thapsigargin and carbonyl-3-chlorophenylhydrazone

We investigated the mechanisms whereby omega-3 and -6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca(2+)](i) and reactive oxygen species (ROS). We applied the C-22, 20, and 18 carbon omega-3 (DHA, EPA, ALA) and omega-6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA-binding dye, propidium iodide. When applied at 20 µM concentration, omega-3 fatty acids killed thymocytes over a period of 1 h with a potency of DHA > EPA > ALA. The omega-6 PUFAs were more potent. The C18 omega-6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca(2+)](i) and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca(2+)](i) nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca(2+)](i) and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca(2+)](i). Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca(2+)](i), but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Cell Death; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Flow Cytometry; Hydrazones; Mice; Mice, Inbred ICR; Mitochondria; Reactive Oxygen Species; Thapsigargin; Thymus Gland; Time Factors; Uncoupling Agents; Vitamin E