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Caveolae and propofol effects on airway smooth muscle
British Journal of Anaesthesia 109 (3): 444–53 (2012) Advance Access publication 27 April 2012 . doi:10.1093/bja/aes130
Caveolae and propofol effects on airway smooth muscle K. J. Grim 1†, A. J. Abcejo 1†, A. Barnes 1, V. Sathish 1, D. F. Smelter1, G. C. Ford2, M. A. Thompson 1, Y. S. Prakash 1,3 and C. M. Pabelick 1,3* 1
Department of Anesthesiology, 2 CTSA Metabolomics Core and 3 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA * Corresponding author. E-mail: [email protected]
Editor’s key points
† Propofol decreased peak intracellular calcium responses to histamine. † When caveolin-1 was blocked with small interference RNA, the effect of propofol was abolished. † Propofol appears to affect airway relaxation via caveolin-1 effects on calcium.
Methods. Caveolar fractions of human ASM cells were tested for propofol content. [Ca2+]i responses of ASM cells loaded with fura-2 were performed in the presence of 10 mM histamine with and without clinically relevant concentrations of propofol (10 and 30 mM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca2+ release were evaluated in zero extracellular Ca2+ using the blockers Xestospongin C and ryanodine. Store-operated Ca2+ entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca2+]i and force regulation were also evaluated. Results. Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 mM propofol form decreased [Ca2+]i peak (but not plateau) responses to histamine by 40%, an effect persistent in zero extracellular Ca2+. Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca2+]i, while propofol blunted [Ca2+]i responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signalregulated kinase phosphorylation. Conclusions. These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca2+]i. Keywords: bronchial smooth muscle; bronchodilatation; calcium regulation; caveolin; intravenous anaesthetic; signalling Accepted for publication: 22 February 2012
Since its invention in 1977, propofol has become the most frequently used i.v. anaesthetic, gaining rapid acceptance for use in sedation and general anaesthesia. Propofol is also known to produce substantial vasodilatation and bronchodilatation.1 Indeed, propofol-induced bronchodilatation has been demonstrated in both healthy and asthmatic patients.2 3 In spite of this clinical evidence, the cellular mechanisms by which propofol produces bronchodilatation are still under investigation. Relaxation of the airway involves a reduction in intracellular Ca2+ concentration ([Ca2+]i) of airway smooth muscle (ASM). We and others have previously demonstrated that in ASM, [Ca2+]i regulation involves both Ca2+ release from sarcoplasmic reticulum (SR) stores and plasma membrane †
Ca2+ influx,4 – 8 with the latter via several mechanisms including controlled influx in response to SR Ca2+ depletion [store-operated Ca2+ entry (SOCE)].4 7 9 Force regulation involves myosin light chain (MLC) and Ca2+ sensitization via the RhoA/Rho kinase pathway.10 11 Thus, there are several potential targets for propofol-induced bronchodilatation. In this regard, propofol has been found to attenuate vagal and methacholine-induced bronchoconstriction, with possible direct actions on muscarinic receptors.12 13 Propofol also decreases ovalbumin-induced contraction of sensitized rat trachea through inhibition of serotonin.14 Whether propofol directly affects ASM has been examined only in a few studies (albeit in varied species). Propofol has been shown to decrease peak [Ca2+]i response to agonists,15 perhaps
These authors contributed equally to this work.
& The Author [2012]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
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† The effect of propofol on intracellular calcium was investigated in human airway smooth muscle cells in vitro.
Background. The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca2+]i. We postulated that propofol-induced bronchodilatation involves caveolar disruption.
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Methods Human ASM cell preparation The techniques for isolation of human ASM cells have been described previously.27 Briefly, bronchi were obtained from lung tissues incidental to patient procedures in thoracic surgery at Mayo Clinic Rochester (de-identified tissues typically from lobectomies and pneumenectomies; approved by the Mayo Institutional Review Board and not considered Human Subjects Research). Tissues were initially placed in Hank’s’ balanced salt solution (HBSS, Invitrogen, Carlsbad, CA, USA) with 2.5 mM extracellular Ca2+. ASM layers were isolated and enzymatically dissociated as described previously.32 Cells were plated in sterile culture flasks and grown in a 95% air/5% CO2 humidified incubator using DMEM F/12 supplemented with 10% fetal bovine serum (FBS). All experiments were performed in cells before the third passage to ensure cell phenotype as verified by immunostaining for smooth muscle actin and myosin and by expression of receptors for commonly used bronchoconstrictor agonists, such as the M3 muscarinic receptor and the H1 histaminergic receptor.
Preparation of caveolar membranes Caveolin-rich membranes were prepared as described previously.27 Briefly, ASM strips were homogenized in cold buffer A (0.25 M sucrose, 1 mM EDTA, and 20 mM Tricine, pH 7.8) until fine emulsion was achieved. The homogenates were sonicated for 10 s each, centrifuged (1000g, 10 min, 48C), the supernatant removed and brought to 5 ml, which was layered onto 30% Percoll, and re-centrifuged (84 000g, 30 min). The plasma membrane fraction was collected and brought to 2 ml with buffer A. A crude membrane fraction was then sonicated, resuspended in OptiPrep, and then placed in a centrifuge tube. A linear 20–10% OptiPrep gradient was layered on top before centrifugation at 52 000g for 90 min. The upper membrane layer containing the caveolae was then collected for analysis. The lower membrane layer was collected as a control.
Measurement of propofol within caveolar membranes by gas chromatography Propofol was measured within caveolar membrane fractions to confirm the connection between lipid-rich caveolae and propofol. Propofol concentrations were measured by gas chromatography –mass spectrometry (GC/MS) using a modified method as described previously.33 34 Briefly, caveolar fractions contained in 0.5 ml sucrose buffer were mixed with 100 ml n-heptane for propofol extraction and the organic phase placed in 200 ml autosampler vials for analysis. GC/MS analysis was performed using a Waters Quattro Micro triple quadruple GC/MS/MS system (Waters, Manchester, UK) operating under electron impact ionization conditions. The column used was a DB-5MS fused silica capillary column (30 m×0.25 mm inside diameter, 0.25 mm film thickness, Agilent Technologies, USA). A temperature programme ramp was performed from 60 to 3008C at 208C min21. The injection temperature was set at 2508C and the transfer line temperature at 2508C. Helium was used as a carrier gas at a flow rate of 1.0 ml min21, and 1 ml of the sample extract was injected. The [M-15]+ fragment ion of propofol at m/z 163.1 was monitored for quantification and the peak area compared with an external calibration curve of propofol, prepared in n-heptane over a concentration range of 0.015 –1.8 ng ml21, 1 ml being injected on-column.
Fluorescence [Ca21]i imaging ASM cells were incubated in 5 mM fura-2 AM (Invitrogen) for 45 min at room temperature and visualized with a fluorescence imaging system (MetaFluor, Universal Imaging, Downingtown, PA, USA) on a Nikon Diaphot inverted microscope. Cells were initially perfused with HBSS (2.5 mM Ca2+, 238C), and baseline fluorescence established. [Ca2+]i responses of 10 cells per chamber were obtained for individual, softwaredefined regions of interest. Fura-2 was alternately excited at 340 and 380 nm, and the ratio of fluorescence emissions at 510 nm was obtained every 0.75 s (Photometric Cascade digital camera system, Roper Scientific, Tucson, AZ, USA).
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through SR Ca2+ release via inositol trisphosphate (IP3) receptor channels,16 but does not appear to affect Ca2+ sensitivity.17 On the other hand, one study suggested that all of the effects of propofol on the airway may be neurally mediated.12 Thus, the mechanisms underlying propofolinduced bronchodilatation remain unclear. A particular feature of propofol (and indeed its preparation) is its highly lipid nature. There is now increasing evidence that the plasma membrane of almost all cell types (including ASM) contain specialized lipid rafts, including 50– 100 nm invaginations called caveolae,18 – 24 likened to be more intricate T-tubules of the striated muscle. Caveolae can facilitate interactions between plasma membrane-regulatory elements and intracellular signalling cascades.18 – 26 We demonstrated in human ASM, that suppression of the constitutional protein caveolin-1 substantially blunts agonist-induced [Ca2+]i responses and alters [Ca2+]i signalling, indicating a role for lipid rafts in ASM contractility.26 27 In human, ASM substantially blunts agonist-induced [Ca2+]i responses and alters [Ca2+]i signalling, indicating a role for lipid rafts in ASM contractility.26 27 Previous studies have shown that local and volatile anaesthetics act via interaction with lipid rafts.28 29 There is also evidence that caveolae specifically are involved in the actions of general anaesthetics.30 31 However, the role of caveolae in mediating the effects of propofol is not known. Given the lipid nature of propofol, we hypothesized that the bronchodilatory effects of this anaesthetic may be mediated (at least in part) by alterations in caveolar signalling. Accordingly, in the present study, we used human ASM cells to examine the potential role of caveolae and caveolin-1 in propofol-induced bronchodilatation.
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Materials Methyl-b-cyclodextrin (CD), Optiprep, and other chemicals were obtained from Sigma (St Louis, MO, USA) unless mentioned otherwise. All tissue culture reagents were obtained from Invitrogen. Primary and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propofol DMSO formulation was obtained from Sigma; propofol emulsion was obtained from the Mayo Clinic Pharmacy.
Statistics Propofol effects on [Ca2+]i responses in human ASM cells were compared with controls using Student’s paired t-tests. At least 10 cells from three ASM isolations (three different patients) were compared. GC measurements were performed in samples from three patients. Values are means (SD) and n is the number of patients. Statistical significance was established at P,0.05.
Results Propofol concentration within caveolae To confirm our hypothesis that propofol induces bronchodilatation via interaction with caveolae, we directly measured propofol concentrations within caveolar membranes using GC. Compared with vehicle control, exposure of ASM to 10 and 30 mM resulted in significantly higher propofol concentrations within caveolar membranes (P,0.05; Fig. 1).
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Fig 1 Propofol concentration within caveolar membranes of human ASM. Exposure of ASM cells to 10 or 30 mM for 5 min significantly increased concentrations within caveolae, as determined using GC (see text for details). Values are mean (SD); n¼3 patients; *significant propofol effect (P,0.05).
Propofol effect on [Ca21]i response to histamine In unstimulated human ASM cells, baseline [Ca2+]i was 100 nM. Upon addition of 10 mM histamine, a typical rapid increase in [Ca2+]i was observed, with a peak response of 514 (43) nM. The subsequent plateau was 223 (53) nM. After a wash in HBSS for 20 min, exposure to 10 or 30 mM propofol for 5 min did not substantially decrease baseline [Ca2+]i levels. However, subsequent stimulation with 10 mM histamine resulted in an 40% decrease in peak [Ca2+]i response (Fig. 2; P,0.05). The plateau [Ca2+]i response was not different between control and propofol-exposed cells.
Effect of caveolin-1 siRNA In ASM cells transfected with caveolin-1 siRNA, exposure to histamine resulted in a considerably smaller [Ca2+]i response, consistent with our previous study.27 In these cells, the addition of 30 mM propofol also decreased peak [Ca2+]i responses to histamine; however, these effects were substantially blunted compared with cells transfected with Lipofectamine alone (Fig. 3; P,0.05 for significant effects of siRNA).
Propofol effect on SR Ca21 release Under conditions of zero extracellular Ca2+ exposure, baseline [Ca2+]i was 80 nM and exposure to 10 mM histamine continued to result in a peak transient [Ca2+]i response [260 (15) nM]. Treatment with 10 mM or 30 mM propofol for 5 min substantially reduced the peak [Ca2+]i response to 10 mM histamine (P,0.05; Fig. 4A). The effects of 10 vs 30 mM propofol were the same.
Effect of propofol on IP3 receptor channels We have previously demonstrated that SR Ca2+ release in ASM involves both IP3 receptor and ryanodine receptor (RyR) channels.35 In one set of experiments, IP3 receptor channels were first blocked with 20 mM Xestospongin C
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Proteins were separated by SDS –PAGE using the Criterion Gel System (Bio-Rad, Hercules, CA, USA) and 15% or 4–15% gradient gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) for 60 min and then blocked for 1 h with 5% milk in Tris-buffered saline containing 0.1% Tween (TBST). Membranes were then incubated with anti-caveolin-1 (1:1000), anti-ERK (1:1000), anti-pERK (1:100), anti-PI3K (1:1000), or anti-RhoA (1:1000) antibodies overnight at 48C. Following three washes with TBST, primary antibodies were detected with horseradish peroxidaseconjugated secondary antibodies and signals developed by Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA).
1.2e+5 Concentration (ng mg–1 protein)
The technique for suppression of caveolin-1 expression in human ASM using small interference RNA (siRNA) caveolin-1 was recently described.27 Briefly, siRNA duplex corresponding to bovine caveolin-1 mRNA targeting against the open reading frame, 223 –241 bases, and a negative control siRNA were selected for caveolin-1 knockdown (Dharmacon, Lafayette, CO, USA). ASM cells at 60% confluence were transfected using 50 nM siRNA and Lipofectamine 2000 (Invitrogen) in DMEM F/12 without FBS. Fresh growth medium was added 6 h after transfection and experiments performed after 48 h. Both Lipofectamine only (vehicle) and scrambled siRNA sequences were used as controls.
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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 mM) on [Ca2+]i responses to histamine was minimal, suggesting that caveolin-1 is important for propofol effect on ASM. Values are mean (SD), n¼3 patients, average of 60 cells per condition; #significant siRNA effect (P,0.05).
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Fig 2 Effect of propofol on [Ca2+]i responses to histamine in human ASM cells. (A) Addition of 10 mM 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 mM propofol DMSO, 30 mM propofol DMSO, 10 mM propofol emulsion or 30 mM 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), n¼3 patients, average of 60 cells per condition. *Significant propofol effect (P,0.05).
(XeC, a cell permeant inhibitor). Cells were stimulated with 10 mM histamine, thoroughly washed, treated with 30 mM propofol for 5 min, and re-stimulated with 10 mM histamine. Although histamine-induced [Ca2+]i responses were decreased by XeC, additional exposure to propofol further decreased this [Ca2+]i response (P,0.05; Fig. 4B).
Effect of propofol on RyR channels We previously demonstrated that RyR channels are blocked by high concentrations of ryanodine.35 In this set of experiments, RyR channels were first blocked with 20 mM ryanodine for 15 min. Cells were stimulated with 10 mM histamine, thoroughly washed, treated with 30 mM propofol for 5 min, and re-stimulated with 10 mM histamine. Although histamine-induced [Ca2+]i responses were decreased by ryanodine, additional propofol did not further decrease this [Ca2+]i response (Fig. 4B).
To further investigate the effects of propofol on SR Ca2+ release via RyR channels, ASM cells were exposed to 5 mM caffeine in the absence or presence of 30 mM propofol. Thirty micromoles of propofol blunted caffeine-induced SR Ca2+ release (Fig. 5; P,0.05).
Effect of propofol on SOCE We have previously demonstrated SOCE in ASM.4 A similar protocol was used here. Extracellular Ca2+ was first removed by perfusion with zero-Ca2+ HBSS. Voltage-gated Ca2+ influx was further inhibited using 1 mM nifedipine and 10 mM KCl. SR Ca2+ stores were then depleted using 10 mM cyclopiazonic acid (an inhibitor of SR Ca2+ reuptake), resulting in a slow increase in [Ca2+]i levels due to continued leak from the SR. After 15 min, extracellular Ca2+ was rapidly re-introduced to trigger SOCE. Cells were then washed thoroughly to refill SR stores, and the protocol repeated with additional exposure to 30 mM propofol 5 min before re-introduction of extracellular Ca2+ (thus avoiding any effect of propofol on the SR release aspect). Exposure to propofol resulted in decreased SOCE in the propofol/ DMSO formulation (P,0.05; Fig. 6), while the propofol emulsion showed no significant changes. Since we postulated that propofol and caveolae or caveolin-1 interact, in an additional set of experiments, the experiments described above were repeated in cells treated with caveolin-1 siRNA. SOCE in cells treated with caveolin-1 siRNA was significantly reduced (P,0.05; Fig. 6B). The addition of propofol now did not further reduce SOCE (Fig. 6B).
Western analyses Given the lipid nature of propofol, we tested whether this anaesthetic decreases caveolin-1 expression, offering an explanation for the observed effects. In whole-cell
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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), n¼3 patients, average of 60 cells per condition; *significant propofol effect; #significant inhibitor effect (P,0.05).
homogenates of human ASM, treatment with either propofol DMSO or emulsion formulations did not significantly alter whole-cell caveolin-1 expression. However, in caveolar fractions enriched in caveolae, propofol decreased caveolin-1 expression (Fig. 7). Previous studies have demonstrated that intracellular mitogen-activated protein kinases such as extracellular signal-regulated kinase (ERK) are involved in
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[Ca2+]i regulation and the effects of inflammation.36 Other studies have shown that caveolin-1 can regulate ERK in smooth muscle.37 – 40 Accordingly, we examined whether ERK expression and phosphorylation are altered by propofol. Total ERK expression was unchanged after propofol treatment of only 5 min. However, the phosphorylated form of ERK (pERK) was significantly lower with propofol treatment
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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 mM propofol, supporting the data in Figure 4 of an effect via RyR channels. Values are mean (SD), n¼3 patients, average of 60 cells per condition; *significant propofol effect.
(Fig. 7). Additionally, there is some evidence that the PI3K/ Akt pathway can indirectly affect ASM [Ca2+]i and force regulation. Caveolin-1 is thought to activate PI3K. Accordingly, we tested whether this pathway is affected by propofol effects. However, the expression of PI3K was unchanged with propofol treatment, regardless of formulation (Fig. 7). Furthermore, Ca2+ sensitization during force production involves the RhoA/ rho kinase pathway. RhoA expression was decreased with propofol treatment with propofol 30 mM concentrations (Fig. 7; P,0.05).
Discussion Propofol is well known to produce bronchodilatation in different species.17 41 42 The results of the present study indicate that propofol-induced bronchodilatation involves direct effects on ASM, potentially via interference with caveolae (specifically the constituent caveolin-1) regulation of [Ca2+]i. In addition, by measuring propofol concentrations within caveolar fractions by GC, we suggest that propofol partitions into caveolae, disrupting caveolar regulation. Given the almost ubiquitous nature of caveolae in different mammalian cell types, these data suggest a potential common
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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), n¼3 patients, average of 60 cells per condition; *significant propofol effect; #significant caveolin-1 siRNA effect (P,0.05).
mechanism by which propofol mediates its effects across different cell types.
Caveolins and [Ca21]i and force regulation in ASM In ASM, the elevation of [Ca2+]i by bronchoconstrictors involves both SR Ca2+ release via IP3 and RyR receptor channels and plasma membrane Ca2+ influx.4 – 7 43 Ca2+ influx can occur through both voltage-gated44 and receptor-gated45 channels. Furthermore, SOCE in response to SR Ca2+ depletion also occurs,4 7 allowing for replenishment of intracellular Ca2+ stores. In a previous study,27 we demonstrated that caveolin-1 is present in the plasma membrane of human ASM and co-localizes with receptors of several bronchoconstrictor agonists. Furthermore, we demonstrated that the disruption of caveolae by CD and the inhibition of caveolin-1 expression via siRNA significantly disrupt baseline [Ca2+]i and the [Ca2+]i responses to agonists. Caveolae (and
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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), n¼3 patients; *significant propofol effect (P,0.05).
caveolin-1) also appear to be important in the regulation of SOCE in ASM. These previous data highlight the importance of caveolae and caveolin-1 in [Ca2+]i regulation in human ASM. Force generation after [Ca2+]i elevation can occur via Ca2+-dependent (MLC phosphorylation) and Ca2+independent mechanisms (Ca2+ sensitization).46 Ca2+ sensitization involves a monomeric GTP-binding protein, RhoA, and its downstream target, Rho-kinase.47 – 49 Phosphorylation by Rho-kinase inhibits MLC phosphatase, thus maintaining MLC phosphorylation and promoting contraction. Rho-kinase inhibitors can attenuate the contraction of ASM in rats, which is further attenuated by the addition of propofol.50 The relevance of the RhoA/Rho-kinase pathway to the present study lies in the fact that caveolins are thought to be essential for the activation of this pathway,51 and caveolin effects on this pathway could modulate force in ASM. In a previous study using porcine ASM, we found that Ca2+ sensitivity via RhoA/ Rho-kinase is indeed altered by CD.52
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Compared with the myriad of studies examining the effect of lipid, volatile anaesthetics on ASM structure and function, relatively few studies have examined the effect of propofol. Propofol has been shown to attenuate vagally induced bronchoconstriction12 and has been suggested to inhibit the actions of serotonin in the ovalbumin-sensitized rat trachea.14 Although one study suggested that propofol may not have any direct effects on ASM,12 in other studies, propofol has been shown to attenuate muscarinic receptormediated13 and ATP-induced ASM contraction.53 Thus, there is some, albeit conflicting, data on the effects of propofol on ASM. The results of the present study indicate that propofol does have effects on [Ca2+]i regulation in ASM. The novel finding here is that perhaps due to its lipid nature, propofol interferes with lipid raft signalling (i.e. caveolae and caveolins). Indeed, our data suggest that while acute propofol exposure does not affect the total amount of caveolin-1 in the cell (which can be partly intracellular), but that propofol does draw out caveolin-1 from caveolae (as shown in the caveolar fractions), an effect similar to cholesterol chelating agents such as CD. In this study, the finding that propofol decreases [Ca2+]i responses to histamine in human ASM cells in vitro, that is, without the confounding effect on airway neurones, underlines a direct effect on ASM itself. In this regard, the relative lack of propofol effects under conditions of suppressed caveolin-1 expression would suggest that propofol effects on the ASM are mediated (potentially in a major way) through lipid rafts. Given our previous data showing the expression of histaminergic and other receptors within caveolae of human ASM, these present data would suggest that the depletion of plasma membrane caveolae may lead to altered receptor expression or potentially disruption of caveolar interactions with intracellular structures such as the SR. In this regard, our finding that propofol effects involve RyR channels, but less so IP3 receptor channels, is interesting. One previous study suggested that propofol affects SR Ca2+ release via IP3 receptor channels.54 However, effects on RyR channels in ASM have not been reported previously. Again, given our previous finding of caveolar interactions with SR Ca2+ regulation,27 perhaps some of these effects are mediated by disruption of such interactions. Further studies (potentially using real-time imaging of caveolin-1) will address this issue. We have previously demonstrated that caveolae in ASM express multiple TRPC isoforms (putative SOCE channels).4 Accordingly, we expected propofol to decrease SOCE, and indeed, we found this to be the case. The mechanisms underlying this effect are not clear, but likely involve caveolae (based on the effect of caveolin-1 siRNA). On the one hand, propofol may simply alter the extent of influx by inhibiting caveolar regulation of SOCE. We have also previously demonstrated that TRPC3 channels are expressed within caveolae.27 Alternatively, propofol may inhibit non-caveolar TRPC isoforms. Finally, if propofol affects SR function (as evidenced
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Methodological issues One potential concern regarding whether propofol has effects on any cell type is the formulation of propofol used. In older studies, metabisulphite, the preservative used in propofol, had also been shown to have bronchodilatory properties. For example, metabisulphite decreases methacholine-induced bronchoconstriction.12 With the newer
propofol formulation, these effects are unlikely. On the other hand, the lipid emulsion may potentially have effects on lipid rafts. However, since our data demonstrate that there is no significant difference in effect between the propofol emulsion and DMSO, this should not be an issue.
Declaration of interest None declared.
Funding This study was supported by grants HL090595 (C.P.) and HL088029 (Y.P.) from the National Institutes of Health, National Heart Lung and Blood Institute, RR024150 (G.F.) from the National Institutes of Health NCRR, Bethesda, MD, USA, and the Department of Anaesthesiology, Mayo Clinic.
References 1 Royse CF, Liew DF, Wright CE, Royse AG, Angus JA. Persistent depression of contractility and vasodilation with propofol but not with sevoflurane or desflurane in rabbits. Anesthesiology 2008; 108: 87–93 2 Conti G, Dell’Utri D, Vilardi V, et al. Propofol induces bronchodilation in mechanically ventilated chronic obstructive pulmonary disease (COPD) patients. Acta Anaesthesiol Scand 1993; 37: 105– 9 3 Burburan SM, Xisto DG, Rocco PR. Anaesthetic management in asthma. Minerva Anestesiol 2007; 73: 357– 65 4 Ay B, Prakash YS, Pabelick CM, Sieck GC. Store-operated Ca2+ entry in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2004; 286: L909– 17 5 Coburn RF, Baron CB. Coupling mechanisms in airway smooth muscle. Am J Physiol 1990; 258: L119– 33 6 Kannan MS, Prakash YS, Brenner T, Mickelson JR, Sieck GC. Role of ryanodine receptor channels in Ca2+ oscillations of porcine tracheal smooth muscle. Am J Physiol 1997; 272: L659– 64 7 Pabelick CM, Ay B, Prakash YS, Sieck GC. Effects of volatile anesthetics on store-operated Ca2+ influx in airway smooth muscle. Anesthesiology 2004; 101: 373– 80 8 Prakash YS, Kannan MS, Sieck GC. Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol 1997; 272: C966–75 9 Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev 2005; 85: 757–810 10 Chiba Y, Goto K, Hirahara M, Sakai H, Misawa M. Glucocorticoids ameliorate antigen-induced bronchial smooth muscle hyperresponsiveness by inhibiting upregulation of RhoA in rats. J Pharmacol Sci 2008; 106: 615–25 11 Witzenrath M, Gutbier B, Owen JS, et al. Role of platelet-activating factor in pneumolysin-induced acute lung injury. Crit Care Med 2007; 35: 1756– 62 12 Brown RH, Greenberg RS, Wagner EM. Efficacy of propofol to prevent bronchoconstriction: effects of preservative. Anesthesiology 2001; 94: 851–5, discussion 6A. 13 Lin CC, Shyr MH, Tan PP, et al. Mechanisms underlying the inhibitory effect of propofol on the contraction of canine airway smooth muscle. Anesthesiology 1999; 91: 750–9 14 Yamaguchi M, Shibata O, Nishioka K, Makita T, Sumikawa K. Propofol attenuates ovalbumin-induced smooth muscle
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by the XeC and ryanodine experiments), it may also modulate regulatory proteins such as STIM1 that are critical to SOCE.55 – 57 These novel mechanisms of propofol action remain to be examined. In our study, we did not find an effect of propofol on baseline [Ca2+]i. However, we have previously shown that baseline [Ca2+]i is affected by caveolar disruption. It is possible that in the normal ASM, caveolar disruption by propofol is not sufficient enough to alter baseline [Ca2+]i. However, under conditions of inflammation and increased baseline ASM [Ca2+]i (and thus airway tone), propofol may very well decrease baseline [Ca2+]i, thus contributing to the bronchodilator effect. In addition to effects mediated by altered Ca2+ regulatory protein expression, caveolae and caveolin-1 may regulate intracellular signalling pathways. In human ASM, the disruption of caveolae using CD or caveolin-1 siRNA causes p42/p44 MAPK (ERK) activation and increased cell proliferation.37 However, studies in other cell types suggest that caveolin-1 actually activates the ERK pathway,38 – 40 which is consistent with our data of propofol effects on ERK. While ERK phosphorylation may be involved, propofol effects do not appear to involve the PI3/Akt pathway, although there are some data that caveolins can interact with this signalling pathway.58 – 60 Accordingly, the activation or inhibition of specific signalling pathways after propofol exposure is likely to be complex and needs further investigation. Overall, the results of the present study suggest that propofol-induced reduction in [Ca2+]i of ASM (and resultant bronchodilatation) involve the disruption of caveolae and caveolin-1 regulation of both plasma membrane and intracellular Ca2+ regulatory mechanisms. Furthermore, propofol could interfere with signalling intermediates that are typically activated by bronchoconstrictors. While further research is needed to examine the molecular mechanisms of action for propofol, an important consideration is that given caveolar expression of muscarinic27 and purinergic receptors,61 caveolae could be important in different targets of propofol in producing bronchodilatation. Furthermore, given the ubiquitous expression of caveolae across cell types, the role of caveolae or caveolin-1 in mediating propofol effects in other smooth muscle types is also warranted. Finally, caveolae may be a common factor in anaesthetic action. For example, previous studies have shown that local anaesthetics and volatile anaesthetics can act via interaction with lipid rafts28 29 and that caveolae are involved in actions of general anaesthetics.30 31
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contraction of the sensitized rat trachea: inhibition of serotonergic and cholinergic signalling. Anesth Analg 2006; 103: 594– 600 Yang M, Ding X, Murray PA. Differential effects of intravenous anesthetics on capacitative calcium entry in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2008; 294: L1007–12 Bai Y, Sanderson MJ. Airway smooth muscle relaxation results from a reduction in the frequency of Ca2+ oscillations induced by a cAMP-mediated inhibition of the IP3 receptor. Respir Res 2006; 7: 34 Hanazaki M, Jones KA, Warner DO. Effects of intravenous anesthetics on Ca2+ sensitivity in canine tracheal smooth muscle. Anesthesiology 2000; 92: 133– 9 Cohen AW, Hnasko R, Schubert W, Lisanti MP. Role of caveolae and caveolins in health and disease. Physiol Rev 2004; 84: 1341– 79 Frank PG, Woodman SE, Park DS, Lisanti MP. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol 2003; 23: 1161– 8 Hnasko R, Lisanti MP. The biology of caveolae: lessons from caveolin knockout mice and implications for human disease. Mol Interv 2003; 3: 445– 64 Razani B, Lisanti MP. Caveolins and caveolae: molecular and functional relationships. Exp Cell Res 2001; 271: 36–44 Schlegel A, Lisanti MP. Caveolae and their coat proteins, the caveolins: from electron microscopic novelty to biological launching pad. J Cell Physiol 2001; 186: 329– 37 Williams TM, Lisanti MP. The Caveolin genes: from cell biology to medicine. Ann Med 2004; 36: 584– 95 Williams TM, Lisanti MP. The caveolin proteins. Genome Biol 2004; 5: 214 Schlenz H, Kummer W, Jositsch G, Wess J, Krasteva G. Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways. Am J Physiol Lung Cell Mol Physiol 2010; 298: L626– 36 Sathish V, Abcejo AJ, Vanoosten SK, Thompson MA, Prakash YS, Pabelick CM. Caveolin-1 in cytokine-induced enhancement of intracellular Ca2+ in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2011; 301: L607–14 Prakash YS, Thompson MA, Vaa B, et al. Caveolins and intracellular calcium regulation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2007; 293: L1118–26 Wang L, Ko KW, Lucchinetti E, et al. Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anaesthetic-induced fuel shift. Anesthesiology 2010; 113: 541– 51 Tsuchiya H, Ueno T, Mizogami M, Takakura K. Do local anesthetics interact preferentially with membrane lipid rafts? Comparative interactivities with raft-like membranes. J Anesth 2010; 24: 639–42 Song JH, Kim M, Park SW, Chen SW, Pitson SM, Lee HT. Isoflurane via TGF-beta1 release increases caveolae formation and organizes sphingosine kinase signalling in renal proximal tubules. Am J Physiol Renal Physiol 2010; 298: F1041–50 Horikawa YT, Patel HH, Tsutsumi YM, et al. Caveolin-3 expression and caveolae are required for isoflurane-induced cardiac protection from hypoxia and ischemia/reperfusion injury. J Mol Cell Cardiol 2008; 44: 123–30 Prakash YS, van der Heijden HF, Kannan MS, Sieck GC. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J Appl Physiol 1997; 82: 1836–43
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56 Pani B, Ong HL, Liu X, Rauser K, Ambudkar IS, Singh BB. Lipid rafts determine clustering of STIM1 in ER-plasma membrane junctions and regulation of SOCE. J Biol Chem 2008; 283: 17333– 40 57 Smyth JT, Dehaven WI, Bird GS, Putney JW Jr. Ca2+-store-dependent and -independent reversal of Stim1 localization and function. J Cell Sci 2008; 121: 762–72 58 Clarke CJ, Ohanian V, Ohanian J. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase. Am J Physiol Heart Circ Physiol 2007; 292: H2248–56 59 Kim S, Lee Y, Seo JE, Cho KH, Chung JH. Caveolin-1 increases basal and TGF-beta1-induced expression of type I procollagen through PI-3 kinase/Akt/mTOR pathway in human dermal fibroblasts. Cell Signal 2008; 20: 1313–9 60 Sedding DG, Hermsen J, Seay U, et al. Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signalling in vitro and in vivo. Circ Res 2005; 96: 635–42 61 Barth K, Kasper M. Membrane compartments and purinergic signalling: occurrence and function of P2X receptors in lung. FEBS J 2009; 276: 341– 53 62 Sathish V, Abecjo AJ, Thompson MA, Sieck GC, Prakash YS, Pabelick CM. Caveolin-1 regulation of store-operated Ca2+ influx in human airway smooth muscle. Eur Respir J 2012. Advance Access published on 12 January 2012, doi: 10.1183/09031936.00090511.
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50 Hanazaki M, Yokoyama M, Morita K, et al. Rho-kinase inhibitors augment the inhibitory effect of propofol on rat bronchial smooth muscle contraction. Anesth Analg 2008; 106: 1765 – 71 51 Hunter I, Nixon GF. Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signalling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways. J Biol Chem 2006; 281: 34705– 15 52 Sathish V, Yang B, Meuchel LW, et al. Caveolin-1 and force regulation in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2011; 300: L920–9 53 Yamaguchi O, Marui E, Kakizaki H, et al. Randomized, doubleblind, placebo- and propiverine-controlled trial of the once-daily antimuscarinic agent solifenacin in Japanese patients with overactive bladder. BJU Int 2007; 100: 579–87 54 Lin K, Sadee W, Quillan JM. Rapid measurements of intracellular calcium using a fluorescence plate reader. Biotechniques 1999; 26: 318–22, 24 –6 55 Lu W, Wang J, Shimoda LA, Sylvester JT. Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 2008; 295: 104– 13
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Caveolae and propofol effects on airway smooth muscle K. J. Grim 1†, A. J. Abcejo 1†, A. Barnes 1, V. Sathish 1, D. F. Smelter1, G. C. Ford2, M. A. Thompson 1, Y. S. Prakash 1,3 and C. M. Pabelick 1,3* 1
Department of Anesthesiology, 2 CTSA Metabolomics Core and 3 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA * Corresponding author. E-mail: [email protected]
Editor’s key points
† Propofol decreased peak intracellular calcium responses to histamine. † When caveolin-1 was blocked with small interference RNA, the effect of propofol was abolished. † Propofol appears to affect airway relaxation via caveolin-1 effects on calcium.
Methods. Caveolar fractions of human ASM cells were tested for propofol content. [Ca2+]i responses of ASM cells loaded with fura-2 were performed in the presence of 10 mM histamine with and without clinically relevant concentrations of propofol (10 and 30 mM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca2+ release were evaluated in zero extracellular Ca2+ using the blockers Xestospongin C and ryanodine. Store-operated Ca2+ entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca2+]i and force regulation were also evaluated. Results. Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 mM propofol form decreased [Ca2+]i peak (but not plateau) responses to histamine by 40%, an effect persistent in zero extracellular Ca2+. Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca2+]i, while propofol blunted [Ca2+]i responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signalregulated kinase phosphorylation. Conclusions. These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca2+]i. Keywords: bronchial smooth muscle; bronchodilatation; calcium regulation; caveolin; intravenous anaesthetic; signalling Accepted for publication: 22 February 2012
Since its invention in 1977, propofol has become the most frequently used i.v. anaesthetic, gaining rapid acceptance for use in sedation and general anaesthesia. Propofol is also known to produce substantial vasodilatation and bronchodilatation.1 Indeed, propofol-induced bronchodilatation has been demonstrated in both healthy and asthmatic patients.2 3 In spite of this clinical evidence, the cellular mechanisms by which propofol produces bronchodilatation are still under investigation. Relaxation of the airway involves a reduction in intracellular Ca2+ concentration ([Ca2+]i) of airway smooth muscle (ASM). We and others have previously demonstrated that in ASM, [Ca2+]i regulation involves both Ca2+ release from sarcoplasmic reticulum (SR) stores and plasma membrane †
Ca2+ influx,4 – 8 with the latter via several mechanisms including controlled influx in response to SR Ca2+ depletion [store-operated Ca2+ entry (SOCE)].4 7 9 Force regulation involves myosin light chain (MLC) and Ca2+ sensitization via the RhoA/Rho kinase pathway.10 11 Thus, there are several potential targets for propofol-induced bronchodilatation. In this regard, propofol has been found to attenuate vagal and methacholine-induced bronchoconstriction, with possible direct actions on muscarinic receptors.12 13 Propofol also decreases ovalbumin-induced contraction of sensitized rat trachea through inhibition of serotonin.14 Whether propofol directly affects ASM has been examined only in a few studies (albeit in varied species). Propofol has been shown to decrease peak [Ca2+]i response to agonists,15 perhaps
These authors contributed equally to this work.
& The Author [2012]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
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† The effect of propofol on intracellular calcium was investigated in human airway smooth muscle cells in vitro.
Background. The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca2+]i. We postulated that propofol-induced bronchodilatation involves caveolar disruption.
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Methods Human ASM cell preparation The techniques for isolation of human ASM cells have been described previously.27 Briefly, bronchi were obtained from lung tissues incidental to patient procedures in thoracic surgery at Mayo Clinic Rochester (de-identified tissues typically from lobectomies and pneumenectomies; approved by the Mayo Institutional Review Board and not considered Human Subjects Research). Tissues were initially placed in Hank’s’ balanced salt solution (HBSS, Invitrogen, Carlsbad, CA, USA) with 2.5 mM extracellular Ca2+. ASM layers were isolated and enzymatically dissociated as described previously.32 Cells were plated in sterile culture flasks and grown in a 95% air/5% CO2 humidified incubator using DMEM F/12 supplemented with 10% fetal bovine serum (FBS). All experiments were performed in cells before the third passage to ensure cell phenotype as verified by immunostaining for smooth muscle actin and myosin and by expression of receptors for commonly used bronchoconstrictor agonists, such as the M3 muscarinic receptor and the H1 histaminergic receptor.
Preparation of caveolar membranes Caveolin-rich membranes were prepared as described previously.27 Briefly, ASM strips were homogenized in cold buffer A (0.25 M sucrose, 1 mM EDTA, and 20 mM Tricine, pH 7.8) until fine emulsion was achieved. The homogenates were sonicated for 10 s each, centrifuged (1000g, 10 min, 48C), the supernatant removed and brought to 5 ml, which was layered onto 30% Percoll, and re-centrifuged (84 000g, 30 min). The plasma membrane fraction was collected and brought to 2 ml with buffer A. A crude membrane fraction was then sonicated, resuspended in OptiPrep, and then placed in a centrifuge tube. A linear 20–10% OptiPrep gradient was layered on top before centrifugation at 52 000g for 90 min. The upper membrane layer containing the caveolae was then collected for analysis. The lower membrane layer was collected as a control.
Measurement of propofol within caveolar membranes by gas chromatography Propofol was measured within caveolar membrane fractions to confirm the connection between lipid-rich caveolae and propofol. Propofol concentrations were measured by gas chromatography –mass spectrometry (GC/MS) using a modified method as described previously.33 34 Briefly, caveolar fractions contained in 0.5 ml sucrose buffer were mixed with 100 ml n-heptane for propofol extraction and the organic phase placed in 200 ml autosampler vials for analysis. GC/MS analysis was performed using a Waters Quattro Micro triple quadruple GC/MS/MS system (Waters, Manchester, UK) operating under electron impact ionization conditions. The column used was a DB-5MS fused silica capillary column (30 m×0.25 mm inside diameter, 0.25 mm film thickness, Agilent Technologies, USA). A temperature programme ramp was performed from 60 to 3008C at 208C min21. The injection temperature was set at 2508C and the transfer line temperature at 2508C. Helium was used as a carrier gas at a flow rate of 1.0 ml min21, and 1 ml of the sample extract was injected. The [M-15]+ fragment ion of propofol at m/z 163.1 was monitored for quantification and the peak area compared with an external calibration curve of propofol, prepared in n-heptane over a concentration range of 0.015 –1.8 ng ml21, 1 ml being injected on-column.
Fluorescence [Ca21]i imaging ASM cells were incubated in 5 mM fura-2 AM (Invitrogen) for 45 min at room temperature and visualized with a fluorescence imaging system (MetaFluor, Universal Imaging, Downingtown, PA, USA) on a Nikon Diaphot inverted microscope. Cells were initially perfused with HBSS (2.5 mM Ca2+, 238C), and baseline fluorescence established. [Ca2+]i responses of 10 cells per chamber were obtained for individual, softwaredefined regions of interest. Fura-2 was alternately excited at 340 and 380 nm, and the ratio of fluorescence emissions at 510 nm was obtained every 0.75 s (Photometric Cascade digital camera system, Roper Scientific, Tucson, AZ, USA).
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through SR Ca2+ release via inositol trisphosphate (IP3) receptor channels,16 but does not appear to affect Ca2+ sensitivity.17 On the other hand, one study suggested that all of the effects of propofol on the airway may be neurally mediated.12 Thus, the mechanisms underlying propofolinduced bronchodilatation remain unclear. A particular feature of propofol (and indeed its preparation) is its highly lipid nature. There is now increasing evidence that the plasma membrane of almost all cell types (including ASM) contain specialized lipid rafts, including 50– 100 nm invaginations called caveolae,18 – 24 likened to be more intricate T-tubules of the striated muscle. Caveolae can facilitate interactions between plasma membrane-regulatory elements and intracellular signalling cascades.18 – 26 We demonstrated in human ASM, that suppression of the constitutional protein caveolin-1 substantially blunts agonist-induced [Ca2+]i responses and alters [Ca2+]i signalling, indicating a role for lipid rafts in ASM contractility.26 27 In human, ASM substantially blunts agonist-induced [Ca2+]i responses and alters [Ca2+]i signalling, indicating a role for lipid rafts in ASM contractility.26 27 Previous studies have shown that local and volatile anaesthetics act via interaction with lipid rafts.28 29 There is also evidence that caveolae specifically are involved in the actions of general anaesthetics.30 31 However, the role of caveolae in mediating the effects of propofol is not known. Given the lipid nature of propofol, we hypothesized that the bronchodilatory effects of this anaesthetic may be mediated (at least in part) by alterations in caveolar signalling. Accordingly, in the present study, we used human ASM cells to examine the potential role of caveolae and caveolin-1 in propofol-induced bronchodilatation.
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Western blot analysis
Materials Methyl-b-cyclodextrin (CD), Optiprep, and other chemicals were obtained from Sigma (St Louis, MO, USA) unless mentioned otherwise. All tissue culture reagents were obtained from Invitrogen. Primary and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propofol DMSO formulation was obtained from Sigma; propofol emulsion was obtained from the Mayo Clinic Pharmacy.
Statistics Propofol effects on [Ca2+]i responses in human ASM cells were compared with controls using Student’s paired t-tests. At least 10 cells from three ASM isolations (three different patients) were compared. GC measurements were performed in samples from three patients. Values are means (SD) and n is the number of patients. Statistical significance was established at P,0.05.
Results Propofol concentration within caveolae To confirm our hypothesis that propofol induces bronchodilatation via interaction with caveolae, we directly measured propofol concentrations within caveolar membranes using GC. Compared with vehicle control, exposure of ASM to 10 and 30 mM resulted in significantly higher propofol concentrations within caveolar membranes (P,0.05; Fig. 1).
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Fig 1 Propofol concentration within caveolar membranes of human ASM. Exposure of ASM cells to 10 or 30 mM for 5 min significantly increased concentrations within caveolae, as determined using GC (see text for details). Values are mean (SD); n¼3 patients; *significant propofol effect (P,0.05).
Propofol effect on [Ca21]i response to histamine In unstimulated human ASM cells, baseline [Ca2+]i was 100 nM. Upon addition of 10 mM histamine, a typical rapid increase in [Ca2+]i was observed, with a peak response of 514 (43) nM. The subsequent plateau was 223 (53) nM. After a wash in HBSS for 20 min, exposure to 10 or 30 mM propofol for 5 min did not substantially decrease baseline [Ca2+]i levels. However, subsequent stimulation with 10 mM histamine resulted in an 40% decrease in peak [Ca2+]i response (Fig. 2; P,0.05). The plateau [Ca2+]i response was not different between control and propofol-exposed cells.
Effect of caveolin-1 siRNA In ASM cells transfected with caveolin-1 siRNA, exposure to histamine resulted in a considerably smaller [Ca2+]i response, consistent with our previous study.27 In these cells, the addition of 30 mM propofol also decreased peak [Ca2+]i responses to histamine; however, these effects were substantially blunted compared with cells transfected with Lipofectamine alone (Fig. 3; P,0.05 for significant effects of siRNA).
Propofol effect on SR Ca21 release Under conditions of zero extracellular Ca2+ exposure, baseline [Ca2+]i was 80 nM and exposure to 10 mM histamine continued to result in a peak transient [Ca2+]i response [260 (15) nM]. Treatment with 10 mM or 30 mM propofol for 5 min substantially reduced the peak [Ca2+]i response to 10 mM histamine (P,0.05; Fig. 4A). The effects of 10 vs 30 mM propofol were the same.
Effect of propofol on IP3 receptor channels We have previously demonstrated that SR Ca2+ release in ASM involves both IP3 receptor and ryanodine receptor (RyR) channels.35 In one set of experiments, IP3 receptor channels were first blocked with 20 mM Xestospongin C
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Proteins were separated by SDS –PAGE using the Criterion Gel System (Bio-Rad, Hercules, CA, USA) and 15% or 4–15% gradient gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) for 60 min and then blocked for 1 h with 5% milk in Tris-buffered saline containing 0.1% Tween (TBST). Membranes were then incubated with anti-caveolin-1 (1:1000), anti-ERK (1:1000), anti-pERK (1:100), anti-PI3K (1:1000), or anti-RhoA (1:1000) antibodies overnight at 48C. Following three washes with TBST, primary antibodies were detected with horseradish peroxidaseconjugated secondary antibodies and signals developed by Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA).
1.2e+5 Concentration (ng mg–1 protein)
The technique for suppression of caveolin-1 expression in human ASM using small interference RNA (siRNA) caveolin-1 was recently described.27 Briefly, siRNA duplex corresponding to bovine caveolin-1 mRNA targeting against the open reading frame, 223 –241 bases, and a negative control siRNA were selected for caveolin-1 knockdown (Dharmacon, Lafayette, CO, USA). ASM cells at 60% confluence were transfected using 50 nM siRNA and Lipofectamine 2000 (Invitrogen) in DMEM F/12 without FBS. Fresh growth medium was added 6 h after transfection and experiments performed after 48 h. Both Lipofectamine only (vehicle) and scrambled siRNA sequences were used as controls.
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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 mM) on [Ca2+]i responses to histamine was minimal, suggesting that caveolin-1 is important for propofol effect on ASM. Values are mean (SD), n¼3 patients, average of 60 cells per condition; #significant siRNA effect (P,0.05).
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Fig 2 Effect of propofol on [Ca2+]i responses to histamine in human ASM cells. (A) Addition of 10 mM 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 mM propofol DMSO, 30 mM propofol DMSO, 10 mM propofol emulsion or 30 mM 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), n¼3 patients, average of 60 cells per condition. *Significant propofol effect (P,0.05).
(XeC, a cell permeant inhibitor). Cells were stimulated with 10 mM histamine, thoroughly washed, treated with 30 mM propofol for 5 min, and re-stimulated with 10 mM histamine. Although histamine-induced [Ca2+]i responses were decreased by XeC, additional exposure to propofol further decreased this [Ca2+]i response (P,0.05; Fig. 4B).
Effect of propofol on RyR channels We previously demonstrated that RyR channels are blocked by high concentrations of ryanodine.35 In this set of experiments, RyR channels were first blocked with 20 mM ryanodine for 15 min. Cells were stimulated with 10 mM histamine, thoroughly washed, treated with 30 mM propofol for 5 min, and re-stimulated with 10 mM histamine. Although histamine-induced [Ca2+]i responses were decreased by ryanodine, additional propofol did not further decrease this [Ca2+]i response (Fig. 4B).
To further investigate the effects of propofol on SR Ca2+ release via RyR channels, ASM cells were exposed to 5 mM caffeine in the absence or presence of 30 mM propofol. Thirty micromoles of propofol blunted caffeine-induced SR Ca2+ release (Fig. 5; P,0.05).
Effect of propofol on SOCE We have previously demonstrated SOCE in ASM.4 A similar protocol was used here. Extracellular Ca2+ was first removed by perfusion with zero-Ca2+ HBSS. Voltage-gated Ca2+ influx was further inhibited using 1 mM nifedipine and 10 mM KCl. SR Ca2+ stores were then depleted using 10 mM cyclopiazonic acid (an inhibitor of SR Ca2+ reuptake), resulting in a slow increase in [Ca2+]i levels due to continued leak from the SR. After 15 min, extracellular Ca2+ was rapidly re-introduced to trigger SOCE. Cells were then washed thoroughly to refill SR stores, and the protocol repeated with additional exposure to 30 mM propofol 5 min before re-introduction of extracellular Ca2+ (thus avoiding any effect of propofol on the SR release aspect). Exposure to propofol resulted in decreased SOCE in the propofol/ DMSO formulation (P,0.05; Fig. 6), while the propofol emulsion showed no significant changes. Since we postulated that propofol and caveolae or caveolin-1 interact, in an additional set of experiments, the experiments described above were repeated in cells treated with caveolin-1 siRNA. SOCE in cells treated with caveolin-1 siRNA was significantly reduced (P,0.05; Fig. 6B). The addition of propofol now did not further reduce SOCE (Fig. 6B).
Western analyses Given the lipid nature of propofol, we tested whether this anaesthetic decreases caveolin-1 expression, offering an explanation for the observed effects. In whole-cell
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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), n¼3 patients, average of 60 cells per condition; *significant propofol effect; #significant inhibitor effect (P,0.05).
homogenates of human ASM, treatment with either propofol DMSO or emulsion formulations did not significantly alter whole-cell caveolin-1 expression. However, in caveolar fractions enriched in caveolae, propofol decreased caveolin-1 expression (Fig. 7). Previous studies have demonstrated that intracellular mitogen-activated protein kinases such as extracellular signal-regulated kinase (ERK) are involved in
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[Ca2+]i regulation and the effects of inflammation.36 Other studies have shown that caveolin-1 can regulate ERK in smooth muscle.37 – 40 Accordingly, we examined whether ERK expression and phosphorylation are altered by propofol. Total ERK expression was unchanged after propofol treatment of only 5 min. However, the phosphorylated form of ERK (pERK) was significantly lower with propofol treatment
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(Fig. 7). Additionally, there is some evidence that the PI3K/ Akt pathway can indirectly affect ASM [Ca2+]i and force regulation. Caveolin-1 is thought to activate PI3K. Accordingly, we tested whether this pathway is affected by propofol effects. However, the expression of PI3K was unchanged with propofol treatment, regardless of formulation (Fig. 7). Furthermore, Ca2+ sensitization during force production involves the RhoA/ rho kinase pathway. RhoA expression was decreased with propofol treatment with propofol 30 mM concentrations (Fig. 7; P,0.05).
Discussion Propofol is well known to produce bronchodilatation in different species.17 41 42 The results of the present study indicate that propofol-induced bronchodilatation involves direct effects on ASM, potentially via interference with caveolae (specifically the constituent caveolin-1) regulation of [Ca2+]i. In addition, by measuring propofol concentrations within caveolar fractions by GC, we suggest that propofol partitions into caveolae, disrupting caveolar regulation. Given the almost ubiquitous nature of caveolae in different mammalian cell types, these data suggest a potential common
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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), n¼3 patients, average of 60 cells per condition; *significant propofol effect; #significant caveolin-1 siRNA effect (P,0.05).
mechanism by which propofol mediates its effects across different cell types.
Caveolins and [Ca21]i and force regulation in ASM In ASM, the elevation of [Ca2+]i by bronchoconstrictors involves both SR Ca2+ release via IP3 and RyR receptor channels and plasma membrane Ca2+ influx.4 – 7 43 Ca2+ influx can occur through both voltage-gated44 and receptor-gated45 channels. Furthermore, SOCE in response to SR Ca2+ depletion also occurs,4 7 allowing for replenishment of intracellular Ca2+ stores. In a previous study,27 we demonstrated that caveolin-1 is present in the plasma membrane of human ASM and co-localizes with receptors of several bronchoconstrictor agonists. Furthermore, we demonstrated that the disruption of caveolae by CD and the inhibition of caveolin-1 expression via siRNA significantly disrupt baseline [Ca2+]i and the [Ca2+]i responses to agonists. Caveolae (and
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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), n¼3 patients; *significant propofol effect (P,0.05).
caveolin-1) also appear to be important in the regulation of SOCE in ASM. These previous data highlight the importance of caveolae and caveolin-1 in [Ca2+]i regulation in human ASM. Force generation after [Ca2+]i elevation can occur via Ca2+-dependent (MLC phosphorylation) and Ca2+independent mechanisms (Ca2+ sensitization).46 Ca2+ sensitization involves a monomeric GTP-binding protein, RhoA, and its downstream target, Rho-kinase.47 – 49 Phosphorylation by Rho-kinase inhibits MLC phosphatase, thus maintaining MLC phosphorylation and promoting contraction. Rho-kinase inhibitors can attenuate the contraction of ASM in rats, which is further attenuated by the addition of propofol.50 The relevance of the RhoA/Rho-kinase pathway to the present study lies in the fact that caveolins are thought to be essential for the activation of this pathway,51 and caveolin effects on this pathway could modulate force in ASM. In a previous study using porcine ASM, we found that Ca2+ sensitivity via RhoA/ Rho-kinase is indeed altered by CD.52
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B
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Compared with the myriad of studies examining the effect of lipid, volatile anaesthetics on ASM structure and function, relatively few studies have examined the effect of propofol. Propofol has been shown to attenuate vagally induced bronchoconstriction12 and has been suggested to inhibit the actions of serotonin in the ovalbumin-sensitized rat trachea.14 Although one study suggested that propofol may not have any direct effects on ASM,12 in other studies, propofol has been shown to attenuate muscarinic receptormediated13 and ATP-induced ASM contraction.53 Thus, there is some, albeit conflicting, data on the effects of propofol on ASM. The results of the present study indicate that propofol does have effects on [Ca2+]i regulation in ASM. The novel finding here is that perhaps due to its lipid nature, propofol interferes with lipid raft signalling (i.e. caveolae and caveolins). Indeed, our data suggest that while acute propofol exposure does not affect the total amount of caveolin-1 in the cell (which can be partly intracellular), but that propofol does draw out caveolin-1 from caveolae (as shown in the caveolar fractions), an effect similar to cholesterol chelating agents such as CD. In this study, the finding that propofol decreases [Ca2+]i responses to histamine in human ASM cells in vitro, that is, without the confounding effect on airway neurones, underlines a direct effect on ASM itself. In this regard, the relative lack of propofol effects under conditions of suppressed caveolin-1 expression would suggest that propofol effects on the ASM are mediated (potentially in a major way) through lipid rafts. Given our previous data showing the expression of histaminergic and other receptors within caveolae of human ASM, these present data would suggest that the depletion of plasma membrane caveolae may lead to altered receptor expression or potentially disruption of caveolar interactions with intracellular structures such as the SR. In this regard, our finding that propofol effects involve RyR channels, but less so IP3 receptor channels, is interesting. One previous study suggested that propofol affects SR Ca2+ release via IP3 receptor channels.54 However, effects on RyR channels in ASM have not been reported previously. Again, given our previous finding of caveolar interactions with SR Ca2+ regulation,27 perhaps some of these effects are mediated by disruption of such interactions. Further studies (potentially using real-time imaging of caveolin-1) will address this issue. We have previously demonstrated that caveolae in ASM express multiple TRPC isoforms (putative SOCE channels).4 Accordingly, we expected propofol to decrease SOCE, and indeed, we found this to be the case. The mechanisms underlying this effect are not clear, but likely involve caveolae (based on the effect of caveolin-1 siRNA). On the one hand, propofol may simply alter the extent of influx by inhibiting caveolar regulation of SOCE. We have also previously demonstrated that TRPC3 channels are expressed within caveolae.27 Alternatively, propofol may inhibit non-caveolar TRPC isoforms. Finally, if propofol affects SR function (as evidenced
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Methodological issues One potential concern regarding whether propofol has effects on any cell type is the formulation of propofol used. In older studies, metabisulphite, the preservative used in propofol, had also been shown to have bronchodilatory properties. For example, metabisulphite decreases methacholine-induced bronchoconstriction.12 With the newer
propofol formulation, these effects are unlikely. On the other hand, the lipid emulsion may potentially have effects on lipid rafts. However, since our data demonstrate that there is no significant difference in effect between the propofol emulsion and DMSO, this should not be an issue.
Declaration of interest None declared.
Funding This study was supported by grants HL090595 (C.P.) and HL088029 (Y.P.) from the National Institutes of Health, National Heart Lung and Blood Institute, RR024150 (G.F.) from the National Institutes of Health NCRR, Bethesda, MD, USA, and the Department of Anaesthesiology, Mayo Clinic.
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by the XeC and ryanodine experiments), it may also modulate regulatory proteins such as STIM1 that are critical to SOCE.55 – 57 These novel mechanisms of propofol action remain to be examined. In our study, we did not find an effect of propofol on baseline [Ca2+]i. However, we have previously shown that baseline [Ca2+]i is affected by caveolar disruption. It is possible that in the normal ASM, caveolar disruption by propofol is not sufficient enough to alter baseline [Ca2+]i. However, under conditions of inflammation and increased baseline ASM [Ca2+]i (and thus airway tone), propofol may very well decrease baseline [Ca2+]i, thus contributing to the bronchodilator effect. In addition to effects mediated by altered Ca2+ regulatory protein expression, caveolae and caveolin-1 may regulate intracellular signalling pathways. In human ASM, the disruption of caveolae using CD or caveolin-1 siRNA causes p42/p44 MAPK (ERK) activation and increased cell proliferation.37 However, studies in other cell types suggest that caveolin-1 actually activates the ERK pathway,38 – 40 which is consistent with our data of propofol effects on ERK. While ERK phosphorylation may be involved, propofol effects do not appear to involve the PI3/Akt pathway, although there are some data that caveolins can interact with this signalling pathway.58 – 60 Accordingly, the activation or inhibition of specific signalling pathways after propofol exposure is likely to be complex and needs further investigation. Overall, the results of the present study suggest that propofol-induced reduction in [Ca2+]i of ASM (and resultant bronchodilatation) involve the disruption of caveolae and caveolin-1 regulation of both plasma membrane and intracellular Ca2+ regulatory mechanisms. Furthermore, propofol could interfere with signalling intermediates that are typically activated by bronchoconstrictors. While further research is needed to examine the molecular mechanisms of action for propofol, an important consideration is that given caveolar expression of muscarinic27 and purinergic receptors,61 caveolae could be important in different targets of propofol in producing bronchodilatation. Furthermore, given the ubiquitous expression of caveolae across cell types, the role of caveolae or caveolin-1 in mediating propofol effects in other smooth muscle types is also warranted. Finally, caveolae may be a common factor in anaesthetic action. For example, previous studies have shown that local anaesthetics and volatile anaesthetics can act via interaction with lipid rafts28 29 and that caveolae are involved in actions of general anaesthetics.30 31
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