AJP - Renal Physiology, Vol 265, Issue 5 723-F728, Copyright © 1993 by American Physiological Society
Role of apical and basolateral secretion in turnover of glutathione in LLC-PK1 cells
R. D. Scott, R. P. Hughey and N. P. Curthoys
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
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.
