A comparison of the induction of immortalized endothelial cell impermeability by astrocytes.

The suitability of various commercially available endothelial cell lines in studies of astrocytic/endothelial cell interactions was assessed. The endothelial-like cell line ECV304 was compared with T24/83, Eahy929, and b.End5 and rat cerebral endothelial cells in their ability, when co-cultured with rat (C6) glioma cells, to form a transendothelial electrical resistance (TEER), an indicator of tight junction formation which is an important property of the blood-brain barrier. As reported previously, the basal TEER of ECV304 cell monolayers was significantly enhanced upon co-culture, an effect reproduced by human 1321N1 astrocytes and primary rat astrocytes. T24/83 cells formed a patchy, gapped monolayer, which produced a poor basal TEER with little in the way of an increase upon co-culture. Similarly, all the other cell monolayers analysed demonstrated poor TEERs that were only moderately increased upon co-culture. These data confirm that while no endothelial cell line with ideal features is available, ECV304 cells remain an appropriate choice especially for studies of astrocyte/endothelial cell interactions.

Decreased endothelial cell glutathione and increased sensitivity to oxidative stress in an in vitro blood-brain barrier model system.

Using a cell culture model of the blood-brain barrier (BBB) we have evaluated the role of endothelial cell glutathione in protecting barrier integrity against nitric oxide (NO)-induced oxidative stress. The co-culture of human umbilical vein endothelial cells (ECV304) with rat (C6) glioma cells, or incubation with glioma cell or primary astrocytic conditioned medium, resulted in a decline in endothelial cell glutathione. Exposure to a single addition of NO gas induced a rapid breakdown in model barrier integrity in endothelial/glioma co-cultures. Addition of NO gas or tumour necrosis factor-alpha (TNF-alpha) also resulted in a loss of membrane integrity, as measured by an enhanced release of lactate dehydrogenase, only from endothelial cells treated with glioma conditioned medium. Furthermore, assessment of viability in endothelial cells grown alone or treated with glioma conditioned medium, by propidium iodide labelled flow cytometry. demonstrated no difference in the number of positively stained cells after NO exposure. These results indicate that when enhanced endothelial monolayer barrier formation occurs via astrocytic-endothelial interactions, cellular glutathione levels are decreased. This renders the barrier cells, under these conditions, more susceptible to oxidative stress but does no necessarily lead to greater cell death.

Upregulation of intercellular adhesion molecule-1 expression on human endothelial cells by tumour necrosis factor-alpha in an in vitro model of the blood-brain barrier.

Adhesion molecules on the endothelial surface of the blood-brain barrier (BBB) play an important role in the pathogenesis of many encephalopathies, including multiple sclerosis (MS) and cerebral malaria (CM). The expression of four surface molecules of relevance to MS and CM on the immortalized human umbilical vein endothelial cell line, ECV304, was investigated using immunofluorescence flow cytometry. We found that ECV304 cells express intercellular adhesion molecule-1 (ICAM-1) and low levels of CD36, but not vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. This expression pattern was unaltered on ECV304 cells which were co-cultured with C6 glioma cells; conditions under which the endothelial cells display enhanced barrier formation. Tumour necrosis factor-alpha (TNF-alpha), which is elevated in MS and CM, decreased the integrity of the barrier in co-cultured endothelial cells and upregulated the expression of ICAM-1 nine-fold. The significance of elevated ICAM-1 expression in relation to the binding of parasitised erythrocytes at the BBB in CM is discussed.

The male antifertility activity of 6-chloro-6-deoxyglucose.

6-Chloro-6-deoxy[U-14C]glucose is not metabolised by mature boar spermatozoa nor has it any specific inhibitory action on their metabolic activity in vitro. The compound is metabolised by the male rat and the identification of two urinary metabolites as alpha-chlorohydrin and 3-chlorolactate confirmed that (S)-3-chlorolactaldehyde is produced by this species in vivo. A tissue distribution study revealed that radioactivity from 6-chloro-6-deoxy[U-14C]glucose was more concentrated in rat caudal spermatozoa than in any other of the major tissues.

Concentrations of quinolinic acid in cerebrospinal fluid measured by gas chromatography and electron-impact ionisation mass spectrometry. Age-related changes in a paediatric reference population.

A simple method for the determination of the excitotoxin, quinolinic acid (QUIN) in cerebrospinal fluid (CSF) is described. QUIN, in lyophilized samples, was silylated by N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide in a single-step reaction at 65 to 70 degrees C to form a di-tert.-butyldimethylsilyl ester. Neither pre-purification of QUIN from CSF nor post-derivatisation sample clean-up was required. The derivatives were analysed by gas chromatography-electron impact mass spectrometry resulting in a prominent and characteristic [M-57]+ fragment ion which was used for quantitation. 2,6-Pyridine dicarboxylic acid, a structural analog of QUIN, was used as the internal standard. The detection limits for injected standards are in the femtomole range. CSF QUIN was found to be age-related and three preliminary reference ranges for CSF QUIN were found: 0 to 1 years, 31 +/- 15 nM QUIN (mean +/- standard deviation): 1.1 to 3 years, 26 +/- 15 nM; 3.1 to 14 years, 14 +/- 9 nM.

Is the nephrotoxicity of (R)-3-chlorolactate in the rat caused by 3-chloropyruvate?

1. When (R, S)-[3-36 Cl]chlorolactate was administered to male rats, two radioactive constituents were excreted in the urine. These were identified as 36Cl- and [3-36 Cl]chlorolactate which was subsequently shown to be essentially the (S)-isomer. 2. Analysis of the urinary oxalate content from rats receiving either (R)- or (S)-3-chlorolactate revealed that elevated levels were produced by the (R)-isomer whereas normal levels followed the administration of the (S)-isomer. 3. Treatment of (R,S)-3-chlorolactate with a modified Fenton's oxidizing system produced oxalate and an intermediate which was identified as 3-chloropyruvate. 4. 3-Chloropyruvate is a potent nephrotoxin in the rat producing a brief phase of diuresis when administered, increasing the urinary excretion of oxalate and inhibiting the oxidative metabolic capability of rat kidney tubules and rat kidney mitochondria in vitro. 5. Both (R)-3-chlorolactate and 3-chloropyruvate were shown to be inhibitors of the commercially-available pyruvate dehydrogenase complex. 6. 3-Chloropyruvate inhibits kidney mitochondrial metabolism possibly at the pyruvate dehydrogenase complex level and appears to be a metabolite of (R)- but not (S)-3-chlorolactate.

Renal and spermatozoal toxicity of alpha-bromohydrin, 3-bromolactate and 3-bromopyruvate.

(R,S)- and (R)-3-Bromopropan-1,2-diol (alpha-bromohydrin) produced diuresis and glucosuria when administered to male rats but had no effect on the metabolic activity of rat kidney tubules in vitro. The oxidation products (R,S)-3-bromolactate and 3-bromopyruvate produced brief phases of diuresis but not glucosuria, and severely inhibited the metabolic activity of rat kidney tubules in vitro. alpha-Bromohydrin had no effect on the metabolic activity of boar spermatozoa whereas the oxidation products severely affected mitochondrial metabolism. 3-Bromopyruvate also inhibited boar spermatozoal glyceraldehyde 3-phosphate dehydrogenase.