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J Appl Physiol 82: 1836-1843, 1997;
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Journal of Applied Physiology
Vol. 82, No. 6, pp. 1836-1843, June 1997
CELLULAR ASPECTS OF LUNG FUNCTION

Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle

Y. S. Prakash1, H. F. M. Van Der Heijden2, M. S. Kannan3, and G. C. Sieck1

1 Departments of Anesthesiology, and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester 55905; 3 Department of Veterinary PathoBiology, University of Minnesota, St. Paul, Minnesota 55108; and 2 Department of Pulmonary Diseases, University Hospital Nijmegen, Nijmegen, The Netherlands NL6500 HB

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, and G. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl. Physiol. 82(6): 1836-1843, 1997.---Relaxation of airway smooth muscle (ASM) by beta -adrenoceptor agonists involves reduction of intracellular Ca2+ concentration ([Ca2+]i). In porcine ASM cells, acetylcholine induces [Ca2+]i oscillations that display frequency modulation by agonist concentration and basal [Ca2+]i. We used real-time confocal microscopy to examine the effect of salbutamol (1 nM to 1 µM), a beta 2-adrenoceptor agonist, on [Ca2+]i oscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of [Ca2+]i oscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 µM. These effects were mimicked by a cell-permeant analog of adenosine 3',5'-cyclic monophosphate. The inhibitory effect of salbutamol was partially reversed by BAY K 8644. Salbutamol reduced [Ca2+]i even when sarcoplasmic reticulum (SR) Ca2+ reuptake and Ca2+ influx were blocked. Lanthanum blockade of Ca2+ efflux attenuated the inhibitory effect of salbutamol on [Ca2+]i. The [Ca2+]i response to caffeine was unaffected by salbutamol. On the basis of these results, we conclude that beta 2-adrenoceptor agonists have little effect on SR Ca2+ release in ASM cells but reduce [Ca2+]i by inhibiting Ca2+ influx through voltage-gated channels and by enhancing Ca2+ efflux.

beta 2-adrenoceptor agonists; confocal imaging; asthma; calcium channel; calcium efflux

INTRODUCTION

ELEVATION OF INTRACELLULAR Ca2+ concentration ([Ca2+]i) plays an important role in the development and maintenance of force in airway smooth muscle (ASM) cells (27). Acetylcholine (ACh) acts as a bronchoconstrictor by elevating [Ca2+]i in ASM cells. Symptomatic treatment of bronchoconstriction in asthma involves the use of beta 2-adrenoceptor-specific agonists. Relaxation of ASM by beta -adrenoceptor agonists involves reduction of intracellular Ca2+ concentration ([Ca2+]i). Studies in different smooth muscle types have demonstrated that stimulation of beta -adrenoceptors may decrease inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) (4, 20), promote Ca2+ reuptake (5, 17), promote Ca2+ efflux (3, 6, 15, 31), and/or inhibit Ca2+ influx (4, 26). Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent hyperpolarization and consequent reduction of Ca2+ influx via activation of large-conductance Ca2+-activated K+ (BKCa) channels have also been demonstrated (16, 24).

In recent studies, we (13, 23) and others (18) have reported that Ca2+ regulation in ASM cells in response to ACh involves repetitive [Ca2+]i oscillations that arise from cyclical SR Ca2+ release and reuptake. Similar [Ca2+]i oscillations have also been previously reported in vascular (2, 7, 9, 21) and gastric smooth muscle cells (19). Nuttle and Farley (22) recently reported that isoproterenol, a beta -adrenoceptor agonist, modulates the frequency of ACh-induced [Ca2+]i oscillations in porcine ASM cells. These investigators proposed that the inhibition of [Ca2+]i oscillations by beta -adrenoceptor agonists involved reduced Ca2+ influx. However, given the fact that beta -adrenoceptor agonists also affect other Ca2+ regulatory sites, modulation of [Ca2+]i oscillations by these agonists might also involve additional mechanisms such as reduced Ca2+ release, increased reuptake, and enhanced efflux.

In the present study, we used real-time confocal imaging to examine the effect of salbutamol, a beta 2-adrenoceptor agonist, on the dynamic [Ca2+]i responses of porcine ASM cells to ACh. Using oscillation amplitude, rise time, fall time, and frequency as parameters, we attempted to elucidate the mechanisms by which salbutamol reduces [Ca2+]i levels.

METHODS

Cell preparation. Porcine trachea were obtained from a local abattoir. The techniques for dissociation of ASM cells have been published previously (11, 12). Briefly, the smooth muscle layer was excised, freed of epithelium, and minced thoroughly in Hanks' balanced salt solution (HBSS) buffered with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4; GIBCO-BRL). ASM cells were dissociated by incubating the tissue with 20 U/ml papain, 2,000 U/ml deoxyribonuclease (Worthington Biochemical), and 1 mg/ml type IV collagenase (Worthington Biochemical). The dissociated cells were then triturated, centrifuged, and resuspended in minimum essential medium with 10% fetal calf serum. The ASM cells were plated on collagen-coated glass coverslips. Trypan blue exclusion was used to assess cell viability (>90% of all cells).

The cells were loaded for 30-45 min at 37°C with 5 µM fluo-3 acetoxymethyl ester (Molecular Probes). The coverslip was then washed in HBSS, mounted on an open microscope slide chamber (RC-25F, Warner Instruments), and perfused at 2-3 ml/min at room temperature.

Real-time confocal imaging. Detailed descriptions of the confocal imaging technique have been recently published (23). An Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI) equipped with an Ar-Kr laser (488-nm line) and mounted on the Nikon microscope was used to visualize fluo-3-loaded ASM cells. The system was controlled through a Silicon Graphics Indy workstation. A Nikon ×40/1.3 oil-immersion objective lens was used, and image size was set to 640 × 480 pixels, with a calibrated pixel area of 0.063 µm2/pixel. Only one fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1,800 of a maximum of 4,096) was used. At this setting, pixel intensities within regions of interest (ROI) ranged between 25 and 255 gray levels (GL). Fluorescence bleaching was found to be ~25% across a 20-min period. However, all experimental protocols were limited to <5 min of continued laser exposure. ROI with a fixed dimension of 5 × 5 pixels (1.5 µm2) were drawn within cell boundaries. The optical section thickness for the ×40 lens was set to 1 µm by controlling the confocal slit size. Therefore, [Ca2+]i measurements were obtained from a volume of 1.5 µm3.

The confocal system is capable of acquiring 480 frames/s. In preliminary studies on fluo-3-loaded ASM cells, we determined that an acquisition rate of 30 frames/s was sufficient to measure the various parameters for [Ca2+]i oscillations without frequency aliasing over the range of the [Ca2+]i response. Accordingly, a fixed acquisition rate of 30 frames/s was used in this study. When necessary, noise reduction was achieved by acquiring the images at 60 or 120 frames/s with frame averaging.

Ca2+ calibrations. At the fixed combination of laser intensity and photomultiplier gain, fluo-3-loaded ASM cells were exposed to Ca2+ ionophore (A-23187) and fixed levels of extracellular Ca2+ ranging from 0 [HBSS with ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid] to 10 µM. The fluorescence intensities at the different Ca2+ concentrations were then measured, and a calibration curve of [Ca2+]i vs. GL was constructed. Based on these calibrations, all GL data in the experimental protocols were converted to nanomoles of Ca2+ before further analyses.

Oscillation parameters. Oscillation amplitude was defined as the difference between the peak of the oscillation and the basal [Ca2+]i. Rise time was defined from basal [Ca2+]i to the peak of an oscillation and was normalized for the amplitude of the rise in [Ca2+]i. Fall time was defined from the peak of the oscillation to the basal level of the next oscillation and was normalized for this amplitude. Oscillation frequency was measured as the inverse of the time between two oscillations.

Effect of salbutamol on [Ca2+]i response to ACh. Before ACh exposure, basal [Ca2+]i levels were not significantly different between cells and varied from 100 to 150 nM within an ROI (122 ± 13 nM; n = 112). On exposure to ACh, [Ca2+]i oscillations originated from one end of the long axis of the cell and spread toward the other end in the form of a propagating wave. Previous studies on ASM cells have shown that the 50% effective dose (ED50) for the [Ca2+]i response to ACh is ~1 µM (24). Accordingly, a fixed ACh concentration of 1 µM was used in all the protocols. After an equilibration period in HBSS of 5 min, ASM cells were exposed to 1 µM ACh to induce [Ca2+]i oscillations. Typically, the [Ca2+]i oscillations induced by ACh displayed a biphasic pattern (Fig. 1). During the first 15-30 s after exposure of the cells to 1 µM ACh, the frequency of [Ca2+]i oscillations increased, while the peak-to-trough amplitude of the oscillations decreased. These initial changes in the frequency and amplitude of ACh-induced [Ca2+]i oscillations were associated with an increase in basal [Ca2+]i level. After 30-60 s of continuous exposure to ACh, the basal [Ca2+]i level in ASM cells decreased to a steady-state value that was higher than that observed before ACh exposure. In association with the relatively constant basal [Ca2+]i level, the frequency and amplitude of [Ca2+]i oscillations also remained relatively constant during this steady-state period for as long as the cells were exposed to ACh.
Fig. 1. Acetylcholine (ACh)-induced intracellular Ca2+ concentration ([Ca2+]i) oscillation in localized region of single porcine airway smooth muscle (ASM) cell. Basal [Ca2+]i initially increased and, in concert, oscillation amplitude decreased while frequency increased. With continued ACh exposure, basal [Ca2+]i decreased, and oscillation amplitude increased while frequency decreased. Both oscillation amplitude and frequency were constant 60-90 s after ACh exposure at a constant basal [Ca2+]i level.
[View Larger Version of this Image (25K GIF file)]

After a steady state was reached, the cells were sequentially exposed to 1 nM, 10 nM, 100 nM, and 1 µM salbutamol (Glaxo-Wellcome) in the continued presence of ACh. Each exposure of salbutamol was maintained for 2 min and was followed by a wash in 1 µM ACh for 2 min. Other ASM cells were continuously exposed to 1 µM ACh for 20 min, and the [Ca2+]i response was evaluated. During periods of equilibration and washing, the cells were not exposed to the laser.

On the basis of the results of this protocol, it was determined that the maximal effect of salbutamol on the [Ca2+]i response occurred at a concentration of 1 µM. Accordingly, a fixed concentration of 1 µM salbutamol was used in subsequent protocols.

Effect of salbutamol on Ca2+ influx. ASM cells were exposed to 1 µM ACh to induce [Ca2+]i oscillations. After a steady state was reached, the cells were exposed to 100 nM BAY K 8644, to promote Ca2+ influx through voltage-gated membrane channels. The effect of 1 µM salbutamol was then evaluated. Another group of ASM cells was preexposed to 10 nM charybdotoxin, a selective inhibitor of BKCa channels. The cells were then exposed to 1 µM ACh and subsequently to 1 µM salbutamol.

Effect of salbutamol on SR Ca2+ release. ASM cells were preexposed to 1 µM salbutamol and subsequently to 5 mM caffeine to induce SR Ca2+ release from ryanodine receptor (RyR) channels (28). In a second set of experiments, ASM cells were preexposed to Ca2+-free HBSS and to 1 mM lanthanum to block both Ca2+ influx and efflux, respectively. The cells were then exposed to 1 µM ACh and subsequently to 1 µM salbutamol.

Effect of salbutamol on Ca2+ efflux. ASM cells were exposed to 1 µM ACh to initiate [Ca2+]i oscillations. After a steady state was reached, the cells were exposed to a Ca2+-free HBSS and to 1 µM thapsigargin to block Ca2+ influx and SR Ca2+ reuptake (27), respectively. Continued stimulation of the cells by ACh in the presence of Ca2+-free HBSS and thapsigargin caused an elevation of basal [Ca2+]i level. The cells were then exposed to 1 µM salbutamol to examine its effect on Ca2+ efflux. Another group of ASM cells was preexposed to Ca2+-free HBSS, thapsigargin, and lanthanum, thus blocking Ca2+ influx, reuptake, and efflux, respectively, after ACh-induced [Ca2+]i oscillations had reached steady state. The cells were then exposed to 1 µM salbutamol.

Effect of 8-bromoadenosine 3',5'-cyclic monophosphate on ACh-induced [Ca2+]i oscillations. ASM cells were exposed to 1 µM ACh to induce [Ca2+]i oscillations, and after a steady state was reached, the cells were exposed to 500 µM 8-bromoadenosine 3,',5'-cyclic monophosphate (8-BrcAMP), a membrane-permeant analog of cAMP.

Data analysis. Each cell was exposed to only one experimental protocol. At least five cells were chosen from each coverslip. Overall, 280 cells were analyzed. Dose-dependence data were compared by using a one-way analysis of variance with salbutamol concentration (a concentration of 0 was treated as control) as the grouping variable. Other data were compared by using t-tests. Bonferroni corrections were applied for repeated comparisons. Statistical significance was tested at a 0.05 level. Data are reported as means ± SE.

RESULTS

Effect of salbutamol on ACh-induced [Ca2+]i oscillations. During the steady-state phase of ACh-induced [Ca2+]i oscillations, exposure of ASM cells to increasing concentrations of salbutamol resulted in a progressive decrease in basal [Ca2+]i level (Fig. 2). This decrease in basal [Ca2+]i level was associated with a decrease in the frequency of [Ca2+]i oscillations (P < 0.05; Fig. 2, Table 1; n = 15). The fall time of [Ca2+]i transients was also increased by exposure to increasing salbutamol concentrations (Table 1). In contrast, the peak-to-trough amplitude and rise time of [Ca2+]i oscillations were unaffected by salbutamol at any concentration (Table 1). Increasing salbutamol concentration beyond 1 µM completely inhibited ACh-induced [Ca2+]i oscillations (Fig. 2). Removal of salbutamol from the extracellular medium restored the ACh-induced [Ca2+]i oscillations to presalbutamol values.
Fig. 2. Concentration-dependent modulation of [Ca2+]i oscillations by salbutamol. Over a range of 1 nM to 1 µM, salbutamol progressively decreased basal [Ca2+]i and frequency of ACh-induced [Ca2+]i oscillations but did not affect oscillation amplitude. A concentration of 1 µM salbutamol completely inhibited [Ca2+]i oscillations.
[View Larger Version of this Image (24K GIF file)]

Table  1.   Effect of salbutamol on ACh-induced [Ca2+]i oscillations
Salbutamol Basal [Ca2+]i, nM Peak-to-Trough Amplitude, nM RT, ms/nM FT, ms/nM Frequency
0 nM (control) 100.0 ± 0.0  100.0 ± 0.0  100.0 ± 0.0  100.0 ± 0.0  100.0 ± 0.0 
1 nM 93.3 ± 4.3* 101.7 ± 6.7  96.9 ± 3.9  104.1 ± 1.4  83.1 ± 1.1*
10 nM 88.2 ± 6.4* 97.2 ± 6.5  88.9 ± 5.9* 115.6 ± 5.9* 77.0 ± 4.4*
100 nM 74.6 ± 3.8* 95.1 ± 6.1* 117.2 ± 10.9* 135.4 ± 7.6* 46.7 ± 4.8*
1 µM 61.2 ± 3.2* Inhibition Inhibition Inhibition Inhibition
Values are means ± SE expressed as %control. [Ca2+]i, intracellular Ca2+ concentration; ACh, acetylcholine; RT, rise time; FT, fall time. * Significant concentration-dependent change, P < 0.05.

Effect of salbutamol on Ca2+ influx. During the steady-state phase of ACh-induced [Ca2+]i oscillations, exposure of ASM cells to BAY K 8644 resulted in a gradual increase in basal [Ca2+]i level (Fig. 3). This increase in basal [Ca2+]i level was associated with an increase in the frequency of [Ca2+]i oscillations and a decrease in oscillation amplitude (Fig. 3A; n = 14). After [Ca2+]i oscillations were induced by 1 µM ACh, exposing ASM cells to 1 µM salbutamol resulted in an inhibition of [Ca2+]i oscillations, which could be reversed by subsequent exposure to BAY K 8644 (Fig. 3B; n = 19). Preexposure of ASM cells to charybdotoxin to inhibit BKCa channels did not significantly affect basal [Ca2+]i levels, nor did it affect the ability of ACh to induce [Ca2+]i oscillations or the subsequent inhibitory effects of salbutamol on [Ca2+]i oscillations (n = 8).
Fig. 3. Effect of salbutamol on Ca2+ influx in ASM cells. After 30 s to 1 min, ACh-induced [Ca2+]i oscillations in ASM cells reached a steady state characterized by a relatively constant frequency and amplitude. Subsequently, exposure to BAY K 8644, an agonist of voltage-gated Ca2+ influx channels, resulted in a progressive increase in basal [Ca2+]i level, an increase in oscillation frequency, and a decrease in oscillation amplitude (A). Exposure to salbutamol during the steady-state phase of ACh-induced [Ca2+]i oscillations (~2 min after ACh exposure) resulted in inhibition of oscillations (B). In continued presence of ACh and salbutamol, exposure to BAY K 8644 resulted in slow increase in basal [Ca2+]i and partial recovery of [Ca2+]i oscillations, suggesting that salbutamol inhibits [Ca2+]i oscillations, at least in part, by inhibiting Ca2+ influx.
[View Larger Version of this Image (38K GIF file)]

Effect of salbutamol on SR Ca2+ release. Exposing ASM cells to 5 mM caffeine induced a transient increase in [Ca2+]i (Fig. 4A). Preexposure of ASM cells to 1 µM salbutamol did not affect this [Ca2+]i response to caffeine (Fig. 4B; n = 22). When Ca2+ influx and efflux were both blocked by preexposing ASM cells to Ca2+-free HBSS and to 1 mM lanthanum, subsequent exposure to 1 µM ACh still induced [Ca2+]i oscillations that were sustained (Fig. 4C; n = 16). Further exposure of these ASM cells to 1 µM salbutamol had no effect on the amplitude or frequency of ongoing [Ca2+]i oscillations (Fig. 4C).
Fig. 4. Effect of salbutamol on sarcoplamic reticulum (SR) Ca2+ release in ASM cells. Exposure of ASM cells to caffeine induced a large, transient elevation of [Ca2+]i (A). Preexposure to salbutamol for 30 s or greater (not shown) did not affect the [Ca2+]i response to caffeine (B). When Ca2+ influx and efflux were both blocked by absence of extracellular Ca2+ and lanthanum, ACh induced stable [Ca2+]i oscillations, representing cyclical SR Ca2+ release and reuptake (C). Subsequent exposure to salbutamol did not affect ongoing [Ca2+]i oscillations, suggesting that salbutamol does not affect SR Ca2+ release. Time and [Ca2+]i calibrations are the same in A-C.
[View Larger Version of this Image (29K GIF file)]

Effect of salbutamol on Ca2+ efflux. During the steady-state phase of ACh-induced [Ca2+]i oscillations, exposure of ASM cells to Ca2+-free HBSS and thapsigargin resulted in a gradual increase in basal [Ca2+]i level (Fig. 5A; n = 16). As basal [Ca2+]i level increased, the ACh-induced [Ca2+]i oscillations were eventually inhibited (Fig. 5A). Subsequent exposure to 1 µM salbutamol decreased basal [Ca2+]i level but did not reinitiate [Ca2+]i oscillations (Fig. 5A). In another group of ASM cells, ACh-induced [Ca2+]i oscillations were not inhibited by exposure to Ca2+-free HBSS and thapsigargin when lanthanum was also present to block Ca2+ efflux (Fig. 5B; n = 19). However, in these cells, the amplitude of ACh-induced [Ca2+]i oscillations progressively decreased while basal [Ca2+]i level increased. In contrast to the effect of salbutamol on [Ca2+]i level in the presence of Ca2+-free HBSS and thapsigargin (Fig. 5A), salbutamol had no effect on basal [Ca2+]i or [Ca2+]i oscillations when lanthanum was also present (Fig. 5B).
Fig. 5. Effect of salbutamol on Ca2+ efflux in ASM cells. A: removal of extracellular Ca2+ and addition of thapsigargin during ongoing ACh-induced oscillations caused an elevation of [Ca2+]i and a decrease in oscillation amplitude. Subsequent exposure to salbutamol decreased [Ca2+]i levels. B: when Ca2+ influx, efflux, and SR reuptake were all blocked by removal of extracellular Ca2+ and addition of lanthanum and thapsigargin, addition of salbutamol had no effect on ACh-induced [Ca2+]i oscillations, confirming the modulation of Ca2+ efflux by salbutamol.
[View Larger Version of this Image (49K GIF file)]

Effect of 8-BrcAMP on [Ca2+]i oscillations. Exposing ASM cells to 500 µM 8-BrcAMP during the steady-state phase of ACh-induced [Ca2+]i oscillations did not affect oscillation amplitude but significantly decreased oscillation frequency (Fig. 6; P < 0.05; n = 18).
Fig. 6. Effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on ACh-induced [Ca2+]i oscillations in ASM cells. Exposure to 8-BrcAMP during the steady-state phase of ACh-induced [Ca2+]i oscillations (~2 min after ACh exposure) resulted in decreased basal [Ca2+]i and frequency of [Ca2+]i oscillations.
[View Larger Version of this Image (45K GIF file)]

DISCUSSION

Exposure of ASM cells to ACh resulted in [Ca2+]i oscillations. Exposure to the beta 2-adrenoceptor agonist salbutamol decreased the frequency, but not the amplitude, of ACh-induced [Ca2+]i oscillations in a dose-dependent fashion, completely inhibiting the oscillations at a concentration of 1 µM. Salbutamol inhibited BAY K 8644-induced elevation of [Ca2+]i, suggesting a blockade of Ca2+ influx via voltage-gated channels. These data are in complete agreement with recent reports by Nuttle and Farley (22) on the effect of isoproterenol on [Ca2+]i in porcine ASM cells. The fact that BAY K 8644 partially restored ACh-induced [Ca2+]i oscillations after their inhibition by salbutamol also suggests that the effects of salbutamol are mediated, at least in part, by inhibition of Ca2+ influx. Even when Ca2+ influx was blocked by the removal of extracellular Ca2+, salbutamol decreased both basal and ACh-induced [Ca2+]i, suggesting that salbutamol also enhances Ca2+ efflux. This was confirmed by the fact that blockade of Ca2+ efflux by using lanthanum prevented reduction of ACh-induced [Ca2+]i by salbutamol. The lack of an effect on the [Ca2+]i response to caffeine suggests that salbutamol may not significantly affect SR Ca2+ release. The results of this study indicate that beta -adrenoceptor stimulation results in altered Ca2+ flux across the membrane of ASM cells, reducing [Ca2+]i by inhibiting influx and enhancing efflux.

The [Ca2+]i oscillations induced by ACh in porcine ASM cells were qualitatively similar to [Ca2+]i oscillations observed in vascular smooth muscle (2, 7, 9), colonic smooth muscle (19), uterine smooth muscle (14), and more recent studies using porcine ASM cells (13, 18, 22, 23). [Ca2+]i oscillations may arise from cyclical Ca2+ influx, mediated via changes in membrane potential, or from cyclical SR Ca2+ release and reuptake. We (13, 23) and others (18, 22) have recently shown in porcine ASM cells that [Ca2+]i oscillations arise from cyclical Ca2+ release and reuptake and may involve IP3-receptor and RyR channels. Ca2+ influx appears to be necessary for the maintenance of [Ca2+]i oscillations, most likely by replenishing SR Ca2+ stores depleted by agonist stimulation (13, 18, 22, 23). In our previous study (23), we demonstrated that the amplitude and frequency of ACh-induced [Ca2+]i oscillations in ASM cells are correlated to basal [Ca2+]i. With increasing basal [Ca2+]i, the amplitude of the [Ca2+]i oscillations decreases, whereas oscillation frequency increases. These relationships between basal [Ca2+]i and the amplitude and frequency of [Ca2+]i oscillations may be a result of the limited SR Ca2+ pool available for release with each oscillation. Basal [Ca2+]i may set the concentration gradient for release of this limited pool and, therefore, amplitude may decrease with increasing basal [Ca2+]i. With increasing basal [Ca2+]i, the frequency of oscillations may increase due to the enhancement of SR Ca2+ reuptake, allowing for faster recycling of the limited Ca2+ pool. Accordingly, various factors that influence either resting or agonist-induced basal [Ca2+]i levels, such as Ca2+ influx, efflux, and reuptake are likely to modulate the amplitude and frequency of [Ca2+]i oscillations. For example, factors that decrease basal [Ca2+]i would tend to increase oscillation amplitude and decrease oscillation frequency. The modulation of oscillation frequency by salbutamol suggests effects on one or more of the factors that influence basal [Ca2+]i.

Relaxation of smooth muscle by beta -adrenoceptor stimulation is thought to be achieved by cyclic nucleotide-dependent and -independent activation of BKCa channels (16, 24). Specific blockers of BKCa channels, such as charybdotoxin, have been shown to inhibit the relaxation induced by beta -adrenoceptor agonists (8, 10). Therefore, it is possible that the inhibition of [Ca2+]i oscillations in ASM cells by salbutamol is achieved by enhanced open probability of BKCa channels with subsequent membrane hyperpolarization and consequent inhibition of Ca2+ influx through voltage-gated Ca2+ channels. However, using patch-clamp techniques, Nuttle and Farley (22) demonstrated that, even when membrane potential is clamped close to the reversal potential for K+, ACh can induce [Ca2+]i oscillations that are blocked by beta -adrenoceptor agonists. Therefore, it is unlikely that the modulation of oscillation frequency or the inhibition of [Ca2+]i oscillations by salbutamol results from a decrease in Ca2+ influx by a mechanism that requires a change in membrane potential. This conclusion is also supported by the results of the present study in which, even in the presence of charybdotoxin, [Ca2+]i oscillations could be induced by ACh, which were then blocked by 1 µM salbutamol.

Although salbutamol may not inhibit Ca2+ influx indirectly via changes in membrane potential, the fact that BAY K 8644 partially reversed the inhibition of [Ca2+]i oscillations by salbutamol indicates that Ca2+ influx through voltage-gated channels is nevertheless affected. This result is in agreement with the previous study by Nuttle and Farley (22), who demonstrated that raising extracellular Ca2+ levels and increasing the inward driving force for Ca2+ restored [Ca2+]i oscillations that had been blocked by isoproterenol. There is currently no precedent for a direct effect of beta -adrenoceptor agonists on Ca2+-influx channels in ASM cells. However, several studies in vascular smooth muscle have demonstrated that beta -adrenoceptors can modulate voltage-dependent Ca2+ channels through a cyclic nucleotide-dependent phosphorylation of the channel or an associated protein (1, 30). A similar mechanism may explain the effect of salbutamol on Ca2+ influx. In the present study, the observation that 8-BrcAMP also modulated the frequency of [Ca2+]i oscillations suggests that cyclic nucleotides may be involved in mediating the effects of beta -adrenoceptors.

The results of the present study clearly demonstrate that, in addition to inhibiting Ca2+ influx, beta -adrenoceptor stimulation results in enhancement of Ca2+ efflux, which would also tend to decrease basal [Ca2+]i, leading to a slowing of oscillation frequency and even an inhibition of oscillations. For example, even when Ca2+ influx and reuptake were blocked by zero extracellular Ca2+ and thapsigargin, respectively, salbutamol decreased basal [Ca2+]i. It was not possible to study directly the effect of salbutamol on [Ca2+]i oscillations under these conditions, because removal of extracellular Ca2+ would itself lead to an inhibition of oscillations (18, 23), and thapsigargin also leads to an elevation of [Ca2+]i and inhibition of oscillations. Nonetheless, the fact that salbutamol had no effect on the stable ACh-induced oscillations, under conditions where Ca2+ influx and efflux were both blocked by the combination of zero extracellular Ca2+ and lanthanum, clearly demonstrates that salbutamol primarily affects Ca2+ flux across the cell membrane. Enhancement of Ca2+ efflux may be achieved by modulation of the Na+/Ca2+ exchanger or by enhanced activity of the plasma membrane adenosinetriphosphatase pump.

Salbutamol did not appear to have any significant effect on SR Ca2+ release. In the present study, we did not specifically examine SR Ca2+ release through both IP3 receptor and RyR channels. The data obtained by using caffeine suggest that Ca2+ release through RyR is unaffected. However, the fact that, when Ca2+ flux across the membrane was blocked by zero extracellular Ca2+ and lanthanum, salbutamol had no effect on ongoing ACh-induced [Ca2+]i oscillations would suggest that SR Ca2+ release is not significantly affected by beta -adrenoceptors. In the present study, we observed that the fall time of ACh-induced oscillations, normalized for amplitude, was prolonged by exposure to salbutamol. This would suggest a partial inhibition of Ca2+ reuptake. Previous studies have suggested that beta -adrenoceptor-agonist stimulation actually increases SR Ca2+ reuptake via a cAMP-mediated pathway. This would lead to an increase in oscillation frequency, which was not observed. Therefore, the results of the present study on oscillation fall time must be interpreted with caution. Because Ca2+ reuptake is directly correlated to basal [Ca2+]i, the prolongation of fall time with salbutamol exposure may be related to the reduction in basal [Ca2+]i.

In conclusion, the results of the present study indicate that increased beta -adrenoceptor-agonist stimulation in ASM cells inhibits [Ca2+]i oscillations in a dose-dependent fashion by decreasing basal Ca2+ levels. This decrease in basal Ca2+ may be achieved by modulating Ca2+ flux across the cell membrane.

ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grant HL-51736 and by the Mayo Foundation. Y. S. Prakash is supported by a fellowship from Abbott Laboratories. H. F. M. van der Heijden is supported by grants from Glaxo-Wellcome, The Netherlands, and by the Van Walree Foundation of the Royal Netherlands Academy of Arts and Sciences.

FOOTNOTES

Address for reprint requests: G. C. Sieck, Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail: sieck.gary{at}mayo.edu).

Received 23 September 1996; accepted in final form 3 February 1997.

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