Biochemical Pharmacology, Vol. 35, No. 24. pp. 4537-4542, 1986. 0006-2952/86 $3.00 + 0.00 Printed in Great Britain. © Pergamon Journals Ltd. PROTECTION AGAINST PARAQUAT-INDUCED INJURY BY EXOGENOUS GSH IN PULMONARY ALVEOLAR TYPE II CELLS TORY M. HAGEN,* LOU ANN BROWNt and DEAN P. JONES*~ Departments of *Biochemistry and +Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. (Received 27 March 1986; accepted 9 June 1986) Abstract--Exogenous GSH provided rat alveolar type II cells with significant protection against injury induced by paraquat. This protection was also observed in cells treated with acivicin to inhibit GSH degradation and buthionine sulfoximine to inhibit GSH synthesis. Exogenous GSH was transported into cells by a Na+-dependent system. Addition of inhibitors of this transport system, 7-glutamyl-glutamate and probenecid, prevented the protection against injury afforded by GSH. Thus, the results indicate that alveolar type II cells can supplement endogenous synthesis of GSH with uptake of exogenous GSH to protect against paraquat-induced injury. Oxidative injury in the lung occurs from inhaled air pollutants, oxygen therapy, antitumor compounds, and other redox cycling agents such as paraquat [1-3]. Considerable effort has been made to under- stand and minimize oxidative injury in lung. However, because of the large number of different cell types, understanding the critical susceptibility of the individual cell types has been difficult. Over the past several years, preparations of some of these cell types have become available. We have focused on the alveolar type II cells because they function in the production and release of surfactant into the alveolar space and also are progenitors of type I cells which form most of the epithelial surface of the alveolus [4, 5]. While type II cells are susceptible to oxidative injury, they are more resistant than are type I cells, but the reasons for this apparent resistance are as yet unclear [6]. Glutathione (GSH) constitutes the major source of low molecular weight thiol in mammalian tissue and functions in detoxification of electrophilic com- pounds and protection against oxidative injury. Numerous reports show that GSH depletion occurs prior to cell injury for several types of toxic sub- stances [7-9]. Much of the effort has focused on hepatic injury, and there is now substantial under- standing of the toxicological consequences involving thiol oxidation, such as inhibition of Ca 2+ ATPases [10], disruption of cytoskeletal elements [11], and alteration of metabolic regulation [12]. Molddus and co-workers [13] found that isolated perfused lung can synthesize GSH from the precursor amino acids and that the lung can also use exogenous GSH for this purpose. However, inhibitor studies indicated that the utilization of exogenous GSH was largely due to extracellular breakdown and sub- sequent resynthesis. We have found recently that renal epithelial cells and small intestinal epithelial cells contain a sodium- Author to whom all correspondence should be sent. dependent uptake system for intact GSH [14, 15]. In the small intestinal cells, exogenous GSH provides effective protection against oxidative injury due to either t-butylhydroperoxide or menadione [15]. In the present study, we investigated whether intact GSH can provide protection in type II cells against the strong oxidant, paraquat, and, if so, the mechanism by which GSH may offer this protection. The results show that intact exogenous GSH was taken up by isolated type II cells and that it provided substantial protection against paraquat-induced injury. MATERIALS AND METHODS GSH, Percoll, paraquat, (1,1-dimethyl-4,4'-bypy- ridinium dichloride), HEPES (N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid), probenecid, ),- L-glutamyl-L-glutamate and heparin were purchased from Sigma. [Glycine-2-3H]GSH (specific activity l.lCi/mmole) was purchased from New England Nuclear. Penicillin G potassium was obtained from the Emory University Hospital Pharmacy. Oph- thalmic acid was purchased from Bachem Inc., Torr- ence, CA. Acivicin [L-(olS,5s)-cr-amino-3-chloro-4,5- dihydro-5-isoxazoleacetic acid; AT-125] was a gift from Dr. J. P. McGovren (Upjohn Co., Kalamazoo, MI). Buthionine-SR-sulfoximine was purchased from the Chemical Dynamics Corp. Alveolar type II cells were obtained from male Sprague-Dawley rats (King outbred albino), weigh- ing approximately 200-250 g, that were kept in air- filtered chambers to reduce the risk of lung infec- tions. Type II cells were isolated by trypsinization and purified by discontinuous albumin density gradi- ent centrifugation as previously described [16]. When examined by polychrome staining [17], 80-85% of the cells contained lamellar bodies, indicating that the preparation is largely type II cells. Primary cul- tures established with this preparation yield approxi- mately 95% type II cells which produce surfactant 4537