Role of apical and basolateral secretion in turnover of glutathione in LLC-PK1 cells.
Previous clearance measurements have established that the rapid turnover of renal proximal tubular glutathione is in part due to apical secretion and degradation by gamma-glutamyltranspeptidase, an ectoenzyme that is primarily associated with the brush-border membrane. The relationship between glutathione turnover and secretion was further characterized using confluent cultures of LLC-PK1 cells grown on nitrocellulose supports. The resulting cell layer was impermeable to [3H]inulin and exhibited a polarized expression of gamma-glutamyltranspeptidase. Incubating cells with 5 mM buthionine sulfoximine, an inhibitor of glutathione synthesis, produced an 86% inhibition of [35S]cystine incorporation into intracellular glutathione. Under these conditions, the prominent intracellular pool of glutathione turns over with an apparent half-life of 4 h and a first-order rate constant of 0.17 h-1. This turnover is unaffected by pretreatment with AT-125, an inhibitor of gamma-glutamyltranspeptidase. The rate of accumulation of glutathione in the apical and basolateral medium of cells pretreated with AT-125 was 22 and 34 nmol.mg protein-1.h-1, respectively. The combined secretion was equivalent to the calculated turnover rate of intracellular glutathione (57 nmol.mg protein-1.h-1). Therefore, the combined processes of apical and basolateral secretion can account for the turnover of intracellular glutathione in LLC-PK1 cells.
Scott
R D
RD
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Hughey
R P
RP
Curthoys
N P
NP
D000818:Animals; D019328:Buthionine Sulfoximine; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005978:Glutathione / Q000378:metabolism*; D006207:Half-Life; D007365:Intercellular Junctions / Q000502:physiology; D007668:Kidney; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013552:Swine
D013462:Sulfur Radioisotopes; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005978:Glutathione