IDENTIFICATION OF A MELANOCORTIN RECEPTOR EXPRESSED BY MURINE BRAIN MICROVASCULAR ENDOTHELIAL-CELLS IN CULTURE

Microvascular endothelial cells were isolated from the brains of C57 m ice and cultured in selective growth media. The isolation and culture techniques employed in this study minimised the contamination by nonen dothelial cells such as astrocytes, pericytes, and smooth muscle cells . Microvascular endothelial cells examined using phase contrast light microscopy grew as small colonies of spindle-shaped cells which merged together to form typical contact-inhibited monolayers. The endothelia l origin of these cells was determined using several established chara cterisation techniques. Preliminary receptor binding studies at 4 degr ees using [I-125-Tyr(2), Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone ([I-125-Tyr(2), Nle(4), D-Phe(7)]alpha-MSH) suggested the pos sibility that melanocortin receptors were present on the surface of br ain microvascular endothelial cells. Subsequent binding isotherms conf irmed that a small. population of high-affinity melanocortin receptors was expressed. The existence of a specific binding site for alpha-MSH was confirmed by photoaffinity labeling with the 4-(1-azi-2,2,2,-trif luoroethyl)benzoic acid (ATE) derivative, [I-125-Tyr(Z), Nle(4), D-Phe (7) (ATB)-Lys(11)]alpha-MSH. SDS-PAGE analysis identified the presence of a specific band with a molecular mass of approximately 45 kDa, whi ch was consistent with previous data on melanoma melanocortin receptor s, and represented a ligand-receptor complex. This study suggests that a receptor for alpha-MSH is expressed on the extracellular surface of murine brain microvascular endothelial cells; however, the physiologi cal role of this receptor is as yet unknown. (C) 1995 Academic Press, Inc.