Biosynthesis and degradation of proteoglycans in cultured smooth muscle cells

Master Thesis


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University of Cape Town

Smooth muscle cells isolated from neonatal rat hearts synthesize and secrete radioactively labelled proteoglycans into two distinct extracellular compartments, the pericellular (cell surface/matrix layer) and the culture medium (extracellular). Cultures grown in the presence of ascorbic acid synthesize proteoglycans that are more highly sulphated than those produced in the absence of ascorbate. The glycosaminoglycan chains associated with the proteoglycans synthesized by rat smooth muscle cells were heparan sulphate, chondroitin sulphate and dermatan sulphate. There was no evidence for the synthesis of hyaluronic acid by these cells. Most of the heparan sulphate was found to be associated with the pericellular and intracellular compartments, whereas the extracellular compartment contained the bulk of the chondroitin sulphate. In the presence of ascorbate there was an increase in dermatan sulphate content of the pericellular compartment at the expense of heparan sulphate, whilst in the absence of ascorbate the heparan sulphate content of this compartment was significantly increased. Hyaluronic acid and the antibiotic Tunicamycin had no effect on the biosynthesis of sulphated macromolecules produced by the rat smooth muscle cells. However, p-nitrophenyl-β-D-xyloside increased by 10-fold the amount of radioactive sulphate incorporation into macromolecules in the extracellular compartment. This increase was due to increased sulphation of glycosaminoglycan chains synthesized in the presence of the exogenous acceptor, as evidenced by the sulphate/ uronate ratio of these sulphated macromolecules. Furthermore, heparan sulphate secretion into the extracellular compartment was decreased whilst dermatan sulphate increased in the presence of xyloside. Pulse-chase experiments with radioactive sulphate were used to study the pathways and kinetics of secretion in the rat smooth muscle cell system. The data from these studies are consistent with a very rapid intracellular sulphation mechanism followed by rapid secretion to the pericellular compartment of macromolecular sulphated proteoglycans. Subsequently some of these molecules then travel to the extracellular compartment. The time that different proteoglycan species remain associated with the pericellular compartment is influenced by the different matrix connective tissue proteins found in this compartment as a result of ascorbate supplementation or deprivation. During the course of these investigations, it was observed that the pericellular compartment contributed to catabolism of sulphated macromolecules. The sulphated proteoglycans associated with this compartment are acted upon by a sulphatase or sulphatases to give rise to free radioactive inorganic sulphate and macromolecules which have been desulphated. That this process occurs in the pericellular compartment only was proven by the use of intracellular lysomotrophic inhibitors and by the continuous exposure of sulphate labelled macromolecules to the extracellular extract. Neither resulted in the release of radiolabelled inorganic sulphate from sulphated macromolecules.