Abstract

The cytotoxicity, uptake, and metabolism of thapsigargin, an inhibitor of the Ca(2+)-ATPases of the sarco- and endoplasmatic reticulum (the SERCA-family), were investigated in suspensions of rat hepatocytes using [3H]thapsigargin labeled at C-8. No effect was observed on the lactate dehydrogenase leakage from the cells or in glucose formation when hepatocytes were incubated with 0.5-25 microM thapsigargin. At 25 microM [3H]thapsigargin the initial rate of uptake into the cells was 471 nmol/10(8) cells/min. Thapsigargin metabolism followed first-order kinetics, with an initial rate of metabolism at 25 microM of 65 nmol/10(8) cells/min. The much faster uptake than metabolism suggests that thapsigargin probably is bound to cellular proteins or trapped in cellular membranes. The metabolites were characterized by normal and reversed phase TLC and by positive and negative FAB/ms. Two labeled products were identified when cells were incubated with 25 microM [3H]thapsigargin. The first product formed was desoctanoyl-thapsigargin, followed by formation of desacyl-thapsigargin. Thapsigargin metabolism was strongly inhibited by diethyl p-nitrophenyl phosphate, indicating that the deacylation of the compound is catalyzed by microsomal carboxylesterases. Upon prolonged incubation, a volatile, tritiated compound appeared, possibly water, due to oxidation of the alcohol group at C-8 of thapsigargin.