Lens capsule basement membrane synthesis. Stimulation by glucose in vitro.

To examine the question of whether hyperglycemia per se can affect basement membrane synthesis, intact rat lenses, which produce basement membrane in vitro, were incubated for 24 h with radioactive proline or lysine and varying concentrations of glucose. Lens capsule basement membrane (LCBM) was subsequently purified and analyzed for radiolabel incorporation and for specific activities of proline, hydroxyproline, lysine, and hydroxylysine. [14C]-proline and lysine incorporation into LCBM was increasingly stimulated in incubations performed with 10 and 20 mM compared with 5 mM glucose. High glucose concentration increased the specific activity of proline and lysine but not hydroxyproline or hydroxylysine, and decreased the ratio of radioactive hydroxyproline to proline. Gel electrophoresis of radiolabeled LCBM prepared after high glucose incubation revealed increased radioactivity in serveral high-molecular-weight components corresponding with the major Coomassie-blue peptide bands. The results indicate that glucose stimulates LCBM synthesis, and suggest that this effect derives from increased production of noncollagenous components.

Nonenzymatic glycosylation of basement membranes: in vitro studies.

Incubation of purified rat glomerular basement membrane (GBM) with [14C]-glucose in vitro resulted in the incorporation of [14C] into acid-precipitable radioactivity in a reaction that was time and temperature dependent. Findings with rat lens capsule basement membrane (LCBM), an anatomically distinct but chemically similar extracellular matrix, incubated for varying times at different temperatures with [14C]-glucose at constant specific activity were similar. Nonenzymatic glycosylation of basement membrane, documented by hydroxymethylfurfuraldehyde generation after incubation with unlabeled glucose, increased in proportion to the ambient glucose concentration over a range of 5--100 mM. Acid-precipitable radioactivity also increased in proportion to [14C]-glucose concentration, although this method overestimated glycosylation about 15-fold at 5--20 mM glucose and 50-fold at 50--100 mM glucose. Coupled with recent in vivo studies, these findings indicate that exposure to increased glucose concentration alters the chemistry of glomerular and other basement membranes. Since accumulation of basement membrane characterizes several of the microangiopathic sequelae of diabetes, the role of increased nonenzymatic glycosylation on the structure, function, and metabolism of basement membrane warrants investigation.

Presence of glycosaminoglycans in retinal capillary basement membrane.

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [35S]sulfate and [14C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase ad chondroitinase ABC. Retinal microvessels also incorporated [35S]- and [14C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinal microvessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.