REGULATION OF GAMMA-GLUTAMYL-TRANSPEPTIDASE AND ALKALINE-PHOSPHATASE ACTIVITIES IN IMMORTALIZED RAT-BRAIN MICROVESSEL ENDOTHELIAL-CELLS

Rat brain microvessel endothelial cells were immortalized by transfect ion with a plasmid containing the E1A adenovirus gene. One clone, call ed RBE4, was further characterized. These cells display a nontransform ed phenotype and express typical endothelial markers, Factor VIII-rela ted antigen and Bandeiraea simplicifolia binding sites. When RBE4 cell s were grown in the presence of bFGF and on collagen-coated dishes, co nfluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the bloo d-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamm aGTP), was expressed in these structures, not in the surrounding monol ayer. Similar results were obtained with the microvessel-related enzym e alkaline phosphatase (ALP). Addition of agents that elevate intracel lular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gammaGTP and ALP acti vities. Such structures, associated with high levels of gammaGTP and A LP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astrogl ial factors for the expression of the blood-brain barrier-related gamm aGTP activity, as well as for ALP activity, and could constitute the b asis of a valuable in vitro model of the blood-brain barrier. (C) 1994 Wiley-Liss, Inc.