Molecular and cellular pharmacology Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress Gabriela Ziomek a , Parisa Cheraghi Zanjani a , Darian Arman a , Cornelis van Breemen a,n , Mitra Esfandiarei a,b,nn a Child & Family Research Institute, Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada b Department of Biomedical Sciences, College of Health Sciences, Midwestern University, Glendale, AZ, USA article info Article history: Received 19 December 2013 Received in revised form 31 March 2014 Accepted 6 April 2014 Available online 25 April 2014 Keywords: Vascular smooth muscle cell Endoplasmic reticulum stress Unfolded protein response Tunicamycin Calcium abstract Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long- term exposure to 10 mM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsi- gargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability. & 2014 Elsevier B.V. All rights reserved. 1. Introduction The endo/sarcoplasmic reticulum (ER/SR) is a complex orga- nelle that, through orchestrating calcium (Ca 2 þ ) signaling, con- trols most “fast” cell functions but also participates in a large variety of “slower” cellular processes, including protein folding and transport, which appear to be related to Ca 2 þ storage (Michalak et al., 2002; Walter and Ron, 2011; Xu et al., 2005). This last function is one of vast importance, particularly in the situation that arises within cells during the stress response. ER/SR stress is a phenomenon characterized by the accumulation of unfolded proteins in the ER/SR lumen (Chao et al., 2012; Rutkowski and Kaufman, 2004; Yoshida, 2007). When the ER/SR is subjected to these circumstances, coordinated efforts are initiated by the cell to correct the problem and alleviate the pathogenic condition by initiating a multi-stage process collectively known as the unfolded protein response (UPR). It is generally accepted that ER/SR stress is associated with a reduction of ER/SR Ca 2 þ content as a result of a vicious cycle initiated by the accumulation of unfolded proteins, which induces Ca 2 þ leakage from the ER/SR, which in turn inhibits calreticulin- controlled protein folding (Ashby and Tepikin, 2001; Foufelle and Ferre, 2007). In our study on vascular smooth muscle cells (VSMCs), which are a potentially important target for disease, we also wished to investigate this relationship. However, Ca 2 þ regulation in the ER/SR of VSMCs is complex, with two parallel pathways involved in the maintenance of a steady state ER/SR Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ejphar European Journal of Pharmacology http://dx.doi.org/10.1016/j.ejphar.2014.04.025 0014-2999/& 2014 Elsevier B.V. All rights reserved. n Corresponding author. Tel.: þ1 604 875 3852. nn Corresponding author at: Department of Biomedical Sciences, College of Health Sciences, Midwestern University, Glendale, AZ 85308, USA. Tel.: þ1 623 572 3666. E-mail addresses: casey@ti.ubc.ca (C. van Breemen), mesfan@midwestern.edu (M. Esfandiarei). European Journal of Pharmacology 735 (2014) 86–96