Inhibitor of Sarco-endoplasmic Reticulum Ca2+-ATPase Thapsigargin Stimulates Production of Nitric Oxide and Secretion of Interferon-gamma

Thapsigargin is a sesquiterpene lactone of guaianolide type isolated from the Mediterranean plant Thapsia garganica L. It is widely used experimentally as a potent and selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) leading to rapid elevation of intracellular calcium [Ca2+]i. Several previous reports have shown that thapsigargin interferes with production of nitric oxide (NO) by mouse peritoneal macrophages and mouse macrophage cell lines. The present data confirm that thapsigargin is a modest inducer of NO in mouse macrophages, production of NO being slightly enhanced by lipopolysaccharide. However, thapsigargin on its own very potently induces NO in macrophages of rats under conditions in vitro. The highest effect was observed after the concentration of 0.25 microM thapsigargin, producing approximately 30 microM accumulation of nitrites in supernatants of cells cultured for 24 h. The aim of our experiments was to investigate immune mechanisms implicated in activation of high-output NO biosynthesis. It has been found that thapsigargin dose-dependently induces secretion of interferon-gamma (IFN-gamma) in macrophages of both rats and mice, and also in human peripheral blood mononuclear cells. The IFN-gamma production was rather low in macrophages of mice while relatively very high levels of IFN-gamma were found in cultures of rat macrophages and human peripheral blood mononuclear cells. The concentration of IFN-gamma produced by 5 microM thapsigargin within the interval of 24 h exceeded 3 ng/ml in rat macrophages and approached 2 ng/ml in cultures of human peripheral blood mononuclear cells. The effects are mediated by mitogen-activated protein kinases (MAPKs) such as p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinases 1/2 (ERK1/2), and by nuclear transcriptional factor NF-kappaB. In summary, the original findings demonstrate immunostimulatory potential of thapsigargin and warrant more detailed preclinical studies.