Optic nerve microvessels: a partial molecular definition of cell surface anionic sites

Abstract

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are considered functionally important (a) in their interaction with circulating blood constituents, and (b) as a determinant of vascular permeability. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anatomical 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 post-embedding techniques, and examined by electron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzymes in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis software. The biotinylated lectin wheat germ agglutinin (WGA) with streptavidin gold was also used to localize specific monosaccharide residues. The 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. Digestion of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective than papain but both produced a significant reduction. Neuraminidase almost completely eliminated labelling. CCG binding to the luminal plasma membrane of optic nerve EC can be significantly reduced with proteolytic and glycolytic enzymes. The results demonstrate that sialoglycoproteins principally constitute these luminal EC anionic sites. Biotinylated WGA-streptavidin gold, which detects both N-acetylneuraminic (sialic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These findings suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylglucosamine.