Aqueous outflow pathway complex carbohydrate synthesis in vitro

A technique is described for examining the in vitro synthesis of glycosaminoglycans and glycoproteins in the aqueous outflow pathway (AOP). New Zealand red rabbit eyes were maintained at near-physiologic conditions and were infused by anterior chamber exchange with 30 muCi [3H] glucosamine and 100 muCi [35S] sulfate. At 0.5, 1.0, and 2.0 hours, the anterior segment tissues were dissected, isolated, and fractionated by gel filtration chromatography on Sephadex G-50 columns. The elution profiles demonstrated an increase of labeled material with time in the glycosaminoglycan (GAG) fractions and in the glycopeptide fractions. The relative rate of synthesis of labeled material was: central cornea greater than peripheral cornea greater than iris-ciliary body greater than AOP greater than anterior sclera. In order to characterize the profiles of each tissue, the isolated material was analyzed for hexosamine, hexuronic acid, and sulfate. The AOP cells synthesized a heterogenous mixture of GAGs and glycoproteins which biochemically appeared to be distinct from other anterior segment tissues. In addition, the percent distribution of GAG polymers in each tissue was determined by selective GAG degradative procedures and by gel filtration chromatography. The AOP cells synthesized four types of GAGs, and the percent distribution of the labeled GAGs was different from the other tissues. The present results suggest that this technique may provide a well-controlled method to probe the metabolic activity of complex carbohydrates in the AOP and in the anterior segment.