Questioning the reliability of p-nitrophenyl-beta-D-xyloside as probe to study the metabolic effects of abrogated proteoglycan synthesis in cultured cells.

p-Nitrophenyl-beta-D-xylopyranoside (PNP-Xyl) and similar aglycone derivatives of xylosides are proposed selective inhibitors of proteoglycan synthesis which are used frequently to analyse the metabolic and cellular effects of abrogated proteoglycan formation and, hence, tentatively, the functions of these complex molecules. Using rat liver fat storing cell (FSC) cultures as a model, the possibility was tested that p-nitrophenol (PNP), which might be generated by the enzymatic hydrolysis of PNP-Xyl, could mediate some of those effects ascribed previously to PNP-Xyl induced inhibition of proteoglycan synthesis. PNP-Xyl and PNP inhibited dose-dependently the proliferation of FSC reaching 50% inhibition at about 1.9 and 0.6 mM, respectively. The inhibition of proliferation was not accompanied by signs of toxic cell damage and was fully reversible after withdrawal of the drugs. After an initial 4-fold stimulation of the formation of [35S]sulfate-labeled medium glycosaminoglycans (GAG) by PNP-Xyl at 0.1 mM, higher concentrations of this compound (about 0.5 mM) but also PNP decreased progressively the synthesis of sulfated medium GAG. A proliferation inhibiting concentration of PNP (0.75 mM) induced disorganization and reduced the expression of desmin- and smooth muscle iso-alpha-actin containing cytoskeletal filaments. These effects were similar to related effects reported previously for PNP-Xyl. Incubation of FSC with 5 mM PNP-Xyl resulted in a time-dependent increase of PNP in medium and cells; intracellular concentrations of PNP were reached sufficient to inhibit the mitotic activity of FSC. In lysates of FSC 0.65 nmol PNP/hr/micrograms DNA or 1 x 10(5) cells were generated from PNP-Xyl (5 mM) added as substrate. Exemplified with PNP-Xyl-treated FSC cultures, the results suggest for other cell and organ systems also that PNP, which is enzymatically cleaved from PNP-Xyl, might mediate at least some of the major effects attributed previously to the inhibition of proteoglycan synthesis. The aglycone may interfere with the effects of PNP-Xyl on proteoglycan metabolism and, therefore, could complicate in an unpredictable manner the interpretation of metabolic inhibitory studies using these compounds.