The basement membrane of bovine corneal endothelial cells in culture with beta-aminopropionitrile: biosynthesis of hexagonal lattices composed of a 160 nm dumbbell-shaped structure.

Bovine corneal endothelial cells maintained in culture produced an extracellular matrix on their basal surface. In the presence of beta-aminopropionitrile (beta-APN) (100-500 micrograms/ml), an inhibitor of cross-linking, this extracellular matrix contained hexagonal lattices of 150 nm periodicity. The lattice was composed of round densities 60 nm in diameter comprising the nodes of the lattice and rod-like structures 40 nm in thickness comprising its sides. This structure is almost identical to that of bovine Descemet's membrane in situ. The lattice was produced only in the presence of beta-APN. By freeze-etch replica and negative staining of 1 day cultures, the unit component of the lattice appeared to be a novel 160 nm long dumbbell-shaped symmetrical structure. The central rod of the dumbbell-shaped structure was 80 nm long and 20 to 50 nm in diameter with round ends (40-50 nm). In the rod 2 nm fibrillar substructures were discerned by negative staining. The rod-like central portion was resistant to trypsin digestion whereas the round ends were digested by it. Both structures disappeared after bacterial collagenase digestion. By immunoblotting the extracellular matrix contained 160kd and 180kd bands which bound anti-type IV collagen antibodies and a 140kd band which bound anti-type III collagen antibodies. The lattice area was also positively stained with these antibodies by immunoperoxidase. The findings strongly suggest that the hexagonal lattice of bovine Descemet's membranes in situ is composed of this collagenous structure.