Ultracytochemical characteristics of cultured sheep brain microvascular endothelial cells

Abstract

This ultrastructural study was undertaken to determine the localization of cytochemically demonstrable blood-brain barrier (BBB)-associated enzymatic activities and of some non-enzymatic constituents in sheep brain microvascular endothelial cells (ECs) growing in vitro. Positive reactions for alkaline phosphatase (AP), 5'-nucleotidase (5'N), transport ATPase (Na+,K+-ATPase), and adenosine diphosphatase (ADPase) were present on both apical and basolateral plasma membranes (PMs) of the ECs. The reaction for calcium-dependent ATPase (Ca2+-ATPase) was less intense and was restricted to basolateral PM and associated plasmalemmal pits. These cells also revealed an abundance of anionic sites labeled with cationic colloidal gold (CCG) and Ricinus communis agglutinin 120 (RCA)-binding sites, specific for β-D-galactosyl residues, on the apical PM. The labeling of the apical PM with Ulex europaeus agglutinin (UEA)-gold complex, specific for α-L-fucosyl residues, was negligible. When compared with results of cytochemical examination of the ECs of sheep brain capillary in vivo, these observations indicate that although cells cultivated in vitro retain at confluence the enzymatic activities typical for BBB-type ECS, they lose their characteristic (polar) localization. This loss is interpreted as a reflection of lost functional polarity of the microvascular endothelium in vitro resulting from deprivation of the normal influence of the components of brain parenchyma.