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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Mechanisms of Interleukin-4 Effects on Calcium Signaling in Airway Smooth Muscle Cells

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

Received November 3, 2004; accepted January 4, 2005.

	Abstract

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In airway smooth muscle cells, interleukin (IL)-4 inhibited both carbachol- and caffeine-induced calcium mobilization from the sarcoplasmic reticulum (SR). Because of the known signaling pathways for IL-4 and importance of calcium uptake in maintaining SR calcium stores shared by agonists and caffeine, it was hypothesized that this rapid inhibitory effect might depend on phosphatidylinositol 3-kinase (PI3K) and on inhibition of calcium uptake by the SR. Enzyme-dispersed bovine trachealis cells were loaded with Fura-2/acetoxymethyl ester, and changes in cytosolic calcium were imaged in single cells. Cells were pretreated with inhibitors of PI3K, either wortmannin (100 nM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (50 µM), or deguelin (100 nM). Calcium transients in response to carbachol (10 µM) were significantly decreased to 0.34 ± 0.10 of control after 20-min treatment with IL-4 but were 1.10 ± 0.26 and 1.08 ± 0.23 when wortmannin or deguelin, respectively, was added along with IL-4. LY294002 alone had nonspecific effects on transients. In other experiments, cyclopiazonic acid (CPA) (5 µM), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 ± 0.08 of control (n = 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 ± 0.05 (n = 6; p < 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 µM) gave similar results. It was concluded that IL-4 did not inhibit transients in the presence of PI3K antagonists but that it did in the presence of CPA. This suggested that IL-4 inhibited calcium transients by mechanisms dependent upon a wortmannin-sensitive PI3K but not by inhibition of calcium uptake into the SR.

In single, bovine trachealis cells, IL-4 alone had modest effects on intracellular calcium concentration ([Ca²⁺]_i) but rapidly (20 min) inhibited both carbachol- and caffeine-stimulated calcium transients (Madison and Ethier, 2001). Since carbachol and caffeine cause rapid release of calcium from a common calcium compartment within the sarcoplasmic reticulum (SR) by different mechanisms involving distinct calcium release channels, the findings suggested that brief treatments with IL-4 reduced the amount of calcium that was available for mobilization from the SR (Janssen and Sims, 1993; Liu and Farley, 1996; Madison et al., 1998; Ethier et al., 2001). The signal transduction pathways that might link IL-4 receptor activation to changes in calcium homeostasis are potentially multiple and could include STAT6 regulation of gene expression, phosphatidylinositol 3-kinase (PI3K) activation, the adaptor protein Shc, and guanine nucleotide exchange factors (Fruman and Cantley, 2002; Kelly-Welch et al., 2003). Since the inhibitory effect of IL-4 on calcium transients was relatively rapid, it was hypothesized that IL-4 stimulated PI3K to modulate calcium signaling. An effect of PI3K on calcium signaling was plausible because prior studies in HepG2 cells linked PI3K to the activation of PLC, a source of inositol 1,4,5-trisphosphate (IP₃) for calcium mobilization (Rameh et al., 1998). To assess the role of PI3K in regulating calcium transients in airway smooth muscle cells, we tested whether IL-4 had inhibitory effects on calcium transients in the presence of PI3K antagonists.

In airway smooth muscle, the main intracellular store for rapidly mobilized calcium is the SR (Madison et al., 1998; Ethier et al., 2001). Store content of calcium is an important determinant of the magnitude of calcium transients and represents a dynamic balance between uptake of calcium via the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) and release of calcium from the SR via inositol 1,4,5-trisphosphate receptor (IP₃R), ryanodine receptor (RyR), and possibly other channels (Pozzan et al., 1994; Minn et al., 1997; Schendel et al., 1997; Liu et al., 2003). To explain the effects of IL-4 on calcium transients, it was hypothesized that IL-4 acted by inhibiting SR uptake of calcium by SERCA. Over approximately 20 min, this would decrease the amount of calcium available for subsequent mobilization by carbachol and caffeine (Madison et al., 1998; Ethier et al., 2001). However, an alternative was that IL-4 acted as a low-efficacy agonist that decreased SR calcium by increasing calcium release rates from the SR. To assess these possibilities, we tested whether IL-4 could still attenuate the magnitude of calcium transients when SERCA was inhibited.

	Materials and Methods

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Cell Isolation. Smooth muscle cells were dispersed from bovine trachealis (Ethier et al., 2001; Madison and Ethier, 2001). Trachealis tissue was minced and placed in 2.5 ml of physiological salt solution (PSS, 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25.6 mM NaHCO₃, 11.1 mM glucose, and 2.5 mM CaCl₂) modified to have no added CaCl₂ and containing collagenase A (6 mg) and elastase (3 mg). The mince was incubated at 37°C for 12 min, and then this treatment was repeated with an additional aliquot of enzyme. The partially digested mince was transferred to 3 ml of PSS modified to contain 0.1 mM CaCl₂ and incubated for 3 to 6 min with constant stirring. The released cells were loaded with 0.5 µM Fura-2/AM in the presence of Pluronic F-127 (0.004%) for 60 min at room temperature, placed in a perfusion chamber, and allowed to attach to glass for 10 min. After attachment, PSS perfused the chamber at 1 ml/min.

Calcium Measurement. Fura-2 was excited by computer-controlled 337- and 380-nm UV light generated by a nitrogen laser and a nitrogen laser-pumped dye laser, respectively (Laser Science, Franklin, MA). Each laser alternately fired pulses (3 ns) at 30 Hz that reached the cells through a 40x objective lens (Nikon, Melville, NY). The fluorescent signals emitted by Fura-2 were passed back through the objective to a 455-nm dichroic mirror, a 475-nm barrier filter (Omega Optics, Brattleboro, VT), and an image intensifier (Xybion Electronic Systems, San Diego, CA) and captured by a Philips-based frame transfer charge-coupled device camera (CCTV, New York, NY). The analog signals from the camera were digitized and stored in an imaging board with digital outputs to a personal computer with software by Recognition Technology, Inc. (Framingham, MA).

As described previously (Ethier et al., 2001; Madison and Ethier, 2001), background from a cell-free region of the coverglass was subtracted before data acquisition, and then an 11 x 11 pixel area was selected over the cell. The fluorescence stimulated by alternating pulses of 337- and 380-nm light was recorded, and their ratios were plotted. Ratios were converted to calcium concentrations (Grynkiewicz et al., 1985): [Ca²⁺]_i = K_D * *(R – R_min)/(R_max – R), where R_max and R_min are the fluorescence ratios measured in high and zero calcium, respectively; is the ratio of fluorescence stimulated by 380-nm light in zero versus high calcium; and K_D is the equilibrium dissociation constant describing calcium binding to Fura-2. Based on an in situ determination in bovine trachealis cells (Kajita and Yamaguchi, 1993), a K_D value of 386 nM was used in converting fluorescence ratios to [Ca²⁺]_i.

Gel Electrophoresis and Western Blotting. For Western blotting, bovine trachealis (300 mg) was minced (1 x 1 mm) and washed four times with ice-cold PSS. Aliquots of tissue were preincubated with wortmannin (100 nM) or vehicle for 30 min at 37°C. Then, IL-4 (50 ng/ml) or vehicle was added to each sample and incubated at 37°C for the specified times (0–60 min). Reactions were terminated by placing tubes on ice and washing twice with ice-cold cytosol buffer (25 mM HEPES, pH 7.0, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1.0 mM sodium vanadate, 0.2 M sucrose, 1 mM dithiothreitol, 1 mM ATP, 0.01 mg/ml tosylargininemethylester, 4 µg/ml leupeptin, 1 mM benzamidine, 1 mM 1,10-phenanthrine, and 0.2 mM phenylmethylsulfonyl fluoride in water). Samples then were suspended in 1 ml of ice-cold lysis buffer (cytosol buffer with 1% Triton X-100), sonicated, and centrifuged at 1000g for 10 min at 4°C. Supernatants were collected, and protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Aliquots of supernatant were diluted 1:10 with detergent stock (10% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, and 1.0 M NaCl in cytosol buffer), mixed with Laemmli buffer, and boiled 5 min.

Samples containing equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Protran; Schleicher & Schuell, Keene, NH). Membranes were blocked for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 2% bovine serum albumin and incubated overnight at 4°C in phospho-Akt (Thr308) antibody (Cell Signaling Technology Inc., Beverly, MA) at a 1:1000 dilution. Secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (Promega, Madison, WI), and this was visualized by enhanced chemiluminescence (PerkinElmer Life and Analytical Sciences, Boston, MA).

Protocols. Cells were stimulated with PSS containing carbachol (10 µM) for 2 min and then washed at least 30 min with PSS before the start of experiments. In all experiments, cell chambers were superfused with PSS containing the specified reagents, and a four-way valve allowed changing of the perfusate without disturbing the recordings. There was cell-to-cell variability in the magnitude of calcium transients; therefore, in all protocols, calcium transients following IL-4 (50 ng/ml, recombinant human IL-4) treatment (S2) were normalized to the magnitude of a carbachol transient in that same cell before IL-4 treatment (S1). An S2/S1 ratio then was calculated for each cell as an index of cell responsiveness. For all experiments, controls were exposed to vehicle, instead of IL-4, for the same lengths of time and according to the same protocols. In measuring S1 and S2 responses, the magnitude of the calcium transient was measured from the peak [Ca²⁺]_i level, and basal [Ca²⁺]_i was subtracted. All data were expressed as a mean ± S.E.M., and n indicates the number of cells studied. Analysis of variance with Newman-Keuls follow-up test was used for multiple comparisons between means. For comparing two groups, an unpaired Student's t test was used.

Materials. Collagenase and elastase were obtained from Roche Diagnostics (Indianapolis, IN). Fura-2/AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). Recombinant IL-4 was obtained from R&D Systems (Minneapolis, MN). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

	Results

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Effects of IL-4 Alone on [Ca²⁺]_i. Cells were loaded with Fura-2/AM, and [Ca²⁺]_i was recorded during application of IL-4 (50 ng/ml) alone. Responses to IL-4 alone were small and variable (Fig. 1). For 7 of 17 cells, [Ca²⁺]_i transiently increased 81 ± 19 nM and then returned to basal levels. In 4 of 17 cells, there was a small (46 ± 14 nM) gradual decrease in [Ca²⁺]_i, and in one cell, a gradual increase of 17 nM. In the remaining five cells, no change in [Ca²⁺]_i was detectable in response to IL-4 alone. For all cells, the mean maximum increase in [Ca²⁺]_i in response to IL-4 alone occurred at t = 141 ± 43 s and was 24 ± 15 nM (n = 17; N.S.). After 20 min in IL-4 alone, mean [Ca²⁺]_i was 289 ± 37 nM, and this was not significantly different than basal (289 ± 28 nM).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Effects of IL-4 alone on [Ca2+]i. Single airway smooth muscle cells were exposed to IL-4 (50 ng/ml) alone and changes in F340/F380 ratio were recorded. Representative traces from three separate cells are shown.

Effect of IL-4 on Transients and the Role of PI3K. Western blot analysis with phospho-Akt (Thr308) antibody was used to assess activation of Akt by IL-4. Bovine trachealis was exposed to IL-4 (50 ng/ml) for 0 to 60 min in the presence or absence of wortmannin (100 nM), a concentration that inhibits PI3K activity (Rameh et al., 1998; Krymskaya et al., 1999; Pasquet et al., 1999). Phospho-Akt was increased at 10 and 20 min, but not 60 min, and this response to IL-4 was inhibited by the presence of the PI3K antagonist wortmannin (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Rapid activation of Akt by IL-4. Trachealis was exposed to IL-4 (50 ng/ml) for 0 to 60 min in the presence or absence of wortmannin (100 nM). Western blots were probed with anti-phospho-Akt as an indicator of PI3K activation. IL-4 increased phospho-Akt at 10 and 20 min but not 60 min, and the PI3K antagonist wortmannin inhibited this effect of IL-4. Data are representative of three independent experiments.

To assess the functional effects that PI3K antagonists have on IL-4 inhibition of calcium transients, cells were stimulated with carbachol (10 µM) (S1) and then washed for 10 min before exposure to IL-4 (50 ng/ml) or vehicle control for 20 min in the presence or absence of wortmannin (100 nM), LY294002 (50 µM), or deguelin (100 nM), concentrations that inhibited PI3K activity in previous studies (Cheatham et al., 1994; Rameh et al., 1998; Krymskaya et al., 1999; Pasquet et al., 1999; Chun et al., 2003). A second response to carbachol (S2) then was recorded (Fig. 3). Treatment with IL-4 decreased carbachol-stimulated calcium transients to 0.34 ± 0.10 (S2/S1) compared with control values of 1.03 ± 0.10 (p < 0.05; n = 7–8) (Fig. 4). When the magnitudes of transients were expressed in absolute terms without S2/S1 normalization, the mean magnitude of S2 transients in controls was 958 ± 137 nM (n = 7) but 296 ± 121 nM (n = 8) after pretreatment with IL-4. Wortmannin (100 nM) and deguelin (100 nM) significantly antagonized this inhibitory effect of IL-4 such that mean responses to carbachol (S2/S1) were 1.10 ± 0.26 and 1.08 ± 0.23, respectively. Neither wortmannin nor deguelin alone had effects on basal [Ca²⁺]_i, and, by themselves, these antagonists had no effect on S2/S1 ratios. LY294002 (50 µM) did not antagonize IL-4 effects, but as previously reported, it did have a nonspecific inhibitory effect on transients by itself (Ethier and Madison, 2002).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Inhibition of calcium transients by IL-4 and the effect of wortmannin. After establishing baseline responsiveness to carbachol (10 µM) (S1), cells were exposed to vehicle alone (control), IL-4 (50 ng/ml) alone, or IL-4 plus wortmannin (wort) (100 nM) for 20 min. Then, a second response to carbachol was elicited (S2). Representative traces from three separate cells are shown. Mean data are shown in Fig. 4.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effects of wortmannin, LY294002, and deguelin on IL-4 inhibition of carbachol-stimulated calcium transients. After S1 responses were recorded, cells were allowed to recover during a 15-min wash. Next, cells were treated with wortmannin (wort) (100 nM), LY294002 (50 µM), or deguelin (100 nM) for 5 min before exposure to IL-4 (50 ng/ml) for 20 min in the continued presence of the PI3K antagonists. IL-4 inhibited the magnitude of calcium transients in response to carbachol (S2/S1), and this inhibition was blocked by wortmannin and deguelin. LY294002 did not block the inhibitory effect of IL-4, but notably, LY294002 alone had an inhibitory effect on carbachol-stimulated calcium transients. Data are mean ± S.E.M. (n = 3–8). *, p < 0.05 and **, p < 0.01 indicate significantly different than control. , p < 0.05 indicates significantly different than IL-4 alone.

Effects of IL-4 When SERCA-Inhibited. When cyclopiazonic acid (CPA) inhibits SERCA, net release of calcium from the SR is increased, and this depletes SR calcium stores (Ethier et al., 2001). For example, in preliminary experiments, cells were stimulated with carbachol (10 µM) to establish the S1 response, washed, and then treated with CPA (5 µM), a concentration that inhibits SERCA in bovine airway smooth muscle (An and Hai, 2000; Ethier et al., 2001), for 0 to 15 min before stimulation by carbachol a second time (S2). The S2 responses decreased during treatment with CPA (Fig. 5). To assess whether IL-4 had detectable effects in the presence of CPA, cells were stimulated with carbachol to establish the S1 response, washed, and then treated for 4 min with either CPA (5 µM) alone or CPA plus IL-4 (50 ng/ml). Cells then were stimulated with carbachol a second time (S2) (Fig. 5). Responses (S2/S1) to carbachol were 0.83 ± 0.08 after CPA alone but 0.51 ± 0.05 after CPA plus IL-4 (p < 0.05; n = 6). This suggested that IL-4 regulates calcium responses by a mechanism other than the inhibition of SERCA.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Effects of CPA on calcium transients. Left, cells loaded with Fura-2 and imaged ratiometrically were stimulated with carbachol (10 µM) to record a calcium transient (S1) in each cell. After S1, the cells were allowed to recover during a 15-min wash. Then, each cell was exposed to CPA (5 µM) for 0 to 15 min before stimulation with carbachol a second time (S2). By inhibiting SERCA activity, CPA caused a net release of SR calcium such that S2/S1 ratios gradually decreased over 15 min. Data are mean ± S.E.M., n = 4 to 8. Right, after establishing responsiveness in each cell (S1), cells were exposed to CPA alone (5 µM) or to CPA plus IL-4 (50 ng/ml) for 4 min. Responses to a second stimulation with carbachol (S2) were recorded. IL-4 had an inhibitory effect on transient magnitude (S2/S1) in the presence of CPA (*, p < 0.05; n = 6).

For additional evidence, a separate series of experiments were designed to assess the effects of a higher concentration of CPA (30 µM) during longer periods of IL-4 exposure (Tao et al., 2000). For these experiments, it was first necessary to find a time point after IL-4 exposure when there was not yet a detectable effect on transients. Therefore, in preliminary experiments, cells were stimulated with carbachol (10 µM) to establish cell responsiveness (S1), washed, and then treated with IL-4 for varying lengths of time before stimulation with carbachol a second time (S2). Cells treated with IL-4 for 10 min had carbachol-stimulated calcium transients of 1.12 ± 0.31 (S2/S1), and this was not significantly different than control of 0.98 ± 0.14 (n = 8). In absolute terms, S2 responses to carbachol were 1169 ± 196 and 1056 ± 194 nM (N.S.). Thus, after up to 10 min of IL-4 exposure, there was not yet detectable inhibition of responses to carbachol. Based on these initial experiments, the following protocol was used to further assess the role of SERCA in mediating IL-4 effects. After the S1 response to carbachol, cells were exposed to IL-4 or vehicle control for 10 min. Then, CPA (30 µM) alone was added for 5 min followed by a second stimulation with carbachol (S2) (Fig. 6). Cells that were not pretreated with IL-4 had responses of 0.59 ± 0.12 (S2/S1), but the IL-4-treated cells had responses of only 0.18 ± 0.05 (p < 0.05; n = 3–4) (Fig. 7). Additionally, the inhibitory effect that IL-4 had in these protocols was blocked by the PI3K antagonist wortmannin (100 nM) (S2/S1 = 0.56 ± 0.09; n = 4) but not by the IP₃R antagonist xestospongin C (3 µM) (S2/S1 = 0.22 ± 0.07), a concentration that inhibits calcium release through IP₃R of the SR (Gafni et al., 1997). Higher concentrations of xestospongin C (10 µM) also failed to inhibit the effects of IL-4 (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Pretreatment with IL-4 augments CPA effects on transients. After establishing baseline responsiveness to carbachol (10 µM) (S1), each cell was allowed to recover during a 15-min wash. Then, cells were treated with either IL-4 (50 ng/ml) alone or with vehicle control for 10 min, treatments that by themselves had no significant effect on responses (see text). Each cell then was exposed to CPA (30 µM) (arrow) for 5 min before stimulation with carbachol (Cch, arrow) a second time (S2). Representative recordings from two separate cells are shown. The dotted lines indicate the magnitude of the S1 response in that cell. Mean data are shown in Fig. 7.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Pretreatment with IL-4 augments the inhibitory effect of CPA on calcium transients: effects of wortmannin and xestospongin C. Cells were treated as described in Fig. 6 and in the presence or absence of wortmannin (wort) (100 nM) or xestospongin C (xesto) (10 µM). Since xestospongin C alone blocked IP3 elicited responses, S2 responses were stimulated with caffeine (10 mM) in these cells. Data are mean ± S.E.M. (n = 3–4), and * indicates values significantly different than CPA alone (p < 0.05).

	Discussion

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For isolated bovine tracheal smooth muscle cells, IL-4 alone had minimal effects on [Ca²⁺]_i, and yet significantly inhibited the magnitude of calcium transients stimulated by carbachol (10 µM). The major conclusions of this study are that this inhibitory effect of IL-4 on calcium transients in response to carbachol (10 µM) was antagonized by wortmannin and deguelin but not by the presence of CPA. These findings are most consistent with a model where IL-4 stimulates a wortmannin-sensitive PI3K to increase the rate of calcium release from the SR. In this model, the net rate of calcium release is slow enough that IL-4 alone causes only small or undetectable effects on [Ca²⁺]_i. However, over 20 min, the increased rate of calcium release is sufficient to attenuate the magnitude of calcium transients. Because xestospongin C did not inhibit the effects of IL-4, the data further suggest that the effects of IL-4 do not depend on IP₃ signaling.

IL-4 alone had only small and variable effects on [Ca²⁺]_i. This suggests that the changes in [Ca²⁺]_i elicited by IL-4 are of small magnitude and/or that whole cell imaging poorly detects localized changes. The source of calcium for IL-4 induced increases in [Ca²⁺]_i is likely the SR because a previous study showed that changes in [Ca²⁺]_i were not detectable after the SR was depleted of calcium by exposure to thapsigargin, an inhibitor of SERCA (Madison and Ethier, 2001). Although the changes in [Ca²⁺]_i caused by IL-4 were small and difficult to detect, IL-4 had a major effect on regulation of SR calcium. This was apparent because 20 min after exposure to IL-4, calcium transient magnitude in response to carbachol (10 µM) was significantly decreased. This concentration of carbachol (10 µM) was used in this study because it consistently produced calcium transients of sufficient magnitude to detect the inhibitory effects of IL-4 (Kajita and Yamaguchi, 1993; Madison and Ethier, 2001) and was a concentration high enough that a single 2-min exposure effectively depleted SR calcium in these cells (Ethier et al., 2001).

The signaling pathways that might underlie regulation of calcium by IL-4 are potentially complex, but two main candidates are STAT6 and PI3K (Kelly-Welch et al., 2003). Because of the relatively rapid time course for the IL-4 effect on calcium transients, it was hypothesized that PI3K mediated the effect of IL-4 on transients. To assess whether IL-4 activated PI3K with a time course compatible with its effects on transients, the time course for phosphorylation of Akt was measured as an indirect indicator of PI3K activation. Consistent with the effects of IL-4 on calcium transients, Akt was phosphorylated within 10 to 20 min, and this phosphorylation was inhibited, as expected, by the PI3K inhibitor wortmannin (Arcaro and Wymann, 1993).

PI3K antagonists then were used to functionally implicate PI3K in the signaling pathways connecting IL-4 to calcium regulation. The main findings of these experiments were that wortmannin and deguelin completely antagonized the ability of IL-4 to inhibit carbachol-stimulated calcium transients, suggesting that a PI3K did mediate the inhibitory effects that IL-4 has on calcium transients. Because deguelin is a putatively selective antagonist of Akt pathways stimulated by PI3K, the data further suggested that Akt may play a role in mediating the effects of IL-4 on calcium transients (Chun et al., 2003). LY294002 was tested in these studies because it is also an antagonist of PI3K. However, it had nonspecific effects on calcium transients by itself, making it poorly suitable for these experiments. Although the mechanism of this nonspecific effect is not known, a previous study showed that LY294002 mobilizes intracellular calcium stores in airway smooth muscle by mechanisms independent of PI3K, IP₃R, and PLC (Ethier and Madison, 2002). Evidence in other cell types also suggests that LY294002 has nonspecific effects on calcium regulation (Pasquet et al., 1999; Straube and Parekh, 2001; Warashina, 2001).

Having implicated PI3K, a separate series of experiments assessed whether IL-4 inhibits transients by inhibiting SERCA. The reason for this is that inhibition of SERCA would be expected to decrease SR calcium content and calcium transient magnitude (Sims et al., 1996; Janssen et al., 1997; Madison et al., 1998; Ethier et al., 2001). Net release of calcium from the SR could be caused by either increasing the rate of calcium release and/or decreasing the rate of calcium uptake. To distinguish between these possibilities, effects of IL-4 on transients were assessed in the presence of CPA, an inhibitor of SERCA. The effect of CPA alone (5 µM) on transients was compared with the effect of CPA plus IL-4 being added simultaneously for 4 min. In these experiments, transients were inhibited by IL-4 despite the presence of CPA. However, these initial findings did not exclude the possibility that inhibition of SERCA might be important during longer exposures to IL-4. Also, at least one study (Tao et al., 2000), but not another (An and Hai, 2000), found higher concentrations of CPA to be maximally effective.

Therefore, to further assess whether an effect of IL-4 could still be detected in the presence of CPA, we designed experiments that incorporated longer exposures to IL-4 and a higher concentration of CPA. In these experiments, cells were treated with vehicle control or IL-4 alone for 10 min, a treatment that, by itself, had no detectable effect on transients. Then, controls and IL-4-treated cells were exposed to CPA (30 µM) alone for 5 min before responses to carbachol were tested. Under these conditions, responses to carbachol still were significantly less in the IL-4-treated cells. Therefore, treatment with IL-4 had a detectable effect on transients 10 to 15 min after the initial exposure to IL-4 and even in the presence of high concentrations of CPA. Notably, wortmannin antagonized the effect of IL-4 in these same protocols. Based on all these results, it was not likely that IL-4 attenuated transients by inhibiting SERCA activity. Instead, the finding was more consistent with IL-4 acting as a low-efficacy agonist that increases calcium release rates from the SR, thereby decreasing the amount of calcium available for subsequent calcium transients. Finding that IL-4 alone caused detectable increases in [Ca²⁺]_i in at least some cells was consistent with IL-4 acting as a weak calcium-mobilizing agent.

A notable feature of the IL-4 effect on transients is that IL-4 did not need to be continually present. Brief (5–10-min) exposures to IL-4 had no immediate effect on transients but were sufficient to inhibit transients 20 min later (Madison and Ethier, 2001). Similarly, in the above-mentioned experiments with CPA, IL-4 did not have to be continually present during the CPA exposure to have a significant effect on transient magnitude. These observations suggested that IL-4 initiates a series of signaling events that are slow to affect calcium regulation but are, nonetheless, significant for calcium signaling because they are sustained even when IL-4 is washed from the media. Potentially, the inhibitory effect of IL-4 on calcium transients in airway smooth muscle cells could modulate contraction, proliferation, apoptosis, migration, and secretion of mediators (Knox et al., 2000; Hirst et al., 2004). However, it is emphasized that this study focused only on the mechanisms by which IL-4 regulates calcium signaling and not on the functional consequences of that regulation.

In some cell types, PI3K activation promotes activation of PLC (Rameh et al., 1998). This is notable because generation of IP₃ by PLC could mechanistically link IL-4 receptor activation to an increased rate of release of calcium through IP₃R. However, xestospongin C, an antagonist of IP₃-mediated calcium mobilization (Gafni et al., 1997), did not inhibit the effect that IL-4 had on transients. Therefore, the findings with xestospongin C suggested that IL-4 effects were not dependent on IP₃ signaling. Possibly, IL-4 causes slow release of calcium through RyR calcium release channels instead, and this hypothesis is currently under investigation. Notably, evidence suggests that xestospongin C is an inhibitor of SERCA as well as IP₃R (De Smet et al., 1999). Because IL-4 inhibits transients in the presence of xestospongin C, the finding further supports the conclusion that IL-4 does not regulate SERCA to inhibit calcium transients.

In summary, for bovine trachealis cells, the relatively rapid inhibitory effect of IL-4 on agonist-stimulated calcium transients is mediated by a wortmannin-sensitive, CPA-insensitive mechanism. The following model is consistent with these and previous results (Madison and Ethier, 2001). IL-4 activates a PI3K that increases the rate of calcium release from the SR. Because of the putative selectivity of deguelin, data suggest that Akt-activated pathways may mediate these effects of PI3K. According to the model, the rates of calcium release are so low that either calcium efflux mechanisms keep increases in [Ca²⁺]_i small and/or that whole cell imaging does not have the spatial resolution needed to detect them. However, over approximately 10 to 20 min, SR calcium content is decreased enough that calcium transients in response to carbachol are significantly attenuated. Because xestospongin C did not inhibit the effects of IL-4 in these experiments, it is speculated that RyR calcium release channels may be involved instead.

	Footnotes

 
This research was supported by National Heart, Lung, and Blood Institute Grant HL-54143.

doi:10.1124/jpet.104.079343.

ABBREVIATIONS: IL, interleukin; [Ca²⁺]_i, intracellular calcium concentration; SR, sarcoplasmic reticulum; STAT, signal transducer and activator of transcription; PI3K, phosphatidylinositol 3-kinase; IP₃, inositol 1,4,5-trisphosphate; IP₃R, inositol 1,4,5-trisphosphate receptor; SERCA, sarco(endo)plasmic reticulum calcium ATPase; RyR, ryanodine receptor; PSS, physiologic salt solution; AM, acetoxymethyl ester; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; CPA, cyclopiazonic acid.

Address correspondence to: Dr. J. Mark Madison, Department of Medicine, LRB Room 319, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605. E-mail: mark.madison{at}umassmed.edu

	References

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