Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx

Abstract

1. The proteolytic enzyme pronase, which degrades the endothelial cell glycocalyx, was perfused through single capillaries of frog mesentery. Hydraulic conductivity (Lp) of each vessel was determined before and after pronase perfusion. In three vessels in which Lp increased, the ultrastructure of interendothelial clefts was examined. In a separate group of frogs the effect of pronase on the endothelial glycocalyx was assessed by using cationized ferritin to label the capillary luminal surface. 2. Control Lp was 2.0 x 10(-7) cm s-1 cmH2O-1 (10 mg ml-1 bovine serum albumin, BSA, in frog Ringer solution). Vessels were then perfused with a solution containing 0.1 mg ml-1 pronase and 10 mg ml-1 BSA for 1 min. Lp measured in the same eleven vessels increased to 4.9 x 10(-7) cm s-1 cmH2O-1 (P less than 0.005). 3. Transverse sections of three of these vessels were examined by transmission electron microscopy at eight sites along each vessel. In these sections a total of 156 interendothelial cell clefts were found and photographed. No morphological features, such as fenestrations, transendothelial channels, or intercellular gaps associated with inflammation, were found which might account for the increases in Lp. 4. Measurement of cleft dimensions yielded a harmonic mean cleft depth (delta x) of 0.32 microns and an arithmetic mean cleft depth of 0.64 microns. Mean width (w) of the clefts outside the tight regions was 0.012 microns and the cleft length per unit area (L) was 1330 cm-1. The mean fractional pore area of vessel wall per unit cleft depth, Ap/delta x, calculated as Lw/delta x, was 48.7 cm-1. 5. There was less cationic ferritin (CF) labelling of the luminal glycocalyx in pronase-perfused than in control capillaries. On average, the proportion of the luminal surface covered by CF was 85% in controls and 42% in pronase-treated capillaries (P less than 0.01). In some vessels the CF pattern was greatly disrupted, indicating large changes in the glycocalyx structure. 6. It is concluded that the moderate increases in Lp induced by pronase perfusion are associated with partial digestion of the endothelial glycocalyx but are not accompanied by changes in the dimensions of the intercellular cleft. These observations support the fibre matrix hypothesis of capillary permeability and suggest that the endothelial glycocalyx contributes as much as 60% of the hydraulic resistance of the capillary wall.