ENHANCEMENT OF GLUTATHIONE CONTENT IN GLUTATHIONE SYNTHETASE-DEFICIENT FIBROBLASTS FROM A PATIENT WITH 5-OXOPROLINURIA VIA METABOLIC COOPERATION WITH NORMAL FIBROBLASTS

Fibroblasts from patients with the disease 5-oxoprolinuria have reduce d glutathione synthetase activity and are thus glutathione (GSH) defic ient. In this study, 5-oxoprolinuria fibroblasts (GM3877 cells) contai ned less GSH than normal diploid fibroblasts as determined by biochemi cal analysis and by flow cytometry using monochlorobimane. They also c ontained lower gamma-glutamylcysteine synthetase activity than normal cells. However, cocultures of GM3877 cells and normal cells displayed either normal or slightly elevated GSH content, depending upon the ass ay used. When differentially labeled with fluorescent beads, coculture d, and then isolated by fluorescence-activated cell sorting, both GM38 77 cells and normal cells had GSH content similar to that of sorted no rmal cells cultured alone, whereas sorted GM3877 cells cultured alone showed depressed GSH content. GM3877 cells had detectable levels of ga mma-glutamylcysteine (gamma-GC) when cultured alone, but gamma-GC was undetectable in these cells when they were cocultured with normal cell s, indicating that it was efficiently metabolized to GSH by the normal cells. These changes in low-molecular-weight thiols were likely to ha ve been mediated by metabolic cooperation across gap junctions because they were dependent upon confluency and because media conditioned by either cell type failed to significantly alter the GSH content of the other cell type. Cocultures exposed to moderate levels of hydrogen per oxide showed less depletion of GSH than GM3877 cells cultured alone, s uggesting that the sharing of low-molecular-weight thiols or other red uctants via metabolic cooperation can protect cells from oxidative str ess. (C) 1994 Academic Press,Inc.