The Use of Cultured Epithelial and Endothelial Cells for Drug Transport and Metabolism Studies

In an effort to develop novel strategies for delivery of drug candidates arising from rational drug design and recombinant DNA technology, pharmaceutical scientists have begun to employ the techniques of cell culture to study drug transport and metabolism at specific biological barriers. This review describes some of the general factors that should be considered in developing a cell culture model for transport studies and metabolism studies. In addition, we review in detail the recent progress that has been made in establishing, validating, and using cell cultures of epithelial barriers (e.g., cells that constitute the intestinal, rectal, buccal, sublingual, nasal, and ophthalmic mucosa as well as the epidermis of the skin) and the endothelial barriers (e.g., brain microvessel endothelial cells).     

Synergistic Efficacy of Concurrent Treatment with Cilostazol and Probucol on the Suppression of Reactive Oxygen Species and Inflammatory Markers in Cultured Human Coronary Artery Endothelial Cells

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3~3 µM) with probucol (0.03~0.3 µM) significantly suppressed TNF-α-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-α release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3~3 µM) with probucol (0.1~0.3 µM) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.     

血脂康对血管紧张素Ⅱ诱导内皮细胞E-选择素表达的影响

目的观察血管紧张素Ⅱ（AngⅡ）对人脐静脉内皮细胞（简称内皮细胞）E-选择素表达的影响以及血脂康对AngⅡ此诱导作用的干预。方法用ELISA法检测AngⅡ对内皮细胞E-选择素表达的影响，用RT—PCR检测AngⅡ对内皮细胞E-选择素mRNA表达的影响，用细胞计数法观察经AngⅡ作用的内皮细胞与单核细胞的粘附率，同时检测血脂康的干预效应。结果四种不同浓度的AngⅡ（10^-8、10^-8、10^-7、10^-6mol／L）能诱导内皮细胞E-选择素及其mRNA的表达，增加内皮细胞与单核细胞的粘附率，且作用呈剂量依赖关系；而血脂康（500ng／ml）能抑制AngⅡ诱导的内皮细胞E-选择素及其mRNA的表达，降低内皮细胞与单核细胞的粘附率。结论血脂康能发挥抗动脉粥样硬化作用，抑制动脉粥样硬化的进程。     

Fluoride,Fluoride Resistance and Glycolysis in Cultured Cells

Abstract Long-term (several years) exposure to fluoride induced decreased production of lactate in cultured, fluoride resistant LS cells. In intact, sensitive cells, 6 mM NaF had no effect on glycolysis, whereas in homogenates, from both resistant and sensitive cells, lactate production was blocked by 6 mM NaF, indicating the cell membrane to be a barrier to fluoride. A lower intracellular rather than extracellular fluoride concentration was found in the sensitive cell with a ratio of 0.4, whereas fluoride resistant cells excluded fluoride from their intracellular milieu.     

Permeability and Mechanism of Albumin,Cationized Albumin,and Glycosylated Albumin Transcellular Transport Across Monolayers of Cultured Bovine Brain Capillary Endothelial Cells

We have measured the permeability and binding characteristics of bovine serum albumin (BSA), cationized BSA (cBSA), and glycosylated BSA (gBSA) to primary cultures of bovine brain capillary endothelial cells (BBCEC). These endothelial cells serve as an in vitro model to study the binding, uptake, and transcellular transport of small and large molecule flux across the blood–brain barrier. The rate of [³H]BSA flux across the cultured BBCEC monolayers grown onto polycarbonate membranes (5-µm pore size) was linear with increasing BSA concentration and the flux could be inhibited by temperature reduction to 0–4°C. The maximal binding of [³H]BSA was 0.04 fmol/mg total cell protein and could not be inhibited by nonradiolabeled BSA. The binding of cBSA and gBSA was rapid and could be inhibited by nonradiolabeled cBSA or gBSA, respectively. The maximal amount bound was 1.8 fmol/mg total cell protein for cBSA and 17.4 fmol/mg total cell protein for gBSA. The dissociation constants (K _d's) were 27 ± 13 and 3.7 ± 1.1 nM for cBSA and gBSA, respectively. The flux rates of cBSA and gBSA across the endothelial cell monolayers were linear with respect to concentration and they were approximately seven times greater than those observed for BSA. Each of the proteins appeared on the antiluminal side of the endothelial cell monolayers primarily (90%) as intact protein as determined by trichloroacetic acid (TCA) precipitations and sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). The results for BSA are similar to those observed for lucifer yellow, a fluid-phase endocytic marker. In contrast to BSA, the binding and transcellular transport of cBSA and gBSA appear to proceed by an adsorptive-phase endocytic mechanism.     

Modification by LY 83583 and methylene blue of relaxation induced by nitric oxide,glyceryl trinitrate,sodium nitroprusside and atriopeptin in aortae of the rat,guinea-pig and rabbit

• 1.1. The relaxation by nitroglycerin (GTN) and nitric oxide (NO) of aortic smooth muscles from rabbit and rat contracted by phenylephrine was inhibited by LY 83583 (LY) and methylene blue (MB) (the same applied to guinea-pig aorta), while the relaxation by SNP was not inhibited in rabbit. The relaxation by ANP was not inhibited.
• 2.2. All these agents produced concentration-dependent increases in cyclic GMP. While the increases by GTN and NO were inhibited by LY and MB, the increases by SNP were inhibited only in rat and those by ANP were not inhibited.
• 3.3. Thus, LY behaved essentially similar to MB, indicating that the substance is an inhibitor of activation of soluble guanylate cyclase by NO and NO-related vasodilators. It was assumed that, like MB, LY facilitated intracellular release of NO from SNP in rabbit.

     

Lipopolysaccharides,but not Angiotensin ll,lnduces Direct Pro‐lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells

Angiotensin II (Ang II) might induce pro‐inflammatory effects directly in the vascular wall independently of its haemodynamic effects. The aim of our study was to investigate the putative direct pro‐inflammatory and vasomotor effects of Ang II and compare to those of lipopolysaccharides (LPS) in mouse isolated mesenteric resistance‐sized arteries (MRA) supported by experiments in cultured human primary endothelial and vascular smooth muscle cells. Results showed that 24‐hr organ culture of mouse MRA with 10 nM Ang II had, unlike 100 ng/mL LPS, no effects on IL‐6 or MCP‐1 secretion, VCAM1 mRNA expression or endothelial function, while Ang II significantly decreased maximal vasomotor responses to phenylephrine. In support, 24‐hr organ culture of mouse MRA significantly suppressed Agtr1a mRNA and augmented Tlr4 mRNA along with attenuated vasomotor responses to Ang II. Moreover, contrary to LPS and TNF‐α, Ang II and [Sar1]‐Ang II had no concentration‐ or time‐dependent effects on IL‐6 and MCP‐1 secretion in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC). AGTR1 or AGTR2 mRNA expression was undetectable in HUVEC, whereas HASMC expressed only AGTR1 mRNA. In summary, contrary to previous studies and the observed effects of LPS, we could not demonstrate direct vascular pro‐inflammatory effects of Ang II ex vivo or in vitro. As indicated by our results, down‐regulation or desensitization of AT₁R during culture may explain our findings.     

缬沙坦在缺氧状态下对冠状动脉内皮细胞NOS和NO的影响

目的：研究缬沙坦、血管紧张素Ⅱ（AⅡ）对缺氧状态下培养的成人冠状动脉内皮细胞一氧化氮合酶（NOS）活力及一氧化氮（NO）含量的影响。方法：将培养的第三代成人冠状动脉内皮细胞分为对照组、AⅡ（10μmol/L）组、缬沙坦（10μmol/L）组及AⅡ（10μmol/L）加上不同浓度缬沙坦（0.1、1、10μmol/L）组。当细胞长满培养瓶面积60/时加入缬沙坦和AⅡ,先后常规培养32h,缺氧培养4h,检测NO含量及NOS活力。结果：与对照组相比,单独应用AⅡ能显著降低NOS活力并抑制NO的合成（P〈0.01）,单独应用缬沙坦对NOS活力及NO的合成均无明显影响（P〉0.05）。缬沙坦和AⅡ合用时可逆转后者对NOS及NO的抑制作用,且呈剂量依赖性（P〈0.01）。结论：AⅡ在缺氧状态下能降低成人冠状动脉内皮细胞NOS活力并抑制NO的生成,缬沙坦在缺氧状态下能逆转AⅡ的上述作用,提示缬沙坦在缺氧状态下可改善成人冠状动脉内皮细胞的功能,从而起到预防或逆转冠状动脉粥样硬化的作用。     

The Disposition of Morphine and Its Metabolites in the In-situ Rat Isolated Perfused Liver

A specific HPLC method with UV detection was used to investigate the disposition of morphine and its metabolites in the in-situ rat isolated perfused liver preparation. Livers of male Sprague-Dawley rats (n = 4) were perfused under single pass conditions with protein-and erythrocyte-free perfusate, containing 2·66 μm morphine, for up to 90 min. The concentration of morphine, normorphine and morphine-3-glucuronide (M3G) in outflow perfusate, and the biliary excretion of M3G and normorphine glucuronide, all reached steady-state levels within 15–20 min after commencing perfusion. At steady-state, the mean (± s.d.) extraction ratio of morphine was 0·87 ± 0·06 and clearance (26·0 ± 1·7 mL min^?1) approached perfusate flow rate (30 mL min^?1). Although M3G was the main metabolite, accounting for 72·8 ± 12·7% of eliminated morphine, a significant proportion (21·6 ± 13·5%) was N-demethylated to normorphine and was recovered as unchanged normorphine in outflow perfusate and normorphine glucuronide in bile. The biliary extraction ratio of hepatically-formed M3G was 0·61 ± 0·31. Results from an additional six experiments, in which livers were perfused with 1·33 and 2·66 μm of morphine for 30 min each in a balanced cross-over manner, indicated that the disposition of morphine and its metabolites was approximately linear within this concentration range.     

水溶性氮酮对体外培养兔角膜内皮细胞活性与超微结构的影响

目的研究水溶性氮酮对体外培养的兔角膜内皮细胞活性与超微结构的影响,确定水溶性氮酮应用于眼部的安全浓度。方法采用消化法培养兔角膜内皮细胞,取第3或第4代细胞以1×104个.L-1的浓度接种,共分8组,其中一组设为对照组,不给予水溶性氮酮,其他7组细胞中水溶性氮酮浓度分别为0.001,0.005,0.010,0.050,0.100,0.500,1.000 g.L-1,24 h后采用MTT法测定细胞活性。另取上述培养的细胞,按1×104个.L-1的浓度接种到载玻片上,分别加入浓度为0.010,0.100,0.500 g.L-1的水溶性氮酮作用10 m in,观察细胞膜超微结构变化。另采用相同浓度的水溶性氮酮作用于体外培养的兔角膜内皮细胞10 m in,观察细胞膜超微结构的变化。结果与对照组比较,浓度≤0.100 g.L-1的水溶性氮酮作用24 h后对兔角膜内皮细胞的活性没有明显影响,浓度≥0.500 g.L-1的水溶性氮酮作用24 h可以引起兔角膜内皮细胞活性下降(P<0.01);经浓度≤0.100 g.L-1水溶性氮酮作用10 m in,兔角膜内皮细胞细胞膜有裂隙形成,但细胞器等结构无明显损害,浓度≥0.500 g.L-1水溶性氮酮可导致兔角膜内皮细胞凋亡。结论浓度<0.100 g.L-1水溶性氮酮可增加角膜内皮细胞对药物的通透性,并有望用于角膜内皮细胞基因转染研究。     

Effects of Flavonoids Isolated from Scutellariae Radix on the Production of Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor-1 Induced by Thrombin and Thrombin Receptor Agonist Peptide in Cultured Human Umbilical Vein Endothelial Cells

The effects of different flavonoids isolated from the roots of Scutellaria baicalensis Georgi on the production of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) induced by thrombin and thrombin receptor agonist peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe, have been examined in cultured human umbilical vein endothelial cells (HUVECs). Thrombin and thrombin receptor agonist peptide induced production of both t-PA and PAI-1 and the elevation of intracellular free calcium concentration ([Ca²⁺]_i). Baicalein isolated from Scutellariae Radix dose-dependently inhibited PAI-1 production induced by thrombin and thrombin receptor agonist peptide; its concentrations for 50% inhibition (IC50) were 6.8 and 3.5 μm , respectively. Other flavonoids had no effect. In contrast, flavonoids isolated from Scutellariae Radix had no effect on production of t-PA induced by thrombin and thrombin receptor agonist peptide. Baicalein inhibited the elevation of [Ca²⁺]; induced by thrombin and thrombin receptor agonist peptide and, at a concentration of 1000 μm , slightly increased t-PA production. These findings suggest that the mechanism by which baicalein inhibits PAI-1 production induced by thrombin and thrombin receptor agonist peptide might be by reduction of [Ca²⁺]_i elevation. The results suggest that baicalein in Scutellariae Radix might be active as a drug in the treatment of arteriosclerosis and thrombosis.     

Cytotoxicity, Accumulation, and Efflux of Cisplatin and Its Metabolites in Human Ovarian Carcinoma Cells

Cisplatin (CP) is one of the most useful antineoplastic drugs. When CP is dissolved in human plasma, different metabolites are formed. Using the OV 2008 human ovarian cancer cell line, we examined the relative cytotoxicities of CP and its metabolites (aquated CP [AP], monomethionine CP [MP], bismethionine CP [BP], and a mixture of CP metabolites in ultrafiltrated human plasma [UP])in vitro.By clonogenic assay, cell survival (percent of mean ± SE) of OV 2008 cells exposed for 1 hr to 6.7 μ of CP was 9.8 ± 0.7 and for its equal platinum contents of AP, MP, BP, and UP solutions were 3.3 ± 0.7, 9.8 ± 0.9, 15.9 ± 1.1, 76.8 ± 2.1, and 13.1 ± 0.7, respectively. AP was the most cytotoxic species, and BP was the least cytotoxic species. Cellular platinum uptake (ng/10⁶cells) after addition of 0.33, 1.6, and 2.5 m of each species for 1 hr was measured and a strong correlation was found between cytotoxicity of each CP metabolite and its corresponding cellular platinum (Pt) uptake (r= 0.997). There was a strong correlation between cytotoxicity of the CP metabolites and their DNA binding. With fractionation of these cells into DNA, nucleoplasm and cytoplasm, the highest platinum content was found on DNA. The most important factor that seems to be responsible for the cytotoxicities of the different CP metabolites is the amount of their associated intracellular accumulation, and particularly the degree of their binding to DNA.     

紫草宁生物合成相关的代谢酶及基因

紫草是一种重要的中草药材，其培养细胞在M9培养基中可以大量生产主要的药物成分紫草宁。本文主要论述了在紫草细胞中与紫草宁形成相关的代谢酶和基因方面取得的研究进展，分别讨论了这些代谢酶和基因克隆的功能及表达特性，对植物次生代谢、紫草宁次生药物的生产调控及其代谢工程等研究具有重要的意义。     

The effects of LY393613, nimodipine and verapamil, in focal cerebral ischaemia

This study evaluates the effects of N-(2-[bis (4-fluorophenyl)methoxy]ethyl)-1-butanamine hydrochloride (LY393613), a novel neuronal (N/P/Q-type) Ca(2+) channel blocker, in ischaemia. For comparison, two commonly used L-type Ca(2+) channel blockers; nimodipine and verapamil were also evaluated. Ischaemia was induced in freely moving rats by micro-injection of endothelin-1 near the middle cerebral artery. In vivo microdialysis, laser Doppler flowmetry and histology were used to monitor ischaemia. Administration of LY393613, before and after the insult, attenuated the ischaemia-induced glutamate release, but not the dopamine release. Both nimodipine and verapamil failed to affect transmitter releases significantly, when administered post-occlusion. None of the compounds tested, produced any significant change in striatal blood flow. Histology showed that ischaemic damage was significantly less in LY393613 pre-treated rats. In conclusion, LY393613, a neuronal N/P/Q-Ca(2+) channel blocker, can attenuate ischaemic brain damage. The protective mechanism appears to be mainly the attenuation of the ischaemia-induced glutamate release, rather than its effect on cerebral hemodynamics.     

Effects of Tamoxifen,Toremifene and Chloroquine on the Lysosomal Enzymes in Cultured Retinal Pigment Epithelial Cells

Abstract: Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-β-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1–40 μM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestro-genic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-β-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-β-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-β-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-β-glucosaminidase.     

Inhibition of Prolactin Secretion and Synthesis by Dopamine,Noradrenaline and Pilocarpine in Cultured Rat Pituitary Tumour Cells

Clonal strains of rat pituitary tumour cells (GH₃-cells) synthesize and secrete prolactin into a chemically defined culture medium. Short time treatment (0.5–2 hrs) of cell cultures with noradrenaline (10^-3 M) and dopamine (10^-3 M) reduced the spontaneous secretion of prolactin by 50 % and 30 %, respectively, while pilocarpine (10^-3 M) had no effect. Long-term treatment (20 hrs) with noradrenaline (10^-3 M) or with pilocarpine (10^-3 M) inhibited prolactin synthesis by 45 % and 65 % of control cultures, respectively, Neither compounds affected cell growth. The inhibitory effect of noradrenaline, but not that of pilocarpine, was completely reversed 4 hrs after cessation of treatment. Adrenaline, dopamine and acetylcholine in concentrations up to 10^-3 M did not change prolactin synthesis. In contrast thyroliberin treatment (2×10^-8 M) caused a 45 % increase in prolactin secretion, and resulted in a 40 % increase in hormone synthesis after treatment for 20 hrs. It is concluded that both noradrenaline and dopamine are able to inhibit prolactin secretion. Prolactin synthesis could be inhibited by noradrenaline and pilocarpine. However, only the effect of noradrenaline is easily reversible on cessation of treatment.     

血管内皮祖细胞在创伤修复与改善皮瓣血运中的研究综述

<正>血管内皮祖细胞(endothelial progenitor cell,EPC)是一种能直接分化为血管内皮细胞的前体细胞。EPC形成于胚胎期,胚外造血启动,卵黄囊的胚外中胚层的一些间充质细胞逐渐聚集成条索或团块状,形成血岛,血岛进一步出现腔隙化改变,中央的细胞分化成造血     

环磷腺苷葡胺对人脐静脉血管内皮细胞增殖、迁移、管腔形成及凋亡的影响

目的 探索环磷腺苷葡胺（meglumine cyclic adenylate,MCA）对人脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVEC）增殖、迁移、管腔形成及凋亡的影响。方法 通过显微镜观察、MTT法、划痕修复试验、管腔形成试验、Western blotting观察不同浓度MCA对HUVEC细胞形态、增殖、迁移、管腔形成能力及凋亡相关蛋白表达的影响。结果 显微镜观察发现,随着MCA剂量增加,HUVEC细胞形态由多角形变为卵圆形。MCA可呈浓度依赖地抑制HUVEC增殖、迁移、管腔形成,并且高剂量的MCA还可诱导细胞凋亡,表现为细胞凋亡数目增加,Bax、Cleaved-PARP蛋白表达上调,PARP、Bcl-2蛋白表达下调。结论 MCA可抑制HUVEC细胞增殖、迁移、管腔形成,并诱导细胞凋亡。     

The group II metabotropic glutamate receptor agonist, LY379268, inhibits both cocaine- and food-seeking behavior in rats

Rationale Group II metabotropic glutamate receptor (mGluR2/3) agonists are proposed to serve as potential treatment for addiction.Objectives The present study examined the hypothesis that mGluR2/3 agonists exert inhibitory effects on cocaine-induced reinstatement of cocaine-seeking.Methods Rats were trained to self-administer either cocaine or control reinforcer (food), then responding on the reinforcer-paired lever was extinguished. Reinstatement of responding was induced by a noncontingent presentation of the self-administered reinforcer (10 mg/kg cocaine, i.p. or 765 mg of food). In one experiment, rats were systemically pretreated with vehicle (Veh) or the mGluR2/3 agonist LY379268 (0.3, 1, or 3 mg/kg, i.p.) 30 min before the reinstatement test session. In a second experiment, Veh or LY379268 (0.05, 0.5, or 5 nmol/side) was microinjected into the nucleus accumbens core (NAc core) 5 min before the reinstatement test session. The effects of LY379268 on cocaine- and food-induced reinstatement on reward seeking were assessed.Results Both systemic and intra-NAc core pretreatment with LY379268 inhibited both cocaine- and food-seeking behavior. However, the effect of LY379268 appeared somewhat more effective for cocaine-seeking than food-seeking.Conclusions These results support a potential therapeutic role for mGluR2/3 agonists on relapse of cocaine-seeking. However, doses that inhibited cocaine-seeking were only threefold lower than those inhibiting food-seeking, indicating possible unacceptable nonspecific effects. In addition, the NAc core is one site of action where the mGluR2/3 agonists elicit effects on reward-seeking behavior.