Brain Microvessel Endothelial Cell Monolayers Research Articles

Relationship of octanol/buffer and octanol/water partition coefficients to transcellular diffusion across brain microvessel endothelial cell monolayers

The importance of lipid solubility on the rate of passage of solutes across the bovine blood-brain barrier (BBB) was studied with an in vitro BBB model. The model consisted of primary cultured bovine brain microvessel endothelial cell monolayers mounted in a side-by-side diffusion cell. Nine compounds (sucrose, mannitol, urea, glycerol, butyrate, thiourea, caffeine, antipyrine, propanolol) with different octanol/water partition coefficients were selected in order to provide a wide range of lipid solubility. Time- and concentration-dependence, and the effect of bovine serum albumin on the rate of transport of these compounds across the monolayers was determined. Consistent with in vivo studies, a significant positive correlation between transcellular diffusion and octanol/buffer or octanol/water partition coefficients was observed. The passage of these compounds across the monolayers was also concentration-dependent, non-saturable, and reduced in the presence of bovine serum albumin. Results demonstrate that these compounds undergo passive diffusion across the monolayers in which the rate of transfer across the monolayers depends directly on their lipid solubility and indirectly upon molecular size. In addition, serum albumin is an apparent factor in determining the passive permeability of the monolayers.

Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers.

The large neutral amino acid (LNAA) transport system, or L (leucine) system, in primary cultures of bovine brain microvessel endothelial cell monolayers has been characterized. The transendothelial transport of leucine in this in vitro blood-brain barrier (BBB) model was determined to be bidirectional and time, temperature, and concentration dependent. Leucine transport was saturable, and the apparent Km and Vmax were determined to be 0.18 mM and 6.3 nmol/mg/min, respectively. Transendothelial transport of leucine was resistant to inhibition by ouabain and sodium azide. Other LNAAs, including the centrally acting drugs alpha-methyl-3,4-dihydroxyphenylalanine (alpha-methyldopa), L-3,4-dihydroxyphenylalanine (L-DOPA), alpha-methyltyrosine, and baclofen, inhibited leucine transport. The leucine carrier system was also found to be stereospecific. Sucrose, used as a diffusion marker, moved across the monolayers slowly, and its concentration was not significant for at least 30 min.

Catecholamine-metabolizing enzymes of bovine brain microvessel endothelial cell monolayers.

The activities of monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), phenol sulfotransferase (PST), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GT), and angiotensin converting enzyme (ACE) were quantitated in primary cultures of bovine brain microvessel endothelial cell monolayers and cerebral gray matter. Significant MAO-A and -B, cytosolic and membrane-bound COMT, PST, AP, GT, and ACE activities are demonstrated in bovine gray matter. By comparison, enzyme activities of the monolayers vary with the age of the monolayer and are generally higher in complete monolayers. Relative to gray matter enzyme activities, the monolayers are enriched with AP, GT, and ACE, enzymes considered to be markers for brain endothelium. Results also indicate that the activities of MAO-A and PST in the monolayers approach those found in the gray matter. Conversely, cytosolic COMT and MAO-B activities in the monolayers are negligible and much lower, respectively, compared to activities in gray matter. Additional studies with both tissues suggest that the PST of both tissues is the thermostable form of the enzyme.