Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels |
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Authors: | Hisashi Fukushima Masafumi Fujimoto Misao Ide |
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Institution: | (1) Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-Ku, 553 Osaka, Japan |
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Abstract: | Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase
(γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured
cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the
cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays
gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured
endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss
due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly
In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for
both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP
activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient
means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes. |
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Keywords: | blood-brain barrier endothelial cells microvessels astroglia pig alkaline phosphatase γ -glutamyltranspeptidase |
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