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Regulation of Airway Smooth Muscle Cell Contractility by Ca²⁺ Signaling and Sensitivity

¹ Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts

Correspondence and requests for reprints should be addressed to Michael J. Sanderson, Ph.D., Department of Physiology University of Massachusetts Medical School Worcester, MA. 01655. E-mail: michael.sanderson{at}umassmed.edu

ABSTRACT

Airway smooth muscle cell contraction is regulated by changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the responsiveness of the airway smooth muscle cell to this Ca²⁺. The mechanism controlling [Ca²⁺]_i primarily involves agonist-induced release of Ca²⁺ from internal stores to generate Ca²⁺ oscillations. The extent of contraction correlates with the persistence and frequency of these Ca²⁺ oscillations. The maintenance of the Ca²⁺ oscillations requires Ca²⁺ influx, but membrane depolarization appears to have a minor role in initiating or sustaining contraction. Contraction also requires agonist-induced Ca²⁺ sensitization, which is mediated mainly by decreases in myosin light-chain phosphatase activity. Although it is not clear if airway hyperresponsiveness associated with asthma results from the specific modulation of these Ca²⁺-based regulatory mechanisms, bronchodilators relax airways by both attenuating the Ca²⁺ oscillations and by decreasing the Ca²⁺ sensitivity.

Key Words: asthma • hyperresponsiveness • Ca²⁺ oscillations • relaxation

Airway hyperresponsiveness (AHR), the excessive contraction of airway smooth muscle cells (SMCs), is characteristic of asthma but is poorly understood. Although asthma involves inflammation, SMC hypertrophy, and hyperplasia, the primary event leading to AHR is the stimulation of SMC contraction. This commonly involves an extracellular agonist or messenger that acts via receptor molecules to initiate a cascade of intracellular events. These events culminate in an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and/or an increase in sensitivity to Ca²⁺ to stimulate force production (1–7).

ANTAGONISTIC REGULATION OF SMC CONTRACTION

At the molecular level, airway SMC contraction is fundamentally regulated by the antagonistic activities of myosin light-chain (MLC) kinase (MLCK) and MLC phosphatase (MLCP) (Figure 1). The net activity of these enzymes determines the phosphorylation state and, thereby, the activity of the regulatory MLC (rMLC). This, in turn, determines the extent of actin–myosin cross-bridge interactions and force production. The regulation of MLCK activity is generally believed to be a function of the [Ca²⁺]_i, although MLCK activity can also be increased by Ca²⁺-independent processes. By contrast, the regulation of MLCP activity has generally been considered independent of [Ca²⁺]_i, but is determined by the complex interactions and activities of a variety of other kinases or phosphatases. This separation of the regulatory mechanisms of MLCP activity from the Ca²⁺ regulation of MLCK means that changes in SMC contraction can occur independently of changes in Ca²⁺ concentration, and this has been referred to as Ca²⁺ sensitivity. However, a broader definition of Ca²⁺ sensitivity is required, because changes in the contractile force at similar Ca²⁺ concentrations can also occur by mechanisms that do not alter the phosphorylation of the rMLC. Because both the Ca²⁺ signaling and Ca²⁺ sensitivity of SMCs are receptor-mediated processes, an alteration of SMC tone in disease may occur by either or both mechanisms in response to a variety of putative or inflammatory signaling molecules (Figure 1). Consequently, the relative contribution and mechanisms of the Ca²⁺ signaling and Ca²⁺ sensitivity of SMCs must be understood in detail.

View larger version (12K): [in this window] [in a new window]   Figure 1. An overview of the major interacting mechanisms that regulate airway smooth muscle contraction via changes in Ca2+ signaling or sensitivity. A relative control is exerted at several different scales. At the molecular level, contractile force is determined by the relative activity of myosin light-chain (MLC) kinase and MLC phosphatase, which are, in turn, dependent on the Ca2+ oscillations and the Ca2+ sensitivity. At the intracellular level, the properties of the Ca2+ oscillations are determined by the relative activities of the inositol trisphosphate receptor and ryanodine receptors and Ca2+ fluxes, whereas the Ca2+ sensitivity is influenced by Rho kinase, protein kinase C, cAMP- or cGMP-dependent kinases, or by phosphorylation-independent alterations of actin–myosin interactions. At the extracellular level, local agonists determine the intracellular response and, at the tissue level, a variety of stimuli are translated into cellular responses. Antagonistic cross-talk occurs to enhance the positive pathway. Dashed arrows, negative effect. 5-HT = 5-hydroxytryptamine; ACh = acetylcholine; β2 = beta 2 receptor; DAG = diacylglycerol; ET = endothelin; IP3 = inositol trisphosphate; IP3R = IP3 receptor; MLCK = MLC kinase; MLCP = MLC phosphatase; NO = nitric oxide; PK = protein kinase; rMLC = regulatory MLC; rMLC–P = rMLC–phosphorylation; RyR = ryanodine receptor; SR = sarcoplasmic reticulum.

Although it has been established that SMC contraction usually requires an increase in [Ca²⁺]_i, our understanding of the nature of this Ca²⁺ increase has improved with time. Initially, agonist-induced Ca²⁺ increases were observed to occur as a whole-cell transient in Ca²⁺ followed by a lower, sustained plateau of Ca²⁺. However, more recently, with higher resolution microscopy, these Ca²⁺ increases have been observed to occur as a series of Ca²⁺ oscillations in airway SMCs of many species, including mouse, pig, and human (7–11). In addition, these Ca²⁺ oscillations have dynamic spatial characteristics, and commonly occur as Ca²⁺ waves that propagate along the full length of the SMCs. Interestingly, the origination site of the Ca²⁺ oscillations is often localized to one end of the cell. Ca²⁺ oscillations have also been frequently observed in systemic blood vessels (8), and we have observed them in intrapulmonary arterioles (9). Importantly, the increasing frequency of these Ca²⁺ oscillations correlates with an increasing agonist concentration and SMC contraction, but the amplitude of the Ca²⁺ oscillations generally remains similar.

In airway and vascular SMCs, agonists initiate, but cannot maintain, contraction in Ca²⁺-free conditions. This response correlates with the initiation, but subsequent run-down, of the Ca²⁺ oscillations, and indicates that Ca²⁺ oscillations are mediated by repetitive release of Ca²⁺ from internal stores that require refilling by Ca²⁺ influx (7, 10–12). The mechanisms by which Ca²⁺ is repetitively released appear to vary between SMCs of different species or from different locations within the airway. However, by understanding and reconciling these variations, the importance of changes in this form of signaling can be evaluated in disease. There are two basic release mechanisms, centered on either the inositol trisphosphate (IP₃) receptor (IP₃R) or the ryanodine receptor (RyR) with Ca²⁺-induced Ca²⁺ release (CICR) occurring via these receptors.

IP₃R-BASED MECHANISMS

One mechanism for Ca²⁺ oscillations, commonly believed to occur in many other cell types, is the activation and sensitization of the IP₃R by the binding of IP₃ (13) (Figure 1). The binding of IP₃ opens the IP₃R to allow a Ca²⁺ efflux from the sarcoplasmic reticulum (SR). The resulting increase in [Ca²⁺]_i promotes the binding of Ca²⁺ to the IP₃Rs, and this enhances their open probability with the result that the [Ca²⁺]_i continues to rise, which, in turn, initiates the propagation of a Ca²⁺ wave by stimulating neighboring IP₃R. However, the additional binding of Ca²⁺ to a second site on the IP₃R reduces its open probability, and this, together with a localized decrease in [Ca²⁺] of the SR, leads to the termination of the Ca²⁺ release. Early evidence for IP₃-based Ca²⁺ oscillations was found in porcine tracheal SMCs (14, 15). Acetylcholine (ACh)-induced Ca²⁺ oscillations, detected, for the most part, by Ca²⁺-dependent Cl^– currents, were inhibited by antibodies to the IP₃R and by the IP₃R antagonist, heparin.

Although Ca²⁺ oscillations are formed by the repetition of this release process, there are a number of schemes that contribute to or regulate the interspike period or oscillation frequency. Because the SR forms the Ca²⁺ reservoir to drive the Ca²⁺ efflux, each Ca²⁺ oscillation reduces the Ca²⁺ content of the SR. Consequently, the time taken to refill the Ca²⁺ store to reprime the SR can limit the Ca²⁺ oscillation frequency (16). This period is primarily a function of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump rates and the amount of Ca²⁺ available for sequestration. Some Ca²⁺ is always lost to the extracellular space as a result of the plasma membrane Ca²⁺ ATPase and Na⁺/Ca²⁺ exchanger. Consequently the Ca²⁺ influx or replenishment mechanisms (see subsequent text) will also contribute to the Ca²⁺ oscillation frequency. Although refilling the SR with Ca²⁺ reestablishes the Ca²⁺ driving force, the SR [Ca²⁺] may also be critical for the regulation of the SR release channel.

In the above scenario, it is implied that the IP₃ concentration is stable. However, in some cells, the activity of phospholipase C(PLC), the enzyme that generates IP₃, is Ca²⁺ sensitive. Consequently, the IP₃ concentration may also oscillate along with the changes in Ca²⁺ to perpetuate the Ca²⁺ oscillations. The feedback mechanisms driving Ca²⁺ oscillations can be distinguished by examining the effect of the fast release (by photolysis) of a bolus of IP₃ (17). In cells displaying Ca²⁺ oscillations, an increase in IP₃ is predicted to increase the frequency of the Ca²⁺ oscillations that rely on the dynamics of Ca²⁺ inhibition of the IP₃R, but to delay the onset of the next Ca²⁺ oscillation that relies on the dynamics of the IP₃ concentration. In airway SMCs, the release of IP₃ increased the frequency of the Ca²⁺ oscillations, and this implies that these oscillations occur by Ca²⁺ feedback dynamics. This is also consistent with the fact that the frequency of Ca²⁺ oscillations mediated by Ca⁺-induced IP₃ production (found in pancreatic cells) are relatively slow (1/min) compared with those of airway SMCs (20/min; induced by ACh) (17). Further evidence for Ca²⁺ oscillations mediated by IP₃ comes from the findings that the photolytic release of IP₃ in airway SMCs, in the absence of agonist, induces both Ca²⁺ oscillations and contraction. In addition, agonist-induced Ca²⁺ oscillations in SMCs of lung slices persist in the presence of ryanodine (18), and are inhibited by 2-aminoethoxydiphenyl borate (2-APB), an antagonist of the IP₃R.

RyR-BASED MECHANISMS

In an alternative scheme, Ca²⁺ oscillations are proposed to be mainly mediated by CICR via the RyR. This idea was proposed because ACh-induced Ca²⁺ oscillations in isolated porcine tracheal SMCs were found to be slowed by ryanodine and inhibited by caffeine, both RyR antagonists (11). A subsequent study with permeabilized cells indicated that sustained Ca²⁺ increases could be induced by IP₃ and that ACh-induced Ca²⁺ oscillations were slowed by heparin, an IP₃R antagonist. However, the same ACh-induced Ca²⁺ oscillations were abolished by ryanodine and ruthenium red (19), another RyR antagonist. Consequently, although there appears to be a requirement for IP₃ in the initiation of the Ca²⁺ oscillations, the maintenance of the Ca²⁺ oscillations appears to require the RyR (19). A similar inhibition of ACh-induced Ca²⁺ oscillations by ryanodine was found in muscle bundles from porcine or human airways (12, 20). Carbachol-induced contraction of mouse tracheal rings was also decreased by ryanodine, but the role of Ca²⁺ oscillations was not addressed (21). Although ryanodine inhibits RyR activity, it may also serve to empty the internal Ca²⁺ stores and thereby affect the IP₃-induced Ca²⁺ oscillations. Other inhibitors of the RyR, such as procaine and tetracaine, also stopped the Ca²⁺ oscillations, but the use of these compounds on whole cells is not compelling, due to their nonspecific actions. A role for IP₃R-based Ca²⁺ oscillations in airway SMCs was argued against by the failure of 2-APB and xestospongin-C (IP₃R antagonists) to inhibit the Ca²⁺ oscillations, especially because 2-APB inhibited phenylephrine-induced Ca²⁺ oscillations in SMCs of the vena cava (22). In view of these data and those examining the role of the IP₃R, it is perplexing that the results of very similar experiments are contrary. In many other cell types, Ca²⁺ oscillations are thought to occur via the IP₃R; therefore, it is essential to understand these differences to fully explain Ca²⁺ oscillations in airway SMCs.

CYCLIC ADENOSINE DIPHOSPHATE RIBOSE AS A SECOND MESSENGER

Based on the concept that IP₃ sensitizes the IP₃R to CICR, and the idea that the RyR underlies Ca²⁺ oscillations in airway SMCs, it has been envisioned that the RyR may be sensitized with the second messenger cyclic ADP-ribose (cADPr) to facilitate the Ca²⁺ oscillations. cADPr was originally found in sea urchin eggs, where it released Ca²⁺ from internal stores via the RyR. A similar Ca²⁺ release in response to exogenous cADPr has been reported in permeabilized porcine tracheal SMCs (23). This response was blocked by RyR inhibitors, but not by IP₃R inhibitors. Interestingly, cADPr was unable to induce Ca²⁺ oscillations alone, but increasing concentrations of cADPr enhanced the frequency of ACh-induced Ca²⁺ oscillations (23). Whereas heparin slowed ACh-induced Ca²⁺ oscillations, 8-bromo-cADPr, an antagonist of cADPr, inhibited ACh-induced Ca²⁺ oscillations. In these cells, IP₃ was also found to release Ca²⁺, but did not induce Ca²⁺ oscillations. However, this is consistent with the facts that the concentration of IP₃ used was relatively high and that high concentrations of IP₃ generally induced plateaus in Ca²⁺ concentrations rather than Ca²⁺ oscillations. An action for cADPr was also found in isolated equine SMCs, where it increased the frequency of spontaneous transient inward currents (usually an indicator of Ca²⁺ sparks). This response was both mimicked and antagonized by tacrolimus (or FK506, a compound that binds RyR-associated proteins, FK binding protein [FKBP]) and lost in FKBP12.6 null (^–/–) mice (24). The response to FK506 was insensitive to heparin, but was abolished by ruthenium red, which implicates the RyR. However, ACh still evoked Ca²⁺ changes in the presence of ruthenium red, suggesting that the IP₃ signaling pathway remained viable. The effects of FK506 appeared to be mediated through the RyR₂, because the RyR₁ and RyR₃ were not abundant (24), although all three RyRs have been shown to be expressed in mouse SMCs (21). In summary, the above studies indicate that ACh-induced Ca²⁺ changes or oscillations may require both IP₃ and cADPr. The IP₃R may initiate the Ca²⁺ oscillations and determine their baseline characteristics, but, in the presence of cADPr, the RyR may amplify or modify the dynamics of the Ca²⁺ response.

The possible involvement of cADPr in Ca²⁺ release of airway SMCs has stimulated a number of studies investigating the relationship between cADPr, its synthesis by the ectoenzyme, CD38, and AHR (25–27). The enzymes necessary for the conversion of nicotinamide adenine dinucleotide to cADPr (mainly, CD38) were found to be present on the membranes of airway SMCs (28). Moreover, the activity and quantity of this enzyme appears to be upregulated by a variety of cytokines that are associated with inflammation (29–31). In cultured human airway SMCs, tumor necrosis factor-, IL-1β, and IFN- increased CD38 expression and ADP-ribosyl cyclase activity. Similarly, IL-13 increased CD38 expression (30). In addition, extracellular cADPr (28) and a variety of cytokines (29, 30, 32, 33) have been reported to augment Ca²⁺ signals and contraction of airway SMCs. However, the effect of these treatments on the Ca²⁺ signaling is not easily interpreted. Although the transient peak response or net response (peak – baseline) to a variety of agonists (ACh, bradykinin, thrombin, histamine) was increased, the sustained response to the agonists appeared similar (29, 30). In these studies, it is not clear if the SMCs displayed Ca²⁺ oscillations, but the net increases appeared to be attenuated by 8-bromo-cADPr. In view of the more recent data that indicate that contraction is related to the sustained frequency of the Ca²⁺ oscillations, the relevance of the net change is unclear.

The idea that cytokines can alter airway responsiveness via CD38 expression has been further supported by studies with CD38-deficient mice (26, 34). In CD38^–/– mice, the airway resistance and lung compliance in responses to methacholine (MCh) was reported to be lower than the changes invoked in control mice. These attenuated whole-lung response correlated with a net or integrated decrease in the Ca²⁺ responses of isolated tracheal SMCs to ACh or endothelin (ET). Although the SMCs displayed Ca²⁺ oscillations in response to ACh and ET, it was reported that there was no difference in the oscillatory behavior between the control and CD38^–/– mice. In addition, the Ca²⁺ responses of the CD38^–/– mice were unaffected by 8-bromo-cADPr. These responses suggested that cADPr production is either not required for, or modulates, ACh-induced Ca²⁺ oscillation in mice. On the other hand, cADPr did appear to influence the initial peak Ca²⁺ response, but, again, it is not clear how this transient Ca²⁺ response relates to sustained SMC contraction implied by changes in airway resistance measurements. The idea that cADPr contributes to airway responsiveness is also implicated in CD38^–/– mice that are subjected to IL-13 exposure. In these mice, IL-13 did not induce the expected increase in sensitivity to MCh normally found in IL-13–exposed control mice, even though the lungs showed similar signs of inflammation (26).

Another important requirement of a second messenger is its production in response to agonists. Consequently, the finding that cADPr levels in porcine SMCs were substantially increased within 1 minute of exposure to the contractile agonists, ACh, bradykinin, ET, and histamine, supports its role in regulating contraction (35). Importantly, the increase in cADPr was sustained (at least for the periods of measurement: 5 min), although a proportional increase related to the concentration of the stimulating agonists or the receptor type was not clearly evident.

The above studies form a consensus that CD38 expression can be increased by inflammation and that this inflammation appears to augment SMC Ca²⁺ signaling in a cADPr-dependent manner; however, the details of these relationships are not clear. The studies have been conducted with human, porcine, and mouse tissues and isolated cells, and this gives rise to inconsistent results and makes the correlation of contraction and Ca²⁺ signaling difficult. Recently, two approaches for examining and correlating cell and tissue responses have been reported using either lung slices, which are compatible for mouse, rat, and hamster tissues, or tracheal strips, which are useful for large airway SMCs from porcine or human tissue. It is likely that, with these techniques, the inferences that cADPr acts via RyRs to modulate agonist-induced Ca²⁺ oscillations and airway SMC contraction can be directly demonstrated.

ELEMENTAL Ca²⁺ SIGNALING

Although Ca²⁺ waves arise from the sequential release of Ca²⁺ via individual receptor-channels, the independent activity of these receptors or elemental Ca²⁺ signaling (36) has been implicated in the regulation of SMC contraction. Elemental Ca²⁺ signals are usually mediated by small groups of receptors, and are termed Ca²⁺ sparks or puffs if predominantly mediated by RyR or IP₃Rs, respectively. Ca²⁺ sparks were initially observed in cardiac cells, whereas Ca²⁺ puffs have been well documented in oocytes. Ca²⁺ sparks often appear to occur spontaneously or in response to low concentrations of caffeine, whereas Ca²⁺ puffs are usually invoked by low concentrations of agonist or cytosolic IP₃.

The correlation of Ca²⁺ sparks with spontaneous transient outward currents mediated by Ca²⁺-activated K⁺ channels has lead to the idea, mainly in systemic vascular SMCs, that Ca²⁺ sparks relax SMC by hyperpolarizing the cell membrane to reduce Ca²⁺ entry (37). This hypothesis may also apply to airway SMCs after the demonstration of Ca²⁺ sparks and spontaneous transient outward current in isolated SMCs in combination with evidence for Ca²⁺ signals mediated via RyR (38, 39).

The idea that airway SMCs are relaxed by hyperpolarization implies that SMC contraction requires membrane depolarization and an associated Ca²⁺ influx, presumably via voltage-gated Ca²⁺ channels. In many studies, KCl has been used to depolarize and contract airway SMCs (40). Because this response is abolished or reduced by the absence of extracellular Ca²⁺ or inhibitors of voltage-gated Ca²⁺ channels in isolated cells (11, 40) and lung slices (10), airway SMC contraction mediated by Ca²⁺ influx appears feasible.

On the other hand, the use of L-type channel blockers to treat AHR in asthma has been without success. Extensive reviews of this concept (6, 41) emphasize that the extent of depolarization of airway SMCs induced by agonists is inadequate to fully activate voltage-gated Ca²⁺ channels to exploit this signaling mechanism. In support of this viewpoint, airway SMCs display strong contraction in response to a wide variety of agonists that induce Ca²⁺ oscillations that are primarily dependent on intracellular Ca²⁺ release. Furthermore, this agonist-induced airway contraction is significantly larger and more stable than KCl-induced contraction in small airways of lung slices. The Ca²⁺ oscillations persist for multiple cycles in the absence of extracellular Ca²⁺ or in the presence of Ca²⁺ channels blockers (e.g., Ni²⁺) (10, 11, 42). Similarly, agonist-induced contraction and/or Ca²⁺ oscillations were unaffected in lung slices (10) and isolated cells (43), or only partially slowed in tracheal strips by L-type calcium channels blockers (20, 44). The conclusion drawn from these data is that airway SMC contraction relies primarily on intracellular Ca²⁺ release mechanisms rather than depolarization-mediated Ca²⁺ influx. However, this does raise the question of why voltage-dependent channels are present. Their role may be related to refilling Ca²⁺ stores (40).

It also follows that relaxation mediated by hyperpolarization and Ca²⁺ sparks is not essential. If this is the case, why are Ca²⁺ sparks commonly observed, and what conditions lead to their appearance? Interestingly, Ca²⁺ sparks associated with airway SMCs have only been observed in isolated tracheal SMCs from guinea pig (39), swine (38), horse (24) and mouse (45). Studies with trachea strips have not reported Ca²⁺ sparks, and, despite many hours of observation with relatively fast (30 Hz) confocal imaging, our group has never observed Ca²⁺ sparks in SMCs of fully relaxed airways or airways contracted with agonist in mouse lung slices. By contrast, Ca²⁺ spark–like events or elemental Ca²⁺ events were observed when airway SMCs were exposed to KCl to investigate the effects of membrane depolarization (10).

Although KCl revealed elemental Ca²⁺ signaling, these events were part of a significantly more global Ca²⁺ response. Contrary to the expected steady state of increased Ca²⁺ due to the opening of voltage-gated channels, KCl induced a series of Ca²⁺ transients. In contrast to agonist-induced Ca²⁺ oscillations that occurred at approximately 20 per minute with each oscillation lasting for less than 1 second, the KCl-induced Ca²⁺ transients occurred with long intervals (1–2 min) and persisted for many seconds. Also, in contrast to agonist-induced Ca²⁺ oscillations, KCl-induced Ca²⁺ transients were acutely dependent on external Ca²⁺, and were inhibited by zero extracellular Ca²⁺ and Ca²⁺ channel blockers (nifedipine and Ni²⁺). However, the KCl-induced Ca²⁺ transients also used intracellular stores, because they were inhibited by caffeine, cyclopiazonic acid (a SERCA pump inhibitor), and ryanodine.

A closer examination of these KCl-induced Ca²⁺ transients revealed that they were preceded by an increasing occurrence of elemental Ca²⁺ signals. The Ca²⁺ transient would then propagate from these signaling sites. Conversely, the elemental Ca²⁺ signaling events vanished immediately after the formation of a Ca²⁺ transient. However, with time, the elemental signals returned before the next Ca²⁺ transient. A similar coalescence of Ca²⁺ sparks followed by a period of quiescence has been observed in isolated tracheal cells (38), and this type of behavior has been commonly observed in cardiac myocytes that are overloaded with Ca²⁺.

Because we only observed Ca²⁺ sparks in intrapulmonary SMCs under conditions of KCl-induced stress (10) where it appeared that Ca²⁺ was leaking into the cell, we believe that the Ca²⁺ store becomes overfilled. This, in turn, leads to the progressive sensitization of the RyR channel, which is visualized by the increase in frequency of the elemental signaling events. When the Ca²⁺ store content is elevated, the heightened Ca²⁺ sensitivity of the RyR allows a single elemental event to "spark" a propagated Ca²⁺ wave via CICR and opening of RyR channels to empty the Ca²⁺ store. The reduced Ca²⁺ content of the store resets the system. This activity is similar to the observation that Ca²⁺ waves in isolated SMCs are initiated from areas of high Ca²⁺ spark activity (7, 38). This sensitization hypothesis was further investigated by mimicking the occurrence of an elemental Ca²⁺ event with the release of caged Ca²⁺. A pulse of Ca²⁺ was found to have little effect on cells under normal conditions. However, in the cells that were exposed to KCl, a similar Ca²⁺ release evoked a propagating Ca²⁺ wave (18).

These data suggest that elemental Ca²⁺ signaling in airway SMCs occurs in response to store filling. Ca²⁺ leakage into the cell could be countered by Ca²⁺ uptake into the internal stores. Once these stores reach their capacity, the release of Ca²⁺ via Ca²⁺ sparks would serve both to stabilize the store Ca²⁺ concentration and minimize further Ca²⁺ influx by their action on Ca²⁺-activated K⁺ channels. In this sense, Ca²⁺ sparks would help prevent an uncontrolled increase in global Ca²⁺ to maintain a relaxed state in times of stress, but, under normal conditions, this process appears to be a secondary control for airway SMC contraction.

Although the physiological role for Ca²⁺ sparks in airway SMCs may be questioned, the value of studying elemental Ca²⁺ signaling with regard to determining receptor identification, participation and regulation in Ca²⁺ oscillations and propagated waves is unchallenged. Indeed, caution in naming the elemental Ca²⁺ signals in SMCs of lung slices arises from the fact that their duration was longer and their size was larger than that associated with Ca²⁺ sparks in other tissues (39). However, the elemental Ca²⁺ events observed in mouse tracheal SMCs (45) were of a similar size and duration as those observed in SMCs of lung slices. The sparks observed in porcine SMCs could be considered to be intermediate between the Ca²⁺ sparks observed in guinea pig tracheal cells and mouse tracheal cells in terms of their dynamics. The difference in the response times of these elemental signals may be the result of a different composition of receptor types. Ca²⁺ spark sites are defined as being associated with clusters of RyR. Although the Ca²⁺ sparks of mouse trachea are, like other Ca²⁺ sparks, inhibited by ryanodine (at relatively high doses), the frequency and amplitude of mouse tracheal sparks are clearly influenced by treatments that alter IP₃ concentrations. This has two major implications: first, the receptor cluster may include IP₃Rs, and their presence prolongs the Ca²⁺ release characteristics; second, a close association of IP₃R and RyRs provides the opportunity for cross-talk between IP₃ signaling mechanisms and Ca²⁺ signaling via RyRs.

This link between IP₃R-based Ca²⁺ signaling and the RyR is also indicated by the response of tracheal SMCs from mice lacking the gene for the RyR binding protein (FK506-binding protein), FKBP12.6 (24). In FKBP12.6^–/– mice, the contractile response to MCh was increased. Because FKBP12.6 appeared to attenuate Ca²⁺ responses via the RyR, this result suggests that Ca²⁺ responses initiated by the IP₃R were amplified through the RyR (24).

REFILLING OF Ca²⁺ STORES BY Ca²⁺ INFLUX

A common feature required to sustain agonist-induced contraction is the presence of extracellular Ca²⁺ (Figure 1). However, voltage-dependent Ca²⁺ channels do not appear to strongly influence SMC contraction (6). We found that agonist-induced contraction and Ca²⁺ oscillations of mouse bronchiole SMCs were unresponsive to L-type Ca²⁺ channel blockers (10). On the other hand, nifedipine either inhibited (43, 46) or reduced the frequency of (12, 20) the Ca²⁺ oscillations of human or porcine and tracheal SMCs, respectively. A potential mechanism for membrane depolarization is the activation of an inward current mediated by Ca²⁺-dependent Cl^– channels as well as nonspecific cation currents (14, 47, 48). However, changes in Cl^– may have an additional electrogenic action to facilitate Ca²⁺ release and uptake across the SR (49). Although these data indicate that there is a minor involvement of membrane depolarization and voltage-gated channels in refilling internal stores, the major conclusion is that additional mechanisms are employed for Ca²⁺ entry to maintain contraction.

A common mechanism believed to stimulate Ca²⁺ entry is the emptying of the internal stores to activate Ca²⁺ influx via membrane channels, called store-operated Ca²⁺ channels (SOCs) (50). Many investigations have explored the proposed mechanism linking store depletion to influx, and recent studies have identified a potential Ca²⁺ sensor in the SR, stromal interacting molecule (STIM) 1, which may communicate with a cell membrane protein or channel (Orai1) (51, 52). STIM1 is a protein that appears to have an SR luminal Ca²⁺ binding domain and, upon store emptying, the distribution of STIM1 changes from diffuse to form clusters adjacent to the plasma membrane. The linkage that STIM1 forms with the membrane channels is not entirely clear. The expression of STIM1 and its role in agonist-induced Ca²⁺ influx has been reported in human cultured airway SMCs (53).

Although Ca²⁺ influx via SOCs appears to be a common mechanism, Ca²⁺ influx also occurs via receptor-operated channels (ROC) or second messenger operated channels. Of particular interest is the arachidonic acid–regulated Ca²⁺ channel (ARC), which, importantly, is activated without store depletion (54). These channels are Ca²⁺ specific and are activated at low agonist levels, which are generally associated with Ca²⁺ oscillations in cells. Interestingly, STIM1 also appears to regulate ARC, but in a store-independent manner (55). This raises the question as to which Ca²⁺ influx channels operate when, because store depletion is not thought to be extensive during Ca²⁺ oscillations. Ca²⁺ influx is further complicated by the potential role of the family of transient receptor potential channels (56). This is a large family of channels that may form ROCs or SOCs, although they do not seem to be ARC channels. With the lack of reliable and specific agonists for many of these channels, their exact identity, distribution, and physiological role in Ca²⁺ influx remains the subject of ongoing research.

The opening of nonspecific cation channels can also lead to an increase in intracellular Na⁺ near the cell membrane, and this is proposed to lead to the reversal of the Na⁺–Ca²⁺ exchanger to increase Ca²⁺ entry (20, 57). Because the maintenance of Ca²⁺ oscillations and tone rely on Ca²⁺ influx, it is essential to understand these various pathways and their relationship to disease.

Ca²⁺ SENSITIVITY OF SMCs

Although the mechanism underlying Ca²⁺ oscillations in airway SMCs is not fully resolved, there appears to be a consensus that SMCs frequency encode their Ca²⁺ signals to regulate contraction (10, 12, 58). Despite the similarity of form and function of the Ca²⁺ oscillations, there are significant differences in the frequency–contractile relationship between different agonists, between airways and intrapulmonary arterioles (9, 10), as well as between airway SMCs of different species (mouse and rat [18]; swine and human [20]) that need to be understood. For example, in the same airway, 5-HT produced slower oscillations than ACh, but induced greater contraction (10). Similarly, 5-HT induced slower oscillations in pulmonary arteriole SMCs than in airway SMCs, but induced greater contraction in the arteriole SMCs (9). We believe that a key component to understanding these differences in the frequency–contraction relationships is the Ca²⁺ sensitivity of the muscle (59) (Figure 1).

Although the mechanisms of Ca²⁺ sensitivity in airway SMCs are not yet fully characterized (59, 60), there have been many studies that indicate that increased Ca²⁺ sensitization most often reflects decreased MLCP activity (61). Although a full review is beyond the scope of this article, there is evidence for two major pathways leading to decreased MLCP activity in airway SMCs. These are phosphorylation and inhibition of the regulatory myosin phosphatase target subunit (MYPT1) of MLCP by Rho kinase and phosphorylation of CPI-17, a 17-kD inhibitory protein of type 1 protein phosphatase, by protein kinase (PK) C. The production of Rho and diacylglycerol (DAG) to active Rho kinase and PKC are G-protein–coupled receptor processes, but depolarization by KCl also appears to sensitize cells to Ca²⁺ (62, 63). Consequently, either the binding of agonist to the SMCs or the depolarization of the SMCs stimulates both an increase in MLCK activity via Ca²⁺ increases and a decrease in MLCP activity via secondary kinases.

In addition to changes in MLCP activity, changes in Ca²⁺ sensitization can also be mediated by modulating the phosphorylation of rMLC by Ca²⁺-independent kinases (i.e., leucine zipper kinase) or by altering the myosin–actin cross-bridge interactions that generate force by actin-binding proteins (i.e., caldesmon).

To investigate the role of Ca²⁺ sensitivity in agonist-induced contraction, it is necessary to gain experimental control of the [Ca²⁺]_i. This has commonly been achieved using either bacterial toxins or detergents to permeabilize the cell membrane (19, 23). However, the disadvantage of these approaches is that the membrane integrity is compromised, and potentially important soluble cell components can be lost. To counter this problem, we have developed a lung slice "model" by simultaneously treating lung slices with caffeine and ryanodine: caffeine is used to open the RyR and ryanodine is used to lock the RyR in an open state (64). The result of this treatment is that the internal SR Ca²⁺ stores are emptied (indicated by no further Ca²⁺ increases in response to caffeine or agonists), which, in turn (as described previously here), activates a Ca²⁺ influx current mediated by SOCs. Because the effects of ryanodine are irreversible, the Ca²⁺ stores remain empty after the removal of caffeine/ryanodine, with the result that the cytosolic [Ca²⁺]_i is a function of external [Ca²⁺]. In the lung slice, this allows the SMC [Ca²⁺]_i to be clamped and correlated with airway contraction, and, because the cell membrane has not been disrupted, these "model" slices are viable for many hours.

Predictably, with this "model" lung slice, the arteriole SMCs contracted in response to a sustained increase in [Ca²⁺]_i, but, surprisingly, the airway SMCs only transiently contracted and then relaxed (64). Furthermore, the subsequent exposure of the slice to a contractile agonist (5-HT) induced a contraction of the airway (usually equal to that induced in a normal slice) and a further contraction of the arteriole without changing the [Ca²⁺]_i in either SMC type. The result that mouse airway SMCs are relaxed by sustained high Ca²⁺ was not expected. In fact, it is likely that this result would be controversial if presented in isolation. However, one of the strengths of the lung slice preparation is that it provides the ability to perform experimental comparative physiology. Consequently, the simultaneous observation of different responses in the airway and arterioles in the same slice under the same conditions is compelling, and immediately emphasizes that the Ca²⁺ sensitivity in bronchioles and arteriole SMCs is very different. Most importantly, the results indicate that agonist-induced Ca²⁺ sensitization is vital for airway contraction.

The characterization of the "model" airway contractile response to increasing concentrations of agonist at fixed [Ca²⁺]_i, or increasing [Ca²⁺]_i at a fixed agonist concentration, each revealed sigmoidal relationships, indicating that airway SMCs contraction is strongly dependent on both the [Ca²⁺]_i and the Ca²⁺ sensitivity. Consequently, in a normal SMC, the contractile response to an agonist results from a dose-dependent change in Ca²⁺ sensitivity and a dose-dependent change in the Ca²⁺ oscillation frequency. Similar conclusions were found with partial agonists of the muscarinic receptor (65). This dual regulation of contraction helps explain why two different agonists can stimulate different levels of contractility at a similar Ca²⁺ oscillation frequency. Similar studies have revealed that rat airways have a greater sensitivity to Ca²⁺ than mouse airways; thus, species differences may also be explained. For us, these results have emphasized the importance of Ca²⁺ sensitivity in agonist-mediated Ca²⁺ signaling, and have convinced us that the contractile process must be examined from both viewpoints before it can be understood.

To explain Ca²⁺-induced relaxation of airway SMCs, we have hypothesized that MLCP can be slowly activated by Ca²⁺ (64). Thus, in response to a step increase in Ca²⁺, the faster-responding MLCK (via activation of Ca²⁺/calmodulin) will initially induce phosphorylation of the rMLC to induce contraction. However, the slower but stronger activation of the MLCP by Ca²⁺ will dephosphorylate rMLC to counter the effects of MLCK and induce relaxation. The subsequent addition of agonist serves to inactivate MLCP by the process of Rho Kinase or PKC activation. Thus, the net MLCP activity is a balance between MLCP inactivation via Rho Kinase/PKC and activation via Ca²⁺. Interestingly, our mathematical model of such a scheme confirms the feasibility of the hypothesis for airway SMCs, but, more importantly, it predicts the response of the arteriole, if the Ca²⁺-dependence of the MLCP is omitted (66).

RELAXATION OF AIRWAY SMCs

The objective of understanding SMC contraction is ultimately to understand how to relax airway SMCs and prevent asthmatic hyperresponsiveness. Clearly, from the rationale described previously here, SMC relaxation must result from either decreased [Ca²⁺]_i or Ca²⁺ sensitization, or both (Figure 1). The mostly commonly used relaxant pathway in asthma control is mediated via increases in cAMP after the activation of adenylyl cyclase by β₂-adrenergic agonists. Surprisingly, in view of the wide-spread use of these drugs, the action of cAMP is not fully defined, and may, in fact, have multiple effects. The most obvious way to induce relaxation would be the reversal of the Ca²⁺ increases. Because SMC Ca²⁺ increases were thought to result directly from Ca²⁺ influx, a focus on Ca²⁺ channels resulted. It was reported that the membrane hyperpolarization resulting from the activation of large-conductance, calcium-activated K (K_Ca) channels by increases in cAMP decreased Ca²⁺ influx (67–70), and antagonists of the K_Ca channels (e.g., iberiotoxin) were reported to antagonize the effects of cAMP (71, 72). Alternative ways to decrease [Ca²⁺]_i include an inhibition of SOCs after Ca²⁺ store release (73), an increase in SERCA pump activity (74, 75), or an increase in Ca²⁺ efflux (76). However, because airway SMC contraction appears to use Ca²⁺ oscillations, we examined the effects of isoproterenol, forskolin, and cAMP analogs on airway SMCs in lung slices, and found that all these agents slowed the Ca²⁺ oscillation and induced relaxation. Similar decreases in Ca²⁺ oscillation frequency without contractile data were reported for isolated tracheal cells (67, 77).

In view of the facts that the frequency of Ca²⁺ oscillations can depend on the refilling of Ca²⁺ stores, and that this refilling can be influenced by Ca²⁺ influx (13), it is possible that both of these mechanisms could contribute to a reduction of the Ca²⁺ oscillation frequency to induce relaxation. To test this idea, we increased the amount of Ca²⁺ available by flash photolysis of caged Ca²⁺, increased the Ca²⁺ influx into the cell with ionomycin, or prevented a decrease in Ca²⁺ influx with iberiotoxin. Surprisingly, these treatments did not increase the frequency of the Ca²⁺ oscillations. By contrast, the oscillations either slowed or stopped and this enhanced the relaxing effects of cAMP rather than antagonizing them. These results suggest that Ca²⁺ availability for refilling stores was not a limiting factor. The similarity of the caffeine-induced Ca²⁺ release from internal stores in the presence or absence of forskolin also supports this idea.

Because the regulation of the Ca²⁺ oscillation frequency is also thought to be related to the sensitization of the IP₃R, we examined the Ca²⁺ response to the photolysis of caged IP₃. An increase in IP₃ was found to increase the frequency of Ca²⁺ oscillations slowed by exposure to forskolin, whereas, at threshold conditions, forskolin inhibited the ability of IP₃ to release Ca²⁺. These results indicate that cAMP inhibits Ca²⁺ release from the IP₃R in mice and rats (18). A similar conclusion, that the action of relaxing agonists is focused more on the internal Ca²⁺ release processes rather than membrane hyperpolarization and Ca²⁺ influx via K_Ca channels, was recently reached from studies with mice (78). We have also found that nitric oxide (NO) strongly attenuates Ca²⁺ oscillations and relaxes airway and arteriole SMCs in mouse lung slices (79). NO acts via guanylyl cyclase, the elevation of cGMP, and activation of cGMP-dependent protein kinase type I (cGKI). NO-induced SMC relaxation appears to be mediated by decreasing [Ca²⁺]_i by inhibiting the IP₃R through the phosphorylation and complex formation with an IP₃R-associated protein (IRAG) (80, 81). This mechanism of action appears similar to the action of cAMP. However, a cAMP analog still relaxed SMCs from IRAG- or cGKI-deficient mice, a result implying that cAMP does not act on the IP₃R via IRAG/cGKI. A slowing of Ca²⁺ oscillations was also induced by NO in isolated porcine tracheal cells (43), but, because the Ca²⁺ oscillations in these cells seem to rely more on the RyR, it is not clear where NO (cGMP) acts.

The complementary mechanism by which cAMP/cGMP can act is by Ca²⁺ sensitization; cAMP- or cGMP-dependent kinases may alter the activity of MLCP or the proteins associated with contraction (72, 75, 82, 83). Using the "model" lung slice, we have found that exposure to forskolin and a phosphodiesterase inhibitor (treatments that increase cAMP) relaxed airways by decreasing the Ca²⁺ sensitivity, as well as by reducing the Ca²⁺ oscillation frequency (64). A similar finding has been concurrently reached with guinea pig trachea (82).

TISSUE RESPONSES INFLUENCING CONTRACTILITY

For the most part, we have considered chemical regulators of contraction. However, mechanical influences appear to have a strong influence on airway SMC tone (Figure 1). In normal lungs, inspiration can protect against airway constriction induced by MCh, whereas, in individuals with asthma, this response is attenuated (84–87). The mechanism underlying this response is poorly understood, but it has been proposed that dynamic stretching plasticizes the SMC cytosol to reduce its stiffness so that breathing forces can dilate the airways. It is not clear how traditional second messengers, such as Ca²⁺, would influence the process. On the other hand, the fact that the stretch–relaxation response is lost in patients with asthma suggests that some pathophysiological process is acting on a key parameter. Acute stretch–induced responses mediated by the opening of ion channels to induce Ca²⁺ influx or activate Rho kinase (88, 89) would appear counterproductive to inducing airway relaxation. An alternative way in which stretch might reduce the Ca²⁺ sensitivity of the SMCs would be the release of NO by epithelial cells. However, the degree of Ca²⁺ sensitization may be adapted over a longer term or genetically encoded, as different strains of mice have been found to respond differently to mechanical ventilation (90). In view of this, it is interesting that different mouse strains also showed different contractile responses to the same Ca²⁺ oscillation frequency (91). This implies that different mouse strains have an inherently different level of Ca²⁺ sensitization, and we have emphasized that this concept seems to apply to a variety of species and agonists.

In summary, the aim of this research is to provide a basic understanding of the Ca²⁺-based regulatory mechanisms of SMC contractility to identify the changes that mediate SMC hyperresponsiveness in asthma. The recognition that the control of airway SMC contraction is dynamic and relies strongly on internal Ca²⁺ release mechanisms and a simultaneous Ca²⁺ sensitization emphasizes the importance of the dual investigation of these process in response to disease conditions, such as airway inflammation.

FOOTNOTES

Conflict of Interest Statement: M.J.S. is the recipient of research grants of $128,000 in 2006 and $125,000 in 2007 from Sepracor. P.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.F.P.-Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form April 28, 2007; accepted in final form May 11, 2007)

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