Ryanodine Receptor Sensitization Results in Abnormal Calcium Signaling in Airway Smooth Muscle Cells

Abstract

Intracellular Ca²⁺ dynamics of airway smooth muscle cells (ASMCs) are believed to play a major role in airway hyperresponsiveness and remodeling in asthma. Prior studies have underscored a prominent role for inositol 1,4,5-triphosphate (IP₃) receptors in normal agonist-induced Ca²⁺ oscillations, whereas ryanodine receptors (RyRs) appear to remain closed during such Ca²⁺ oscillations, which mediate ASMC contraction. Nevertheless, RyRs have been hypothesized to play a role in hyperresponsive Ca²⁺ signaling. This could be explained by RyRs being “sensitized” to open more frequently by certain compounds. We investigate the implications of RyR sensitization on Ca²⁺ dynamics in ASMC using a combination of mathematical modeling and experiments with mouse precision-cut lung slices. Caffeine is used to increase the sensitivity of RyRs to cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and sarcoplasmic reticulum Ca²⁺ ([Ca²⁺]_SR). In ASMCs, high caffeine concentrations (>10 mM) induce a sustained elevation of [Ca²⁺]_i. Our mathematical model accounts for this by the activation of store-operated Ca²⁺ entry that results from a large increase in the RyR sensitivity to [Ca²⁺]_SR and the associated Ca²⁺ release, which leads to a reduction of [Ca²⁺]_SR. Importantly, our model also predicts that: (1) moderate RyR sensitization induces slow Ca²⁺ oscillations, a result experimentally confirmed with low concentrations of caffeine; and (2) high RyR sensitization suppresses fast, agonist-induced Ca²⁺ oscillations by inducing substantial store-operated Ca²⁺ entry and elevated [Ca²⁺]_i. These results suggest that RyR sensitization could play a role in ASMC proliferation (by inducing slow Ca²⁺ oscillations) and in airway hyperresponsiveness (by inducing greater mean [Ca²⁺]_i for similar levels of contractile agonist).

Clinical Relevance

Our work shows that increased ryanodine receptor (RyR) sensitivity can underlie unexpected complications in airway smooth muscle cell Ca²⁺ signaling and therefore in the downstream physiology. This suggests RyR as a target for therapeutic drugs.

Intracellular Ca²⁺ dynamics control a wide variety of cellular processes. In particular, airway smooth muscle cell (ASMC) contraction is mediated by oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which correspond to the cyclic release and reuptake of Ca²⁺ from the sarcoplasmic reticulum (SR). The release of Ca²⁺ from the SR occurs via two types of channels, the inositol 1,4,5-triphosphate (IP₃) receptor (IP₃R) and the ryanodine receptor (RyR), whereas Ca²⁺ reuptake into the SR is performed by sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases (SERCAs). Ca²⁺ may also enter the cell via receptor-operated, store-operated, and voltage-operated Ca²⁺ channels, and be extruded from the cell across the plasma membrane by Ca²⁺ ATPases (PMCAs). In ASMCs, G protein–coupled receptor stimulation (by agonists) and membrane depolarization (by KCl) induce two types of Ca²⁺ oscillations (1, 2). KCl-induced Ca²⁺ oscillations have a low frequency (∼3/min) as compared with faster agonist-induced Ca²⁺ oscillations (∼10/min in human and ∼30/min in mouse at room temperature; these are even faster at 37°C). In mouse ASMCs, KCl-induced Ca²⁺ oscillations induce twitching of the ASMCs and substantially less airway contraction as compared with agonist-induced contraction, but the relative magnitudes of KCl versus agonist-induced contraction is species dependent (2, 3).

We have previously (4) proposed a mechanism for how IP₃R and RyR interact to give these two oscillatory behaviors. The pathways are summarized in Figure 1. In brief:

• •Agonist stimulation, and subsequent production of IP₃, leads to release of Ca²⁺ through the IP₃R, and a cycle of Ca²⁺ release and reuptake to and from the SR, via the mechanism of Ca²⁺-induced Ca²⁺ release. This causes sufficient depletion of the SR to inactivate the RyR, which thus play no significant role in on-going agonist-induced Ca²⁺ oscillations (see Figure E1A in the online supplement).
• •KCl stimulation causes overfilling of the SR, which activates the RyR and leads to Ca²⁺ release from the SR, again in a process of Ca²⁺-induced Ca²⁺ release (Figure E1B).
• •The IP₃R and RyR interact via their joint effects on [Ca²⁺]_i and sarcoplasmic reticulum Ca²⁺ ([Ca²⁺]_SR). The fact that RyRs become desensitized when the SR is depleted plays a crucial role in determining the relative contributions of IP₃R and RyR to the control of Ca²⁺ oscillations.

Figure 1.

Schematic diagram of the major pathways involved in the model of Ca²⁺ dynamics in airway smooth muscle cells (ASMCs). Solid black lines denote Ca²⁺ fluxes. Stimulation of cell surface G protein–coupled receptors (GPCR) leads to production of inositol 1,4,5-triphosphate (IP₃) and activation of the IP₃ receptor (IP₃R). Subsequent release of Ca²⁺ from the sarcoplasmic reticulum (SR) leads to Ca²⁺-induced Ca²⁺ release (CICR) through either IP₃R or ryanodine receptors (RyRs). Depletion of the SR induces the opening of store-operated Ca²⁺ channels (SOCCs) in the plasma membrane but also inactivates RyR. Ca²⁺ is removed from the cytoplasm by plasma membrane ATPase (PMCA) or by sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA). Addition of extracellular KCl leads to membrane depolarization and opening of voltage-operated Ca²⁺ channels (VOCCs). The consequent increase in Ca²⁺ influx causes (in the absence of agonist) overfilling of the SR and activation of RyR. IP₃R and RyR interact via their effects on the concentration of Ca²⁺ ([Ca²⁺]) in the cytosol and the SR. Here, we focus on the predicted effects of caffeine (and other compounds), which sensitizes RyR to cytosolic and luminal Ca²⁺.

As such, increasing the Ca²⁺ sensitivity of RyR (which we refer to in this work as RyR “sensitization”) could have important implications for the participation of RyRs in Ca²⁺ dynamics in hyperresponsive conditions. In addition to agonist-induced calcium signaling being intrinsically altered in asthmatic airway (e.g., Ref. 5), proinflammatory factors present in asthma enhance [Ca²⁺]_i responses of (nonasthmatic) ASMCs to agonist stimulation and/or to store depletion (6–12). There is evidence that the RyR could play a role in these augmented Ca²⁺ responses (13, 14). This role for RyR sensitivity can be explored experimentally with compounds, such as caffeine, that increase the sensitivity of RyR to [Ca²⁺]_SR and [Ca²⁺]_i (15, 16). Therefore, in this work, we integrate both IP₃R and RyR dynamics into an extension of our previous mathematical model of Ca²⁺ dynamics in ASMC (17), and experimentally test the predictions of this model. The consequences of RyR sensitization on ASMC Ca²⁺ dynamics is evaluated in the absence and presence of agonist stimulation.

Materials and Methods

Experimental Methods

Precision-cut lung slices (PCLSs) were prepared from female BALB/c mice (7–12 wk old) as described in Section E1.1 of the online supplement. All experiments were conducted at body temperature (37°C) in a custom-made, temperature-controlled microscope chamber as described in (3).

Measurement of Ca²⁺ oscillations: PCLSs were incubated in Hanks' buffered salt solution supplemented with 20 mM HEPES (sHBSS) containing 20 μM Oregon Green 488 BAPTA-1-AM (Invitrogen, Carlsbad, CA), a Ca²⁺-sensitive dye, 0.1% Pluronic F-127 (Invitrogen), and 200 μM sulfobromophthalein in the dark at 30°C for 1 hour. Subsequently, the PCLSs were incubated in 200 μM sulfobromophthalein for 30 minutes. Slices were mounted on a cover glass and held down with 200-μm nylon mesh. A smaller cover glass was placed on top of the mesh and sealed at the sides with silicone grease to facilitate solution exchange. Mounted lung slices were continuously perfused with sHBSS or sHBSS containing the required compounds. ASMC Ca²⁺ signals were examined with a custom-built two-photon scanning laser microscope with a 40× oil immersion objective (NA 1.35) and images recorded at 15 or 30 images s⁻¹. Changes in fluorescence intensity (which represent changes in [Ca²⁺]_i) were analyzed in an ASMC of interest by averaging the gray value of up to a 10 × 10-pixel region using custom written software. Relative fluorescence intensity was expressed as a ratio of the fluorescence intensity (F_t/F₀; i.e., fluorescence intensity at a particular time [F_t] normalized with respect to the initial fluorescence intensity [F₀]).

Statistical Analysis

Results on Ca²⁺ oscillation frequency are shown as mean (±SEM). Student’s t test was used to compute the 95% confidence interval of the mean (mean ±t_0.975^n − 1 SEM, where $\underset{0.975}{\overset{k}{t}}$ denotes the quantile at 97.5% of the student distribution with k degrees of freedom), and to evaluate the significance of the difference between means at different caffeine concentrations.

Mathematical Model

The model is an extension of previous work (17). In brief, the dynamics of c = [Ca²⁺]_i and c_s  = [Ca²⁺]_SR are governed by:

$$\begin{array}{l}\frac{dc}{dt}=J_{in}-J_{PMCA}+J_{rel}-J_{SERCA},\\ \frac{d{c}_s}{dt}=\gamma \left(J_{SERCA}-J_{rel}\right),\end{array}$$ (1)

where J_in models Ca²⁺ influx (principally through store-operated Ca²⁺ channels), J_PMCA and J_SERCA are the Ca²⁺ fluxes through the PMCA and SERCA pumps, respectively, and J_rel models the flux out of the SR through IP₃R and RyR. Full details of each of these fluxes are given in the online supplement (Section E1.2).

For our purposes, the most important term is J_rel, which contains a model of the RyR flux. In this flux, the open probability of the RyR is assumed to be given by:

$$P_{RyR}\left(c,c_s\right)=\frac{c^3}{K_{cR}^3+c^3}\frac{c_s^4}{K_{sR}^4+c_s^4}.$$ (2)

Thus, the RyR sensitivity to cytosolic and luminal Ca²⁺ is governed by the two parameters, K_cR and K_sR, respectively.

Results

Effect of RyR Sensitization on Resting Ca²⁺

Model predictions.

Sensitization of the RyR is obtained in the model by decreasing the cytosolic Ca²⁺ activation threshold (K_cR) or the luminal Ca²⁺ activation threshold (K_sR) from their resting values (Equation 2 and Table E1). We define the indices, cytosolic sensitivity (r_c) and luminal sensitivity (r_s), to reflect the degree of cytosolic and luminal sensitization, respectively, as follows: r_c = (K_cR0 − K_cR)/K_e and r_s = (K_sR0 − K_sR)/K_s, where K_cR0 and K_sR0 are the resting values of K_cR and K_sR in Table E1. Thus, increasing values of r_c and r_s represent increasing cytosolic and luminal sensitization, respectively, of RyR to Ca²⁺.

Figures 2A and 2B show the effect of increasing r_c and r_s, respectively, on [Ca²⁺]_i dynamics. Both moderate cytosolic and luminal sensitization of RyR induce low-frequency RyR-mediated Ca²⁺ oscillations (panels i and ii), with a frequency that increases with increased sensitization. We note that the induction of Ca²⁺ oscillations by an increase in r_s can be understood as the mirror mechanism by which KCl induces Ca²⁺ oscillations: the increase in [Ca²⁺]_SR induced by membrane depolarization, which activates RyR (Figure E1B), is effectively replaced by a decrease in the [Ca²⁺]_SR half-activation threshold, K_sR (i.e., overcoming the threshold is equivalent to lowering it sufficiently).

Figure 2.

(A) Simulations of the effect of increasing levels of cytosolic RyR sensitization (r_c) on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) dynamics, starting from (r_c, r_s) = (0, 1) (red dot in Figure E3A). (B) Simulations of the effect of increasing levels of luminal RyR sensitization (r_s) on [Ca²⁺]_i dynamics, starting from (r_c, r_s) = (0, 0) (no sensitization; black dot in Figure E3A). (C) Relative fluorescence signals indicating the [Ca²⁺]_i responses induced by caffeine in ASMCs of mouse lung slices (see Materials and Methods) for (i) 1 mM caffeine, (ii) 3 mM caffeine, and (iii) 10 mM caffeine. (D) The predicted sarcoplasmic reticulum Ca²⁺ ([Ca²⁺]_SR) associated with the [Ca²⁺]_i dynamics modeled in B. (E) The predicted fraction of open RyRs (magenta) and open SOCCs (blue) associated with the [Ca²⁺]_i dynamics in B.

At higher RyR sensitization (Figures 2A and 2B [iii]), the Ca²⁺ oscillations disappear and are replaced by a steady [Ca²⁺]_i, with one important difference: the steady [Ca²⁺]_i induced by high cytosolic RyR sensitization remains close to the resting [Ca²⁺]_i, whereas the steady [Ca²⁺]_i induced by high luminal sensitization is substantially elevated. This is worth noting for comparison with experimental observation (Ref. 1 and Figure 2C [iii]; see subsequent text). The model indicates that this difference in steady state is due to the different levels of store-operated Ca²⁺ entry (SOCE) activation. Indeed, as we have previously shown (17), a sustained increase in Ca²⁺ influx (i.e., a Ca²⁺ flux from the extracellular medium into the cytosol) is required for a sustained increase in [Ca²⁺]_i. An increase in RyR r_s, but not r_c, induces a sustained Ca²⁺ influx, because it allows RyR to remain open at lower [Ca²⁺]_SR, and thus SOCE is substantially activated. Figure 2E illustrates this mechanism: with increased RyR luminal sensitivity, the RyRs open more frequently (magenta curve), because the [Ca²⁺]_SR (Figure 2D) reaches the lower threshold for RyR activation sooner. Simultaneously, because the [Ca²⁺]_SR remains lower, a larger fraction of the store-operated Ca²⁺ channel remains open (Figure 2E, blue curve). The results of Figures 2A and 2B are summarized and extended in the online supplement (Figure E3).

Experimental validation.

Caffeine has been shown by other groups to increase RyR sensitivity to both cytosolic and luminal Ca²⁺ in artificial lipid bilayers (15, 16). In addition, in ASMCs, high caffeine concentrations (20 mM) induce a sustained, elevated [Ca²⁺]_i (1), which is a signature for luminal RyR sensitization according to our model (Figure 2B [iii]). Consequently, to validate the predictions of our model that RyR sensitization influences the Ca²⁺ dynamics of ASMCs, we used varying concentrations of caffeine to sensitize the RyR of ASMCs in mouse lung slices (Figure 2C).

Stimulation of ASMCs with 1–4 mM caffeine (at 1-mM intervals) induced low-frequency [Ca²⁺]_i oscillations (Figures 2C [i and ii]). The experimental dose–response curve for caffeine (for a total of four different caffeine concentrations) is given in Figure E4. It can be seen that the oscillation frequency increases as a function of caffeine concentration. Specifically, the mean oscillation frequency increased from 1.4 (±0.49)/min for 1 mM caffeine, to 3.5 (±1.1)/min for 3 mM caffeine, the increase between the two means being statistically significant at the 1% level. This is consistent with an increase in RyR sensitization, as shown by Figures 2A and 2B. We note, however, substantial variation in the Ca²⁺ response, shape, and frequency between cells (Figure E5). A similarly large cell variability is observed for KCl-induced Ca²⁺ oscillations (Figure E6).

By contrast, stimulation of ASMCs with higher concentrations of caffeine (>6 mM caffeine; 10 mM shown in Figure 2C [iii]) induced a steady, elevated [Ca²⁺]_i. These results are explained by the model predictions for luminal sensitization of RyR (Figure 2B [iii]), and are similar to prior results obtained with 20 mM caffeine (1). In summary, these experimental results and model predictions indicate that RyR sensitization can lead to significant changes in the Ca²⁺ dynamics of ASMCs in resting conditions.

Effect of RyR Sensitization on Agonist-Induced Ca²⁺ Oscillations

Because ASMCs use Ca²⁺ oscillations to induce contraction, and this contraction may be related to airway hypersensitivity, we used our model to explore how RyR sensitivity may alter agonist-induced Ca²⁺ oscillations. There is evidence that [Ca²⁺]_SR is reduced during agonist-induced Ca²⁺ oscillations (Ref. 4 and Figure E1A). We therefore expect that cytosolic sensitization of RyR, if not accompanied by substantial luminal sensitization, will have negligible effect on agonist-induced Ca²⁺ oscillations. This is because, in the absence of luminal sensitization, the opening probability of RyR decreases quickly with decreasing [Ca²⁺]_SR. As a consequence, we begin the discussion of our study of the effect of RyR sensitization on agonist-induced Ca²⁺ oscillations by assuming luminal RyR sensitization only, and then discuss how these results are modified if there is concomitant cytosolic RyR sensitization.

We simulate pre-existing luminal RyR sensitization by setting r_s to specific values (Figures 3A and 3B) and mimic agonist application by increasing the IP₃ concentration (p) from zero to either 0.5 (Figure 3A) or 1.5 (Figure 3B) at a t of 200 seconds. These simulations show that low or moderate luminal RyR sensitization has little effect on Ca²⁺ oscillations induced by low IP₃ concentrations (p = 0.5); a small effect on Ca²⁺ oscillation frequency is noticeable with an r_s of 3 (compare middle and bottom panels in Figure 3A; see also red curve in Figure 3C).

Figure 3.

(A and B) Effect of agonist application on ASMC Ca²⁺ dynamics with different levels of pre-existing luminal RyR sensitization (r_s; increasing from top to bottom—red to blue to green), in the absence of cytosolic RyR sensitization (r_c = 0). (A) Low [IP₃] (p = 0.5), and (B) high [IP₃] (p = 1.5), applied at t of 200 seconds (p = IP₃ concentration). (C and D) Long-term mean [Ca²⁺]_i (black) and Ca²⁺ oscillation frequency (red) as a function of luminal RyR sensitization for the two agonist levels in A and B.

By contrast, moderate luminal RyR sensitization (r_s = 3) has a greater effect on Ca²⁺ oscillations induced by higher IP₃ concentration (p = 1.5, Figure 3B). The Ca²⁺ oscillations are abolished and replaced with sustained elevated [Ca²⁺]_i (blue trace). With higher levels of luminal RyR sensitization (r_s = 4.25), the substantially elevated [Ca²⁺]_i that follows the few initial slow Ca²⁺ oscillations is not significantly affected by an increase in [IP₃] (green trace). Unfortunately, our experimental approach to test these predictions of RyR sensitization effect on agonist-induced Ca²⁺ oscillations with caffeine were confounded by the inhibitory action of caffeine on the IP₃R (18, 19) (see also Discussion). We found the frequency of agonist-induced Ca²⁺ oscillations to be smaller (instead of larger) in the presence of caffeine than in its absence (Figures E7–E9), even at high agonist concentrations (800 nM methacholine [MCh]). This outcome can be reproduced with the model by assuming that caffeine inhibits the IP₃R (e.g., decreases IP₃R conductance) in addition to increasing luminal RyR sensitization (simulations not shown).

A summary of the interaction between luminal RyR sensitization and agonist stimulation is shown in Figures 3C and 3D. The frequency of Ca²⁺ oscillations (red curve) at low or high IP₃ concentrations is relatively constant for increasing RyR sensitivity up to an r_s of 2.5. The same applies for mean [Ca²⁺]_i (dashed black curve). However, at higher RyR sensitivities (r_s > 3), the mean [Ca²⁺]_i increases gradually with increasing r_s, and Ca²⁺ oscillations are suppressed.

Figure 4 shows how Figure 3A (middle panel) and Figure 3C are modified if we assume cytosolic RyR sensitization (r_c = 0.5 or r_c = 1) in addition to luminal RyR sensitization (r_s = 3 in Figure 4A). Moderate cytosolic sensitization (r_c = 0.5) increases the frequency of agonist-induced Ca²⁺ oscillations (Figure 4A, cyan trace), whereas large cytosolic sensitization (r_c = 1) suppresses the Ca²⁺ oscillations (Figure 4A, magenta trace). Figure 4B shows that cytosolic sensitization reduces the range of luminal sensitization (r_s) in which agonist-induced Ca²⁺ oscillations persist, and renders the Ca²⁺ oscillation frequency sensitive to luminal sensitization (compare with Figure 3C).

Figure 4.

Effect of simultaneous cytosolic and luminal RyR sensitization on agonist-induced Ca²⁺ oscillations for low [IP₃] (p = 0.5). (A) Increasing levels of cytosolic sensitization (from top to bottom) with RyR luminal sensitization (r_s = 3; top panel is identical to middle panel in Figure 3A). (B) Long-term mean [Ca²⁺]_i (black curves) and oscillation frequency (red curve) as a function of luminal RyR sensitization for the two non-zero sensitization levels used in A (compare with Figure 3C).

Figure 5 underscores the important implications of our model findings by showing how the mean [Ca²⁺]_i as a function of [IP₃] is modified by increasing RyR sensitization. For any given agonist concentration (dotted vertical lines represent p = 0.5 and 1.5, used in Figure 3), the mean [Ca²⁺]_i is always higher in the presence of RyR sensitization; that is, the greater the RyR sensitization, the greater the mean [Ca²⁺]_i. These response curves also show that the range of IP₃ concentrations that can induce Ca²⁺ oscillations (dashed lines) shrinks and disappears as luminal RyR sensitization is increased. In particular, the overlap between Ca²⁺ oscillations for an r_s of 0 (dashed blue curve) and steady Ca²⁺ for an r_s of 3 (solid red curve) over a large range of [IP₃] (p = 0.8–2.3 for r_c = 0) implies that moderate changes in RyR sensitivity can have a large impact on the dynamics of ASMC Ca²⁺ signaling. In summary, increasing luminal RyR sensitization progressively impairs agonist-induced Ca²⁺ oscillations and increases mean [Ca²⁺]_i. A simultaneous increase in cytosolic RyR sensitization accelerates the suppression of the Ca²⁺ oscillations, but does not induce an additional increase in mean [Ca²⁺]_i at high IP₃ concentrations, where [Ca²⁺]_i is already steady (compare blue and magenta curves).

Figure 5.

Effect of different levels of RyR sensitization (increasing as indicated by the arrow) on the entire dose–response curve relating mean [Ca²⁺]_i to [IP₃] during agonist stimulation. Dashed curves correspond to Ca²⁺ oscillations and solid curves to steady Ca²⁺. Dotted vertical lines indicate the [IP₃] values used in Figures 3A and 3B. The bottom (red) curve is the dose–response curve in the absence of sensitization; hence, it corresponds to the response curve in Figure E2A. Gray curves represent unstable solutions.

Discussion

In this work, we have investigated the effect of RyR sensitization on Ca²⁺ dynamics in ASMCs using a dual approach of mathematical modeling and experimental observation of ASMCs in mouse lung slices. Our objective was to use our model to explore the hypothesis that an increase in RyR sensitivity, which may result from on-going airway inflammation, will alter the basic Ca²⁺ dynamics of ASMCs and their responses to contractile agonists. Any changes in the Ca²⁺ dynamics of ASMCs would be expected to have consequences for ASMC proliferation and airway hyperresponsiveness that are typical of asthma.

A key advance in our model of the Ca²⁺ dynamics of ASMCs that allows us to explore the effect of RyR sensitization is the incorporation of SOCE and the interaction of the luminal Ca²⁺ concentration of the SR with RyR. The regulation of SOCE is determined by [Ca²⁺]_SR, which, in turn, is highly dependent on the open probability of the RyR, which is itself dependent on [Ca²⁺]_SR. Agonists that produce IP₃ cause Ca²⁺ release from the SR, a gradual decrease of [Ca²⁺]_SR and gradual buildup of SOCE. KCl, on the other hand, causes increased Ca²⁺ influx, overloading of the SR, and release of Ca²⁺ through activated RyR. In this latter case, the SR is overloaded, and so SOCE does not occur; even the Ca²⁺ spikes do not deplete the SR enough to cause significant SOCE. Caffeine, however, enables RyR activation in the absence of SR overloading. Hence, the Ca²⁺ spikes induced by caffeine may deplete the SR sufficiently to result in increased SOCE. Thus, although caffeine and KCl both induce RyR opening, they have quite different effects on SOCE.

It is also important to note that, in our model, SOCE acts like a low-pass filter, responding not to each individual Ca²⁺ spike, but to mean levels of SR depletion or overfilling. Thus, individual Ca²⁺ spikes do not activate spikes of Ca²⁺ entry through store-operated Ca²⁺.

The idea that the RyR may be involved in on-going inflammatory processes and may influence airway responsiveness has been previously suggested. For example, the cytokines, TNF-α and IL-13, were reported to increase the [Ca²⁺]_i responses of ASMC to contractile agonists, a mechanism that correlates with activation of CD38, the production of cyclic ADP-ribose, and the modification of the RyR (8, 12). Experimental results obtained with β-escin–permeabilized ASMCs suggest that cyclic ADP-ribose has a direct effect on RyR (13).

RyR Sensitization Stimulates Slow Ca²⁺ Oscillations

The first prediction of the model that is experimentally confirmed is that small increases in cytosolic and/or luminal RyR sensitivity result in low-frequency Ca²⁺ oscillations. Although the consequences of these Ca²⁺ oscillations for ASMC physiology are not currently known, we propose two possibilities. The first and most obvious would be an increase in ASMC tone. However, in mouse airways, low-frequency Ca²⁺ oscillations do not induce substantial contraction; each ASMC is observed to twitch asynchronously. This is a result of the combination of a low sensitivity of mouse ASMCs to Ca²⁺ and the short duration of myosin light-chain kinase activation. By contrast, other species, including human, show greater contraction to low-frequency Ca²⁺ oscillations. The second, but less obvious, consequence of low-frequency oscillations could be a change in gene expression. In previous studies, Ca²⁺ oscillations were generated with the repetitive photolytic release of IP₃ (20) or the alternate application of calcium and calcium-free solutions (21) to demonstrate that gene expression was initiated by low-frequency Ca²⁺ oscillations. Ca²⁺ oscillations generated by IP₃ at 1/min stimulated more gene expression than at 0.5/min or 2/min or by a sustained plateau (20). The effectiveness of Ca²⁺ oscillations with a period of over 100 seconds declined when using alternating Ca²⁺ solutions (21). In a similar manner, Ca²⁺ oscillations may also affect enzyme activity as part of a frequency-modulated control system (22). Thus, the stimulation of Ca²⁺ oscillations may have significant effects on the basic phenotype of the ASMCs. The idea that slow Ca²⁺ oscillations are associated with asthma is supported by the finding of spontaneous Ca²⁺ oscillations in human ASMCs from biopsies (23).

RyR Sensitization Increases Mean [Ca²⁺]_i

A second major prediction of the model is that moderate or high luminal RyR sensitization results in higher mean (or elevated) [Ca²⁺]_i when the ASMCs are exposed to any concentration of contractile agonist. This prediction is consistent, irrespective of whether luminal RyR sensitization is moderate, and does not abolish the agonist-induced Ca²⁺ oscillations, or whether the RyR sensitization is high and converts the Ca²⁺ oscillations into a sustained [Ca²⁺]_i elevation. Cytosolic RyR sensitization can enhance the effect of luminal RyR sensitization on mean [Ca²⁺]_i when luminal sensitization is moderate and not capable by itself of abolishing agonist-induced Ca²⁺ oscillations. The immediate implication of a higher mean [Ca²⁺]_i is that, in the presence of agonist, RyR sensitization could generate greater ASMC contraction. This, in turn, suggests a possible mechanism for airway hyperresponsiveness.

The concept that increased (sustained) mean [Ca²⁺]_i results in increased contraction is demonstrated in Ca²⁺-permeabilized human lung slices (2), where, for a given agonist concentration, airway contraction increases with constant [Ca²⁺]_i imposed. Thus, with RyR sensitization, the mean [Ca²⁺]_i would be higher than normal, resulting in additional contraction.

Another potential consequence of the replacement of agonist-induced Ca²⁺ oscillations with a sustained elevation in [Ca²⁺]_i is stimulation of Ca²⁺-dependent mechanisms that have slow response kinetics. It is already well known that the frequency and shape of Ca²⁺ oscillations are important controllers of contraction (24). However, there are potentially many other ways in which the frequency of the Ca²⁺ oscillations can control cellular processes; in the presence of RyR sensitization, recurrent exposure to agonist is likely to stimulate the ASMCs in ways other than simple contraction.

Unfortunately, however, we were unable to test this second prediction. Although caffeine is known to increase the sensitivity of RyR to luminal Ca²⁺, it also inhibits the IP₃R. It is, thus, not possible to test the effects of RyR sensitization in the absence of other confounding effects. Experimental results are shown in Figures E7–E9. Addition of MCh and caffeine results in a plateau of increased Ca²⁺, but this plateau has superimposed slow oscillations, a result that does not agree with our model predictions. It is possible to modify our model to include the potential effects of caffeine inhibition of the IP₃R, and simulations with such a modified model display much better agreement with the experimental results shown in Figures E7–E9. However, such modifications are speculative and outside the scope of the present study. It is left to future work to develop, more rigorously, a model of caffeine inhibition of IP₃R.

Given the impossibility of testing the effect of RyR sensitization on agonist-induced Ca²⁺ oscillations with caffeine, due to its inhibitory effect on the IP₃R, we have looked for other drugs that could possibly sensitize RyR without inhibiting IP₃R. We found that pentifylline induces slow Ca²⁺ oscillations similar to those induced by caffeine (Figures E10 and E11), but it is not clear whether pentifylline does so via luminal sensitization of RyR (which is necessary to prevent RyR inhibition during agonist-induced Ca²⁺ oscillations) or via another mechanism.

However, like caffeine, pentifylline impairs agonist-induced Ca²⁺ oscillations (Figures E12 and E13); this could be due to an inhibition of IP₃R, similar to that induced by caffeine, or possibly to the absence of luminal RyR sensitization. We have also tried the drug, chlorocresol (25), but it did not induce Ca²⁺ oscillations.

Limitations of the Model

In this work, we have modeled ASMC Ca²⁺ dynamics in a deterministic and homogenous manner, although Ca²⁺ channels operate stochastically, and are not distributed homogeneously on the SR membrane. The extent to which this stochasticity translates into “macroscopic” (cell-level) Ca²⁺ dynamics depends on the cell type, the Ca²⁺ channels involved, and the level of stimulation (e.g., agonist concentration). The spatial distribution of Ca²⁺ channels on the SR membrane could also be modified in disease. In ASMCs, as well as in other cell types, the distribution of the occurrence of Ca²⁺ spikes during agonist-induced Ca²⁺ oscillations is well described by a Poisson process, characterized by a linear dependence between the SD and the mean of the interspike interval (26). However, the main properties of these stochastic Ca²⁺ oscillations can be predicted by a deterministic mathematical model (27). Hence, the stochastic nature of the process does not preclude the value of a deterministic description of Ca²⁺ dynamics as used in this study.

The mathematical model used here was originally designed to account for agonist-induced Ca²⁺oscillations and SOCE in ASMCs of human lung slices at room temperature. These Ca²⁺ oscillations are much slower than Ca²⁺ oscillations in mice (10/min versus 30/min). The model is therefore qualitative in this respect. Similarly, the RyR-mediated Ca²⁺ oscillations have a profile that is not fully reproduced by the model. Some differences in the Ca²⁺ profile of the model and the data are likely the result of the affnity of Oregon Green BAPTA-1-AM (OGB) that was used to report changes in Ca²⁺ (K_d ∼ 0.2 μM). This reporter is saturated at [Ca²⁺] above 1 μM. Hence, peak amplitudes of Ca²⁺ oscillations are unlikely to be reproduced in the fluorescent signal. Similarly, the kinetics of OGB may not be fast enough to track the rapid changes in [Ca²⁺]_i. This could explain the elevated fluorescent baseline observed during agonist-induced Ca²⁺ oscillations, which is not apparent during RyR-mediated Ca²⁺ oscillations.

However, our model accounts for a key feature of Ca²⁺ oscillation frequencies in ASMCs; that is, IP₃R-mediated Ca²⁺ oscillations are faster than RyR-mediated Ca²⁺ oscillations. This is because IP₃R gating is mainly governed by inhibition of the receptor by [Ca²⁺]_i, whereas RyR gating is essentially governed by the level of Ca²⁺ store depletion. Because the refilling of the SR with Ca²⁺ occurs on a longer timescale than recovery of IP₃R from inhibition, RyR-mediated oscillations have a lower frequency than IP₃R-mediated oscillations. The frequencies of these two types of oscillations are closer in human ASMCs (10/min [IP₃R] versus 3/min [RyR]) than in mouse ASMC (30/min [IP₃R] versus 3/min [RyR]), which suggests that the effect of RyR sensitization on agonist-induced Ca²⁺ oscillations could be larger in human ASMCs (because oscillations of closer frequency are more likely to interact).

Scope of the Experimental Results

As mentioned previously, we have used mouse ASMCs in this study. These exhibit faster IP₃R-mediated Ca²⁺ oscillations than human ASMCs upon agonist stimulation, but both species exhibit maximum contraction (∼55% in both cases) at the highest end of the frequency spectrum (e.g., figure 6 in Ref. 28). Hence, the two species respond differently to a given Ca²⁺ oscillation frequency. Our predictions are, however, independent of this quantitative difference, because, in both cases, we expect RyR sensitization–induced Ca²⁺ oscillations to enhance agonist-induced Ca²⁺ oscillations; the size of the effect will, of course, depend on the species, but the prediction is qualitatively the same.

The key observation, that increased RyR sensitivity can underlie unexpected complications in ASMC Ca²⁺ signaling and downstream physiology, suggests RyR as a target for therapeutic drugs. However, the exact nature of such a drug is diffcult to predict, as the mechanism of luminal Ca²⁺ sensitivity is not well understood. Compounds that could prevent Ca²⁺ binding to RyR would serve to reduce its Ca²⁺ sensitivity. This might take the form of a Ca²⁺ buffer within the SR; the protein, calsequestrin, serves such a process in the SR. Because the SR Ca²⁺ content is a key parameter, drugs reducing SERCA pump activity or SOCE may also counteract increased RyR sensitivity by helping to maintain reduced SR Ca²⁺ content.