In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca(2+) stores by increasing Ca(2+) "leak." Accordingly, SR replenishment becomes dependent on Ca(2+) influx. Depletion of SR Ca(2+) stores triggers Ca(2+) influx via specific plasma membrane channels, store-operated Ca(2+) channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca(2+) "leak," preventing SR replenishment and enhancing ASM relaxation.
In porcine ASM cells, SR Ca was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca(2+), nifedipine and KCl (preventing Ca(2+) influx through L-type and SOCC channels). Extracellular Ca(2+) was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined.
Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca(2+) rapidly increased [Ca(2+)]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni and blockade of inositol 1,4,5-triphosphate-induced SR Ca(2+) release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca(2+) influx when extracellular Ca(2+) was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca(2+) influx.
Volatile anesthetics inhibit a Ni/La-sensitive store-operated Ca(2+) influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.
VOLATILE anesthetic-induced bronchodilation involves a reduction in intracellular Ca2+([Ca2+]i) that is key to development and maintenance of force in airway smooth muscle (ASM) cells.1–5These effects of anesthetics are partly attributable to an inhibition of Ca2+influx via L-type Ca2+channels.5–7We1,2and others8have shown that anesthetics also deplete Ca2+stores of the sarcoplasmic reticulum (SR) by increasing Ca2+“leakage.” Such anesthetic-induced SR Ca2+leak occurs via both inositol 1,4,5-trisphosphate or ryanodine receptor (RyR) channels.2
[Ca2+]iregulation in ASM is mediated by both extracellular Ca2+influx and SR Ca2+release. Both inositol 1,4,5-trisphosphate receptor9and RyR10channels play important roles in ASM Ca2+regulation. Ca2+influx occurs through both voltage-gated11and receptor-gated channels.12There is now considerable evidence from a variety of cell types that controlled Ca2+influx through store-operated Ca2+channels (SOCC; also termed capacitative Ca2+entry) occurs in response to SR Ca2+depletion, thus allowing for replenishment of intracellular Ca2+stores.13–16Such influx does not occur through either L-type or receptor operated channels.13
There is now considerable evidence for SOCC in vascular13,17–20and other smooth muscle types.21Previous studies suggested that SOCC-mediated Ca2+influx is restricted to the replenishment of inositol 1,4,5-trisphosphate-sensitive Ca2+stores.13,22Recent studies in tissues other than ASM have suggested that Ca2+release through RyR channels may also trigger SOCC.21,23The role of SOCC in ASM had been suggested by experiments in guinea pigs based on comparisons of contractions induced by agonists versus inhibitors of the SR Ca2+adenosine triphosphatase.24Furthermore, SR Ca2+adenosine triphosphatase inhibitors have been shown to increase [Ca2+]iin human bronchioles and bovine ASM, suggesting the involvement of SOCC.25However, characterization of SOCC-mediated Ca2+influx in ASM is relatively lacking.
Volatile anesthetic-induced SR Ca2+leak (and eventual depletion) may be expected to trigger SOCC-mediated Ca2+influx in ASM cells. However, we hypothesize that volatile anesthetics will also inhibit such influx, thus accentuating the state of SR depletion.
Materials and Methods
Details of the ASM cell preparation technique have been previously described.10,26Porcine tracheae were obtained from a local abattoir. The smooth muscle layer was excised, minced in Hanks’ balanced salt solution (HBSS) with 10 mm HEPES (pH 7.4; Invitrogen, Carlsbad, CA), and then incubated first for 2 h with 20 U/ml papain and 2000 U/ml DNase (Worthington Biochemical Corp., Lakewood, NJ) and subsequently for 1 h at 37°C with 1 mg/ml type intravenous collagenase (Worthington). The sample was triturated, centrifuged, and resuspended in minimum essential medium with 10% fetal calf serum. Isolated cells were plated on collagen-coated glass coverslips, and incubated for 1 h in 5% CO2at 37°C.
Real-Time Confocal Imaging
Details of the confocal imaging technique for ASM cells have also been previously published.10,26Briefly, cells attached to coverslips were loaded with 5 μm fluo-3 am (Molecular Probes, Eugene, OR) for 30–45 min at 37°C in HBSS, washed, mounted on an open slide chamber (RC-25F, Warner Instruments, Hamden, CT), and perfused with HBSS at room temperature. An Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI) with an Ar-Kr laser (488 nm line) and a Nikon 40×/1.3 oil-immersion objective lens was used to visualize ASM cells. Using manufacturer-provided software, regions of interest encompassing single cells were outlined for measurements of [Ca2+]i. Empirical calibration of fluo-3 fluorescence concentrations for [Ca2+]ibased on exposure to known Ca2+concentrations was performed as described previously.26
Administration of Volatile Anesthetics
As previously described,2calibrated online vaporizers were used to add halothane (Wyeth-Ayerst Laboratories, Philadelphia, PA), isoflurane, and sevoflurane (Abbott Laboratories, Deerfield, IL) to the aerating gas mixture. Aqueous anesthetic concentrations equivalent to 1 and 2 adult porcine minimum alveolar concentration (MAC) at room temperature (25°C) were determined for halothane and isoflurane by gas chromatography and electron capture detector (Hewlett-Packard 5880A; Hewlett-Packard, Sunnyvale, CA) and for sevoflurane by a flame ionization detector. Concentrations were halothane 1 MAC 0.35 ± 0.09 mm, 2 MAC 0.53 ± 0.10 mm, isoflurane 1 MAC 0.4 ± 0.09 mm, 2 MAC 0.6 ± 0.11 mm, and sevoflurane 1 MAC 0.50 ± 0.10 mm, 2 MAC 0.68 ± 0.10 mm.
Baseline Ca2+concentrations were measured while ASM cells were perfused with HBSS. Extracellular Ca2+([Ca2+]o) was then removed by exposure to zero-Ca2+HBSS (5 mm EGTA). In the continued absence of [Ca2+]o, cells were also exposed to 1 μm nifedipine and 10 mm KCl to ensure that L-type Ca2+channels were not activated during the protocol. Cells were then rapidly exposed to 1 μm cyclopiazonic acid (CPA) in zero-Ca2+HBSS. This technique has been frequently used to induce passive SR depletion by inhibiting SR Ca2+adenosine triphosphatase with continued SR Ca2+leak, presumably from both inositol 1,4,5-trisphosphate- and ryanodine-sensitive SR stores.13,16Continued SR Ca2+leak resulted in increased [Ca2+]iconcentrations that typically reached a plateau concentration or started to trend down (reflecting increasing Ca2+efflux via the plasma membrane). At this point, 2.5 mm [Ca2+]owas rapidly reintroduced (in the continued presence of CPA). The observed [Ca2+]iresponse was then characterized using various pharmacological manipulations.
In separate sets of ASM cells, we performed additional studies using caffeine for selective depletion of ryanodine-sensitive Ca2+stores and the assessment of any interactions between Ca2+release via RyR channels and SOCC. The protocol involved replacing CPA in the above experiments with 5 mm caffeine. As with the CPA protocols, pharmacological manipulations were used to characterized SOCC.
Effect of Volatile Anesthetics on Store-Operated Ca2+Influx
SOCC in ASM cells was first established by performing a control protocol using CPA or caffeine in the presence of nifedipine and KCl (as above). Once SOCC-mediated Ca2+influx was observed, the cells were washed for 15–20 min with HBSS to remove CPA (or caffeine) and to replenish SR Ca2+stores. [Ca2+]owas then removed and the cells exposed to either CPA or caffeine. Once a [Ca2+]iresponse was observed (slow development of plateau with CPA or a transient with caffeine), the cells were exposed for 1 min to 1 or 2 MAC halothane, isoflurane, or sevoflurane in the continued presence of CPA or caffeine. This technique ensured that the anesthetics did not influence the SR Ca2+release process itself, but were present in sufficient concentration before activation of SOCC. In the continued presence of CPA (or caffeine), and anesthetic, [Ca2+]owas rapidly reintroduced and the [Ca2+]iresponse was recorded. In control experiments, the SOCC protocol was performed twice without anesthetic, with an intervening wash in HBSS.
Interactions Between Acetylcholine and Volatile Anesthetic Effects on Store-Operated Ca2+Influx
As above, SOCC in ASM cells was first established by performing a control protocol using CPA or caffeine in the presence of nifedipine and KCl. Cells were then washed in HBSS. [Ca2+]owas removed, nifedipine and KCl were added, and SR Ca2+release was induced by CPA or caffeine. In the continued presence of CPA or caffeine, the cells were exposed to 10 nm, 100 nm, or 1 μm acetylcholine (Ach) for 2 min. This ensured activation of muscarinic receptors occurred after SR Ca2+release had already occurred. Activation of muscarinic receptors occurred with both protocols; however, in the case of CPA, SR Ca2+depletion is passive, whereas for caffeine, complete release through RyR channels occurs, although inositol 1,4,5-trisphosphate-induced release is dependent on ACh concentration. In the continued presence of CPA (or caffeine) and ACh, [Ca2+]owas rapidly reintroduced to activated SOCC. Cells were then washed for a second time, and [Ca2+]oremoved (with addition of nifedipine and KCl). After CPA or caffeine exposure, ACh was added as above and after 1 min, cells were exposed to 1 or 2 MAC halothane, isoflurane, or sevoflurane for an additional 1 min. This ensured that muscarinic activation occurred before anesthetic exposure and that confounding effects of anesthetic interactions with the receptor itself were avoided. [Ca2+]owas then rapidly reintroduced. In control protocols, after a control SOCC activation, the ACh protocol was performed twice, without anesthetic.
For each ASM cell, comparisons before and after exposure to a drug were made using paired Student t test. Repeated measures analysis of variance was used for multiple comparisons with post hoc Bonferroni and Sheffé F tests. All experimental protocols were not performed in the same cells. Results were replicated in at least 3–5 cells obtained from each of five animals (paired comparisons within cells, independent testing across cells). Statistical significance was tested at P < 0.05. Values are reported as mean ± SD.
Establishment of Store-Operated Ca2+Influx
At the beginning of experiments, baseline [Ca2+]iconcentrations of ASM cells ranged from 70 to 100 nm (78 ± 10 nm; n = 128). Neither removal of [Ca2+]onor addition of nifedipine and KCl significantly affected [Ca2+]iconcentrations. In the absence of [Ca2+]o, 1 μm CPA resulted in increased [Ca2+]iconcentrations that reached a plateau (228 to 518 nm; n = 65). Subsequent rapid reintroduction of [Ca2+]oresulted in a further, sustained increase of [Ca2+]i(fig. 1A; 340 to 660 nm; P < 0.05 when compared to the first plateau). In contrast to CPA, exposure to 5 mm caffeine in the absence of [Ca2+]oresulted in a transient [Ca2+]iresponse (fig. 1B; n = 63). Reintroduction of [Ca2+]oresulted in a relatively sustained increase in [Ca2+]iranging from 110 to 386 nm (compared to a peak caffeine response ranging from 455 to 890 nm, the influx response was ∼25% of the caffeine peak). The influx observed with reintroduction of [Ca2+]oafter caffeine exposure was significantly smaller than that after CPA exposure (P < 0.05).
Characterization of Store-Operated Ca2+Influx
It has been previously reported that SOCC-mediated Ca2+influx to be inhibited by Ni2+and La3+, at least in tissues other than ASM. After control activation of SOCC with CPA (n = 10) or caffeine (n = 10), and HBSS wash, ASM cells were exposed to nifedipine, KCl and 1 μm NiCl2or LaCl3in HBSS. [Ca2+]owas then removed and, in the continued presence of nifedipine, KCl and Ni2+(or La3+), cells were reexposed to CPA or caffeine, with subsequent reintroduction of [Ca2+]o. In the presence of either 1 μm Ni2+or La3+, reintroduction of [Ca2+]odid not significantly increase [Ca2+]i(Table 1; P < 0.05 compared to control) indicating that SOCC-mediated Ca2+influx in ASM cells is sensitive to both ions.
To determine whether the observed Ca2+influx was mediated by mechanisms other than SOCC (because L-type Ca2+channels were already inhibited by nifedipine and clamped membrane potential), inhibitors of store-operated Ca2+influx (SKF-96365; 10 μm) and Na+/Ca2+exchange (KBR-7943; 10 μm) were tested. Although the bidirectional plasma membrane Na+/Ca2+exchanger may not play a major role in most smooth muscle types, expression of this protein has been found in ASM.27When operating in the “reverse” or influx mode, the exchanger can bring Ca2+intracellularly, in exchange for Na+. After control SOCC activation with CPA (n = 11) or caffeine (n = 11) and HBSS wash, ASM cells were exposed to nifedipine, KCl, and 10 μm SKF-96365 or KBR7943 in HBSS. The SOCC protocol was then repeated in the continued presence of these agents. In 90% of cells, SKF-96365 inhibited SOCC-mediated Ca2+influx with both CPA and caffeine protocols (Table 1; P < 0.05 compared with controls). In contrast, the presence of KBR-7943 had no effect on the [Ca2+]iresponse upon reintroduction of [Ca2+]o(Table 1), demonstrating that Na+/Ca2+exchange did not substantially contribute to the observed Ca2+influx.
Effect of ACh on Store-Operated Ca2+Influx
Compared with controls where no ACh was present, preexposure of ASM cells to either 100 nm or 1 μm ACh significant increased Ca2+influx with reintroduction of [Ca2+]ofor both CPA and caffeine protocols (P < 0.05; Table 1). In contrast, in other controls where the ACh protocol was repeated, there was ∼5–7% decrease in the Ca2+influx upon repetition (not shown).
Effect of Volatile Anesthetics on Store-Operated Ca2+Influx
In ASM cells where SOCC-mediated Ca2+influx was first established, repetition of the protocol (CPA or caffeine) resulted in a 5–8% decrease in the observed Ca2+influx (rundown control). In contrast, exposure to halothane, isoflurane, or sevoflurane all resulted in significant decreases in the extent of Ca2+influx after reintroduction of [Ca2+]o(P < 0.05 compared to control for each anesthetic at 1 or 2 MAC, both same cell and rundown control; fig. 2). For halothane and isoflurane, the effects at 1 MAC were significantly less than at 2 MAC, whereas for sevoflurane, the effects were comparable between 1 and 2 MAC. Among the three anesthetics, sevoflurane caused the least inhibition. At 2 MAC, halothane and isoflurane produced comparable inhibition (fig. 2). Between the CPA and caffeine protocols, for each volatile anesthetic, the effect on Ca2+influx was significantly greater for CPA compared to caffeine (P < 0.05).
Interaction Between ACh and Volatile Anesthetic Effects on Store-Operated Ca2+Influx
In ASM cells in which both SOCC-mediated Ca2+influx and an ACh-induced augmentation of influx were first established using the CPA or caffeine protocols, exposure to halothane and isoflurane, but not sevoflurane, resulted in significant decreases in the Ca2+influx after reintroduction of [Ca2+]o(P < 0.05 compared to both same cell and ACh rundown control for 1 or 2 MAC; fig. 3). Compared to anesthetic effects on Ca2+influx in the absence of ACh, anesthetic inhibition of ACh-induced augmentation of influx was significantly greater (P < 0.05). For halothane and isoflurane, the effects at 1 MAC were significantly less than at 2 MAC, with halothane causing greater inhibition. For both halothane and isoflurane, the effect on ACh augmentation of Ca2+influx was significantly greater for the CPA protocol (P < 0.05).
Volatile anesthetic effects on ASM have been previously shown to involve inhibition of Ca2+influx via L-type Ca2+channels5–7as well as increased SR Ca2+leak via both inositol 1,4,5-trisphosphate and RyR channels.1,2,8Such a leak may be expected to trigger SOCC-mediated Ca2+influx in ASM cells, thus providing a mechanism for replenishment of depleted SR Ca2+stores. However, in support of our hypothesis, the results of this study demonstrate that volatile anesthetics also inhibit such store-operated Ca2+influx, thus accentuating the state of SR depletion. The results further demonstrate that volatile anesthetics, especially halothane and isoflurane, interfere with ACh-induced enhancement of SOCC-mediated Ca2+influx, potentially maintaining bronchodilation even during agonist stimulation.
[Ca2+]iRegulation in ASM
Regulation of [Ca2+]iin ASM is mediated by both extracellular Ca2+influx and SR Ca2+release via both inositol 1,4,5-trisphosphate receptor9and RyR10channels. Previous studies have demonstrated that Ca2+influx in ASM occurs through both voltage-gated11and receptor-gated channels.12,28–30Agonists such as ACh are known to produce SR Ca2+release via both inositol 1,4,5-trisphosphate and RyR channels.10Indeed, it is thought that the initial [Ca2+]iresponse to ACh stimulation is SR Ca2+release, whereas maintenance of ASM [Ca2+]iand thus muscle tone is thought to involve sustained Ca2+influx. However, we have previously shown that the [Ca2+]iresponse to ASM is oscillatory, mediated by repetitive SR Ca2+release and reuptake, where initiation of oscillations is dependent on Ca2+release through inositol 1,4,5-trisphosphate receptor channels, but sustenance of oscillations occurs through Ca2+-induced Ca2+release mechanisms via RyR channels.10,26The amplitude of [Ca2+]ioscillations represent SR Ca2+content, and frequency depends on sensitivity for Ca2+release via RyR channels. These studies also showed that Ca2+influx serves to maintain and replenish SR Ca2+because oscillations were not sustained in the absence of extracellular Ca2+or in the presence of inhibitors of Ca2+influx.10,26Our previous study on SOCC31as well as the current report emphasize the importance of this SR- and [Ca2+]o-dependent mechanism in maintaining the repletion state of the SR in ASM.
Effect of Volatile Anesthetics on [Ca2+]iin ASM
Volatile anesthetics have been shown to target several [Ca2+]iregulatory mechanisms in ASM at both SR and the plasma membrane. In terms of SR Ca2+release, we and others have shown that volatile anesthetics deplete SR Ca2+by decreasing SR Ca2+content (store) via increased “leak” through both inositol 1,4,5-trisphosphate and RyR channels.2,32–35We have previously demonstrated that clinically relevant concentrations of halothane decrease both amplitude and frequency of ACh-induced [Ca2+]ioscillations. We interpreted these findings as a decrease in SR Ca2+content and reduced sensitivity for Ca2+induced Ca2+release by volatile anesthetics.35These data collectively demonstrate that volatile anesthetics deplete SR Ca2+stores, thus preventing an increase of [Ca2+]iin response to agonist stimulation. Anesthetic-induced depletion of SR Ca2+then raises the question of whether Ca2+influx via SOCC (if present) is then activated.
Given the existence of voltage-gated and receptor-operated Ca2+influx mechanisms in ASM, there is already considerable evidence in the literature that the decrease in [Ca2+]iby volatile anesthetics involves inhibition of Ca2+influx3,5through voltage-gated L-type Ca2+channels.5,6These studies underline the inhibitory effect of volatile anesthetics on plasma membrane Ca2+regulatory proteins. Accordingly, the results of the current study demonstrating inhibition of SOCC in the plasma membrane are consistent. Functionally, such inhibition only emphasizes the accentuation of anesthetic effects on [Ca2+]iin ASM because prevention of influx would only maintain the SR in a state of depletion, thus keeping [Ca2+]ilow even with agonist stimulation.
SOCC-Mediated Ca2+Influx and Volatile Anesthetics Effects
In addition to previously established modes of Ca2+influx, i.e. , voltage-gated11and receptor-gated channels,12,28–30in a recent study, we31found that Ca2+influx in ASM occurs in response to SR Ca2+depletion. Such a mechanism for influx in response to the state of SR Ca2+stores was initially reported in parotid acinar cells and vascular smooth muscle in which influx was found to be dependent on [Ca2+]obut not L-type or receptor operated channels.13,36Other studies have demonstrated that SR Ca2+adenosine triphosphatease inhibitors (such as thapsigargin, similar to CPA) increase Ca2+influx or smooth muscle tone.20,37–40In these studies, whereas [Ca2+]ois necessary for maintenance of muscle tone or [Ca2+]i, Ca2+-channel blockers do not consistently prevent influx, suggesting that both voltage-gated Ca2+channels and SOCC contribute to smooth muscle Ca2+regulation and tone. Studies to date suggest that SOCC may play a significant role in tonic muscles such as blood vessels.13,17,18Only a few studies, including our own, have reported the existence of SOCC in ASM cells.31,41
Although the channels that represent SOCC have not been definitively identified, members of the tryptophan family have been implicated in several studies, and mammalian tryptophan genes have been identified.15,42Some of these isoforms have been shown to be involved in SOCC.15,43We have previously verified the expression of several tryptophan C isoforms in porcine ASM,31although other studies have reported tryptophan C expression in guinea pig ASM.44Therefore, it is possible that SOCC in porcine ASM is mediated via tryptophan C proteins.
There are scarce data on anesthetic effects on SOCC (or capacitative Ca2+influx per some authors). Horibe et al. 45found that propofol inhibits capacitative Ca2+entry in pulmonary artery smooth muscle. Tas et al. 46found that in primary human endothelial cells, isoflurane inhibits histamine induced Ca2+influx via SOCC. However, this group also found that enflurane enhances Ca2+influx in rat glioma cells.47Pochet et al. 48reported that trichloroethanol, the active metabolite of chloral hydrate, inhibits SOCC-mediated Ca2+influx in submandibular acinar cells. The current study is the first to report the effects of anesthetics on SOCC in airway smooth muscle.
Studies from several cell types have now shown that SOCC does not appear to be activated by changes in membrane potential, thus making this mechanism different from L-type Ca2+channels.13In accordance, in our previous study, we found that changes in membrane potential (induced by altering extracellular K+concentration) did not affect SOCC-mediated Ca2+influx in ASM.31Furthermore, Ca2+influx after SR Ca2+depletion by CPA was found to be insensitive to nifedipine, confirming that L-type Ca2+channels do not mediate the observed influx. However, the existence of L-type Ca2+channels in ASM has already been established,11,49as has the inhibitory effect of volatile anesthetics.5,6Certainly, volatile anesthetic inhibition of L-type channels would also prevent SR repletion, and blunt the [Ca2+]iresponse to ACh. Therefore, in the current study, we used nifedipine to block the confounding effect of this mechanism on the observed influx and volatile anesthetic effects. Addition of KCl to the extracellular medium also helped maintain membrane potential at a value where L-type Ca2+channels were unlikely to be activated.
Consistent with our previous study, and with studies in other smooth muscle types, we found that SOCC is blocked by Ni2+17,19,21,31,41and La3+.17,21,31Although both ions are capable of nonspecifically inhibiting influx via several mechanisms, inhibition of influx by μM concentrations of either ion strongly indicate that the observed influx is mediated via SOCC.
Previous studies in tissues other than ASM have found that SOCC-mediated Ca2+influx occurs after depletion of inositol 1,4,5-trisphosphate-sensitive SR Ca2+stores.13,22In our previous study, we found that SOCC in ASM is triggered after SR Ca2+release via inositol 1,4,5-trisphosphate receptor channels.31It has been previously demonstrated that volatile anesthetics markedly increase inositol 1,4,5-trisphosphate concentrations in ASM cells, even in the absence of agonist stimulation2(but see Yamakage et al. 8). Accordingly, anesthetic exposure should enhance SOCC-mediated Ca2+influx. However, we31and others14have found that inositol 1,4,5-trisphosphate by itself does not trigger SOCC-mediated Ca2+influx even in the presence of agonist stimulation, and it is SR Ca2+release via inositol 1,4,5-trisphosphate receptor channels that is important. In this regard, previous studies have demonstrated that volatile anesthetics enhance SR Ca2+leak via inositol 1,4,5-trisphosphate receptor channels.2,32–35This should lead to enhanced SOCC-mediated Ca2+influx in the presence of anesthetics. However, the current study demonstrates that despite this potential enhancement, volatile anesthetics can inhibit Ca2+influx.
In addition to triggering of SOCC after release via inositol 1,4,5-trisphosphate receptor channels, there are now data in skeletal muscle50,51and in ASM31demonstrating SOCC activation in response to depletion of caffeine-sensitive SR Ca2+stores and subsequent introduction of [Ca2+]o. Again, it has been previously demonstrated that volatile anesthetics increase SR Ca2+leak via RyR channels,2,52,53suggesting that in the presence of anesthetics, SOCC-mediated influx may be enhanced. However, as with inositol 1,4,5-trisphosphate receptor channels, the data from the current study (i.e. , the caffeine protocol), demonstrates that volatile anesthetics overwhelm any potential enhancement of influx resulting from depletion of caffeine-sensitive Ca2+stores.
In ASM, exposure to ACh appears to enhance SOCC-mediated influx. However, it is not entirely clear whether agonist stimulation modulates SOCC because influx is confounded by the presence of mechanisms for Ca2+influx that are neither voltage-gated nor mediated by SOCC, i.e. , G-protein coupled receptor-operated mechanisms.41,54,55Whether such a mechanism is involved in ASM is also not clear.41In our previous study,31we found that selective activation of the non-SOCC component by preventing SR Ca2+depletion resulted in significantly less enhancement of Ca2+influx by ACh compared with when SR depletion was present, suggesting the existence of both SOCC and noncapacitative Ca2+influx in ASM. Regardless, the data at least suggests that ACh does modulate SOCC in ASM.
Muscarinic modulation of SOCC has the potential for at least partial inhibition of anesthetic blockade of SOCC; however, it is possible that anesthetic effects on SOCC more likely help maintain bronchodilation, even during agonist stimulation. Furthermore, volatile anesthetics also inhibit muscarinic receptor activation.56,57Accordingly, even in the presence of ACh, volatile anesthetics likely maintain their inhibition of SOCC.
Significance of SOCC and Volatile Anesthetic Effects
Increased [Ca2+]iis key to force generation in ASM. SOCC-mediated Ca2+influx has been shown to produce ASM contraction.25Accordingly, anesthetic inhibition of SOCC has the potential to prevent SR Ca2+repletion and agonist-induced bronchoconstriction. Volatile anesthetics are known to affect other mechanisms that regulate both [Ca2+]i(e.g. , SR and voltage-gated Ca2+channels) and force (e.g. , Ca2+sensitivity of force generation58), albeit with different relative potencies (e.g. , sevoflurane has a much smaller effect on influx, but is more potent than isoflurane in decreasing Ca2+sensitivity). Nonetheless, anesthetic inhibition of SOCC may significantly contribute to the overall bronchodilatory effect of these agents by decreasing overall [Ca2+]iavailability and thus help to maintain bronchodilation.
The authors thank Larry Hunter, M.S. (Associate in Research, Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota) and Thomas Keller (Technician, Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota) for their superb technical assistance.