The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca²⁺ signaling of airway smooth muscle cells

Abstract

The relative contribution of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) and ryanodine receptors (RyRs) to agonist-induced Ca²⁺ signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca²⁺ oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca²⁺ oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca²⁺ waves generated by the photolytic release of IP₃. However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP₃-induced Ca²⁺ oscillations or Ca²⁺ wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca²⁺ oscillations in a concentration-dependent manner. However, tetracaine did not affect IP₃-induced Ca²⁺ release or wave propagation nor the Ca²⁺ content of SMC Ca²⁺ stores as evaluated by Ca²⁺-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca²⁺ oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca²⁺ oscillations of SMCs were also observed at 37°C. In Ca²⁺-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca²⁺ levels indicating that relaxation also resulted from a reduction in Ca²⁺ sensitivity. These results indicate that agonist-induced Ca²⁺ oscillations in mouse small airway SMCs are primary mediated via IP₃Rs and that tetracaine induces relaxation by both decreasing Ca²⁺ sensitivity and inhibiting agonist-induced Ca²⁺ oscillations via an IP₃-dependent mechanism.

in airway smooth muscle cells (SMCs), agonist-induced contraction is associated with an elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, this increase of [Ca²⁺]_i is not steady but occurs as repetitive Ca²⁺ waves propagating along the length of the SMCs (6, 33, 43, 44). The basic mechanism underlying these Ca²⁺ oscillations consists of a cyclic Ca²⁺ release from, and reuptake to, the sarcoplasmic reticulum (SR) via specialized receptor/Ca²⁺ channels and Ca²⁺ pumps together with a Ca²⁺ influx to replace any Ca²⁺ lost across the cell membrane to the extracellular space (8).

There are two major types of Ca²⁺ release channels on the SR of airway SMCs, inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) and ryanodine receptors (RyRs) (1, 2, 31). Each type of receptors exists as three different isomers, all of which have been identified in the airway SMCs (19, 24, 31, 55). Although these receptors differ in structure and molecular weight, their functional activity, to mediate Ca²⁺ release from the SR, is similar. However, each receptor type is regulated in a different manner. IP₃Rs are activated by IP₃ (22), produced from phosphatidylinositol 4,5-bisphosphate (PIP₂) by Gα_q-activated PLCβ in response to G protein-coupled receptor (GPCR) agonists. In addition, the open probability of the IP₃R is also modulated by [Ca²⁺]_i; increasing [Ca²⁺]_i serves to enhance IP₃R opening, but high [Ca²⁺]_i can reduce the open probability of some IP₃Rs (10, 22, 27). In contrast to the IP₃R, the opening of the RyRs is primarily modulated by [Ca²⁺]_i. However, SR luminal Ca²⁺ concentration may also serve to regulate the RyR and IP₃R (21, 22). In addition, the second messenger, cyclic ADP-ribose (cADPR), also has been implicated in the activation of RyRs in association with agonist-induced Ca²⁺ signaling (29).

To explore the regulation of agonist-induced Ca²⁺ oscillations of SMCs, it is crucial to first understand the mechanisms mediating the cyclic Ca²⁺ release from SR. Generally, it is acknowledged that the initiation of SMC Ca²⁺ oscillations is mediated by Ca²⁺ release from SR via the IP₃R (7, 9, 37). However, it has been a long-standing debate in airway or other types of SMCs as to whether RyRs are involved and are required to maintain cyclic Ca²⁺ release and the propagation of Ca²⁺ waves by Ca²⁺-induced Ca²⁺ release (CICR) (14, 31, 44, 46, 57) or, alternatively, if the entire process was exclusively accomplished by activated IP₃R without significant RyRs involvement (34, 36, 50).

The aim of the present research is to determine the relative contribution of the IP₃Rs and RyRs to agonist-induced intracellular Ca²⁺ signaling of airway SMCs. Most previous investigations have relied on pharmacological approaches, using RyR inhibitors, such as ryanodine, dantrolene, and tetracaine, and IP₃R inhibitors, such as xestospongin C (XeC) and 2-aminoethoxydiphenyl borate (2-APB). Unfortunately, all of these drugs have some nonspecific effects that will confound results if the assays are not sufficiently specific (49). Consequently, to diminish the misinterpretation of experimental data and obtain a consistent mechanism, we have examined the action of these pharmacological agents on the IP₃Rs or RyRs in a number of different conditions.

A second type of Ca²⁺ signaling occurs in airway SMCs, namely agonist-independent, KCl-induced Ca²⁺ oscillations. From our previous studies (43), we found that KCl induced slow frequency, long-lasting Ca²⁺ oscillatory waves that result from the sequential events of depolarization-induced Ca²⁺ influx, overload of the Ca²⁺ store, and subsequent Ca²⁺ release via sensitized RyRs. Because these Ca²⁺ signals do not appear to involve the IP₃R, they serve as a useful comparison for agonist-induced Ca²⁺ oscillations.

For these studies, we have used mouse lung slices to simultaneously measure Ca²⁺ signaling and airway contraction. We found that the IP₃R antagonist, 2-APB, could relax airways contracted with methacholine (MCh) or 5-hydroxytryptamine (5-HT) and inhibit agonist-induced Ca²⁺ oscillations. The inhibitory effect of 2-APB was also confirmed by its ability to inhibit IP₃-induced Ca²⁺ release. On the other hand, the RyR antagonist, ryanodine had little effect on agonist-induced airway contraction or Ca²⁺ oscillations or IP₃-induced Ca²⁺ release. However, ryanodine did inhibit KCl-induced slow Ca²⁺ oscillations. By contrast, tetracaine, another reported RyR antagonist, clearly inhibited agonist-induced airway contraction and Ca²⁺ signaling but had little effect on IP₃-induced Ca²⁺ release or the Ca²⁺ content of the SR. Moreover, tetracaine significantly reduced the agonist-induced increase in Ca²⁺ sensitivity of the SMCs. Therefore, it appears that tetracaine, although often used as a RyR inhibitor in channel studies, inhibits agonist-induced airway contraction by modulating receptor-G protein interaction to decrease both IP₃ production and Ca²⁺ sensitivity. From these data, we suggest that in mouse airway SMCs the IP₃R serves as the primary receptor in the initiation and maintenance of intracellular Ca²⁺ signaling during agonist-induced airway contraction. An involvement of RyRs is not required in agonist-induced Ca²⁺ responses in normal physiological conditions. However, it is not known whether the RyR may become involved and change IP₃R-mediated Ca²⁺ signaling of airway SMCs in disease conditions such as airway hyperresponsiveness.

MATERIALS AND METHODS

Materials.

Cell culture reagents were obtained from GIBCO-Invitrogen; Oregon Green 488 BAPTA-1 AM was obtained form Molecular Probes-Invitrogen; and caged-iso-Ins(1,4,5)P₃/propionoxymethyl ester (PM) was obtained from Alexis Biochemicals (San Diego, CA) and dissolved in DMSO, aliquoted, and frozen at −80°C. Other reagents were obtained from Sigma-Aldrich or Calbiochem. Hanks-Balanced Salt Solution (HBSS) was supplemented with 20 mM HEPES buffer (sHBSS) and adjusted to pH 7.4.

Lung slices.

The preparation of lung slices has been previously described in detail (43). Briefly, male BALB/c mice (7–10 wk; Charles River Breeding Laboratories, Needham, MA) were killed by intraperitoneal injection of 0.3 ml of pentobarbital sodium (Nembutal) as approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. After the thoracic cavity was opened, the lungs were inflated with ∼1.3-ml warm (37°C) ∼2% agarose in sHBSS via a catheter cannulated into the trachea. The agarose was followed by a final injection of a bolus of air (∼0.2 ml) to flush the agarose out of the airways and into the distal alveoli. After the agarose was gelled with cold sHBSS (∼4°C), the lung lobes were removed and sectioned into ∼140-μm thick slices with a vibratome. Slices were maintained in DMEM and antibiotics at 37°C and 10% CO₂ for up to 3 days.

To conduct experiments at 37°C, the perfusion system and microscope were enclosed in a Plexiglas chamber and heated with a custom-built heater consisting of two heater elements (AF20-300-120-1D-K-10-3.1; Farnam Custom Products, Philadelphia, PA) coupled with circulating fans (1976K37; McMaster-Carr Supply, Chicago, IL). The temperature was monitored and regulated by a T thermocouple (HTTC36-T-14G-6; Omega Engineering, Stamford, CT) and a thermal controller (CC-A10; Farnam Custom Products).

Measurement of airway contraction and relaxation.

Lung slices were transferred to a large cover glass on a Plexiglas support, and a nylon mesh with a small hole cut in it, to expose the selected airway, was placed over the slice to hold it in position. A second, smaller cover glass, edged with silicone grease, was placed on the top of nylon mesh to form a sealed chamber. Experimental solutions, selected by electronic valves under computer control, were perfused through the chamber under gravity flow. Experiments were performed at either RT or 37°C. Phase-contrast images of airway contraction were collected with an inverted microscope (IX71; Olympus America, Melville, NY) with a ×10 objective, a zoom adapter, and a CCD camera (RS-170 video in conjunction with a frame grabber; Picolo; Euresys, Itasca, IL) at 1 frame every 2 s. Images were captured by Video Savant (IO Industries, London, Ontario, Canada), an image-acquisition software package, and stored on a hard drive. Airway contraction was measured by determining the change in lumen area, with respect to the initial lumen area, by pixel summing with custom-written macros (43).

Measurement of intracellular Ca²⁺ signaling.

Approximately 15 lung slices were placed in 2 ml of sHBSS containing 20 μM Oregon Green 488 BAPTA-1 AM, 100 μM sulfobromophthalein (an inhibitor of anion exchanges to reduce dye extrusion), and 0.1% Pluronic F-127 and incubated in the dark for 1 h at 30°C. The slices were washed in sHBSS with 100 μM sulfobromophthalein for an additional 30 min at 30°C to allow for dye deesterification. The lung slices were placed in the perfusion chamber as described above. The intracellular Ca²⁺ signaling was examined with either a custom-built video-rate confocal microscope or 2-photon scanning laser microscope as previously described (4, 43). Fluorescent images were recorded at a rate of 15 or 30 images per second. Fluorescence intensity of the SMCs was determined with respect to time (image number) from the average gray value of user-selected regions of interest (ROI; 6 × 6 pixels) within the cells with custom-written software. Fluorescence intensity was expressed in absolute units or in relative terms as a fluorescence ratio (F/F₀) normalized to the initial fluorescent intensity (F₀).

Flash photolysis of caged-IP₃.

Flash photolysis of caged-IP₃ was used to experimentally increase the [IP₃]_i. Lung slices were initially loaded with Oregon Green 488 BAPTA-1 AM as described above and subsequently incubated with 2 μM caged-IP₃ for 1 h in sHBSS containing 0.1% Pluronic F-127 and 100 μM sulfobromophthalein followed by deesterification for 30 min in sHBSS containing 100 μM sulfobromophthalein. The details of the flash photolysis setup have also been previously described (2). Briefly, a UV flash was generated by an electronic shutter from a filtered (330 caged-IP₃ nm) mercury arc lamp and focused as point source into the microscope. The intensity of flash was regulated by neutral density filters. The spot size could be adjusted with an iris diaphragm.

Preparation of Ca²⁺-permeabilized lung slices.

To clamp the [Ca²⁺]_i of airway SMCs at a constant level, it is necessary to increase the permeability of the plasma membrane to Ca²⁺. Typically, this has been achieved by the application of detergents or toxins. Unfortunately, these agents frequently increase membrane permeability to other cellular constituents. To overcome this problem, we have developed an alternative approach using caffeine and ryanodine to only increase the permeability of cell membrane to Ca²⁺; the use and verification of this method has been previously described in detail (4). Briefly, lung slices were simultaneously exposed to 20 mM caffeine and 50 μM ryanodine for 5 min. This treatment is believed to lock the RyRs in the open state and thereby brings about the depletion of the SR Ca²⁺ store. This, in turn, leads to a persistent Ca²⁺ influx via store-operated channels to elevate the [Ca²⁺]_i. As a result, the extracellular Ca²⁺ concentration is used to determine the [Ca²⁺]_i. The constant elevated [Ca²⁺]_i of permeabilized cells was monitored and confirmed by Ca²⁺ imaging. Lung slices treated with caffeine and ryanodine remain viable for experimentation for >5 h, and during this time the effect of the treatment was irreversible.

Data analysis.

Results are expressed as means ± SE. Statistical significance was determined by using Student's or paired Student's t-test.

RESULTS

Experiments were only performed on intact airways (100- to 200-μm diameter, midposition of the respiratory tract; Ref. 5) with an epithelium displaying active ciliary beating and with a lumen free of agarose (Fig. 1A). We examined the airway responses to two different agonists (MCh and 5-HT) and KCl to avoid the possibility of characterizing mechanisms that were only specific to single agonist. In addition, because experiments have the best chance of detecting changes in behavior if they are performed in the responsive region of the concentration-response curves, we used agonist concentrations (200 nM) that we (2, 6, 43) have previously found to induce nonsaturated responses of airway contraction and SMC Ca²⁺ signaling.

Fig. 1.

The effect of 2-aminoethoxydiphenyl borate (2-APB) on agonist-induced airway contraction. A: phase-contrast images of the same airway (at times indicated by numbered arrows in B) showing 1) the relaxed initial state of the airway, 2) the contractile state of the airway induced by 200 nM methacholine (MCh), and 3) the relaxation induced by 100 μM 2-APB. B: the change in airway area with respect to time showing the airway contraction in response to 200 nM MCh and the subsequent relaxation induced by either 50 or 100 μM 2-APB. The magnitude of airway relaxation increased as the concentration of 2-APB increased. C: summary of the relaxation induced in airways contracted with 200 nM MCh or 200 nM 5-hydroxytryptamine (5-HT) by 50 and 100 μM 2-APB. The magnitude of the relaxation was measured after 10 min of 2-APB exposure and is expressed as a percentage of the contracted luminal area induced by agonists. Each column represents the mean ± SE from 6 different experiments on different slices from 3 mice.

Effect of 2-APB on agonist-induced airway contraction.

To explore the role of the IP₃R in agonist-induced airway contraction, the airway responses to the IP₃R antagonist 2-APB were examined. When exposed to 200 nM MCh, the airways responded by contracting and reducing their luminal area within 2 min, after which the contractile state stabilized (at 40–50% of initial luminal area) while MCh was perfused. Removal of MCh resulted in airway relaxation (Fig. 1, A and B). When the same airways, again contracted with MCh, were exposed to 2-APB, they quickly relaxed; relaxation was fast in the first 2 min but subsequently slow (Fig. 1B). The airways recontracted when 2-APB was removed. The amount of relaxation induced by 2-APB is expressed as the percentage increase in luminal area normalized to the contracted area induced by MCh. After 10 min, the airways relaxed 28.44 ± 0.83 and 52.33 ± 5.13% in response to 50 and 100 μM 2-APB, respectively (Fig. 1C). Similar responses of airway contraction (52.0 ± 8.0% related to initial area) and 2-APB-induced relaxation (26.43 ± 5.51 and 55.25 ± 4.72% of contracted area at 50 and 100 μM) were obtained when the contractile agonist was changed to 5-HT (200 nM) (Fig. 1C). These results suggest that the activity of the IP₃R contributes to airway contraction.

Effect of ryanodine and tetracaine on agonist-induced airway contraction.

To similarly explore the role of the RyR in agonist-induced airway contraction, the airway responses to antagonists of the RyR (ryanodine and tetracaine) were examined. After the airways were contracted with 200 nM MCh, the addition of ryanodine, at concentrations ranging from 1 to 50 μM, had little effect on the airway contractile response (Fig. 2). During the course of these experiments, the airway showed a small, steady relaxation rate (∼8% over 10 min), but this process was not altered by the addition of increasing concentrations of ryanodine (Fig. 2, A and C). Similarly, ryanodine (1, 10, or 50 μM) had little effect on 5-HT-induced (200 nM) contractile responses (Fig. 2C). These results suggest that the RyR is not involved in agonist-induced airway contraction.

Fig. 2.

Effects of ryanodine and tetracaine on agonist-induced airway contraction. Representative experiments showing airway contraction in response to 200 nM MCh and the subsequent responses to different concentrations of ryanodine (Rya; A; 1, 10, or 50 μM) or tetracaine (Tetra; B; 1, 10, or 50 μM) are shown. At all concentrations tested, ryanodine had no obvious effect on MCh-induced contraction. Because the effects of ryanodine are irreversible, 3 individual lung slices were used to test the effects of each concentration of ryanodine. By contrast, tetracaine relaxed MCh-contracted airways in a concentration-dependent manner. Because the effects of tetracaine are reversible, the same lung slice was used to examine the effects of multiple concentrations of tetracaine. A summary of the airway responses to ryanodine (C) and tetracaine (D) in the presence of MCh or 5-HT is shown. Relaxation was measured after 10 min of exposure to ryanodine or tetracaine and is expressed as a percentage of the contracted luminal area induced by the specific agonist. Each column represents the mean ± SE from ≥6 different experiments on different slices from >3 mice.

Surprisingly, in contrast to the effects of ryanodine, tetracaine, an antagonist used in other studies to implicate the RyR in airway contraction, induced a significant relaxation of both MCh- and 5-HT-induced airway contraction. In response to tetracaine, contracted airways quickly relaxed to a plateau level within 3 min in a concentration-dependent manner; a near full relaxation was induced by 50 μM tetracaine (Fig. 2, B and D). This tetracaine effect was readily reversible; the airways quickly recontracted when tetracaine was removed (Fig. 2B).

Effect of 2-APB on agonist-induced Ca²⁺ oscillations.

In response to 200 nM MCh, airway SMCs displayed an initial elevation of [Ca²⁺]_i followed by cyclic increases of [Ca²⁺]_i in the form of oscillatory Ca²⁺ waves. These repetitive Ca²⁺ oscillations usually originated from one end of the SMC and propagated the full length of the cell. The frequency of Ca²⁺ oscillations stabilized after ∼2 min of MCh exposure. These changes in the intracellular Ca²⁺ signaling have been previously shown to closely correlate with the contractile response of the airway SMCs (2, 43).

The subsequent exposure to 2-APB gradually slowed the Ca²⁺ oscillations (from 20.5 ± 1.8 to 15.8 ± 2.6 min⁻¹ after 5 min of exposure at 50 μM, n = 4 experiments from different airways from 3 mice; Fig. 3B) or nearly stopped the Ca²⁺ oscillations (at 100 μM; Fig. 3C). When 2-APB was removed, the Ca²⁺ oscillations resumed and gradually increased their frequency to approach the original rate after 3 min. The time course of this inhibition (∼2 min) and restoration of the Ca²⁺ signaling correlates with the time taken for the airways to relax and recontract in response to 2-APB (Fig. 3C). A similar cessation of 5-HT-induced (200 nM) Ca²⁺ oscillations by 100 μM 2-APB was observed in three different airways from two mice.

Fig. 3.

The effect of 2-APB on the Ca²⁺ signaling of airway smooth muscle cells (SMCs) induced by 200 nM MCh. A: a fluorescence image of part of an airway obtained by confocal microscopy. L, lumen; ECs, epithelial cells; SL, scan line. B: the intracellular Ca²⁺ signaling occurring within airway SMC [recorded from a region of interest (ROI) of about 6 × 6 pixels as represented by the white square indicated in A] in response to 200 nM MCh and 50 μM 2-APB; a representative trace from 4 slices from 3 mice. C: line-scan (top, constructed by sequentially aligning the pixels from the SL shown in A) and the Ca²⁺ signaling (bottom) showing the effect of 100 μM 2-APB on 200 nM MCh-induced contraction and Ca²⁺ signaling of airway SMCs. Representative data are from 8 different slices from 4 mice. MCh induced high-frequency Ca²⁺ oscillations within the SMC and airway contraction. 2-APB at 50 μM (B) or 100 μM (C) either slowed or nearly stopped the Ca²⁺ oscillations and induced relaxation (Relax). On 2-APB removal, the Ca²⁺ oscillations resumed.

Effect of ryanodine and tetracaine on agonist-induced Ca²⁺ oscillations.

In keeping with the observation that ryanodine had no effect on airway contraction, ryanodine (examined with 1, 10, or 50 μM) had no significant effect on the frequency of MCh-induced Ca²⁺ oscillations (Fig. 4B) during the 5 min of observation. The Ca²⁺ oscillation frequency was 20.9 ± 2.8 min⁻¹ in MCh alone compared with 19.5 ± 2.3 min⁻¹ after 5-min exposure of 50 μM ryanodine in presence of MCh (n = 10 different airways from 5 mice). Similarly, the propagation of the oscillatory Ca²⁺ wave, as examined by line-scan analysis along the longitudinal axis of SMC (Fig. 4C), was not altered by 5 min of ryanodine exposure. Likewise, the propagation velocity of Ca²⁺ wave, determined by the time taken for the wave to propagate between two ROIs (e.g., Fig. 4D, left, “a” and “b”), was not influenced by ryanodine. In response to 200 nM MCh, Ca²⁺ wave velocity was 27.63 ± 2.76 and 27.60 ± 3.37 μm/s in the absence or presence of 50 μM ryanodine, respectively (n = 5 SMCs from different slices from 2 mice). Ryanodine also had no significant effect on the frequency of the Ca²⁺ oscillation of SMCs induced by 200 nM 5-HT. The Ca²⁺ oscillation frequency was 20.2 ± 2.7 min⁻¹ before and 19.2 ± 3.0 min⁻¹ after 5-min exposure to 50 μM ryanodine (n = 4 airways from different slices from 2 mice).

Fig. 4.

The effect of 50 μM ryanodine on Ca²⁺ signaling induced by 200 nM MCh in airway SMCs. A: a fluorescence image of part of an airway obtained by confocal microscopy under resting conditions. B: changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with respect to time (from a ROI; white square in A) in a SMC in response to 200 nM MCh and 50 μM ryanodine. MCh induced high-frequency Ca²⁺ oscillations that were unaffected by the addition of ryanodine. Representative data are from ≥10 different slices from 5 mice. C: line-scan plots of the propagated changes in [Ca²⁺]_i (Ca²⁺ waves) following exposure to MCh (0–30 s) and ryanodine (360–390 s). Line-scans were constructed by sequentially aligning the pixels from the SL shown in A, parallel to the longitudinal axis of the SMC. D: the propagation velocity of the Ca²⁺ waves was measured by determining the period (P) between sequential changes in Ca²⁺ in regions “a” and “b” (right) of the same SMCs (left). Ryanodine (50 μM) had no significant effect on the frequency of MCh-induced Ca²⁺ oscillations, the appearance of the Ca²⁺ waves, or the propagation velocity of the Ca²⁺ waves.

By contrast, but consistent with its relaxant action, tetracaine at 10 μM slowed the frequency of MCh-induced (200 nM) Ca²⁺ oscillations within 2 min (Fig. 5A; from 23.2 ± 3.5 to 14.6 ± 1.0 min⁻¹ after 5 min of exposure). In addition, the period and waveform of these slowed Ca²⁺ oscillations also became irregular. Higher concentrations (50 μM) of tetracaine stopped the Ca²⁺ oscillations after 1 min. Airway relaxation was tightly coupled to this cessation of the Ca²⁺ oscillations (Fig. 5B). The Ca²⁺ oscillations were restored to normal on tetracaine removal (n = 4, from different slices from 3 mice). A similar complete inhibitory action of 50 μM tetracaine on 5-HT-induced Ca²⁺ oscillations was observed (data not shown).

Fig. 5.

The effect of tetracaine on Ca²⁺ signaling in airway SMCs induced by MCh. High-frequency Ca²⁺ oscillations induced in airway SMCs by 200 nM MCh were either slowed by 10 μM tetracaine (A) or stopped by 50 μM tetracaine (B, bottom). The line-scan plot (B, top) shows that the cessation of the Ca²⁺ oscillations (white vertical lines) is associated with the relaxation of the SMC (upward deflection of oscillation trace). The resumption of the Ca²⁺ oscillation is correlated with the recontraction of the SMC. Representative data are from 4 different slices from 3 mice.

Second messenger mediating Ca²⁺ oscillations.

In many cases, Ca²⁺ oscillations, involving repetitive Ca²⁺ release and reuptake from the SR, are mediated by agonist-induced production of IP₃ acting on the IP₃R. A similar process of sensitizing the RyR with the second messenger cADPR can also occur and has been proposed as a regulatory mechanism of agonist-induced Ca²⁺ oscillations in airway SMCs. Because both the IP₃R and RyR are implicated in airway SMC Ca²⁺ signaling, we performed studies to determine the predominant messenger mediating agonist-induced Ca²⁺ oscillations in mouse airway SMCs.

We examined the effect of the photolytic release of IP₃ (uncaging) on airway SMCs and found that the increase in [IP₃]_i, following exposure to UV light, induced repetitive Ca²⁺ waves that propagated from the illuminated area to the rest of the SMC (Fig. 6A). The frequency and number of these Ca²⁺ oscillations was increased by an increase in the UV exposure time (Fig. 6B). These IP₃-induced Ca²⁺ oscillations mimicked concentration-dependent agonist-induced Ca²⁺ oscillations and were accompanied by a contractile response.

Fig. 6.

Effect of photolytic release of inositol 1,4,5-trisphosphate (IP₃) from caged-IP₃ on Ca²⁺ signaling of airway SMCs. A: fluorescent images of part of an airway obtained by confocal microscopy showing the intracellular Ca²⁺ signaling of airway SMC before (left) and after (right) the UV flash. Dashed white oval represents the position and size of the zone of UV illumination. B: intracellular Ca²⁺ oscillatory waves induced by photolysis of caged-IP₃. When the flash time was extended from 0.5 to 1.5 s, the Ca²⁺ signaling increased from a single Ca²⁺ transient to multiple Ca²⁺ oscillations. F/F₀, fluorescence ratio.

Because cADPR is not cell-permeable, similar sensitization experiments of the RyR with cADPR were not possible. Consequently, we took the opposite approach and applied the cell-permeable competitive inhibitor of cADPR, 8-bromo-cADPR (8-Br-cADPR), and examined its effect on MCh-induced SMC Ca²⁺ signaling and airway contraction. No significant changes in the Ca²⁺ oscillations or contractile responses of the airways were observed during 20-min incubation with 100 μM 8-Br-cADPR (data not shown). Collectively, these results with caged-IP₃ and 8-Br-cADPR suggest that IP₃ is the primary intracellular mediator responsible for the generation of Ca²⁺ oscillations in mouse airway SMCs.

Effect of 2-APB on IP₃-induced Ca²⁺ release.

In view of our earlier result that 2-APB inhibited agonist-induced Ca²⁺ oscillations (Fig. 3), we investigated the effect of 2-APB on IP₃-induced Ca²⁺ release. It is important to note that in these experiments, the duration of the UV flash used for photolysis was reduced to 0.2 s so that it only induced a single Ca²⁺ wave within the SMC (Fig. 6B). This adjustment allowed us to release a small but similar amount of IP₃ multiple times in the same SMC and thereby compare changes in the Ca²⁺ signaling of the same SMC before and after application of 2-APB.

Under these conditions, the first UV flash induced a single Ca²⁺ transient in the SMC (Fig. 7). However, after 3 min of exposure to 100 μM 2-APB, the elevation of [Ca²⁺]_i in response to a second photolysis of caged-IP₃ was virtually eliminated, and no Ca²⁺ wave was observed in SMCs. When the 2-APB was removed, the third UV flash of similar intensity once again induced a transient increase in [Ca²⁺]_i and a propagating Ca²⁺ wave that was indistinguishable from that induced by the first UV flash (Fig. 7, A and C).

Fig. 7.

Effect of 2-APB on Ca²⁺ waves induced by the flash photolysis of caged-IP₃. The Ca²⁺ response of the SMC induced by the repetitive flash photolysis (0.2-s exposure; lightning bolts) of caged-IP₃ in lung slices incubated with 100 μM 2-APB (A) or zero extracellular Ca²⁺ (0-Ca²⁺; B) for 3 min is shown. Summary of the changes in SMC Ca²⁺ signaling (peak florescence intensity) induced by IP₃ in the presence of 2-APB (C) or absence of extracellular Ca²⁺ (D) is shown. Exposure to 2-APB virtually abolished the Ca²⁺ response to IP₃, whereas the removal of extracellular Ca²⁺ had little effect on IP₃-induced Ca²⁺ signaling. Data represent the means ± SE from 5 different slices from 2 mice.

In addition to its action on the IP₃R, 2-APB also appears to inhibit store-operated Ca²⁺ entry (SOC) (11), and this could reduce the amount of Ca²⁺ stored within the SR. Consequently, to rule out the possibility that emptying of internal Ca²⁺ stores during the course of experiments was responsible for the abolishment of IP₃-induced Ca²⁺ release by 2-APB, we repeated the above experiments in the absence of Ca²⁺ entry by the removal of extracellular Ca²⁺. As shown in Fig. 7, B and D, the absence of Ca²⁺ entry had little effect on the magnitude of the Ca²⁺ transient induced by a second release of IP₃. However, the duration of the IP₃-induced Ca²⁺ transient was shortened in absence of Ca²⁺ entry (Fig. 7B). This result indicated that some emptying of Ca²⁺ store had occurred due to Ca²⁺ leakage, and this resulted in the earlier termination of the Ca²⁺ release from SR. An alternative explanation is that Ca²⁺ removal from the cytoplasm was enhanced by the increased gradient between the intracellular and extracellular Ca²⁺ concentration. A second possible action of 2-APB is the inhibition of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), which again could empty Ca²⁺ stores. However, we found by using caffeine to induce the release of internal Ca²⁺ that the Ca²⁺ store retained 85.1 ± 3.7% of its Ca²⁺ charge after 5 min in 2-APB (by comparing the peak value of the caffeine-induced Ca²⁺ wave before and after exposure to 2-APB, n = 4 different airways from 2 mice). These results are consistent with the hypothesis that 2-APB acts primarily by inhibiting IP₃-induced Ca²⁺ release via the IP₃R.

Effect of ryanodine and tetracaine on IP₃-induced Ca²⁺ release.

The idea that IP₃ is releasing Ca²⁺ via the IP₃R was further corroborated by the observations that the extended incubation of airway SMCs with 50 μM ryanodine (>5 min; Fig. 8A) and 50 μM tetracaine (Fig. 8B) had no effect on repetitive flash-induced Ca²⁺ transients or the ability to propagate Ca²⁺ waves in SMC. These results also indicated that the RyRs do not contribute to the Ca²⁺ transients initiated by IP₃ and were consistent with the action of ryanodine on Ca²⁺ oscillations. In addition, it also implies that the inhibitory effect of tetracaine on agonist-induced Ca²⁺ oscillations was not mediated by inhibiting Ca²⁺ release via the IP₃R. Alternative explanations are that tetracaine leads to the depletion of the internal Ca²⁺ store (25) or that it decreases the production of IP₃.

Fig. 8.

Effect of ryanodine and tetracaine on Ca²⁺ waves induced by flash photolysis of caged-IP₃. Representative experiments showing the Ca²⁺ changes induced by repetitive flash-photolysis of caged-IP₃ of airway SMCs in lung slices incubated with 50 μM ryanodine (A) and 50 μM tetracaine (B) are shown. Summary of changes in the Ca²⁺ signals (peak fluorescence intensity, expressed as the ratio of the Ca²⁺ response before and after drug exposure) induced by IP₃ in the presence of 50 μM ryanodine (C) or 50 μM tetracaine (D) is shown. Neither ryanodine nor tetracaine blocked the Ca²⁺ increases induced by IP₃. Each column represents the mean ± SE from 5 different slices from 2 mice.

Effect of tetracaine on refilling of the internal Ca²⁺ store.

To investigate the effect of tetracaine on the Ca²⁺ capacity of SR, we examined the contractile responses and Ca²⁺ signaling of airway SMCs to repetitive stimuli of caffeine (Fig. 9). In these experiments, we found that the airway contractile response to 20 mM caffeine was similar before, during, or after exposure of the airways to 50 μM tetracaine (Fig. 9A). A comparable set of results was obtained with respect to the Ca²⁺ release induced by caffeine (Fig. 9B). Tetracaine (50 μM) was found to have little effect on the caffeine-induced Ca²⁺ transients. These results indicated that tetracaine did not reduce the Ca²⁺ capacity of the SR, and this argues against the possibility that tetracaine inhibited agonist-induced Ca²⁺ oscillations by the depletion of internal Ca²⁺ stores.

Fig. 9.

The effect of tetracaine on caffeine (caf)-induced Ca²⁺ release. Representative experiments demonstrating contraction (A) and Ca²⁺ signaling (B) of airway SMCs induced by repetitive exposure to 20 mM caffeine in the presence of 50 μM tetracaine are shown. Tetracaine had no significant effect on caffeine-induced contraction or Ca²⁺ release. Representative data are from 4 different slices from 2 mice.

So far, we have demonstrated that tetracaine does not inhibit Ca²⁺ oscillations by inactivating IP₃Rs or by depleting Ca²⁺ stores. The alternative possibility that tetracaine is acting as a RyR antagonist to reduce agonist-induced Ca²⁺ oscillations also appears unlikely in view of our findings that ryanodine, a well-documented specific antagonist of the RyR, had no effect on agonist-induced Ca²⁺ oscillations. As a result, we are forced to hypothesize that, in intact SMCs, tetracaine acts via a mechanism other than RyR inhibition even though tetracaine may serve as a RyR antagonist in studies with isolated channels or cell extracts.

Consequently, we hypothesized that tetracaine decreases the amount of IP₃ available to drive the Ca²⁺ oscillations. To test this idea, we used photolysis to increase the [IP₃]_i in cells in which the agonist-induced oscillations had been inhibited with tetracaine (Fig. 10). In response to flash photolysis, the Ca²⁺ oscillations were reinitiated and lasted for ∼1 min. This Ca²⁺ response was associated with a transient recontraction of the SMCs.

Fig. 10.

Effect of flash photolysis of caged-IP₃ on SMCs treated with MCh and tetracaine. A: a fluorescence image of part of an airway obtained by confocal microscopy under resting conditions. Dashed white oval represents the position and size of the zone of UV illumination. The intracellular Ca²⁺ signaling of a SMC in response to a UV flash (1.0 s) in the presence of 200 nM MCh and 50 μM tetracaine is represented by a line-scan plot (B), constructed by sequentially aligning the pixels along a SL (white line indicated in A) across the SMC and ECs and changes in [Ca²⁺]_i in respect to time (C; from a ROI, white square in A). Photolysis of caged-IP₃ reinitiated Ca²⁺ oscillations and a transient contraction in the presence of MCh and tetracaine. Representative data are from 4 different airways from 2 mice.

Effect of tetracaine on agonist-induced contraction of permeabilized SMCs.

The idea that tetracaine acts by another mechanism is further supported by the observation that a greater airway relaxation is induced by tetracaine compared with 2-APB (at low concentrations; Figs. 1B and 2B). The fact that the Ca²⁺ oscillations are slowed to a similar extent by tetracaine and 2-APB (Figs. 3B and 5A) implies that the Ca²⁺ sensitivity of the airway has been decreased by tetracaine. To investigate this possibility, we examined the responses of lung slices permeabilized to Ca²⁺ with caffeine and ryanodine.

After permeabilizing the SMCs to Ca²⁺, their [Ca²⁺]_i was set at an elevated level by perfusion with sHBSS (extracellular Ca²⁺ = 1.3 mM). Under conditions of elevated [Ca²⁺]_i, these permeabilized mouse airways are fully relaxed. This response is fully described in previous work (4) and has been ascribed to a Ca²⁺-dependent decrease in Ca²⁺ sensitivity. Importantly, during the subsequent contractile and relaxant responses induced by agonists or tetracaine, there was no significant change in the intracellular Ca²⁺ signal in these permeabilized SMCs (Fig. 11, C and D). When the permeabilized airways were exposed to MCh, they responded with a significant contraction. Because the Ca²⁺ levels did not change, this contraction is attributed to a MCh-induced increase in Ca²⁺ sensitization. In the presence of MCh, exposure to tetracaine relaxed the airway in a concentration-dependent manner, from 2.10 ± 0.42% at 1 μM tetracaine to 60.78 ± 4.09% at 50 μM tetracaine (Fig. 11, A and B). This relaxation indicated that tetracaine also acts by reducing airway Ca²⁺ sensitivity.

Fig. 11.

The effect of tetracaine on MCh-induced contraction and the [Ca²⁺]_i of permeabilized airway SMCs. The airway SMCs were permeabilized to Ca²⁺ by treatment with 20 mM caffeine and 50 μM ryanodine and washed with HBSS supplemented with 20 mM HEPES buffer (sHBSS) for 10 min. A representative trace (A) and summary data (B) showing the effect of tetracaine on the airway contracted with 200 nM MCh are shown. Increasing concentrations of tetracaine relaxed the airway. The extent of relaxation is expressed as a percentage of contracted lumen area induced by MCh. Data represent the means ± SE from 4 different slices from 2 mice. A representative trace (C) and summary data (D) demonstrate that in permeabilized SMCs, the [Ca²⁺]_i remained at a high constant level throughout the experiments (the [Ca²⁺]_i was expressed as ratio of the levels before and after drug exposure in D). Data represent the means ± SE from 4 different slices from 2 mice.

Effect of ryanodine and tetracaine on KCl-induced airway contraction and Ca²⁺ signaling of SMCs.

Although ryanodine and tetracaine had no effect on IP₃-induced Ca²⁺ release in airway SMCs, it is necessary to demonstrate, if we are to imply that RyRs are not involved, that these antagonists can block the Ca²⁺ signals and contractile responses of SMCs when RyRs do play a major role in the underlying mechanism. Therefore, we examined the effect of ryanodine and tetracaine on the Ca²⁺ and contractile responses of SMCs induced by membrane depolarization. We (43) have previously demonstrated, and as shown in Fig. 12A, that membrane depolarization in response to KCl (50 or 100 mM) induced very slow frequency and prolonged Ca²⁺ oscillations (2–3 min⁻¹). We concluded these Ca²⁺ oscillations resulted from the overloading of the SR with Ca²⁺ due to a continuous Ca²⁺ influx stimulated by depolarization. When the SR Ca²⁺ content was high, spontaneous RyR openings were amplified into whole cell Ca²⁺ waves by CICR. In presence of 50 μM ryanodine, these KCl-induced Ca²⁺ oscillations slowed down and stopped after 4 min, whereas the basal level of [Ca²⁺]_i gradually increased (Fig. 12A). Tetracaine also blocked the KCl-induced Ca²⁺ oscillations, but this stoppage was not accompanied by an elevation of baseline level of Ca²⁺ (Fig. 12C).

Fig. 12.

Effect of ryanodine and tetracaine on KCl-induced Ca²⁺ oscillations and contraction of airway SMCs. A and C: representative data showing that slow frequency Ca²⁺ oscillations are induced in airway SMCs in response to 50 mM KCl and that these Ca²⁺ oscillations are inhibited by 50 μM ryanodine (A) or 50 μM tetracaine (C). The baseline [Ca²⁺]_i remained elevated in response to ryanodine but not tetracaine. B and D: representative data showing effect of ryanodine and tetracaine on KCl-induced changes in airway lumen area (top traces) and unsynchronized transient SMC contractions or twitching (small white arrows in the bottom line-scan image). Tetracaine had a significant relaxant effect on KCl-induced airway contraction, whereas ryanodine did not. Both tetracaine (reversibly) and ryanodine (irreversibly) inhibited SMC twitching. The line-scan images were obtained from phase-contrast images along a SL across the airway wall and lumen. Representative data are from 4 different slices from 2 mice.

These slow frequency, unsynchronized KCl-induced Ca²⁺ oscillations resulted in the twitching of individual SMCs and, because of a lack of a coordinated contractile effort, this resulted in a minimal airway contraction (∼20% reduction of luminal area). In the presence of either ryanodine or tetracaine, the twitching of individual SMC stopped. However, the airway only relaxed in the presence of tetracaine (Fig. 12, B and D). These responses are consistent with the observation that ryanodine, but not tetracaine, induced an elevation of basal [Ca²⁺]_i after the cessation of the KCl-induced Ca²⁺ oscillations. In keeping with the irreversible binding of ryanodine to the RyR, twitching of the SMCs did not reoccur on the removal of ryanodine (Fig. 12B). By contrast, SMC twitching resumed after tetracaine removal, a result indicating a reversible weak binding to the RyR (Fig. 12D). These results demonstrate the effectiveness of ryanodine and tetracaine in blocking KCl-induced Ca²⁺ signaling.

Influence of temperature on the activity of IP₃Rs and RyRs.

To rule out the possibility that the insensitivity of the RyR to antagonists is a temperature-dependent effect, we examined the contribution of the IP₃Rs and RyRs to MCh-induced Ca²⁺ oscillations and contraction in airway SMCs at 37°C.

In response to 200 nM MCh at 37°C, the extent of airway contraction was similar to that observed at RT (Fig. 13A; ∼45%; Ref. 3). This agonist-induced contraction was associated with a similar elevation of [Ca²⁺]_i in the form of Ca²⁺ oscillations (Fig. 14A). However, the frequency of the Ca²⁺ oscillations induced by 200 nM MCh at 37°C was much higher (∼67 min⁻¹) than that at RT (∼20 min⁻¹) (3). These repetitive Ca²⁺ waves started frequently from one end of the SMC and propagated along the entire cell. The propagating velocity of the waves was 94.6 ± 6.9 μm/s (n = 5 different slices from 3 mice), much greater than that at RT (P < 0.05). The fast Ca²⁺ oscillations were clearly observed in the presence of MCh and persisted while MCh was present for >5 min.

Fig. 13.

Influence of temperature on the action of 2-APB and ryanodine. Representative responses showing the change in airway area with respect to time on exposure to 200 nM MCh and subsequently 50 and 100 μM 2-APB (A) or 1 μM (light gray line), 10 μM (gray line), or 50 μM (black line) ryanodine (C) at 37°C are shown. Summaries of the relaxation induced in airways contracted with 200 nM MCh by 50 and 100 μM 2-APB (B) or 1, 10, or 50 μM ryanodine (D) at 37°C. The extent of relaxation (percent) was measured after 10 min of 2-APB or ryanodine exposure. Each column represents the mean ± SE from ≥5 different experiments from 3 mice.

Fig. 14.

Influence of temperature on MCh-induced Ca²⁺ signaling. Representative responses showing the intracellular Ca²⁺ signaling of airway SMCs in response to 200 nM MCh (A) and subsequently 100 μM 2-APB (B) or 50 μM ryanodine (C) at 37°C are shown. MCh induced rapid (∼60 min⁻¹) and sustained Ca²⁺ oscillations. After the MCh-induced Ca²⁺ oscillations had stabilized (∼3 min), 2-APB or ryanodine was added to the lung slice. The Ca²⁺ oscillations were inhibited by 2-APB (B) but unaffected by ryanodine (C) after 3 min. Representative data are from ≥6 slices from 3 mice for each antagonist. In contrast, Ca²⁺ oscillations induced by KCl (50 mM) were rapidly stopped by 50 μM ryanodine (D), and this was associated with an increase in baseline Ca²⁺. Representative data are from ≥5 different airways from 3 mice.

When the IP₃R antagonist 2-APB was added to the MCh (200 nM)-contracted airway SMCs at 37°C, a concentration-dependent relaxation was observed (Fig. 13, A and B). A relaxation of 19.3 ± 2.1 and 46.8 ± 3.8% was induced by 50 and 100 μM 2-APB, respectively, after 10 min. In keeping with this relaxation response, 100 μM 2-APB, at 37°C, slowed the fast MCh-induced Ca²⁺ oscillations of airway SMCs from 69.3 ± 5.2 to 33.5 ± 4.3 min⁻¹ after 3 min of exposure (Fig. 14B; n = 6 different airways from 3 mice). When 2-APB was removed, the frequency of the Ca²⁺ oscillations gradually increased to the original rate. Although 2-APB reduced both MCh-induced airway contraction and Ca²⁺ oscillations at 37°C, this inhibitory action was reduced compared with its action at RT (compare Figs. 1 and 3 with Figs. 13 and 14; P < 0.05 for 50 and 100 μM 2-APB). This apparent attenuation of the inhibitory effect of 2-APB most likely results from the relative increase in the frequency of the Ca²⁺ oscillations at an equal concentration of MCh at 37°C.

When airways contracted with MCh (200 nM) at 37°C were exposed to ryanodine (1, 10, or 50 μM), airway contraction was not substantially affected by any concentration of ryanodine (Fig. 13, C and D). Similarly, 50 μM ryanodine did not significantly alter the frequency of MCh-induced Ca²⁺ oscillations (71.7 ± 6.2 min⁻¹) after 3 min (68.5 ± 5.0 min⁻¹, n = 9 different airways from 4 mice; Fig. 14C) following the propagation of >200 Ca²⁺ waves along the SMCs. This is equivalent to a period of ∼10 min at RT where the Ca²⁺ oscillations have a frequency of ∼20 min⁻¹. These results suggest the RyR does not take part in nor is influenced by agonist-induced Ca²⁺ oscillations.

However, over a longer time (>5-min exposure to 50 μM ryanodine at 37°C), the Ca²⁺ waves associated with the Ca²⁺ oscillations became less regular and did not propagate the full length of the cell. In addition, the baseline [Ca²⁺]_i was slowly elevated. These changes in the Ca²⁺ signaling were not associated with relaxation of SMCs. By contrast, 50 μM ryanodine quickly stopped KCl-induced Ca²⁺ oscillations (original frequency = 5.0 ± 0.6 min⁻¹, n = 5 slices from 3 mice at 37°C) after the propagation of 3–5 Ca²⁺ waves with the last Ca²⁺ wave occurring at 82.5 ± 8.0 s after ryanodine exposure (Fig. 14D), a response similar to that found at RT. This fast action of ryanodine on KCl-induced oscillations indicates that ryanodine interacts quickly with Ca²⁺-activated RyRs and, importantly, implies that the much delayed changes in the form of the agonist-induced Ca²⁺ waves result from an indirect action of ryanodine. The increased baseline of [Ca²⁺]_i indicates a Ca²⁺ leakage and subsequent depletion of Ca²⁺ from the SR. We suggest the most likely explanation for this is a temperature-dependent increase in the random opening of the RyR that allows ryanodine binding. Therefore, at 37°C, a faster accumulation of RyRs, in an open state can occur to drain the SR of Ca²⁺.

DISCUSSION

In this study, we examined the contribution of IP₃R and RyR to agonist-induced Ca²⁺ signaling and contraction of airway SMCs. The IP₃Rs and RyRs appear to be colocalized in SMCs (7, 39), and, as a result, these receptors may interact during SMC Ca²⁺ signaling. Consequently, pharmacological agents that selectively block either the IP₃Rs or RyRs have been frequently used to determine the role of each receptor. However, the interpretation of such experiments requires careful consideration in view of the nonspecific effects of these agents. For example, although ryanodine can increase or decrease the conductance of the RyR in a concentration-dependent manner, this action can also alter the Ca²⁺ content of the SR (4, 34, 53). As a result, the impact of ryanodine on Ca²⁺ signaling may be due to a direct action on the RyR or indirect action on IP₃R-mediated Ca²⁺ signals that use the SR Ca²⁺ store. Similarly, tetracaine, in addition to inhibiting RyR, acts as a local anesthetic and has been reported to block N-methyl-d-aspartate receptors (41) and K⁺ channels (32). Likewise, in addition to acting as a cell-permeant IP₃R antagonist, 2-APB may also inhibit SOC and SERCA activity, which has the result of increasing Ca²⁺ leakage from the SR (40, 42). In addition to nonspecific effects associated with the IP₃R antagonist XeC (16, 48), another difficulty associated with XeC is its low solubility. In previous studies, we (6) reported that the prior prolonged incubation of lung slices with XeC prevented agonist-induced Ca²⁺ signaling. However, in our short-term perfusion experiments reported here with the maximal possible concentration of XeC (20 μM), we did not observe an inhibition of MCh-induced airway contraction (data not shown). This suggests that low concentrations of antagonist are insufficient to immediately compete with high local concentration of agonist-generated IP₃. Because experiments with a long-term exposure to XeC are more susceptible to nonspecific effects, we believe short-term responses are likely to be more reliable. Heparin, another commonly used IP₃R antagonist, has the disadvantages of membrane impermeability, an action on RyRs and the ability to uncouple G protein signaling pathways (15, 20, 49). Although there is clearly no exclusive experimental drug or method, if we are going to understand airway hyperresponsiveness and how it develops in asthma, it is imperative that we understand the contribution of IP₃Rs and RyRs to airway contraction. Consequently, we have carefully examined the effects of inhibiting IP₃Rs and RyRs in response to a variety of different stimuli, including agonists, caged-IP₃, and membrane depolarization, to control for the nonspecific effects of these receptor antagonists.

We and others have shown that airway SMC contraction, induced by agonists, is mediated via Ca²⁺ oscillations (29, 47). Consequently, we initially examined the effect of IP₃R or RyR antagonists on these two basic responses. When 2-APB was applied to airways contracted with agonist, the airways displayed a concentration-dependent relaxation. This relaxation was associated with a slowing or near cessation of the Ca²⁺ oscillations. It was also noticed that the airway was not completely relaxed when a high concentration of 2-APB was applied and the intracellular Ca²⁺ signaling was significantly diminished. This phenomenon can be interpreted by the high Ca²⁺ sensitivity in the presence of agonist. Because 2-APB had no effect on Ca²⁺ sensitization, the airway could maintain a significant contraction with a small elevation of [Ca²⁺]_i. This relaxant effect is different to that of β₂-agonists that inhibit both Ca²⁺ signaling and Ca²⁺ sensitivity. By contrast, the addition of ryanodine to agonist-contracted airways had no effect on either their contractile state or the frequency of the Ca²⁺ oscillations in their SMCs. We anticipated that the velocity of the Ca²⁺ waves would either slow or break up into discrete Ca²⁺ domains (i.e., Ca²⁺ puffs) if a contribution by the RyRs was inhibited. However, the velocity or form of the propagating Ca²⁺ waves associated with the Ca²⁺ oscillations was unaffected by ryanodine. These results strongly indicate that the IP₃Rs primarily contribute to the generation and propagation of agonist-induced Ca²⁺ oscillations. This conclusion is consistent with the finding that the number of IP₃Rs exceeds that of RyRs by 10-fold in SMCs (54). In addition, under physiological conditions, [Ca²⁺]_i only has to increase by submicromolar concentrations (<1 μM) to activate the IP₃R in the presence of IP₃ (30). By contrast, RyRs often require [Ca²⁺]_i increases up to 15 μM for activation (12, 21). Therefore, the propagation of Ca²⁺ waves by CICR via sensitized IP₃Rs is likely to occur at lower thresholds of Ca²⁺.

We recognize that the noninvolvement of the RyR in agonist-induced Ca²⁺ signaling is in contrast to current ideas forwarded by other investigators from studies with airway SMCs from mouse bronchi (19), rabbit trachea (28), porcine trachea (14, 31, 44, 45), and canine (23) or human bronchi (13). Although it is appealing to attribute this variation to fundamental differences in SMCs between species or even between different airway generations, the more likely scenario is, unfortunately, the misleading, nonspecific effects of the pharmacological agents used to obtain the experimental results. To resolve this conundrum, our aim here has been to address, in detail, the known nonspecific effects of the antagonists and the implications that they raise for Ca²⁺ signaling in SMCs.

The first important response is the inhibition of the IP₃R with 2-APB. The inhibition of Ca²⁺ oscillations by 2-APB, supported by the observations with flash photolysis that 2-APB inhibited IP₃-induced Ca²⁺ release, is strong evidence for the expected action of 2-APB on the IP₃R. However, 2-APB has been reported to significantly inhibit SOC (11, 42) and thereby the replenishment of Ca²⁺ stores. We investigated this idea and found some effect of 2-APB on Ca²⁺ stores, as judged by caffeine-induced Ca²⁺ release. Therefore, to ensure that the slowing of the Ca²⁺ oscillations or the failure of IP₃ to evoke a Ca²⁺ transient was not the result of store depletion, we examined the ability of IP₃ to release Ca²⁺ from the SR under conditions that would mimic the full abolishment of SOC, i.e., zero extracellular Ca²⁺. In the absence of extracellular Ca²⁺, for an equal amount of time as 2-APB exposure, the photolytic release of IP₃ elicited a similar Ca²⁺ response, confirming that SOC inhibition was not a concern. Similarly, we considered the action of 2-APB on the depletion of the Ca²⁺ store via an inhibition of SERCA activity and ruled out this possibility by demonstrating that the Ca²⁺ store was sufficiently replenished to maintain a caffeine-induced Ca²⁺ response in presence of 2-APB. In addition, compared with our previous studies, 2-APB inhibited the Ca²⁺ oscillations more quickly than by Ca²⁺-store depletion induced by zero extracellular Ca²⁺. Consequently, these experiments collectively confirm the primary function of 2-APB as an IP₃R antagonist and emphasize the major role of IP₃R in airway SMC Ca²⁺ oscillations.

On the other side of the hypothesis, the failure of ryanodine to influence the Ca²⁺ oscillations in any way indicates that there is little involvement of RyRs in agonist-induced Ca²⁺ oscillations of mouse intrapulmonary airway SMCs. An important feature of the action of ryanodine is that it can only combine with an open RyR, a process called use dependency (21). The fact that no effects were observed with ryanodine implies that the RyRs were not activated (or open) at any time during the Ca²⁺ oscillation process. If RyRs made a major contribution, we would have expected to see similar results to those we have obtained with caffeine and ryanodine treatment of airway SMCs, i.e., the RyRs will be activated by caffeine and locked in the open state by ryanodine, which would lead to the depletion of the internal store and continuous Ca²⁺ influx. Under these conditions, the Ca²⁺ oscillations would stop, and [Ca²⁺]_i would remain at a sustained high level. If RyR made a minor contribution, this might be observed in the characteristics of the propagated Ca²⁺ waves, but again no changes were observed. We also confirmed that ryanodine had no effect on IP₃-induced Ca²⁺ release and Ca²⁺ wave propagation with the photolytic release of IP₃. Collectively, all of these results exclude the direct involvement of RyRs in IP₃-induced CICR and Ca²⁺ waves in airway SMCs. A similar conclusion was also reached in colon SMCs where localized IP₃-mediated Ca²⁺ release could not propagate as Ca²⁺ waves by RyRs in the vicinity (38).

Although we have ruled out a direct action of ryanodine on IP₃-induced Ca²⁺ signaling, it is worth emphasizing that ryanodine may indirectly inhibit IP₃R-mediated Ca²⁺ release. A prolonged exposure to ryanodine, especially at 37°C, which would enhance random channel gating, would provide sufficient time for the stochastic ”trapping“ of the RyRs in the open state (random openings of the RyR followed by ryanodine binding). This would lead to the gradual depletion of the Ca²⁺ content of SR to which IP₃Rs and RyRs have common access and the interference of IP₃-induced Ca²⁺ oscillations. This indirect inhibitory effect of ryanodine may also account for our original observations (45-min exposure time; Ref. 6) and those by others for the inhibitory effect of ryanodine on agonist-induced Ca²⁺ oscillations.

An alternative mechanism for the activation of RyRs in airway SMCs is an agonist-stimulated increase in cADPR. Acting as a functional equivalent of IP₃ for IP₃R, cADPR generated from β-NAD by ADP-ribosyl cyclase located as a cell surface receptor (CD38) is believed to facilitate the activation of RyRs via FK506-binding protein 12.6 (FKBP12.6) (17, 18, 45, 52). The cADPR/CD38 pathway of Ca²⁺ release is thought to be recruited by receptor-coupled G proteins (29), but the details are largely unknown. Although the poor membrane permeability of cADPR precluded its direct use in this study, a membrane-permeable competitive inhibitor of cADPR (8-Br-cADPR) had no effect on the MCh- or 5-HT-induced contractile responses or Ca²⁺ signaling of mouse airway SMCs in lung slices. Therefore, the contribution of cADPR-RyR pathway to agonist-induced Ca²⁺ oscillations under normal physiological conditions appears to be minimal in mouse airway SMCs.

Another means to activate RyRs is overloading the internal Ca²⁺ stores (21, 35). This condition can be experimentally induced by KCl-mediated membrane depolarization to stimulate Ca²⁺ influx via voltage-dependent Ca²⁺ channels. The consequence of a cyclic Ca²⁺ store overload coupled to RyR activation and store emptying was slow frequency Ca²⁺ oscillations (43). As might be expected, ryanodine abolished these KCl-induced slow Ca²⁺ oscillations of airway SMCs by blocking the activated RyR. It also increased the baseline [Ca²⁺]_i by eliciting additional Ca²⁺ leakage from SR via the open RyRs. The importance of this result is that it indicates that the inhibitory effects of ryanodine can be observed in lung slices and that the lack of a response to ryanodine in experiments with agonists is not the result of drug impotency. The positive inhibitory effect of ryanodine is also confirmed by its joint action with caffeine in inhibiting the RyR in the open state.

In addition to demonstrating that IP₃R is the major contributor to Ca²⁺ oscillations in mouse airway SMCs, our data also indicate that agonist-induced Ca²⁺ oscillations are qualitatively similar at RT and 37°C. Only the speed of the Ca²⁺ oscillations increased, suggesting the fundamental mechanisms underlying the Ca²⁺ oscillations are not altered by temperature, although it is likely that the kinetics of the process are changed. As a result, the immediate effects of ryanodine or 2-APB on agonist-induced Ca²⁺ oscillations were similar at RT and 37°C. This behavior is in contrast to vascular SMCs in which agonist-induced Ca²⁺ oscillations occurred at RT but changed to a uniform increase in [Ca²⁺]_i at higher temperature (32°C) in association with the development of myogenic tone in SMCs (56). The higher [Ca²⁺]_i was believed to inactivate the IP₃R.

A surprising finding in our study was the inhibitory effects of tetracaine on agonist-induced Ca²⁺ signaling. The conventional inhibitor effect of tetracaine on RyRs was confirmed in airway SMCs by the cessation of KCl-induced Ca²⁺ oscillations, although this action was relatively weak because it could be reversed with high concentrations of caffeine. More importantly, tetracaine appeared to mediate its action by altering Ca²⁺ signaling and Ca²⁺ sensitization. In other studies, tetracaine has been suggested to block IP₃-mediated Ca²⁺ release at high concentrations (1 mM) (36, 51). However, we found that 50 μM tetracaine does not affect Ca²⁺ release in response to IP₃. Therefore, we suggest the cessation of agonist-induced Ca²⁺ oscillations in presence of tetracaine results from an absence of IP₃. This idea is supported by the reinitiation of Ca²⁺ oscillations when IP₃ was made available by flash photolysis. Another nonspecific effect of tetracaine is inhibition of membrane ion channels and Ca²⁺ influx, but this appears to be minimal in airway SMCs because repetitive exposures to caffeine in the presence of tetracaine induced similar contractile and Ca²⁺ responses and had no effect on the [Ca²⁺]_i of permeabilized SMCs. Consequently, we propose that tetracaine acts at the membrane level to inhibit the receptor-coupled dual pathways that regulate the activation of PLCβ for IP₃ production and G protein-coupled activation of Rho kinase to modulate Ca²⁺ sensitization. Similar actions of tetracaine have been reported previously (26) to explain how tetracaine may act as a local anesthetic. Collectively, these results indicated that tetracaine, and other similar local anesthetic agents, such as procaine, inhibit the Ca²⁺ responses of mouse airway SMCs in a manner unrelated to the RyRs. More importantly, these conclusions emphasize that caution is required when explaining the effects of tetracaine on airway SMC physiology of other species. In conclusion, our experiments demonstrate that the IP₃R plays the major role in GPCR agonist-induced Ca²⁺ oscillations and that RyR-mediated CICR does not appear to be involved in normal physiological conditions.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute Grants HL-71930 and HL-087401 to M. J. Sanderson.