The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells

Abstract

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca²⁺ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 μm) evoked a slow and progressive increase in intracellular free Ca²⁺ ([Ca²⁺ ]_i ) towards a plateau. The effect of the compound on Ca²⁺ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca²⁺ -ATPase inhibitor thapsigargin (1 μm), in the absence of Ca²⁺ in the extracellular medium, induced a transient increase in [Ca²⁺ ]_i . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca²⁺ . Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca²⁺ . In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.

Keywords: calcium; cell viability; luzindole; pancreatic stellate cells; reactive oxygen species.