ROLE OF APICAL AND BASOLATERAL SECRETION IN TURNOVER OF GLUTATHIONE IN LLC-PK(1) CELLS

Previous clearance measurements have established that the rapid turnov er of renal proximal tubular glutathione is in part due to apical secr etion and degradation by gamma-glutamyltranspeptidase, an ectoenzyme t hat is primarily associated with the brush-border membrane. The relati onship between glutathione turnover and secretion was further characte rized using confluent cultures of LLC-PK1 cells grown on nitrocellulos e supports. The resulting cell layer was impermeable to [H-3]inulin an d exhibited a polarized expression of gamma-glutamyltranspeptidase. In cubating cells with 5 mM buthionine sulfoximine, an inhibitor of gluta thione synthesis, produced an 86% inhibition of [S-35]cystine incorpor ation into intracellular glutathione. Under these conditions, the prom inent intracellular pool of glutathione turns over with an apparent ha lf-life of 4 h and a first-order rate constant of 0.17 h-1. This turno ver is unaffected by pretreatment with AT-125, an inhibitor of gamma-g lutamyltranspeptidase. The rate of accumulation of glutathione in the apical and basolateral medium of cells pretreated with AT-125 was 22 a nd 34 nmol . mg protein-1 . h- 1, respectively. The combined secretion was equivalent to the calculated turnover rate of intracellular gluta thione (57 nmol . mg protein-1 . h- 1). Therefore, the combined proces ses of apical and basolateral secretion can account for the turnover o f intracellular glutathione in LLC-PK1 cells.