Store-operated Ca²⁺Influx in Airway Smooth Muscle: Interactions between Volatile Anesthetic and Cyclic Nucleotide Effects

As described in several of our previous studies (e.g. , Kannan et al. ,261996), porcine ASM cells were enzymatically dissociated in Earle’s balanced salt solution (10 mm HEPES, pH 7.4; Invitrogen, Carlsbad, CA) using papain, DNase, and type IV collagenase (Worthington Biochemical Corp., Lakewood, NJ). Isolated cells were plated on collagen-coated glass coverslips, loaded with 5 μm fura-2 AM (Molecular Probes, Eugene, OR) for 30–45 min at room temperature in Hanks balanced salt solution (HBSS), washed, mounted on an open slide chamber (RC-25F; Warner Instruments, Hamden, CT), and imaged at room temperature. Cells were visualized using a MetaFluor real-time fluorescence imaging system (Universal Imaging, Downingtown, PA) on a Nikon Diaphot inverted microscope (Fryer Instruments, Edina, MN). Image pairs (excitation 340 and 380 nm) were obtained once every second, and 340/380 nm fluorescence ratiometric data were calibrated for Ca²⁺levels using previously published techniques.27 

Cells attached to coverslips were loaded with 5 μm fura-2 AM for 30–45 min at 37°C in HBSS, washed, mounted on an open slide chamber (RC-25F), and perfused with HBSS at room temperature. Fura-2–loaded cells were visualized using a MetaFluor real-time fluorescence imaging system on a Nikon Diaphot inverted microscope (40×/1.3 NA lens). Pairs of images for excitation wavelengths 340 and 380 nm and common emission of 510 nm were obtained once every second, and the ratio of emissions for 340 versus  380 nm was calculated. The ratios were calibrated for [Ca²⁺]ⁱlevels using previously published techniques.9,27The calibration technique relies on calculation of the ratios at minimal [Ca²⁺]ⁱconcentration and with maximum [Ca²⁺]ⁱobtained with a Ca²⁺ionophore. We have previously calculated the actual calibration constant in freshly dissociated porcine ASM cells.

Perfusion media were bubbled with volatile anesthetics using calibrated vaporizers (halothane: Wyeth-Ayerst Laboratories, Philadelphia, PA; isoflurane and sevoflurane: Abbott Laboratories, Deerfield, IL).2Aqueous anesthetic concentrations equivalent to 1 adult porcine minimum alveolar concentration (MAC) at room temperature (25°C) were determined by gas chromatography and electron capture detector (halothane, isoflurane: Hewlett-Packard 5880A; Hewlett-Packard, Sunnyvale, CA) or flame ionization detector (sevoflurane). Concentrations were 1 MAC 0.32 ± 0.07 mm halothane, 1 MAC 0.38 ± 0.08 mm isoflurane, and 1 MAC 0.48 ± 0.11 mm sevoflurane.

Store-operated Ca²⁺entry was evaluated using previously described protocols.8,9,24Briefly, ASM cells were washed with 0 Ca²⁺HBSS (5 mm EGTA) for 5 min to remove extracellular Ca²⁺([Ca²⁺]^o). In the continued absence of [Ca²⁺]^o, 1 μm nifedipine and 10 mm KCl were added to inhibit L-type Ca²⁺channels and “clamp” the membrane potential (KCl) respectively, and the SR was passively depleted by 1 μm cyclopiazonic acid (CPA; inhibitor of the SR Ca²⁺adenosine triphosphatase). As reported in our previous studies, CPA exposure caused a gradual elevation of [Ca²⁺]ⁱ(reflecting progressive SR Ca²⁺depletion) and an eventual plateau level (or occasionally a gradual trend down reflecting Ca²⁺efflux mechanisms).8,9At this point, 2.5 mm [Ca²⁺]^owas rapidly reintroduced in the continued presence of CPA, nifedipine, and KCl to trigger SOCE.

The protocol for examining volatile anesthetic effects on SOCE has also been previously described.9Cells in which SOCE was first demonstrated using CPA (control response) were washed for 15–20 min with HBSS to remove CPA and to replenish SR Ca²⁺stores. [Ca²⁺]^owas then removed, and the cells were reexposed to CPA in the presence of nifedipine and KCl. When the [Ca²⁺]ⁱresponse representing SR Ca²⁺depletion was observed, cells were exposed for 1 min to 1 MAC halothane, isoflurane, or sevoflurane in the continued presence of CPA, nifedipine, and KCl, thus ensuring that the anesthetics did not influence SR Ca²⁺release itself. In the continued presence of CPA and anesthetic, SOCE was then triggered by reintroducing [Ca²⁺]^o. In control experiments, the SOCE protocol was performed twice without anesthetic, with an intervening wash in HBSS.

Acetylcholine was used as an ASM agonist to examine interactions with volatile anesthetics. Ca²⁺influx via  SOCE was first established (see “Store-operated Ca²⁺Entry”). Cells were then washed with HBSS for 15–20 min, and the protocol was repeated with the introduction of 1 μm acetylcholine for 2 min before reintroduction of [Ca²⁺]^o(but after CPA exposure, ensuring that activation of muscarinic receptors after SR Ca²⁺release had already occurred). Cells were then washed for a second time, and the SOCE protocol was then performed with the introduction of both acetylcholine and 1 MAC halothane, isoflurane, or sevoflurane before activation of SOCE by reexposure to [Ca²⁺]^o. Volatile anesthetics are known to interfere with muscarinic receptor stimulation.28Acetylcholine was introduced first, followed by anesthetic for 1 min (in the continued presence of acetylcholine), ensuring that muscarinic activation occurred before anesthetic exposure. If anesthetics were introduced first, it would have been difficult to distinguish between anesthetic effects on SOCE versus  on muscarinic receptor activation itself.

We have previously reported the inhibitory effects of cyclic nucleotides on SOCE in ASM.24In this study, we first performed the SOCE protocol in the presence of drugs that increase cAMP or cGMP to verify SOCE inhibition. The same cells were then washed with HBSS, and the SOCE protocol was repeated in the combined presence of these drugs as well as volatile anesthetics. Briefly, after SOCE was first established by performing a control protocol using CPA, ASM cells were washed for 15–20 min with HBSS, [Ca²⁺]^owas then removed, nifedipine and KCl were added, and the cells were reexposed to CPA (in the continued presence of nifedipine and KCl). When an [Ca²⁺]ⁱresponse was observed (thus ensuring SR Ca²⁺depletion first), cells were exposed for 5 min to either 100 μm dibutryl cAMP (dbcAMP; a membrane permeable cAMP analog) or 100 μm 8-Br-cGMP (cell-permeable cGMP analog). [Ca²⁺]^owas then rapidly reintroduced to reactivate SOCE. Cells were then thoroughly washed in HBSS for up to 15 min to ensure sufficient time for washout of dbcAMP or 8-Br-cGMP as well as refilling of SR Ca²⁺stores (in pilot control experiments where the SOCE protocol in the presence of these drugs was performed twice in the same cells, we found that 15 min was sufficient to result in less than 10% difference in amplitudes of two subsequent SOCE responses). After the second wash, extracellular Ca²⁺was removed, and cells were exposed to CPA, nifedipine, and KCl. Subsequently, cells were first exposed to the same drug as in the previous SOCE activation (dbcAMP or 8-Br-cGMP) and then to 1 MAC halothane, isoflurane, or sevoflurane. [Ca²⁺]^owas then introduced to activate SOCE.

Statistical comparisons were performed for the same ASM cell before and after exposure to a drug and/or anesthetic using the paired Student t  test. Comparisons involved absolute values or amplitudes of responses. Where appropriate, multiple comparisons (e.g. , across anesthetics) were performed using analysis of variance and Bonferroni corrections. All experimental protocols were not performed in all cells. Results were replicated in at least five cells obtained from at least four animals (paired comparisons within cells, independent testing across cells). Statistical significance was tested at the P < 0.05 level. Values are reported as mean ± SE.

Baseline [Ca²⁺]ⁱlevels in ASM cells ranged from 85 to 125 nm (93 ± 8 nm; n = 74) and decreased slightly or did not change significantly with initial removal of [Ca²⁺]^o, addition of nifedipine, and addition of KCl. Under these conditions, 1 μm CPA elevated [Ca²⁺]ⁱ, resulting in a plateau (fig. 1). In some cells, there was a gradual decrement in [Ca²⁺]ⁱafter the plateau, presumably reflecting unopposed efflux mechanisms such as plasma membrane Ca²⁺adenosine triphosphatase. Rapid reintroduction of [Ca²⁺]^oresulted in a sustained elevation of [Ca²⁺]ⁱ(P < 0.05 compared with the first plateau) reflecting SOCE (as reported in our previous studies). We have previously characterized SOCE in ASM, including pharmacologic inhibition versus  activation, as well as time dependence,8,9and therefore, these studies were not repeated here.

In control ASM cells, SOCE varied less than 10% with repeated activation (rundown control). In the absence of [Ca²⁺]^o, exposure to 1 MAC halothane, isoflurane, or sevoflurane did not significantly alter the plateau [Ca²⁺]ⁱlevel in the presence of CPA. However, with reintroduction of [Ca²⁺]^oin the presence of any of the three anesthetics, SOCE was significantly diminished compared with the previous control activation in the same cell (P < 0.05 for each anesthetic, compared with both same cell and rundown control; fig. 2). Halothane produced significantly greater inhibition, compared with isoflurane or sevoflurane, the latter causing the least inhibition.

In cells where 1 μm acetylcholine produced augmentation of SOCE after CPA exposure, exposure to halothane and isoflurane, but not sevoflurane, significantly decreased Ca²⁺influx after reintroduction of [Ca²⁺]^o(P < 0.05 for both same cell and acetylcholine rundown control; fig. 2). The inhibition of SOCE by volatile anesthetics was significantly greater in the presence of acetylcholine than in its absence (i.e. , volatile anesthetics alone; P < 0.05).

In absence of [Ca²⁺]^o, exposure to either cyclic nucleotide did not significantly alter the [Ca²⁺]ⁱresponse to CPA (data not shown). However, in the presence of either 100 μm dbcAMP (fig. 3) or 8-Br-cGMP (fig. 4), subsequent reintroduction of [Ca²⁺]^oresulted in a significantly smaller SOCE compared with control in the same cells (P < 0.05; figs. 3 and 4).

In the presence of dbcAMP, exposure to 1 MAC halothane resulted in almost complete inhibition of SOCE when [Ca²⁺]^owas reintroduced in the combined presence of both dbcAMP and halothane (P < 0.05 compared with control; fig. 3). Indeed, only 10% of the cells showed any SOCE under these conditions. Compared with the effects of halothane alone or dbcAMP alone, SOCE inhibition in the presence of both agents was significantly greater (P < 0.05 for both halothane and dbcAMP). As with dbcAMP, in the presence of 8-Br-cGMP, 1 MAC halothane produced significantly greater inhibition of SOCE compared with control, halothane alone, or 8-Br-cGMP alone (P < 0.05; fig. 4).

Similar to halothane, 1 MAC isoflurane–induced inhibition of SOCE in the presence of dbcAMP or 8-Br-cGMP was significantly greater than that by isoflurane, dbcAMP, or 8-Br-cGMP alone (P < 0.05; figs. 3 and 4). Overall, isoflurane effects were smaller than those of halothane. In contrast to halothane or isoflurane, sevoflurane did not produce any greater inhibition of SOCE in the presence of dbcAMP (fig. 3). However, with 8-Br-cGMP, significantly greater inhibition was observed (P < 0.05; fig. 4).

Both cyclic nucleotides (cAMP and cGMP)19,20and volatile anesthetics1,2,29,30relax ASM, by interference with mechanisms that would normally elevate [Ca²⁺]ⁱ.29,31–33Anesthetics have been previously shown to increase SR Ca²⁺leak via  both inositol 1,4,5-triphosphate and ryanodine channels.1,2,18Although such a leak would be expected to trigger SOCE, our studies show that volatile anesthetics also inhibit SOCE, thus accentuating the state of SR depletion. As in our previous study, we found that cyclic nucleotides also inhibit SOCE. Given that SR Ca²⁺release is an important contributor to total [Ca²⁺]ⁱin ASM, SOCE inhibition would only lead to less available SR Ca²⁺release during agonist stimulation and thus to enhanced relaxation of ASM. The data presented in the current study demonstrate that in the simultaneous presence of both anesthetics and agents that elevate cyclic nucleotides, there is an additive effect (disproving our hypothesis of antagonism), resulting in almost complete SOCE inhibition, especially with halothane. These data suggest that the combined effects of anesthetics and cyclic nucleotides may be a potent mechanism for inducing bronchodilation.

[Ca²⁺]ⁱregulation in ASM involves both extracellular Ca²⁺influx and SR Ca²⁺release34,35channels. Ca²⁺influx occurs through voltage-gated,5receptor-gated,6,7,36,37and store-operated8,9channels. SOCE has now been reported in a multitude of cell types, including vascular smooth muscle,14,38where muscle tone is increased with SOCE activation, indicating a physiologic role.12–14In previous studies, we have shown that Ca²⁺influx serves to maintain and replenish SR Ca²⁺stores. Accordingly, SOCE represents an important SR- and [Ca²⁺]^o-dependent mechanism for maintenance and repletion of SR in ASM, and thus modulating the [Ca²⁺]ⁱ(and force) response of ASM to agonist.

Volatile anesthetics are known to target several ASM [Ca²⁺]ⁱregulatory mechanisms. For example, anesthetics inhibit Ca²⁺influx3,29through voltage-gated L-type Ca²⁺channels.29,39We and others have shown that anesthetics deplete SR Ca²⁺via  increased “leak” through both inositol 1,4,5-triphosphate and ryanodine channels.1,2,40–42Anesthetic-induced SR Ca²⁺depletion would be expected to enhance SOCE. However, in a recent study, we found that clinically relevant concentrations of volatile anesthetics inhibit SOCE.8The results of the current study (repeated to establish one set of “controls” for examining interactions with cyclic nucleotides) are completely consistent with our previous studies. SOCE inhibition has been in addition reported in pulmonary artery smooth muscle with propofol43and histamine-induced SOCE in human endothelial cells with isoflurane.44Functionally, SOCE inhibition underlines accentuation of anesthetic effects on [Ca²⁺]ⁱ, maintaining the SR in a state of depletion and keeping [Ca²⁺]ⁱlow even with agonist stimulation.

As with previous studies, we found that exposure to acetylcholine enhances SOCE. Muscarinic receptor stimulation induces SR Ca²⁺release and consequently SR depletion, thus enhancing SOCE. Furthermore, SOCE has been linked to agonist receptor interactions in other cell types.45Accordingly, receptor activation (and potentially G-protein involvement) may modulate SOCE. Such muscarinic modulation has the potential for at least partial reversal of anesthetic blockade of SOCE. However, it is possible that anesthetic effects on SOCE maintain bronchodilation, even during agonist stimulation. Furthermore, anesthetics are known to inhibit muscarinic receptor activation.46Accordingly, even in the presence of agonists such as acetylcholine, volatile anesthetics likely maintain their inhibition of SOCE.

Comparing the effects across volatile anesthetics, halothane and isoflurane seemed to have similar inhibitory effects on SOCE, whereas sevoflurane demonstrated the least effect on SOCE. Such differences are consistent with our previous work on SOCE.8In addition, sevoflurane had no effect on SOCE during muscarinic receptor stimulation. Differences in the chemical structure between sevoflurane versus  halothane/isoflurane may be the reason for these findings. On the other hand, the effects of isoflurane and sevoflurane on SOCE in the presence of either cyclic nucleotide were comparable. Whether different anesthetic agents produce different conformational changes in proteins mediating SOCE or alter other mechanisms that regulate SOCE remains to be determined.

Cyclic nucleotides are known to produce smooth muscle relaxation.19,20cAMP, activated by β-adrenoreceptor agonists (e.g. , albuterol, isoproterenol), is coupled to adenylate cyclase, which in turn activates protein kinase A. cAMP-mediated actions in smooth muscle reflect down-regulation of [Ca²⁺]ⁱ-elevating mechanisms, such as increased inositol 1,4,5-triphosphate hydrolysis, inhibited inositol 1,4,5-triphosphate–induced SR Ca²⁺release, increased calcium uptake by internal stores,31and inhibited Ca²⁺influx via  membrane hyperpolarization.22,23,32In a recent study, we found that both β-adrenoceptor activation via  isoproterenol and direct cAMP elevation by dbcAMP significantly inhibited SOCE in ASM.24The results of the current study with dbcAMP are completely consistent with previous results. Similar inhibitory effects of cAMP on SOCE have been reported in vascular smooth muscle.25,47In our previous study, we demonstrated that cAMP effects on SOCE are largely mediated via  protein kinase A. Given the well-recognized bronchodilatory role of β-adrenoceptor agonists, SOCE inhibition would be expected to lead to lesser available SR Ca²⁺release during agonist stimulation and thus enhanced ASM relaxation.

In our previous study, we found that cGMP, via  protein kinase G, also inhibits SOCE in ASM.24Similar inhibitory effects are suggested by studies in vascular smooth muscle48and endothelial cells.49Interestingly, we found that in comparison to cAMP, cGMP (at least via  a nitric oxide donor) is more potent in inhibiting SOCE. However, in the current study, direct cGMP elevation with 8-Br-cGMP actually resulted in less SOCE inhibition compared with dbcAMP. Whether such differences translate to differential bronchodilatory responses of drugs acting via  the cAMP versus  cGMP pathways remains to be determined.

The discussion above indicates that volatile anesthetics and cyclic nucleotides differently affect the SR, in that anesthetics in general deplete the SR of Ca²⁺stores, whereas cyclic nucleotides (especially cAMP) enhance mechanisms that should increase SR Ca²⁺stores overall (e.g. , enhanced SR Ca²⁺reuptake, inhibited release). Accordingly, the simultaneous presence of both anesthetics and cyclic nucleotides (or agents that increase their levels) may be expected to balance the effects of acetylcholine on the SR. However, both do decrease [Ca²⁺]ⁱlevels. More importantly, both anesthetics and cyclic nucleotides individually inhibit SOCE in a significant fashion. Furthermore, the data of the current study demonstrate that the inhibitory effect of anesthetics on SOCE in the presence of cyclic nucleotides is greater than that with either agent alone. Accordingly, regardless of whether the airway is exposed to anesthetics first (depleting SR) followed by cyclic nucleotides (attempting to refill the SR) or to cyclic nucleotides followed by anesthetics (resulting in SR depletion anyway), there is unlikely to be substantial Ca²⁺available for SR replenishment in the face of inhibited Ca²⁺influx. In this regard, it is interesting to note that anesthetic agents differ in their interactions with cyclic nucleotides in terms of SOCE inhibition. Halothane, as with its effects on several other [Ca²⁺]ⁱregulatory mechanisms, displayed the strongest interactions with both cAMP and cGMP in inhibiting SOCE. However, given the already potent bronchodilatory effect of halothane, it is unlikely that the more pronounced inhibition in the presence of cAMP (and to a lesser extent in cGMP) is of much clinical significance. On the other hand, it is interesting to note that the combination of isoflurane with either cAMP or cGMP results in a significantly greater SOCE inhibition. On the other hand, sevoflurane displayed no significant interaction with cAMP, and thus the combination of the two may not be of much added benefit compared with each agent alone. Interestingly, both isoflurane and sevoflurane had significant interactions with cGMP in inhibiting SOCE. Although cGMP-elevating agents such as nitric oxide or nitrates are not commonly used clinically for bronchodilation, such interactions provide insight into potential alternative therapies in refractory airway hyperreactivity. In this regard, an important finding in both vascular smooth muscle33and in our recent study24in ASM has been that cAMP and cGMP cross-activate protein kinases A and G, resulting in common effects on smooth muscle. Such cross-activation is of significant interest to clinical therapy given the potential for pathways activated by nitric oxide donors or atrial natriuretic peptide to enhance effects of β-adrenoceptor agonists in the treatment of bronchoconstriction. Furthermore, drugs that activate one pathway may still be effective in decreasing [Ca²⁺]ⁱin ASM, even when the other pathway is inhibited by other drugs.

In summary, our study demonstrates that the combination of cyclic nucleotides and volatile anesthetics leads to greater inhibition of SOCE in ASM than each agent alone. Even under conditions of muscarinic receptor stimulation with acetylcholine, volatile anesthetics inhibit SOCE. Overall, halothane has the most profound effect on SOCE, and sevoflurane has the least. These findings suggest that a combination of cyclic nucleotides and anesthetics may be beneficial in the treatment of persistent bronchoconstriction in disease states such as asthma.

The authors thank Larry Hunter, M.S., (Associate in Research), and Thomas Keller, A.B. (Research Technician), both from the Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, for superb technical assistance.