Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture.

Abstract

Abstract. Arterial proteoglycans have been implicated in several important physiological processes ranging from lipid metabolism to regulation of smooth muscle cell growth. Vascular smooth muscle (VSM) cells are the major producers of proteoglycans in the medial layer of blood vessels. To study functional consequences of alterations in VSM proteoglycan metabolism we used 4-methylumbelliferyl-~-D-xyloside to inhibit proteoglycan synthesis in primary and early passage cultures of rat aortic smooth muscle cells. Biochemical analysis of cultures labeled with 35SO, showed the drug inhibited synthesis of different classes of proteoglycans by 50 to 62 %. Inhibition of proteoglycan synthesis resulted in reduced accumulation of extracellular matrix, as shown by immunofluorescent staining with antibodies to chondroitin sulfate, fibronectin, thrombospondin, and laminin. There was also an inhibition of postconfluent (multilayered) growth of the smooth muscle cells, and a change in the morphology of the cells, with no apparent effect on subconfluent growth. In addition, in drug-treated cells there was a reduction in the number of cytoskeletal filaments that contained ot-actin, the actin subtype synthesized by differentiated VSM cells. This occurred even though the total content of c~-actin in the cells was not reduced. The effects of the inhibitor on growth and morphology could be reversed by switching the cultures to normal medium and could be prevented by growing the cells on preformed VSM extracellular matrix. These observations suggest the vascular extracellular matrix may play a role in regulating the growth and differentiation of smooth muscle cells.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

“…By inspection, cells were usually confluent on day 3, at which time 1 mM 4-methylumbelliferyl-pI-Dxyloside and 2.5 mM D( + )-galactosamine were added to the medium. These agents have been shown to inhibit proteoglycan synthesis (8,9). Pilot studies conducted in this lab have demonstrated a marked improvement in the rate of successful seal formation in IMCD cells so treated.…”

“…Given this strategically important location, it is not surprising that several humoral and 1. Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; cpt-cAMP, 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; ISC, short circuit current; LAA-94, 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-I -oxo-I H-inden-5-yl)oxy ] acetic acid; IBMX, isobutyl methyl xanthine; IMCD, inner medullary collecting duct; NMDG, N-methyl-D-glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; RT, transmonolayer electrical resistance. autocrine systems have been shown to be involved in the regulation of ion transport (see review by Zeidel [1]).…”

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

“…In addition to the steroids, 1 mM 4-methylumbelliferyl-D-xyloside and 2.5 mM D(+)-galactosamine were added. We have found that these compounds greatly enhance the rate of seal formation (36), probably due to their ability to inhibit proteoglycan synthesis (37,38). Pilot studies have shown that they do not alter the qualitative response of the IMCD monolayers to steroids and that they retain their sensitivity to benzamil (data not shown).…”

Interactions of Fibrillin-1 with Heparin/Heparan Sulfate, Implications for Microfibrillar Assembly

“…To shed light on this question, we have treated fibroblasts with ␤-D-xyloside derivatives, which reduce the amount of glycosaminoglycan chains in proteoglycans. Since ␤-D-xylosides are typically not completely specific in inhibition of just one type of glycosaminoglycan chain (43)(44)(45), a mixture of 4-methylumbelliferyl-␤-D-xylopyranoside and p-nitrophenyl-␤-D-xylopyranoside was used to obtain a maximum reduction of glycosaminoglycan chains on core proteins. Through this treatment, heparan sulfate and chondroitin sulfate are not biosynthesized on the protein cores of proteoglycans (45,46).…”