Presence of store-operated Ca2+ entry in C57BL/6J mouse ventricular myocytes and its suppression by sevoflurane  A. Kojima, H. Kitagawa, M. Omatsu-Kanbe, H. Matsuura, S. Nosaka  British Journal of Anaesthesia  Volume 109, Issue 3, Pages 352-360 (September 2012) DOI: 10.1093/bja/aes212 Copyright © 2012 The Author(s) Terms and Conditions

Fig 1 Schematic diagram illustrating the superfusion protocol used for fluo-3 fluorescence Ca2+ measurements. (a) Ca2+ concentrations of the superfusate and the period of bath application of thapsigargin (Tg, 5 μM) and angiotensin II (Ang II, 1 μM) are shown. Fluo-3-loaded ventricular myocytes were continuously superfused with normal Tyrode solution (which contained 1.8 mM Ca2+; denoted as 1.8 mM) and nominally Ca2+-free Tyrode solution (which contained 0 Ca2+; denoted as 0 Ca2+), as indicated. (b) The period of bath application of verapamil (10 μM), KB-R7943 (KBR, 5 μM), 2-APB (20 μM), or sevoflurane (SEVO; 1%, 2%, 3%, or 5%) is shown. British Journal of Anaesthesia 2012 109, 352-360DOI: (10.1093/bja/aes212) Copyright © 2012 The Author(s) Terms and Conditions

Fig 2 Elevation of the cytosolic Ca2+ levels associated with SR Ca2+ store depletion in mouse ventricular myocytes. (a) and (b) Time courses of changes in fluo-3 fluorescence intensity during successive superfusion with nominally Ca2+-free Tyrode solution (denoted as 0 Ca2+) and normal Tyrode solution (denoted as 1.8 mM), with (a) and without (b) the combined application of thapsigargin (Tg, 5 μM) and angiotensin II (Ang II, 1 μM). Inset shows fluorescence images of ventricular myocytes taken at the time points indicated by corresponding letters (a–c). (c) Upper panel, time courses of changes in mag-fluo-4 fluorescence during Ca2+-free superfusion, with (filled blue circles) and without (empty green circles) the combined addition of thapsigargin (Tg, 5 μM) and angiotensin II (Ang II, 1 μM). Lower panel, summarized data for mag-fluo-4 F/F0 measured at the end of the 10 min of Ca2+-free superfusion, with and without the addition of thapsigargin and angiotensin II as indicated in the upper panel. *P<0.05. (d) Time courses of changes in the fluo-3 fluorescence intensity during superfusion with normal Tyrode solution with (filled blue circles) and without (empty green circles) the combined addition of thapsigargin (Tg, 5 μM) and angiotensin II (Ang II, 1 μM). *P<0.05 between the two groups at each time point. Error bars are 95% confidence intervals. British Journal of Anaesthesia 2012 109, 352-360DOI: (10.1093/bja/aes212) Copyright © 2012 The Author(s) Terms and Conditions

Fig 3 Inhibition of SOCE by 2-APB in mouse ventricular myocytes. (a) Time course of changes in F/Fbase, calculated from fluo-3 fluorescence images of ventricular myocytes, in the absence (Control) and presence of verapamil (10 μM), KB-R7943 (KBR, 5 μM), or 2-APB (20 μM). (b) The effects of various compounds on the relative fluo-3 fluorescence Fpeak/Fbase, where Fpeak represents the peak fluo-3 fluorescence obtained after the addition of extracellular Ca2+. *Significant difference (P<0.05) compared with the value measured 3 min after starting the Ca2+-free superfusion in the Control. §Significant difference (P<0.01) between the Control and 2-APB groups. Error bars are 95% confidence intervals. British Journal of Anaesthesia 2012 109, 352-360DOI: (10.1093/bja/aes212) Copyright © 2012 The Author(s) Terms and Conditions

Fig 4 Inhibitory effects of sevoflurane on SOCE. (a) Time course of changes in F/Fbase in the absence (Control) and presence of sevoflurane (SEVO) at 1%, 2%, 3%, or 5% plus with thapsigargin (5 μM) and angiotensin II (1 μM). (b) Fpeak/Fbase was plotted as a function of molar concentrations of sevoflurane and was fitted with a Hill equation, yielding an IC50 of 137 μM (which corresponds to 0.96%) and nH of 1.73. *Significant difference (P<0.05) compared with the value measured 3 min after starting the Ca2+-free superfusion in the Control. ¶, †, and ‡Significant difference (P<0.01) between Control and sevoflurane groups at concentrations of 2%, 3%, and 5%, respectively. Error bars are 95% confidence intervals. British Journal of Anaesthesia 2012 109, 352-360DOI: (10.1093/bja/aes212) Copyright © 2012 The Author(s) Terms and Conditions

Fig 5 Inhibition of SOCE current by sevoflurane. (a, d, and g) Time course of changes in membrane currents measured at +50 and −100 mV in the voltage-ramp protocol, during the addition of various compounds, as indicated. (b, e, and h) I–V relationships of membrane currents recorded at time points indicated by letters in a, d, and g, respectively. (c, f, and i) I–V relationships of difference currents obtained by digital subtraction of current traces shown in b, e, and h, as indicated, respectively. (j) Slope conductance of SOCE current in the presence of each concentration of sevoflurane (c–a, in f and i) was normalized to that of SOCE current activated by thapsigargin and angiotensin II (b–a, in f and i), and plotted as a function of sevoflurane concentration. The smooth curve represents a least-squares fit of a Hill equation, yielding an IC50 of 144 μM (which corresponds to 1.0%) and nH of 1.73. Significant differences (*P<0.05; ¶, †, and ‡P<0.01) between the Control and sevoflurane groups at 1%, 2%, 3%, or 5%. Error bars represent 95% confidence intervals. British Journal of Anaesthesia 2012 109, 352-360DOI: (10.1093/bja/aes212) Copyright © 2012 The Author(s) Terms and Conditions