Caveolae and propofol effects on airway smooth muscle K.J. Grim, A.J. Abcejo, A. Barnes, V. Sathish, D.F. Smelter, G.C. Ford, M.A. Thompson, Y.S. Prakash, C.M. Pabelick  British Journal of Anaesthesia  Volume 109, Issue 3, Pages 444-453 (September 2012) DOI: 10.1093/bja/aes130 Copyright © 2012 The Author(s) Terms and Conditions

Fig 1 Propofol concentration within caveolar membranes of human ASM. Exposure of ASM cells to 10 or 30 μM for 5 min significantly increased concentrations within caveolae, as determined using GC (see text for details). Values are mean (sd); n=3 patients; *significant propofol effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 2 Effect of propofol on [Ca2+]i responses to histamine in human ASM cells. (a) Addition of 10 μM histamine resulted in a typical transient peak [Ca2+]i response followed by a lower plateau. After a wash (20 min), cells were exposed to either 10 µM propofol DMSO, 30 µM propofol DMSO, 10 µM propofol emulsion or 30 µM propofol emulsion (5 min). Subsequent determination of the [Ca2+]i response to histamine showed reduced peak [Ca2+]i levels compared with vehicle (buffer, intralipid, or DMSO) controls. Plateau [Ca2+]i levels were not affected by propofol. Values are means (sd) in the summary bar graph of (b), n=3 patients, average of 60 cells per condition. *Significant propofol effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 3 Effect of caveolin-1 siRNA on propofol effects. In human ASM cells, inhibition of caveolin-1 expression using siRNA blunted the normal [Ca2+]i responses to histamine. Under these conditions, the effect of propofol (30 µM) on [Ca2+]i responses to histamine was minimal, suggesting that caveolin-1 is important for propofol effect on ASM. Values are mean (sd), n=3 patients, average of 60 cells per condition; #significant siRNA effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 4 Role of SR Ca2+ release channels in propofol effects on [Ca2+]i. (a) In human ASM cells exposed to zero extracellular Ca2+, propofol continued to decrease the peak [Ca2+]i response to histamine. (b) With inhibition of IP3 receptor channels by XeC, [Ca2+]i responses to histamine were decreased (as expected); however, additional exposure to propofol continued to inhibit [Ca2+]i responses to histamine. In contrast, inhibition of RyR channels with ryanodine decreased [Ca2+]i response to histamine, but prevented propofol-induced decrease in the response. Values are mean (sd) in the summary bar graphs of (b), n=3 patients, average of 60 cells per condition; *significant propofol effect; #significant inhibitor effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 5 Effect of propofol on caffeine-induced SR Ca2+ release via RyR channels. The [Ca2+]i response to 10 mM caffeine was significantly blunted in the presence of 30 μM propofol, supporting the data in Figure 4 of an effect via RyR channels. Values are mean (sd), n=3 patients, average of 60 cells per condition; *significant propofol effect. British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 6 Effect of propofol on SOCE in human ASM cells. Ca2+ influx via SOCE was initiated as described previously.4 (a) Exposure to propofol before re-introduction of extracellular Ca2+ resulted in decreased SOCE with propofol in DMSO, while the propofol emulsion did not show significant changes (data not shown). (b) In cells transfected with caveolin-1 siRNA, SOCE was significantly reduced (as shown previously).62 However, in the presence of caveolin-1 siRNA, both formulations of propofol (DMSO or emulsion) did not further reduce SOCE. Values are mean (sd), n=3 patients, average of 60 cells per condition; *significant propofol effect; #significant caveolin-1 siRNA effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions

Fig 7 Effect of propofol on signalling mechanisms in ASM. In human ASM cells, exposure to propofol did not decrease total cellular caveolin-1, but did decrease caveolar expression of caveolin-1. With prolonged propofol exposure, caveolar expression of caveolin-1 recovered partially (data not shown). While total ERK and PI3K levels were unaffected, phosphorylation of ERK was decreased by propofol. RhoA levels (involved in Ca2+ sensitization) were decreased by propofol at 5 min. Values are mean (sd), n=3 patients; *significant propofol effect (P<0.05). British Journal of Anaesthesia 2012 109, 444-453DOI: (10.1093/bja/aes130) Copyright © 2012 The Author(s) Terms and Conditions