OPTIC-NERVE MICROVESSELS - A PARTIAL MOLECULAR DEFINITION OF CELL-SURFACE ANIONIC SITES

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are cons idered functionally important (a) in their interaction with circulatin g blood constituents, and (b) as a determinant of vascular permeabilit y. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anato mical location. Luminal anionic sites have not been characterized for EC of optic nerve. Optic nerves were removed from Sprague-Dawley rats previously fixed by vascular perfusion. EC anionic sites were labelled with the probes cationic colloidal gold (CCG) and cationic ferritin ( CF), using the pre- and postembedding techniques, and examined by elec tron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzyme s in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis softwa re. The biotinylated lectin wheat germ agglutinin (WGA) with streptavi din gold was also used to localize specific monosaccharide residues. T he luminal front of intraneural EC showed a uniform labelling with CCG and CF which was greater than on the abluminal surface. Extracellular matrix components and basal laminae were moderately labelled. Digesti on of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective t han papain but both produced a significant reduction. Neuraminidase al most completely eliminated labelling. CCG binding to the luminal plasm a membrane of optic nerve EC can be significantly reduced with proteol ytic and glycolytic enzymes. The results demonstrate that sialoglycopr oteins principally constitute these luminal EC anionic sites, Biotinyl ated WGA-streptavidin gold, which detects both N-acetylneuraminic (sia lic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These finding s suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylgl ucosamine. (C) 1995 Academic Press Limited