range of detectable drug fluorescent cells was 34% – 99%. A retrospective analysis of disease outcome in these patients who were treated with a daunorubicin or doxorubicin containing induction regimen indicated the following: (a) 8 of 11 patients who entered complete remission had > 80% of leukemic blast cells accumulating detectable amounts of daunorubicin; and (b) 3 of 4 patients who did not respond to chemotherapy had < 40% of the leukemic blast cells accumulating detectable amounts of daunorubicin. These results suggest that laser flow cytometry may be useful in determining qualitative and quantitative differences in daunorubicin levels in heterogeneous tumor cell populations.     

Fluorescein uptake by a monocarboxylic acid transporter in human intestinal Caco-2 cells.

The fluorescein transport characteristics of the human intestinal epithelial Caco-2 cell line were examined in monolayer cultures. The initial uptake rate was concentration-dependent and saturable; the Michealis constant and the maximum velocity were 0.40 mM and 1.32 nmol/min/mg protein, respectively. A protonophore, carbonyl cyanide m-chlorophenyl-hydrazone, reduced uptake significantly. The replacement of extracellular sodium ions by lithium ions did not alter the initial uptake rate. These facts imply that the transport is driven by a proton gradient. The initial uptake rate was strongly dependent upon extracellular pH, and the uptake was optimal at approximately pH 5.5. Based on the protolytic constants, the main species of fluorescein in the pH range of 5.5 to 6.0 was calculated to be a monoanion, suggesting that fluorescein was taken up by Caco-2 cells as a monocarboxylate. The following findings support this conclusion: the uptake was inhibited significantly by monocarboxylate compounds such as salicylate and pravastatin, but not by di- or tricarboxylic acids or by acidic amino acids. Furthermore, salicylate-preloaded cells showed remarkably enhanced uptake of fluorescein, indicating that monocarboxylates and fluorescein share a common transport carrier. The transporter has a wide spectrum of substrate recognition and seems likely to be different from MCT1.     

Recombinant human erythropoietin stimulates vascular endothelial growth factor release by glomerular endothelial cells.

We designed the present study to address the question of whether recombinant human erythropoietin stimulates DNA synthesis and vascular endothelial growth factor (VEGF) secretion in vitro using cultured bovine glomerular endothelial cells (GENs). Recombinant human erythropoietin dose-dependently stimulated the proliferation of GENs in culture, and this proliferative effect was inhibited by 1 microg/ml anti-VEGF antiserum. The increase in VEGF concentrations in the supernatants containing 10 U/ml rHuEpo was abolished by incubation with 10 microg/ml of anti-human rHuEPO antiserum, 0.2 microg/ml actinomycin D or 10 microg/ml cycloheximide. Taken together, rHuEpo stimulates GEN proliferation in vitro and VEGF release from these cells is associated with stimulation of RNA-dependent DNA and protein synthesis.     

Effect of electromagnetic field on endocytosis of cationic solid lipid nanoparticles by human brain-microvascular endothelial cells

This study investigates the electromagnetic field (EMF)-regulated transport of cationic solid lipid nanoparticles (CSLNs) across human brain-microvascular endothelial cells (HBMECs). The positive charge of CSLNs was from dioctadecyldimethyl ammonium bromide and stearylamine, and radiofrequency EMF was applied to HBMECs for promoting uptake of CSLNs. Immunochemical staining revealed that the expression of clathrin on the membrane of HBMECs enhanced during vesicular endocytosis of CSLNs. However, CSLNs and EMF slightly affected the expression of P-glycoprotein on the membrane of HBMECs. An exposure to EMF yielded negligible increase in the permeability of free saquinavir (SQV) across the HBMEC monolayer. Nevertheless, the permeability of SQV across the HBMEC monolayer increased about 17-fold when SQV was entrapped in CSLNs. Moreover, the permeability of SQV across the HBMEC monolayer increased about 22-fold by applying the CSLN encapsulation and EMF exposure. CSLNs and EMF could produce synergistic effect on improving the brain-targeting delivery.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.     

Cholesteryl butyrate solid lipid nanoparticles inhibit adhesion of human neutrophils to endothelial cells

1. Adhesion of polymorphonuclear cells (PMNs) to vascular endothelial cells (EC) is a critical step in recruitment and infiltration of leukocytes into tissues during inflammation. High doses of butyric acid have been shown to ameliorate inflammation in inflammatory bowel diseases (IBD). Cholesteryl-butyrate solid lipid nanoparticles (chol-but SLN) as prodrug are a possible delivery system for butyric acid. 2. Sodium butyrate or chol-but SLN were coincubated with human PMNs and human umbilical vein EC (HUVEC); adhesion was quantified by computerized microimaging fluorescence analysis. Both chol-but SLN and sodium butyrate displayed antiadhesive effects on FMLP- and IL-1beta-stimulated cells in a concentration-response curve (10(-8)-10(-5) M), but chol-but SLN were in all cases more active. Moreover, chol-but SLN inhibited FMLP-induced adhesion of PMNs to FCS-coated plastic wells, thus showing a direct effect on PMNs, while sodium butyrate had little effect. Confocal microscopy showed that fluorescent SLN entered PMNs and HUVEC after 10 min incubation. Chol-but SLN acted either on activated PMN or HUVEC. 3. Chol-but SLN inhibited O2-* production and myeloperoxidase release by PMNs evoked by FMLP, in a dose-dependent, but not time-dependent, manner and were more active than sodium butyrate. 4. In conclusion, in all tests chol-but SLN were more active than sodium butyrate. Thus, chol-but SLN might be a valid alternative to sodium butyrate in the anti-inflammatory therapy of ulcerative colitis, avoiding complications related to the administration of sodium butyrate.     

Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes: a flow cytometric study

Since some of organotins, accumulated in edible mollusks of aquatic environments, exert a variety of toxic actions on experimental animals, it causes concern for the health of humans. We examined the effects of tri-n-butyltin chloride (TBT) and other organotins (triethyltin chloride, trimethyltin chloride, triphenyltin chloride and tetrabutyltin) on cellular content of glutathione (GSH) in rat thymocytes using a flow cytometer to further characterize the toxicity of TBT. When the cells were incubated with TBT at concentrations of 3 nM or more for 15 min, the cellular content of GSH dose-dependently decreased. However, it completely or partly recovered until 180 min even in the continued presence of TBT. This recovery was temperature-sensitive, suggesting an involvement of metabolic process. The efficacy of TBT to decrease the cellular content of GSH was greater than those of other organotins. Results suggest that TBT and some organotins at environmentally relevant (nanomolar) concentrations significantly reduce the cellular content of GSH, suggesting that they increase the vulnerability to some biological and chemical insults.     

Inhibition of LPS-induced chemokine production in human lung endothelial cells by lipid conjugates anchored to the membrane

1. In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A(2) (sPLA(2)) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA(2) in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA(2) inhibitors, specifically, the extracellular PLA(2) inhibitors (ExPLIs), composed of N-derivatized phosphatidyl-ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non-pancreatic sPLA(2). 2. ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL-8, Gro-alpha and ENA-78, as well as of the adhesion molecules ICAM-1 and E-selectin. Concomitantly, ExPLIs inhibited the LPS-induced activation of NF-kappaB by LPS but not its activation by TNF-alpha or IL-1. 3. Endotoxin mediated chemokine production in LMVEC seems not to involve PLA(2) activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA(2). It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA(2) inhibiting capacity. This was supported by the finding that the LPS-induced chemokine production was not affected by the selective sPLA(2) inhibitor LY311727. 4. It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin-induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.     

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food‐related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric‐based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma–mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma–mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration‐dependent micronucleus formation sevenfold at 10 µg ml^–1 concentration in attached cell conditions and 1.3‐fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2‐fold at a concentration of 10 µg ml^–1 both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver‐induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Cyclosporine uptake by cultured human proximal tubule cells

Characteristics of cyclosporine A (CsA) uptake in cultured normal human proximal tubule cells (PTC) have been studied. The uptake was very rapid, concentration dependent, and reached a steady state level within 10 min. Kinetic analysis yielded an apparent Km of 5.0 μM and V_max of 66.7 pmoles/min μg DNA. Verapamil, a calcium channel blocker, at 0.1, 0.5, and 1.0 mM had no inhibitory effect on CsA uptake. Subcellular distribution of CsA following 1, 2, 5, and 10 min incubation of PTC suspension was determined by digitonin differential solubilization. The cytosol fraction contained 26% and the nuclear fraction contained 74% of CsA at all time periods studied. These studies demonstrate that in normal human renal PTC, uptake of CsA is rapid and saturable. Most of the CsA, once taken up by the human PTC, is concentrated in the nucleus.     

Aldosterone augments LOX-1-mediated low-density lipoprotein uptake in human umbilical artery endothelial cells

Aldosterone and oxidized low-density lipoprotein (oxLDL) are recognized risk factors for cardiovascular disease and atherosclerosis. LOX-1 is a multi-ligand receptor originally identified as the endothelial oxLDL receptor, which mediates the uptake of oxLDL and plays a role in early atherosclerosis. The present study aimed to investigate the pathophysiological relevance of LOX-1 in aldosterone-induced atherosclerosis. The effect of aldosterone on LOX-1 expression and LDL uptake in primary cultures of human umbilical artery endothelial cells (HUAECs) was investigated in the absence and presence of the mineralocorticoid receptor (MR) antagonist spironolactone (Spiro). Aldosterone increased both mRNA and protein expression of LOX-1 in a dose-dependent manner with a maximum effect reached 24 h after treatment. Increased LOX-1 expression was associated with an augmented uptake of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (Dil)-labeled LDL (5 μM/ml, 3h). However, pretreatment with Spiro (1 μΜ) almost reduced these effects. Additionally, an increase in MR expression was detected in response to aldosterone in HUAECs. Collectively, our study demonstrates that aldosterone promotes LOX-1-mediated LDL uptake in human endothelial cells, and Spiro effectively inhibited these effects, suggesting that MR inhibition may be considered as a new anti-atherosclerotic therapy.     

The metabolism of N-acetylcysteine by human endothelial cells

When human umbilical endothelial cells were depleted of their glutathione by incubation in a sulfur amino acid-free medium, subsequent incubation of the cells with this deficient medium supplemented with N-acetylcysteine resulted in a dose-dependent stimulation of the synthesis of cellular glutathione. Similarly, the inclusion of N-acetylcysteine in the medium during the period of depletion of glutathione caused a dose-dependent retardation of the depletion kinetics. In contrast, the incubation of control cells in normal medium supplemented with N-acetylcysteine did not increase cellular glutathione levels above controls. These observations indicate the presence of an N-deacetylase in/on the cells with specificity for N-acetylcysteine. Due to the large surface area of the endothelium in the vasculature it seems likely that endothelial cell N-deacetylation plays a role in the metabolic disposition of N-acetylcysteine, particularly when administered intravenously. N-Acetylcysteine is, however, a relatively poor precursor to glutathione biosynthesis in comparison to cystine. Thus, any cytoprotective, antioxidant effect exerted by N-acetylcysteine on the human endothelium is likely to be due to direct scavenging of reactive intermediates rather than by stimulated glutathione synthesis in the endothelial cells themselves.     

PAF-acether-induced synthesis of prostacyclin by human endothelial cells

Platelet activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) is a potent platelet-stimulating agent formed by most circulating cells. Added to cultured human endothelial cells, PAF-acether induced a dose-dependent synthesis of 6-keto-PGF1 alpha, the major stable metabolite of prostacyclin (PGI2), with a maximal effect at 100 nM, a concentration equivalent to that which aggregates human platelets. No release of von Willebrand factor (vWF) was noted under the same conditions. The poorly active PAF-acether analogue, methoxy-PAF failed to stimulate 6-keto-PFG1 alpha synthesis and the PAF-acether antagonists 48740 RP, BN 52021 and Ro 19-3704, prevented the stimulatory effect of PAF-acether. Methyl-carbamate-PAF, an equieffective analogue of PAF-acether on platelets, also stimulated 6-keto-PGF1 alpha production. After a first stimulation by PAF-acether or methyl-carbamate-PAF, no response was detected when endothelial cells were re-exposed to either agonist, indicating auto- and cross-desensitization as described for other cells. Since PAF-acether stimulated [3H]arachidonate release from pre-labelled endothelial cells, our results suggest that it stimulates phospholipase activity, which accounts for the increased PGI2 synthesis. The auto- and cross-desensitization between PAF-acether and methyl-carbamate-PAF, ineffectiveness of methoxy-PAF and inhibition by selective antagonists strongly suggest interaction with specific membrane receptors.     

雌激素功能性调节人内皮细胞的生长调节致癌基因α表达

目的：研究雌激素在体外调节人脐带静脉内皮细胞（HUVEC）的生长调节致癌基因α（GROα）。方法：以体外培养的HUVEC为模型,Northern法检测CXC族趋化因子GROα mRNA;ELISA方法检测细胞表面的GROα蛋白表达;静态细胞粘附实验测定细胞表面的GROα蛋白的生理意义。结果：17β－雌二醇（0．05μmol/L）明显抑制HUVEC产生GROα mRNA和蛋白表达水平;而且17β－雌二醇抑制其蛋白表达水平显示剂量依赖性关系;雌激素受体α拮抗剂他莫昔芬（0．1μmol/L）单独使用不影响其蛋白表达,但可显著逆转17β－雌二醇抑制的GROα蛋白表达,显著逆转17β－雌二醇抑制单核细胞系细胞U937细胞粘附的HUVEC的作用达。结论：通过内皮细胞上雌激素受体α,雌激素可能功能性调节人内皮细胞的GROα的表达。     

Zolmitriptan uptake by human intestinal epithelial Caco-2 cells

The oral uptake of zolmitriptan, a novel and highly selectively 5-HT 1B/1D receptor agonist, was evaluated in the human epithelial cell line caco-2 that possesses intestinal enterocyte-like properties when cultured in vitro. The study demonstrated that zolmitriptan uptake significantly depended upon the extracelluar temperature and pH in the Caco-2 cell. The zolmitriptan uptake at 39 degrees C was 2.1 fold as that at 23 degrees C and the zolmitriptan uptake at pH 8.0 was 2.7 fold as that at pH 6.0. The uptake rates of zolmitriptan on both sides increased with increasing zolmitriptan concentration from 0.1 to 10 mmol x L(-1), and it shows concave concentration-dependency at high concentration. The uptake rates of zolmitriptan on the basolateral side (BL) were 3-7 times higher than that on the apical side (AP). Verapamil, nimodipine, nifedipine, flunarizine, amiloride and sumatriptan significantly increased the uptake rates of zolmitriptan on the apical sides. Propafenone significantly inhibited the uptake rate of zolmitriptan on both sides. Propranolol and aspirin have no significant effect. The results indicated that the zolmitriptan uptake in Caco-2 cells was temperature, pH and concentration dependent, and was partially counteracted by the action of an outwardly directed efflux pump, presumably p-glycoprotein. Absorption interactions should be considered when P-gp substrates or inhibitors, Na+ -H+ exchange inhibitors, P-gp ATPase agonists or inhibitors are co-administered with zomitriptan in clinical practice.