Transport of methotrexate by primary cultures of rat hepatocytes: Stimulation of uptake in vitro by the presence of hormones in the medium

Abstract

Rat hepatocytes in primary monolayer culture exhibit an energy-dependent, concentrative accumulation of methotrexate. This transport system appears not to be involved in the uptake of the reduced folate derivatives. The capacity to accumulate methotrexate is rapidly diminished during culture and several lines of evidence indicate that this is due to the nonphysiologic environment provided by the culture system. The uptake system is partially stabilized by including glucagon, hydrocortisone, and tocopherol in the medium; these factors increase both the rate and extent of accumulation of methotrexate and result in intracellular levels that are approximately 250% of control (4 h incubation) after 24 and 48 h in culture. The site of hormonal stimulation of uptake is not completely understood, but several features of this process are apparent. Including the additives in the medium does not alter either the metabolism of methotrexate to polyglutamates or its intracellular binding. Thus, the main difference between the control and treated hepatocytes appears to be the capacity to accumulate free intracellular methotrexate. These observations suggest that the hormones act in some manner to stabilize the plasma membrane, and more specifically, the transport system for methotrexate. A similar effect has been observed with the hepatic transport system for asialoglycoproteins. ( A. L. Tarentino and J. H. Galivan, 1980, In Vitro, 16, 833–846). Some understanding of the mechanism of the factors in stimulating methotrexate uptake can be achieved from these studies. Glucagon appears to be acting at least in part through adenosine 3′:5′-monophosphate since it can be replaced by either isoproterenol and theophylline or N 6,O 2′ -dibutyryl adenosine 3′:5′-monophosphate. Dexamethasone can replace hydrocortisone, showing that the metabolism of hydrocortisone is not necessary for it to be active. Tocopherol may be acting as an antioxidant since it can be totally replaced by ascorbic acid. None of the data indicate whether these factors act directly on the plasma membrane or by altering the level of various intracellular metabolites which in turn stabilize the membrane. They do suggest that losses in function that are observed in the transfer of hepatocytes from the intact organ to culture may be prevented by adding factors, particularly hormones, that the cells normally encounter in vivo, and that the restoration of function may in part reflect a physiological function of the hormones.