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Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, Miami, Florida 33136
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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2-Adrenergic agonists
stimulate ciliary beat frequency (CBF), an integral part of mucociliary
clearance. To evaluate the differential effects of albuterol
enantiomers and their racemic mixture on ciliary function, CBF and
intracellular calcium were measured at room temperature from single
ovine airway epithelial cells with use of digital
videomicroscopy. Baseline CBF was 7.2 ± 0.2 (SE) Hz
(n = 80 measurements). R-albuterol
(10 µM to 1 mM) stimulated CBF in a dose-dependent manner to
maximally 24.4 ± 5.4% above baseline. Racemic albuterol
stimulated CBF to maximally 12.8 ± 3.6% above baseline, a
significantly lower increase compared with R-albuterol
alone, despite identical R-enantiomer amounts in both
groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase
in response to R-albuterol was mediated through
-receptors and stimulation of protein kinase A, in a
calcium-dependent and -independent fashion. S-albuterol had
a negligible effect on CBF and did not change intracellular calcium.
Together, these results suggest that R-albuterol is more
efficacious than racemic albuterol in stimulating CBF. Thus
S-albuterol may interfere with the ability of
R-albuterol to increase CBF.
airway; calcium; beta-agonists; signaling
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-AGONISTS
HAVE BEEN SHOWN to stimulate the mucociliary transport rate
(4, 5, 22), and thus they are used clinically to improve
mucociliary clearance (10, 14, 15, 18), a major airway
host defense mechanism. Ciliary beating is an integral part of the
mucociliary transport apparatus, and it has been well established that
2-adrenergic agonists stimulate ciliary beat frequency
(CBF) in a variety of airway epithelial cells (11, 27, 33,
34). Therefore, the increase in CBF is believed to be the main
contributor to higher mucociliary transport rates seen after
2-agonist administration. Albuterol, the most commonly used 2-agonist in a clinical setting, possesses two
stereoisomers due to an asymmetric carbon adjacent to the aromatic
ring. The R-enantiomer binds to the
2-adrenoceptor, leading to cAMP production, whereas the
S-enantiomer has an over 100-fold lower affinity to the
receptor. In addition, some recent data indicate that the S-enantiomer may have properties not usually associated with
2-agonists, such as smooth muscle contraction through
intracellular calcium concentration ([Ca2+]i)
elevation (13) and other side effects (for review, see
Ref. 8). Usually, racemic albuterol mixtures are
used in a clinical setting. These mixtures bring with them the
potential for divergent responses of different cellular functions for
each of the enantiomers. We therefore conducted a study to compare the
effects of the two enantiomers as well as the racemic mixture of
albuterol on CBF in ovine tracheal epithelial cells. Because
[Ca2+]i is a regulator of CBF and because the
S-enantiomer has been shown to increase
[Ca2+]i in airway smooth muscle cells
(13), we also measured [Ca2+]i
from single cells simultaneously with CBF.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chemicals
DMEM, Ham's nutrient F-12, and Hanks' balanced salt solution (HBSS) were purchased from GIBCO, Life Technologies (Grand Island, NY). ATP and propranolol were from Sigma-Aldrich (St. Louis, MO). Myristoylated protein kinase A (PKA) inhibitory peptide-(14
22) [PKI-(1422)] was purchased from Calbiochem (La Jolla, CA) and the
acetomethyl ester form of fura 2 (fura 2-AM) from Molecular Probes
(Eugene, OR). Albuterol enantiomers and racemic albuterol were
from Sepracor (Marlborough, MA).
Preparation of Submerged Tracheal Epithelial Cultures
Primary cultures of tracheal epithelial cells were prepared as previously described (23). Briefly, the mucosa from freshly obtained trachea was dissected from the underlying cartilage under sterile conditions and incubated in 0.05% protease (type XIV, Sigma Chemical) in DMEM overnight at 4°C. Ovine tissue was obtained from adult ewes that were euthanized (30 mg/kg iv pentobarbital sodium) according to protocols approved by the National Institutes of Health and the local animal care use committee. After protease treatment, epithelial cells were released by vigorous shaking and harvested by centrifugation. They were plated on collagen-coated glass coverslips (type VI human placental collagen, Sigma Chemical) at a density of 0.5 × 106 cells/cm2 in a minimal volume of 100 µl/cm2 (= 1 ml per 35-mm dish). The culture media consisted of 50% DMEM, 50% Ham's nutrient F-12 supplemented with 10 µg/ml insulin, 5 µg/ml transferrin, 0.36 µg/ml hydrocortisone, 20 ng/ml triiodothyronine, 7.5 µg/ml endothelial cell growth supplement, 100 U/ml penicillin, and 100 µg/ml streptomycin. Media were exchanged every other day. Cells from these cultures were used for measurements 3-5 days after plating because the2-agonist effect on ciliary
beating usually faded away after this time period.
Measurement of CBF
Cells grown on coverslips were mounted at room temperature onto the stage of a Nikon Eclipse E600FN upright water-immersion lens microscope in a Warner Instrument RC-25F perfusion chamber with a 150-µl working volume and perfused constantly with HBSS buffered with HEPES, pH 7.4 (HBSS-HEPES). The role of kinases in CBF regulation can be assessed at room temperature, as evidenced by multiple publications (2, 3, 11, 29, 36, 37), and if increases are expressed in percent above baseline with-agonist
addition, there is no significant difference between data obtained at
20 vs. 37°C (11). All measurements were performed during
continuous, unidirectional perfusion with HBSS-HEPES at a flow rate of
250 µl/min during baseline recordings, while drug addition or removal
was achieved at a flow rate of 1,000 µl/min for 5 min. Ciliated cells
were imaged with infrared differential interference contrast optics
with an optical gain of ×600. For online CBF measurements, the light
path was directed to a charge-coupled device (CCD) video camera (CCD
100, Dage MIT, Michigan City, IN), and a box of three by three pixels
from the live, digitized, contrast-enhanced video image was selected
(where 1 pixel samples an area of 180 × 180 nm). The magnitude
spectra from a fast Fourier transform (FFT) of each of the pixel's
intensity signals were computed on-line and displayed on the monitor
for immediate adjustments. The intensity signals were recorded and
later used for analysis according to published methods
(24) by using a sliding FFT window approach (128 frames
per FFT, sliding the FFT window through the data set by 100 frames at a
time), providing a frequency resolution of at least 0.23 Hz and a time
resolution of ~3 s. The individual FFT magnitude spectra were peak
extracted for graphing (24).
Measurement of [Ca2+]i
Incubation protocols. Coverslips were rinsed three times with HBSS-HEPES and loaded at room temperature with 4 µM fura 2-AM in HBSS-HEPES containing 2.5% fetal calf serum (Hyclone, Logan, UT) for 60 min on a rocking table. The dishes were washed three times with HBSS-HEPES and used for measurements after a minimum of 30 min.
Imaging hardware and software. For fura-2 excitation, a lambda DG4 system (Sutter, Novato, CA) was used, which was controlled by "ratio-tool" software from Inovision (Durham, NC). Ratiometric calcium estimates were made by using 10-nm-wide filters centered on 340 and 380 nm (Chroma Technology, Brattleboro, VT), capturing the emitted light (510-600 nm) at each excitation wavelength for 600 ms through a ×60 water immersion objective (Nikon) and directing it to a cooled CCD camera (Quantix, Photometrics, Tucson, AZ). With use of Inovision's ratio-tool software, individual cells were identified as regions of interest (ROIs). The ratio within each ROI was computed from images obtained at 340- and 380-nm excitation wavelength, neglecting pixels that failed to reach a threshold value and subtracting the appropriate background fluorescence at each wavelength. Ratios were computed every 10 s. Average ratio values for each ROI were written to disk for later analysis and graphing.
Simultaneous measurement of CBF and [Ca2+]i. Both the CBF analysis software as well as ratio-tool were running on an SGI O2 workstation (Silicon Graphics, Mountain View, CA). By using a dual-image module (Nikon) and guiding the infrared signal for CBF measurements to the CCD camera while sending all fluorescence signals (<600 nm) to the cooled CCD camera, we were able to measure CBF and [Ca2+]i of the same single cell simultaneously.
Extracellular calibration and computation of free [Ca2+]i. Because we were not able to perform reliable calibrations of the calcium indicator dye fura 2 intracellularly in ciliated cells (cells exposed to ionomycin did not change their [Ca2+]i when exposed to different extracellular calcium concentrations), conversions of the fura-2 ratio data into [Ca2+]i was done by using a simpler in vitro calibration procedure. The fluorescence intensity at each wavelength was measured with a calcium-free (1 mM EGTA buffered) and a saturating-calcium aliquot of 10 µM fura 2 (K+ salt) in 150 mM KCl and 10 mM HEPES, pH 7.4. With these values, the data were transformed into [Ca2+]i by the equation of Grynkiewicz et al. (7) assuming a dissociation constant (Kd) of 250 nM. The plotted [Ca2+]i values are thus only an approximation because the true Kd of the dye in the cytoplasm of these cells is unknown and no corrections for cytoplasmic viscosity have been made (20).
Experimental Procedures
All groups of experiments contained cells from at least three different sheep, and coverslips were never reused. All baseline measurements were performed during continuous perfusion with HBSS-HEPES at a flow rate of 250 µl/min. After baseline signals were recorded for ~5 min, racemic albuterol or each albuterol enantiomer was added for 5 min at a flow rate of 1,000 µl/min and then washed away with buffer at flow rates of 1,000 µl/min for 5 min. Flow rates were changed to accelerate the full exchange of the bathing solution during short-term agonist application (shown to be complete after 1 min). These changes in flow rates did not influence either CBF or [Ca2+]i. As a control, the cells were exposed to 10 µM ATP at the end of each experiment to ascertain their response to a known stimulator of CBF.Albuterol was used at different concentrations to establish a
dose-response curve for each enantiomer and for the racemic form. The
concentration of the racemic form was calculated by using twice the
molecular weight of each enantiomer. This ensured that, at all
concentrations used, each enantiomer was present in the same amount in
the racemic form and in the single enantiomer form. To dissect the
signal-transduction pathway of R-albuterol signaling, PKA
was inhibited with cell membrane-permeable PKI-(1422) (16,
21). PKI-(1422) was used at 1 µM (~30-fold excess of estimated inhibition constant) in HBSS-HEPES. Cells were preincubated with this inhibitor for 60 min at room temperature and perfused during
the entire experiment. -Receptors were inhibited by propranolol (1 mM), which was perfused during the entire experiment.
Statistics
Statistical analysis used a one-way ANOVA to compare the means of more than two groups (for instance racemic vs. S- vs. R-albuterol at the same concentration of agonist) by using JMP software from SAS Institute (Cary, NC). If a significant difference was found, a group × group comparison was done by using the Tukey-Kramer honestly significant difference test. P < 0.05 was considered to be significant. Data are expressed as means ± SE. Each n indicates one measurement from a single ciliated cell on one coverslip.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CBF was recorded from single ovine tracheal epithelial cells
during exposures to both albuterol enantiomers as well as racemic albuterol. The identity of the different enantiomers was blinded to the
person performing the experiments, and the code was broken only after
all data were obtained and analyzed. 2-Agonists were applied after a stable baseline was obtained for ~5 min. Exchanges of
the chamber volume with HBSS alone had no influence on CBF at all the
used perfusion rates, ruling out the possibility that changes in CBF
were due to mechanical disturbance of cilia by flow as described
previously by others (28). Cells were exposed to the
agonist for 5 min. Approximately 5 min after cessation of exposure to
the 2-agonist, 10 µM ATP were applied for 2 min (Fig.
1).
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Baseline CBF was 7.2 ± 0.2 (SE) Hz (n = 80) and
did not differ between groups (P > 0.05). More details
can be obtained from Fig. 2.
R-albuterol maximally stimulated CBF in a dose-dependent manner to 9.4 ± 3, 12.9 ± 2.6, and 24.4 ± 5.4% above
baseline at 10 µM, 100 µM, and 1 mM, respectively (all
n > 7; all P < 0.05 compared with
baseline). Racemic albuterol stimulated CBF to 10.1 ± 2.7 and
12.8 ± 3.6% above baseline at 100 µM and 1 mM, respectively (all n > 9; all P < 0.05 compared
with baseline). The increases achieved with racemic albuterol were
lower than the ones seen with R-albuterol alone, indicating
a higher efficacy of R-albuterol compared with racemic
albuterol at the highest concentration used. This finding was
surprising because of identical R-enantiomer amounts in both
the R-albuterol and racemic albuterol groups (Fig. 2).
S-albuterol had a negligible effect on CBF, not reaching a statistically significant change from baseline at any concentration tested (maximum stimulation of 6.4 ± 1% above baseline at 1 mM; all n > 6; all P > 0.05 compared with
baseline).
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The duration of CBF elevation above baseline (within 0.3 Hz) was
significantly different between the R-albuterol and the
racemic albuterol group: CBF remained elevated in 64% of the cells (16 of 25) in the R-albuterol group until the addition of ATP
(10 min after initial -agonist addition) but in only 24% (7 of 29 cells) in the racemic group (P < 0.05). However, there
was no significant difference between the different doses of the same agonist with respect to the duration of CBF elevation.
The cells were exposed to 10 µM ATP to ascertain their response to a known, but different, stimulator of CBF (9, 32, 39). The maximal increases in CBF with ATP exposure were 22.4 ± 2.5% above the pre-ATP baseline in the R-albuterol group (n = 25; no significant differences between the increases at each R-albuterol concentration), 22.1 ± 2.7% above pre-ATP baseline in the S-albuterol group (n = 21), and 18.4 ± 2.3% above pre-ATP baseline in the racemic albuterol group (n = 29). These increases were not different between the two albuterol enantiomers and the racemate group (P > 0.05).
To examine whether the observed increases in CBF were associated with
changes in [Ca2+]i, we measured CBF and
[Ca2+]i simultaneously from single ovine
tracheal cells (Fig. 3). With R-albuterol exposure (0.1 mM),
[Ca2+]i increased transiently in five of
eight measured cells. CBF increased in all cells, however, irrespective
of [Ca2+]i. The increase in CBF was faster
with R-albuterol addition when an initial transient
[Ca2+]i increase was present. This suggests
that the initial increase in CBF was due to an increase in
[Ca2+]i in cells that exhibited such a
[Ca2+]i transient. However, all cells
exhibited a prolonged increase in CBF, which was calcium independent.
In contrast, ATP increased CBF in a strictly calcium-coupled fashion.
S-albuterol did not change either CBF or
[Ca2+]i at 0.1 mM (n = 5).
However, the cells still responded to 10 µM ATP with a strictly
calcium-dependent increase in CBF (Fig. 3). In an additional 15 ciliated cells, 0.1 mM S-albuterol did not elicit any
calcium response.
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To dissect the signal transduction pathway of R-albuterol,
-receptors were inhibited with 1 mM propranolol (Fig.
4). This antagonist prevented 1 mM
R-albuterol from increasing either CBF or
[Ca2+]i (n = 5). After PKA
was inhibited with 1 µM PKI-(1422), 1 mM R-albuterol
did not increase CBF or [Ca2+]i during
simultaneous measurements of both signals (n = 5; Fig. 4). However, all cells responded as predicted with a strictly calcium-coupled increase in CBF with 10 µM ATP exposure. These results show that R-albuterol signals, via -receptors,
stimulate PKA, which in turn increases CBF. They also suggest that PKA
activation may be necessary to increase
[Ca2+]i with -receptor activation because
none of the cells treated with PKI-(1422) responded to
R-albuterol with a transient
[Ca2+]i increase, confirming data obtained by
others in epithelium from frog esophagus (1).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study revealed several interesting findings regarding
2-agonist stimulation of mammalian CBF. First, it showed
that both R-albuterol and racemic albuterol stimulated CBF
in a dose-dependent fashion and that the S-enantiomer was
inactive. Second, racemic albuterol was less efficacious than the
R-enantiomer in stimulating CBF at the highest concentration
used despite the same amount of R-enantiomer present in both
the racemic form and the single-enantiomer form (calculating molarity
of the racemate by using twice the molecular weight of each
enantiomer). This suggests that S-albuterol may interfere
with the ability of R-albuterol to increase CBF. Third, the
R-enantiomer stimulated CBF mainly through a
calcium-independent mechanism and partially, but less consistently,
through a calcium-coupled pathway as previously shown for rabbit airway
epithelial cells (11). Finally, the
S-enantiomer did not elicit any calcium response in ciliated
cells, and thus the pharmacological effect of S-albuterol in
these cells differs from airway smooth muscle cells where the S-enantiomer has been shown to increase
[Ca2+]i (13).
The increase in CBF reported here, expressed as percent above baseline, is similar to results obtained with isoproterenol in rabbit tracheal explants (11) and lies somewhere in the middle of the reported relative changes in CBF with stimulation by a variety of different compounds in cell culture, whether measured at 20 or at 37°C (e.g., Refs. 6, 11, 30). These different changes in CBF with different albuterol enantiomer stimulation are biologically relevant because studies in bovine trachea have shown that these albuterol enantiomers have the same differential effect on mucociliary clearance (Y. Schwartz, Sepracor, personal communication).
In airway epithelial cells, 2-agonists are thought to
stimulate CBF through a signaling cascade involving the
2-receptor adenylyl cyclase, cAMP (3, 31),
cAMP-dependent kinase (PKA) (38), and the phosphorylation
of a ciliary target protein called p26 in ovine airway epithelia
(26). However, a more complex -adrenergic signaling
pathway to stimulate CBF has been delineated in rabbit tracheal
explants where CBF increased both through an initial calcium-coupled
and a prolonged cAMP-dependent mechanism (11). In an
additional study, activation of PKA was needed to cause calcium release
from intracellular stores (1). Our ovine cells confirm
these results; five of eight cells responded to R-albuterol
with an initial calcium-coupled CBF response and then a sustained,
calcium-independent response, whereas three cells responded only with a
calcium-independent response (Fig. 3). Both responses were dependent on
-receptor activation because propranolol blocked them, and both were
dependent on PKA activation because PKI-(1422) blocked them as well
(Fig. 4). In summary, 2-receptor activation in ovine
ciliated cells causes CBF increases through an initial calcium-coupled
mechanism in some cells, as well as a prolonged, calcium-independent
mechanism in all cells. Both mechanisms depend on the activation of PKA.
The concentrations of albuterol tested may seem high considering its
affinity to the 2-receptor. One explanation may be that ciliated cells in culture reduce the number of
2-receptors, as indicated by the waning response to
-agonists over time. Thus higher concentrations of agonist may be
necessary to stimulate a sufficient number of receptors to elicit a
physiological response. In fact, relatively high EC50
values have been reported for isoproterenol (EC50 ~5
µM) in rabbit tracheal explants (27, 33) and for fenoterol (EC50 ~10 µM) in beagle trachea in vivo
(35). Clinically, the necessity for such high doses is not
an issue because millimolar concentrations of albuterol are potentially
encountered by ciliated cells after nebulization of the drug into the
airways (the concentration of nebulized 2-agonists is
>1 mM). Therefore, such high concentrations are meaningful to study in
our culture system.
S-albuterol did not stimulate CBF significantly at any
concentration tested. This was by itself not surprising because it has
a very low affinity to the -receptor (19). It has been reported to increase [Ca2+]i in airway smooth
muscle cells through a muscarinic mechanism involving inositol
trisphosphate (13). Although the major muscarinic receptors on both smooth airway muscle cells and ciliated cells that
work through such a pathway are of the muscarinic receptor M3 subtype (12, 25), S-albuterol
did not elicit any [Ca2+]i response in
ciliated cells. The different measurement temperatures (37°C for
smooth muscle and room air ovine airway epithelial cells) are unlikely
to account for these differences because acetylcholine elicits
transient [Ca2+]i increases in ovine airway
epithelial cells via an inositol trisphosphate mechanism also at room
temperature (25). Therefore, other mechanisms must explain
this discrepancy. However, S-albuterol was not inert in our
assay. In the racemate, it decreased the ability of
R-albuterol to stimulate CBF, at least at the highest concentrations tested. This is similar to previously published findings
of the bronchodilatory efficacy of the enantiomers vs. the racemic
mixture (17). The postulate that the
S-enantiomer is essentially a weak (i.e., with a high
Kd) agonist at the -receptor with no
intrinsic activity may explain these data. S-albuterol acting as a pharmacological agent via another mechanism, however, cannot be ruled out. Whether the observed differences in efficacy between the R-enantiomer and racemic albuterol to stimulate
CBF have clinical relevance remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gregory E. Conner, Richard J. Bookman, and Adam Wanner for valuable comments and support. The elegant programming of Nenad Amodaj is gratefully acknowledged.
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FOOTNOTES |
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This work was funded by a grant from Sepracor, Inc.
Address for reprint requests and other correspondence: M. Salathe, Div. of Pulmonary and Critical Care Medicine (R-47), Univ. of Miami School of Medicine, 1600 NW 10th Ave., RMSB 7063, Miami, FL 33136 (E-mail: msalathe{at}miami.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 8, 2002;10.1152/japplphysiol.00755.2001
Received 23 July 2001; accepted in final form 4 February 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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, R-albuterol; 
, racemic
albuterol; 
, S-albuterol. A:
baseline CBF for each experimental group at all concentrations of
agonist tested. There was no significant difference between baselines
as evaluated by ANOVA. B: maximal CBF increases with
albuterol additions for all experimental groups. Regular dose-response
curves were fit to the data. * P < 0.05 compared
with baseline. ++ P < 0.05, R-albuterol compared with both racemic and
S-albuterol. + P < 0.5, R-albuterol and racemic albuterol compared with
S-albuterol.
