Introduction

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Racemic albuterol has been one of the most widely used ₂-adrenoceptor agonists for the relief of the symptoms of asthma. However, the use of ₂ agonists has been linked to bronchial hyperresponsiveness (Kerrebijin et al., 1987; Sears et al., 1990; Taylor et al., 1993; Wahedna et al., 1993) and a paradoxical increase in the mortality rate known as the "asthma paradox" (Sears et al., 1990; Grainger et al., 1991; Spitzer et al., 1992; Suissa et al., 1994; Barrett and Strom, 1995). The mechanism underlying this ₂-adrenergic agonist-induced hyperresponsiveness is not known. The ₂ agonists generally possess two stereoisomers due to an asymmetrical carbon adjacent to the aromatic ring. The R-enantiomer binds to the ₂-adrenoceptor, promoting formation of cAMP and bronchodilation, whereas the S-enantiomer has been considered to be a less effective agonist (Johnson et al., 1993). Therefore, tachyphylaxis to ₂ agonist has been suggested to be the cause for the loss of the protective effects of the ₂ agonist after prolonged or excessive use of a ₂ agonist (Gibson et al., 1978; Cockcroft et al., 1993). Desensitization of ₂-adrenoceptors through receptor internalization (Straser et al., 1985), together with subsequent reduction in receptor mRNA expression (Nishikawa et al., 1994), offers one plausible explanation for the loss of protective effects. However, a recent in vivo study in guinea pigs suggests that acute airway hyperresponsiveness may be characteristic of the S-enantiomers of ₂ agonists (Mazzoni et al., 1994). Because the in vitro effects of the S-enantiomer of ₂ agonists on [Ca²⁺]_i are unknown and the effector of the spasmogens is airway smooth muscle, we studied the effects of (S)-albuterol in detail at the cellular level to understand its action in dissociated smooth muscle cells; the purpose of the current study was to gain further insight into the possible role of (S)-albuterol in airway hyperresponsiveness.

	Materials and Methods

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Chemicals and reagents. We obtained (S)-, (R,S)-, and (R)-albuterol from Sepracor (Marlborough, MA). (S)-Isoproterenol was purchased from Sigma Chemical. ICI 118,551 was purchased from Tocris Cookson (St. Louis, MO). Nimodipine, U73,122, atropine, and 4-DAMP were purchased from Research Biochemicals (Natick, MA). [³H]QNB was purchased from DuPont-New England Nuclear (Boston, MA). The Ins(1,4,5)P₃ assay kit was from Amersham Life Science (Clearbrook, IL).

Cell isolation. Tracheal smooth muscle cells were dispersed from small pieces (1 × 1 × 5 mm) of bovine trachealis cut while under observation with the use of a dissecting microscope. Approximately 0.5 g of tissue was placed into 2.5 ml of nominally Ca²⁺ free solution, which consisted of PSS (containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25.6 mM NaHCO₃,11.1 mM glucose, 2.5 mM CaCl₂) without 2.5 mM CaCl₂, plus 2.5 mg of collagenase (Boehringer-Mannhein Biochemicals, Indianapolis, IN), 2 mg of elastase (Boehringer-Mannheim), and 0.5 mg of DNase (Boehringer-Mannheim). The mixture was stirred gently with a microstirrer for 15 min at 37°. The tissue slices then were transferred to fresh enzyme solution and gently stirred for an additional 15 min. The digested pieces were recovered by a nylon mesh (hole size, 0.5 mm) and resuspended in Ca²⁺-free PSS after repeated washing. Gentle spinning of the tissue in Ca²⁺-free PSS for 3 min released 1-3 × 10⁵ cells/ml, which were equilibrated in low Ca²⁺ (0.1 mM) PSS.

Ca²⁺ measurements. The measurement of [Ca²⁺]_i was carried out with fluorescent imaging microscopy as described previously (Kajita and Yamaguchi, 1993; Yamaguchi et al., 1995). The dispersed smooth muscle cells were loaded with 0.5 µM Fura-2 acetoxymethyl ester for 60 min at room temperature. Fura-2-loaded cells were transferred to a recording chamber (volume, 150 µl) and allowed to settle to the bottom coverglass before superfusion of normal PSS at 37°. These cells were 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, Newton, MA). Each laser alternately fired short pulses (3 nsec) at 30 Hz. These alternating pulses of light were guided by a bifurcated quartz fiber to a neutral density filter at the epiport of the microscope and then focused on cells through a 40× lens (NA, 1.3; Nikon). The emitted fluorescent signals were passed back through the objective to a 455-nm dichroic mirror and a 475-nm barrier filter (Omega Optics, Brattleboro, VT) and imaged by a frame transfer, charge-coupled device camera (FTM 800; Philips Component, Slatersville, RI). The signals were digitized and stored in an imaging board (Recognition Technology, Westborough, MA), and the digital outputs from the board were transferred to a personal computer. For each set of experiments, the responses from one to seven cells were taken from one animal, and the final data usually represented the measurements from at least two animals.

Binding protocol. Membranes were prepared from trachealis trimmed of mucosa and connective tissue, minced with scissors in ice-cold phosphate-buffered saline, and repeatedly washed with phosphate-buffered saline. The minced tissue then was homogenized in two volumes of homogenization buffer (containing 5 mM Tris·HCl, pH 7.4, 2 mM EDTA, 0.5 mM dithiothreitol) and 0.2 mM phenylmethylsulfonyl fluoride (Sigma Chemical, St. Louis, MO) using an Ultraturrax Tissuemizer (Tekmar, Cincinnati, OH). The crude homogenate was filtered through cheesecloth and centrifuged twice at 900 × g for 10 min to remove undisrupted cells and nuclei. The supernatant was centrifuged twice at 40,000 × g for 40 min. The pellet was resuspended in a buffer containing 30 mM Tris·HCl, pH 7.4, 10 mM MgCl₂, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, and 25% sucrose (w/v) at an approximate protein concentration of 1 mg/ml; frozen rapidly; and stored at 80° until use. Protein concentration was determined according to the method of Bradford (1976) with bovine serum albumin as standard.

Ins(1,4,5)P₃ measurements. Dispersed cells were placed into six-well culture plates (10⁵ cells/well). These cells were incubated with serum-free RPMI media in an incubator for 4 hr and equilibrated with 95% air/5% CO₂ at 37°. The reactions were performed on a warm plastic plate. The agonist was added to a final concentration of 10 µM. The generation of Ins(1,4,5)P₃ was followed at different time periods ranging from 10 sec to 10 min. At each time point, the reaction was stopped by the addition of an equal volume of 10% ice-cold trichloroacetic acid (Sigma Chemical), which subsequently was removed before assay by partitioning three times with five volumes of diethyl ether. After adjustment of the samples to pH 7.5 with 1 M NaHCO₃, 25- and 100-µl volumes of each sample were assayed by using a radiometric Ins(1,4,5)P₃ assay kit. The assays were done in triplicate, and data are presented as percentage increase over the basal value.

Data analysis. All values are expressed as mean ± standard error. Statistical significance was assessed by paired or unpaired Student's t test, and p < 0.05 was considered significant.

	Results

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The effects of (S)-, (R,S)-, and (R)-albuterol on [Ca²⁺]_i in tracheal smooth muscle cells were determined by using an imaging microscope. The response to superfusion of either (S)- or (R,S)-albuterol was characterized as an increase in [Ca²⁺]_i as shown in Fig. 1, A and B. The increased in [Ca²⁺]_i occurred at (S)-albuterol concentrations of >10 pM and increased maximally by 149.6 ± 16.1 nM (12 cells) at 1 µM with an EC₅₀ value of 3.3 nM (Figs. 1A and 2C). Likewise, (R,S)-albuterol increased [Ca²⁺]_i at concentrations of >1 nM and had the maximum increase of 99.6 ± 16.6 nM at 100 µM with an EC₅₀ value of 12.9 nM (Fig. 1B). At 1 and 10 µM (S)-albuterol, a fraction of the cells (10% and 25%, respectively) induced Ca²⁺ oscillations that reached 1-2 µM (Fig. 1C). In contrast, (R)-albuterol decreased [Ca²⁺]_i in these cells (Fig. 1D). At a concentration of 10 µM (S)-albuterol, the increases in [Ca²⁺]_i were accompanied by cell shortening. The effect was observed in all cells, averaging 10.1 ± 0.96% (range, 5-21%; 20 cells) of the preexposure length as determined from stored images before and after a 5-min exposure to the S-enantiomer. The dose response for (R)-albuterol is not shown because the affinity sites we detected for the decrease in [Ca²⁺]_i should themselves be the subject of detailed study. However, the cells responded with a decrease in [Ca²⁺]_i at all concentrations of >5 nM, with a maximum decrease in the range of 105-106 nM.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A, (S)-Albuterol-induced increase in [Ca2+]i. B, (R,S)-Albuterol-induced increase in [Ca2+]i. C, [Ca2+]i oscillations induced by (S)-albuterol. D, (R)-Albuterol (10 µM)-induced decrease in [Ca2+]i. In all cases, a 10-µM concentration of the S- or R-enantiomer was superfused over the cells attached to the coverglass. The delay of application due to the dead space was corrected. The expanded vertical scale of [Ca2+]i below 150 nM reflects the nonlinear nature of calibration curves in these concentrations. E, Dose-response curve of (R,S)- and (S)-albuterol. Data represent net increases in steady state [Ca2+]i above basal levels (, [Ca2+]i) and exclude Ca2+ oscillations observed with 1 and 10 µM (S)-albuterol (one and seven cells, respectively). EC50 values for (R,S)- and (S)-albuterol are 7.89 and 8.48, respectively. Solid line, drawn from the fit of sigmoid curve by the InPlot (GraphPAD Software).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A, Effect of (S)-albuterol in Ca2+-free PSS. (S)-Albuterol (10 µM) induced only a transient rise in [Ca2+]i in nominally Ca2+-free PSS, which was followed by a decreased [Ca2+]i, representing steady state levels in Ca2+-free PSS. B, Changes in [Ca2+]i in response to simultaneously applied (S)-albuterol (10 µM) and U73,122 (10 µM). C, Effect of the PLC inhibitor U73,122 on (S)-albuterol-induced increase in [Ca2+]i, showing the reversal of (S)-albuterol (10 µM)-induced increase in [Ca2+]i to the prestimulation level on the addition of 10 µM U73,122. D, Changes in [Ca2+]i in response to simultaneously applied calphostin C (100 nM) and (S)-albuterol (10 µM). E, Changes in peak transient [Ca2+]i (top) and steady state [Ca2+]i (bottom) induced by 10 µM (S)-albuterol in Ca2+-free PSS, U73,122, and calphostin C. Bars, number of cells used for experiments. The statistical significance was assessed only for the peak Ca2+ increase in Ca2+-free [p > 0.05 (NS)], U73122 (p < 0.001), and calphostin C (p < 0.001), in comparison to control response by 10 µM (S)-albuterol in PSS (141 ± 9.9 nM, 15 cells). F, Ins(1,4,5)P3 generation by 10 µM (S)-albuterol. Ins(1,4,5)P3 levels were measured with radioimmunoassay in cells exposed to (S)-albuterol at various time points (0, 10, 30, 90, and 300 sec). An Ins(1,4,5)P3 increase was significant in cells exposed for 30 sec (p < 0.05, four cells).

A number of mechanisms may be involved in the elevation of [Ca²⁺]_i by (S)-albuterol: (1) Ins(1,4,5)P₃-mediated Ca²⁺ release (Berridge, 1993), (2) depletion-induced influx of Ca²⁺ (Putney, 1986; Randriamampita and Tsien, 1993), and (3) activation of voltage-gated Ca²⁺ channels through modulation of the membrane potential. To investigate these potential mechanisms, we examined the response to (S)-albuterol under various conditions, including Ca²⁺-free PSS and in the presence of a PLC or PKC inhibitor. When (S)-albuterol (10 µM) was superfused in nominally Ca²⁺-free PSS, there was only a transient increase in [Ca²⁺]_i (133 ± 12.6 nM, six cells) and the steady state levels were decreased by 72.3 ± 12.5 nM (six cells) below the levels before the test (Fig. 2A). To test the possibility of phosphatidyl-inositol turnover and Ins(1,4,5)P₃-mediated Ca²⁺ release, we examined the effects of the PLC inhibitor U73,122 (Bleasdale et al., 1990). Fig. 2B illustrates the simultaneous application of 10 µM U73,122 and (S)-albuterol, showing the decrease in steady state [Ca²⁺]_i. This approach was successful because the mean onset time for the (S)-albuterol (10 µM)-induced response from randomly chosen cells was 29.6 ± 2.6 sec (17 cells), which was 15 sec slower than that for carbachol. Fig. 2C shows another Ca²⁺ response to (S)-albuterol that was reversed by subsequent U73,122. We further tested the effects of calphostin C, the blocker of PKC on (S)-albuterol-induced Ca²⁺ response. When applied simultaneously with (S)-albuterol (10 µM), calphostin C (100 nM) induced only an transient rise in Ca²⁺ and eliminated the anticipated Ca²⁺ increase. Instead, the steady state [Ca²⁺]_i was decreased (Fig. 2D). The histograms summarize the effects of (S)-albuterol on the transient (peak) and steady state [Ca²⁺]_i in the presence of U73,122 and calphostin C and with Ca²⁺-free solution (Fig. 2E).

To obtain evidence for the phosphatidyl-inositol turnover, we measured Ins(1,4,5)P₃ levels in smooth muscle cells treated with (S)-albuterol at various times. Exposure to 10 µM (S)-albuterol for 30 sec increased Ins(1,4,5)P₃ by 213 ± 34.4% (p < 0.05, four experiments) (Fig. 2F).

To assess the role of extracellular Ca²⁺, we measured the response to (S)-albuterol in the presence of nimodipine, the antagonist of L-type Ca²⁺ channels. Nimodipine (100 nM) alone decreased basal [Ca²⁺]_i from 134.3 ± 23.4 to 100.6 ± 20.7 nM (p > 0.05) within 3 min, but when tested at 5 min after the introduction of nimodipine, the (S)-albuterol (10 µM)-induced net increase in [Ca²⁺]_i was decreased from a paired control value of 99.7 ± 10.2 to 9.6 ± 12.5 nM (p < 0.001, six cells).

The affinity of (S)-albuterol for -adrenoceptor was determined by competition binding experiments using ¹²⁵I-cyanopindolol, a nonspecific -adrenoreceptor ligand, with a bovine tracheal membrane preparation. The IC₅₀ value of (S)-albuterol for displacement of ligand (average, 293 µM; range, 263-323 µM; three cells) was >100-fold higher than that of (R)-albuterol (average, 1.66 µM; range, 0.5-5.5 µM; three cells), indicating that (S)-albuterol has very low affinity for -adrenoreceptors in these cells (Fig. 3A).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   A, Displacement of 125I-cyanopindolol with (S)- or (R)-albuterol. Values represent the average of data measured in triplicate. The IC50 values for (R)- and (S)-albuterol are 5.78 and 3.53, respectively. B, Displacement of [3H]QNB by atropine and (S)-albuterol. The IC50 values are 43 nM and 21 µM for atropine and (S)-albuterol, respectively. C, Decrease in [Ca2+]i in response to simultaneously applied (S)-albuterol (10 µM) and atropine (100 nM). D, Decrease in [Ca2+]i in response to simultaneously applied (S)-albuterol (10 µM) and 4-DAMP (500 nM). E, Blockade of changes in [Ca2+]i in response to simultaneously applied (S)-albuterol (10 µM), atropine (100 nM), and ICI 118,551 (10 µM). F, Bars, numbers of cells used for experiments. Steady state [Ca2+]i induced by (S)-albuterol (10 µM) simultaneously applied with atropine (p < 0.001), 4-DAMP (p < 0.001), ICI 118,551 (10 µM, p > 0.05), and atropine plus ICI 118,551 (p < 0.001). They illustrate that a decrease in [Ca2+]i is sensitive to 2-adrenoceptor antagonist, but an increase in [Ca2+]i was insensitive to ICI 118,551.

To identify possible receptors for (S)-albuterol, we considered muscarinic receptors. The competition binding against [³H]QNB with atropine or (S)-albuterol is shown in Fig. 3B. The IC₅₀ value for (S)-albuterol was 21 µM. We previously found that the IC₅₀ values for atropine and 4-DAMP in tracheal smooth muscle membranes and single cells were 10 and 45 nM, respectively (Lucchesi et al., 1990); therefore, we used 100 nM atropine or 500 nM 4-DAMP to determine whether these antagonists had an effect on (S)-albuterol-induced changes in [Ca²⁺]_i. Fig. 3C shows the effect of the simultaneous superfusion of 100 nM atropine and 10 µM (S)-albuterol on [Ca²⁺]_i; the increase in [Ca²⁺]_i was absent, and steady state [Ca²⁺]_i decreased. We observed a similar response with 500 nM 4-DAMP (Fig. 3D). We then tested whether the (S)-albuterol-induced increase in [Ca²⁺]_i was sensitive to the specific ₂-adrenoceptor antagonist ICI 118,551(Bilski et al., 1983). In the presence of 10 µM ICI 118,551, which alone had no effect on steady state [Ca²⁺]_i, (S)-albuterol (10 µM) still increased [Ca²⁺]_i by 170.5 ± 43.5 nM (10 cells), a rise virtually identical to that of controls (153.5 ± 41.1 nM, 10 cells). The additional test was conducted to determine whether the decrease in [Ca²⁺]_i observed with atropine was sensitive to ICI 118,551. In seven cells, a decrease in [Ca²⁺]_i was prevented when the ₂-adrenoceptor antagonist ICI 118,551 was applied to cells with atropine and (S)-albuterol (Fig. 3E). These results of the response to 10 µM (S)-albuterol with atropine, 4-DAMP, ICI 118,551, and ICI 118,551 plus atropine are summarized in Fig. 3F.

	Discussion

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Our data indicate that the albuterol stereoisomers [(S)- and (R)-] exhibited opposite effects on [Ca²⁺]_i in airway smooth muscle cells. The unexpected finding was that (R,S)- and (S)-albuterol increased [Ca²⁺]_i, and, in particular with (S)-albuterol, the increase in [Ca²⁺]_i invariably was accompanied by shortening of all cells. In contrast, (R)-albuterol decreased [Ca²⁺]_i and exhibited no shortening effect. These differences between the albuterol stereoisomers can affect the action of contractile agonists; for example, with muscarinic agonist, carbachol administered simultaneously with (S)-albuterol exacerbated Ca²⁺ mobilization in bovine tracheal cells, but carbachol administered simultaneously with (R)-albuterol diminished Ca²⁺ mobilization (Yamaguchi and McCullough, 1996). These results suggest that (S)-albuterol directly opposes the beneficial effects of (R)-albuterol at a critical step for generation of force.

(S)-Albuterol has been considered to be a less effective ₂ agonist because of its effects on cAMP formation (Johnson et al., 1993). The results of our study are inconsistent with the S-enantiomer as a ₂ agonist because not only were the effects of (S)-albuterol on [Ca²⁺]_i insensitive to high doses of ICI 118,551 but also Ins(1,4,5)P₃ generation and shortening of the cell did not fit the known effects of ₂-adrenoceptor activation in smooth muscle. The ₂ agonists and their second messenger decrease [Ca²⁺]_i and relax muscle tone in tracheal and vascular smooth muscle (McDaniel et al., 1991; Yamaguchi et al., 1995), whereas we observed the opposite effects with (S)-albuterol. On the other hand, a decrease in [Ca²⁺]_i by (S)-albuterol in the presence of U73,122 (Fig. 2B), atropine, or 4-DAMP (Fig. 3 AB) was believed to result from ₂-adrenoceptor activation because with atropine, the response was blocked by ICI 118,551 (Fig. 3E). This effect can be attributed to contaminated (R)-albuterol and may explain why cAMP was increased by the S-enantiomer in the previous study.

Functional tests revealing blockade of the increased [Ca²⁺]_i with atropine and 4-DAMP implicate muscarinic receptors in (S)-albuterol-induced response. The finding prompted us to examine whether muscarinic receptor antagonists had direct effects on Ca²⁺ stores. We found that atropine (100 nM) exerted no effects on caffeine (10 mM)-induced Ca²⁺ release (ratio of atropine plus caffeine/caffeine, 1.07 ± 0.2; five cells). Although this was not the direct test on Ins(1,4,5)P₃-mediated stores, the total lack of response in the presence of atropine led us to believe that inhibitory effects of the muscarinic antagonists must have resulted from interactions with the receptors. The competition binding with [³H]QNB may provide additional evidence for interaction of the S-enantiomer with muscarinic receptors (Fig. 3B). The mean K_d value from three such experiments was 8.17 ± 1.1 µM. Considering the high affinity of the EC₅₀ value in the functional responses (Fig. 1D), (S)-albuterol is a highly efficient agonist for a muscarinic receptor. Muscarinic effects in airway smooth muscle, including Ca²⁺ mobilization (Yang et al., 1993) and contraction (Eglen et al., 1990), are known to be mediated by M₃ receptors. The blockade of 4-DAMP on (S)-albuterol-mediated increase in [Ca²⁺]_i in this study is consistent with the idea of the M₃ muscarinic receptors being involved with the response to (S)-albuterol. Our data suggest that (S)-albuterol cross-reacts with muscarinic receptors; however, they do not rule out a possibility that the S-enantiomer has its own receptors in these cells. To discover the universal nature of the effects of the S-enantiomer among ₂ agonists, we also tested the effect of (S)-isoproterenol on [Ca²⁺]_i in bovine tracheal smooth muscle cells. (S)-Isoproterenol (10 µM) increased [Ca²⁺]_i by 98.7 ± 24.5 nM (nine cells). Thus, the increase in [Ca²⁺]_i essentially was similar to that observed with (S)-albuterol. The two drugs share a chemical structure of HOCHCH₂NH adjacent to the phenol ring.

The data for U73,122 and calphostin C suggest that Ca²⁺ mobilization by (S)-albuterol involves (1) activation of PLC and resulting Ins(1,4,5)P₃ production that induces an initial transient release of Ca²⁺ from stores and (2) PKC to induce a sustained influx of Ca²⁺ from the extracellular medium, probably through L-type Ca²⁺ channels because it is sensitive to nimodipine. A complementary mechanism for slow Ca²⁺ elevation by membrane-permeant 1,2-dioctanoyl-sn-glycerol was observed previously in bovine tracheal cells, providing a basis for PKC to play a role in the sustained elevation of [Ca²⁺]_i (Kajita and Yamaguchi, 1993).

Our finding that the S-enantiomer of albuterol primarily functions as a Ca²⁺ agonist follows a well known example of dihydropyridine enantiomers. The S-enantiomer of the dihydropyridine, 202-791, or Bay K-8644 enhances L-type Ca²⁺ current and acts as Ca²⁺ agonists primarily by stabilizing the open state of the channel, whereas the R-enantiomer favors the inactivated state and reduces whole-cell L-type Ca²⁺ current (Kokubun et al., 1986; Hamilton et al., 1987). Taken together with the current results, the future studies of racemic drugs may require identification of the pharmacological and physiological effects of each enantiomer.

In conclusion, our study revealed that (S)-albuterol had characteristics of a typical contractile agonist. This is mostly due to a PLC activation, which results in phosphatidyl-inositol turnover with increased Ins(1,4,5)P₃ levels. The phosphatidyl-inositol cascade further induces PKC translocation and results in nimodipine-sensitive Ca²⁺ influx, possibly through L-type Ca²⁺ channels. Many of these properties are shared by other contractile agonists, such as histamine and carbachol (Chilvers et al., 1990; Berridge, 1993; Yang et al., 1993; Kajita and Yamaguchi, 1993). This enhanced Ca²⁺ mobilization may be one important factor in the hyperresponsiveness associated with the clinical use of racemic ₂-agonists. By exhibiting deleterious effects on airway smooth muscle, the use of S-enantiomer albuterol may have profound clinical implications because 50% of racemic albuterol consists of (S)-albuterol.