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Asthma-associated airway hyper-responsiveness (AHR) is primarily mediated by excessive airway smooth muscle cell (SMC) contraction, yet the mechanisms responsible for this behavior remain unknown. A hypothesis commonly favored to explain excessive force production by individual SMCs is that the intracellular calcium concentration ([Ca2+]i) that regulates SMC contraction is abnormally elevated in asthmatic airway SMCs. However, in view of the importance of Ca2+ as a second messenger, an elevated [Ca2+]i would have other wide-ranging ramifications for SMC physiology and this may be expressed in terms of altered cell proliferation or phenotype. SMC contraction is also determined by the sensitivity of the SMC contractile apparatus to the [Ca2+]i and AHR may be explained by an abnormal increase in Ca2+ sensitivity. Similarly, airway constriction also depends on the number and size of the SMCs and the resistive load experienced by these SMCs. As a result, increased SMC proliferation and airway wall remodeling, processes commonly associated with airway inflammation, are believed to significantly contribute to AHR.
A common theme linking these varied processes is that each one is regulated or influenced by [Ca2+]i which, in turn, is determined by the activity of a wide variety of ion channels, in balance with membrane Ca2+ pumps. This increase in [Ca2+]i can be achieved in two fundamental ways; by the release of Ca2+ from internal stores via specialized receptor/channels on the sarcoplasmic reticulum or by Ca2+ influx from the extracellular space via a variety of plasma membrane ion channels The objective of this review is to focus on the nature of this channel activity and how it relates to changes in [Ca2+]i and the regulation of SMC contraction, proliferation and secretion, with the idea that ion channels may serve as a therapeutic targets for AHR.
Agonist-induced SMC contraction is predominately dependent on increases in [Ca2+]i resulting from the release of Ca2+ from internal stores, namely the sarcoplasmic reticulum. Importantly, this increase in [Ca2+]i is not static, but rhythmically increases and decreases in the form of Ca2+ oscillations. This reliance on internal stores is emphasized by the observations that both agonist-induced airway SMC contraction and Ca2+ oscillations can occur in the absence of extracellular Ca2+ [1, 2] and that, inhibitors (cyclopiazonic acid and thapsigargin) of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) that lead to the run-down or emptying of the internal Ca2+ store, abolish Ca2+ oscillations [2, 3].
Agonist-induced Ca2+ oscillations (using ACh or 5HT) occur in tracheal, bronchial and small airways of mice [1, 4–6], pigs [2, 7, 8], and humans . Importantly, the frequency of these Ca2+ oscillations generally increases with the concentration of the agonist [1, 5, 10]. However, the Ca2+ oscillations are usually initiated at a frequency slightly higher than the steady frequency that is subsequently expressed in the continual presence of agonist. Ca2+ oscillations commonly occur as Ca2+ waves in SMCs. Increases in [Ca2+]i are often initiated at one end of the SMC and propagate, as a Ca2+ wave, along the entire length of the cell. However, the position of the initiation site can fluctuate and relocate to the opposite end of the cell, with the consequence that the direction of the Ca2+ wave propagation reverses [1, 3, 11]. The velocity of the propagating Ca2+ wave is relatively constant but may increase with agonist concentration in range from 8 to 24 μm/s in tracheal SMCs  and from 27 to 35 μm/s in mouse small airways [1, 3].
The model for the generation and maintenance of Ca2+ oscillations in airway SMCs appears to be similar to many other cell types [12, 13]. The activation of G-protein coupled receptors (GPCR) by agonists, such as ACh or 5HT, in airway SMCs leads to Gαq-mediated activation of phospholipase C (PLC) to synthesize the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol from plasma membrane phosphatidylinositol 4,5-bisphosphate [14–16]. Subsequently, IP3 diffuses through the cytosol and binds to IP3 receptors (IP3R) in the sarcoplasmic reticulum. The IP3R is a specialized Ca2+ channel with a gating mechanism regulated by IP3 and Ca2+ (Figure 1).
In response to the initial rise of [IP3]i, cytoplasmic Ca2+ opens the IP3R at a local initiation site (generally situated close to one end of the SMC) to release Ca2+ from the sarcoplasmic reticulum. The released Ca2+ diffuses away and activates neighboring IP3-sensitized IP3Rs to initiate the further release of Ca2+. In this manner, a wave of increased [Ca2+]i propagates through the cell. The resulting local increase in [Ca2+]i promotes the binding of Ca2+ to a second site on the IP3R which inactivates and closes the receptor to terminate Ca2+ release. In addition, the reduction in the [Ca2+] within the sarcoplasmic reticulum also contributes to the termination of the Ca2+ release process . The subsequent decrease in [Ca2+]i results from the combined effects of SERCA activity in the sarcoplasmic reticulum to replenish the Ca2+ stores, the extrusion of Ca2+ by plasma membrane Ca2+-ATPases and Na+/Ca2+ exchangers and the binding of Ca2+ to protein buffers in the cytosol. In addition, the reduction of [Ca2+]i leads to the release of Ca2+ from the IP3R to reset the receptor. In this manner, repetitive Ca2+ oscillations result from the cyclic Ca2+-induced activation and deactivation of IP3-sensitized IP3Rs (Figure 1).
It seems clear that IP3Rs participate in agonist-induced generation of Ca2+ oscillations in airway SMCs: The inhibition of IP3R with heparin (an IP3R antagonist) or with an IP3R monoclonal antibody blocks Ca2+ oscillations (recorded as Ca2+-activated Cl− currents) . However, the additional contribution of a second sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor (RyR), to Ca2+ oscillations and the propagation of Ca2+ waves appears to vary between SMCs at different locations in the airway or with the species. In this alternative model of Ca2+ oscillations, the participation of both the IP3R and the RyR is suggested because ryanodine or ruthenium red (RyR antagonists) blocked Ca2+ oscillations whereas heparin, only partially inhibited Ca2+ oscillations induced by ACh  in pig airway SMCs. By contrast, ryanodine was unable to inhibit or alter the properties of the Ca2+ oscillations of SMCs from mice or rats suggesting that Ca2+ oscillations and waves are dependent only on IP3Rs in bronchioles of these animals .
By measuring airway SMC contraction and Ca2+ signaling, it has been found that increased contraction of small airways and tracheal SMC strips in response to increasing concentrations of agonists correlated with an increase in the frequency, but not the amplitude, of Ca2+ oscillations [1, 5, 19]. This observation supports the hypothesis that airway SMC contraction is regulated by the frequency of the Ca2+ oscillations. In addition, mathematical modeling of the contractile responses of airway SMCs upholds the idea that Ca2+ oscillations are more effective than steady-state Ca2+ for the generation of contraction . However, further analysis suggested that the frequency component of the Ca2+ oscillations alone may not be responsible for this effect. These mathematical models showed that it is more likely that the change in the average [Ca2+]i resulting from the integration of the Ca2+ oscillations has more influence in determining the extent of contraction, a conclusion supporting previous suggestions . In addition, there are significant differences in the contraction-frequency relationship induced by different agonists and between SMCs from different species. For example, 5HT induced Ca2+ oscillations with slower frequencies in bronchiole airway SMCs than those induced by an equal concentration of ACh, but the associated contraction in both cases was similar . Likewise, the frequency of Ca2+ oscillations induced by 5HT was approximately three times slower in the SMCs of intrapulmonary arterioles than it was in airway SMCs, but the arterioles contracted more than the airways . These differences in the contraction-frequency relationships suggested differences in the Ca2+ sensitivity of the SMCs. This idea was confirmed with Ca2+ permeabilized cells in which the [Ca2+]i was set at a high steady level; under these conditions of constant [Ca2+]i, exposure to equal concentrations of different agonists (e.g. methacholine or 5HT) resulted in different amounts of contraction [20, 21]. These observations indicate that, in addition to the frequency of Ca2+ oscillations, the Ca2+ sensitivity of SMCs is a major factor contributing to the regulation of SMC contraction .
With this insight into airway SMC contraction, we can turn our attention to the important mechanism of broncho-dilation that must be acutely induced to counter airway hyper-responsiveness occurring during an asthma attack. Over the longer term, the therapeutic aim is the prevention of airway hyper-responsiveness. These goals require the relaxation and desensitization of the airway SMCs. A current primary approach used to relax airway SMCs is the inhalation of β2-agonists that mediate their effects via increases in cAMP and the activation on protein kinase A [18, 23]. However the mechanism of SMC relaxation is not well understood. Activation of β2-receptors with agonists, such as, salbutamol , isoproterenol  and albuterol , reduces the frequency of agonist-induced Ca2+ oscillations in SMCs and this response correlates with airway relaxation. Airway relaxation and a similar reduction of the Ca2+ oscillation frequency is induced by nitric oxide acting via cGMP [25, 26]. As outlined above, Ca2+ oscillations primarily rely on internal Ca2+ release. Consistent with this idea, is the observation that the slowing of the frequency of the Ca2+ oscillations appears to mainly result from the inhibition of Ca2+ release from internal stores by blocking the IP3R [18, 26]. The inhibition of Ca2+ release by NO/cGMP has been shown to involve protein kinase G-mediated phosphorylation of an IP3R associated protein, but it is not clear if cAMP in airway SMCs also acts via protein kinase A-mediated phosphorylation of a similar associated protein [27–30]. Ca2+ oscillations could also be slowed by a reduction in the [IP3]i, but β2-agonists, forskolin or 8-Br-cAMP have been reported to not interfere with the synthesis of IP3 [31, 32]. In addition, β2–agonists have been reported to activate KCa channels which, as discussed later, would hyperpolarize the cell membrane and reduce Ca2+ entry through voltage activated Ca2+ channels . A limited Ca2+ entry would be expected to lead to a decrease in Ca2+ oscillation frequency by depleting and delaying the re-loading of the internal Ca2+ stores .
Airway SMC relaxation mediated by Ca2+ de-sensitization, commonly involves an increase in the activity of myosin light chain phosphatase to antagonize the activating phosphorylation of the myosin light chain by myosin light chain kinase. In acute responses, β2-agonists and cAMP analogs have been shown to reduce the Ca2+ sensitivity of airway SMC [18, 23, 35]. However, NO does not seem to have a large effect on the Ca2+ sensitivity of mouse airway SMCs .
Over the long-term, it is not clear how the SMCs become hyper-responsive, but stimulation of the mechanisms of Ca2+ release and re-uptake could result in an increase the frequency of agonist-induced Ca2+ oscillations which, in turn, would increase the myosin light chain kinase activity, myosin phosphorylation and force production. In addition, hyper-responsiveness could also result from an increase in Ca2+ sensitivity which may be mediated by alterations in myosin phosphatase activity (inhibition) or one of its regulators such as Rho-kinase [22, 36]. There are a number of additional factors that could also be part of the problem of airway hyper-responsiveness, for example the accumulation of more SMCs in the airway wall. This form of sensitization is often countered by steroid therapy but, as mentioned later, the inhibition of KCa channels to reduce cell proliferation may be a viable alternative.
In view of the excess of extracellular Ca2+, especially in comparison to the limited Ca2+ of the internal stores of SMCs, an obvious alternative way to increase [Ca2+]i in SMCs is by the influx of Ca2+. Despite the wide variety of different channel types in the plasma membrane through which this can occur, a clear correlation of a specific Ca2+ influx pathway with a physiological function of SMC, other than refilling of Ca2+ stores, is, at present, lacking. A common idea has been that Ca2+ influx directly contributes to airway SMC contraction, but, as described above, Ca2+ release from internal stores in the form of Ca2+ oscillations appears to be more important in agonist-induced SMC contraction. However, even though Ca2+ oscillations repetitively utilize much of the Ca2+ released from the sarcoplasmic reticulum, it is inevitable that some Ca2+ is lost to the extracellular environment. Consequently, for sustained Ca2+ oscillations and contraction, it is essential to replenish the [Ca2+] of the sarcoplasmic reticulum and this requires Ca2+ influx.
Ca2+ influx through plasma membrane ion channels may also have other important physiological functions in SMCs. For example, increases in [Ca2+]i caused by Ca2+ influx can lead to alterations in proliferation and secretion (see later). Furthermore, an absence of Ca2+ influx will eventually result in the depletion of the Ca2+ of the sarcoplasmic reticulum. This will affect many of the fundamental functions of the sarcoplasmic reticulum, related to protein folding and maturation, stress responses and apoptosis . It is therefore essential to characterize and determine the molecular identity of the various ion channels involved in Ca2+ influx to understand their role in the physiological and pathophysiological processes of airway SMCs.
The process of replenishing the [Ca2+] of the sarcoplasmic reticulum by agonist-induced Ca2+ influx is termed capacitative calcium entry or store-operated calcium entry and, as the name implies, Ca2+ influx occurs upon the emptying of the internal Ca2+ stores via the activation of store-operated channels in the plasma membrane [38, 39]. However, agonists acting on membrane receptors can also initiate Ca2+ entry by stimulating the production of diacylglycerol or its derivatives to directly activate membrane ion channels called receptor-operated channels (Figure 1). Ca2+ entry via these receptor-operated channels may accompany or precede Ca2+ influx via store-operated channels [40–43]. Even though opinions differ with respect to the contribution of receptor-operated and store-operated Ca2+ channels to store refilling, both processes have been shown to be necessary to sustain Ca2+ oscillations in various cell types [39, 44]. Other routes for Ca2+ entry are voltage-gated Ca2+ channels and the sodium-calcium exchanger operating in the reverse-mode . However, the activation of voltage-operated Ca2+ channels for long time periods requires successive cycles of membrane depolarization and hyperpolarization in order avoid channel inactivation. Although oscillations in membrane voltage during agonist stimulation have been observed in airway SMCs, the changes in membrane potential are usually inadequate to stimulate the opening of L-type Ca2+ channels . In addition, the contribution of voltage-gated Ca2+ channels appears to be minimal because inhibitors of these channels had little effect on agonist-induced SMC contraction or Ca2+ oscillations  or as therapies for AHR .
The identities of the channels involved and the mechanism of store-operated channel activation have been long-standing questions since the discovery of store-operated Ca2+ entry . Multiple mechanisms of channel activation via direct contact with store-associated Ca2+ sensing molecules or by soluble factors have been proposed but without much consensus. Therefore, it was an important advance when the molecular identities of store-operated channels and their regulatory proteins were associated with Orai family proteins [48–50] and stromal interacting molecule 1 (STIM1), respectively [51–53]. Receptor-operated channels may also involve these proteins [54–56]. Orai1 proteins form a highly Ca2+ selective store-operated ion channel in the plasma membrane that is responsible for the well characterized Ca2+ release-activated Ca2+ current, ICRAC. STIM1 is Ca2+-binding membrane protein present in the sarcoplasmic reticulum where it appears to function as a Ca2+ sensor and transmits information regarding the Ca2+ content of the sarcoplasmic reticulum to plasma membrane store-operated channels (Figure 1). The fall in Ca2+ in the internal store leads to the aggregation of STIM1 into punctate structures within the sarcoplasmic reticulum, but close to the plasma membrane, where it can interact with the Orai1 or other store-operated channels [57, 58]. siRNA mediated knock-down of STIM1 significantly reduced store-operated Ca2+ entry following Ca2+ store depletion by histamine but not bradykinin in human airway SMCs . In addition, it has been shown that Orai1 and potentially Orai3 contribute to store-operated channels in these cells . Importantly, Orai1, -2, -3 as well as STIM1 have been found to be expressed in human airway SMCs.
The molecular identities of all store-operated and receptor-operated channels in SMCs are not yet fully characterized and the non-selective characteristics of the Ca2+ influx mechanism suggests the involvement, in addition to Orai1, of other protein channels. In this respect, it appears that the transient receptor potential (TRP) proteins constitute some or parts of these channels [61, 62]. The mammalian TRP protein super-family is comprised of multiple subfamily members . In airway SMCs, several members of the three biggest and most characterized subfamilies (TRPC, TRPM, TRPV) have been proposed to form agonist-activated, cation permeable channels that contribute to Ca2+ influx to regulate contraction and proliferation . Furthermore, members of all three families are reported to be expressed in human airway SMCs including TRPC1, -3, -4, -5, -6, TRPM4, -7 and TRPV1, -2, -4 [65–69].
TRP proteins assemble into homo- and/or heteromeric tetramers, generating cation permeable channels  which are permeable to a range of mono- and divalent cations. However, permeability differs greatly between the various family members. The TRPCs form largely non-selective cation channels with the selectivity for Na+ and Ca2+ being determined by the specific components of the hetero-multimers . TRP channels are activated by a wide variety of stimuli including mechanical, osmotic and thermal stress and chemical compounds and the expression of some TRP channels may be regulated by growth factors and cytokines [68, 71, 72].
The TRPC family is the subfamily classically associated with store-operated and receptor-operated Ca2+ entry. They all appear to be activated by agonist stimulation via the PLC pathway , but there is considerable controversy regarding the relationship of TRPC channels to store-operated and receptor-operated channels. On the basis of over-expression systems, it has been suggested that the activating mechanism may depend on the expression level [73, 74]. The TRPC1 channel is important for agonist-induced Ca2+ entry in SMCs. Generally, TRPC1 has been proposed to form a store-operated channel [66, 75], except in vascular SMCs, where store-operated channels appear to be independent of TRPC1 even though the channel is present and functional . In airway SMCs TRPC1 has been reported to be involved in proliferation and therefore may play a role in airway remodeling .
By over-expression of STIM1 in HEK293 cells, STIM1 has been shown to interact with all TRPC proteins except TRPC7, binding directly to TRPC1, 4 and 5, and promoting hetero-multimerisation of TRPC3 and 1, and TRPC4 and 6. STIM1 seems necessary for these channel to act as store-operated channels, although it does not appear to be required for channel function per se . This may be consistent with the findings in vascular SMCs where STIM1 is required for store-operated channel, but TRPC1 is not .
TRPC3 is also expressed in vascular SMCs where its activation results in Ca2+ influx and vasoconstriction, whereas its inhibition attenuates depolarization and contraction . The channel is thought to be primarily receptor-operated  although in human airway SMCs the expression of TRPC3 channels can be increased by treatment with TNF-α which leads to increased store-operated Ca2+ entry in response to GPCR agonists such as ACh and bradykinin . Dietrich et al., suggested that TRPC3 channel homo-multimers may be constitutive active in vascular and airway SMCs, whereas channels formed of TRPC3 and TRPC6 hetero-multimers require activation via the PLC pathway .
There has been considerable focus on TRPC6 channels in pulmonary vascular SMCs where they are believed to be involved in cell proliferation and may contribute to pulmonary arterial hypertension  and hypoxic pulmonary vasoconstriction . Interestingly, TRPC6-deficient mice showed enhanced agonist-induced contraction of aortic rings, a response perhaps explained by a compensating up-regulation of TRPC3. These results highlight some of the complexity in studying the role of these channels . From these findings, it has been proposed in human airway SMCs that TRPC6 serve as a receptor-operated channel, activated by diacylglycerol and its analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) . However, in guinea pig airway SMCs, TRPC6 knock-down did not affect OAG-induced Ca2+ influx .
In summary, human airway SMCs express several members of the TRPC family, which have been shown to be associated with Ca2+ influx, contraction and proliferation in other SMC types. However the functional importance of TRPC as well as Orai proteins in human airway SMCs still needs to be confirmed, although progress towards this goal is hampered by the lack of specific channel agonists and antagonists. In addition, the findings that TRPC proteins can form hetero-multimers and the suggestions that Orai1 proteins can interact with TRPCs  raises the possibility that several different store- and receptor-operated channels exist in airway SMCs. These could also be dynamically regulated by factors that change their expression (e.g. cytokines and growth factors) during disease. In view of this association of TRP channels with Ca2+ influx, contraction and proliferation, TRP channels may be potentially valuable pharmaceutical targets for the treatment of asthma.
Airway SMCs are exposed to various physical forces such as gravity, compression and rhythmic mechanical strain. Because these forces act on the plasma membrane, Ca2+-permeable cation channels, activated by mechanical stretch or stretch-activated channels provide the SMC with the ability to respond to mechanical stimuli [84–87] (Figure 1). Importantly, these Ca2+ permeable stretch-activated channels occur in human airway SMCs .
The mechanisms by which stretch activates these channels are not fully understood, but a direct and indirect mechanism has been proposed . The finding that Gd 3+ blocks stretch-induced Ca2+ influx in various cells, including airway SMCs, supports the idea of direct activation [88–90]. Currently, there are two models to explain direct gating of stretch-activated channels; a “bilayer model” in which plasma membrane tension itself is sufficient to gate the mechano-sensitive channel and a “tethered model” in which mechanical force is transmitted to the ion channel through a linkage formed by the extracellular matrix, integrin proteins and cytoskeleton [86, 87]. For the indirect mechanism, it has been proposed that the activation of PLC or the release of another signaling molecule (e.g. ATP) following application of mechanical stress elicits intracellular Ca2+ mobilization and subsequent activation of the channels at the plasma membrane [87, 91]. It is also possible that Ca2+ influx via stretch-activated channels secondly activates voltage-dependent channels by causing membrane depolarization [85, 87]. In human ASM cells, stretch-induced Ca2+ influx is not blocked by the PLC inhibitor, U-73122, the IP3R blocker, xestospongin C, or the voltage-gated channel blocker, nifedipine ; these result suggest that Ca2+-permeable stretch-activated channels in human airway SMCs are activated directly by stretch rather than indirectly via intracellular messenger production or voltage-dependent channel gating.
The TRPV subfamily members are activated by a wide variety of stimuli including agonists, heat and changes in osmolarity [61, 92] and there is increasing evidence that a putative mechano-sensitive channel is one of the members of the TRPV subfamily [93–95]. Human airway SMCs express TRPV1, -2 and -4 genes [67, 88] and it appears that TRPV2 and -4 mediate Ca2+ influx in response to membrane stretch or hypo-osmotic induced cell swelling [67, 95, 96]. Stretch-induced Ca2+ influx is inhibited by ruthenium red, a TRPV inhibitor, but not by capsazepine, a TRPV-1 inhibitor . Therefore, TRPV2 and -4 are candidate genes for stretch-activated channels in airway SMCs. However, it should be noted that TRPC6  and TRPM7  channels have also been linked to the stretch-induced Ca2+ influx in other cell types. TRPV4 can also be activated by moderate heat (>24°C), 4α-phorbol 12,13-didecanoate (4α-PDD), and endogenous substances like endocannabinoid anadamide, arachidonic acid and 5′,6′-EET  and this suggests a role in thermosensing and Ca2+ homeostasis. TRPV2 is also activated by insulin growth factor (IGF-1) by the insertion of the channel into the cell membrane. Intracellular vesicles, that arise from the Golgi and incorporate the channel, fuse with the plasma membrane upon stimulation with IGF-1 and this process may underlie IGF-1-induced contraction and proliferation of airway SMCs . TRPV2 might also be upregulated in capsaicin-induced asthma and it can be upregulated by growth factors .
The lungs are continuously exposed to a mechanically dynamic environment during breathing  and excessive strain and pressure occurs in the airway walls during bronchoconstriction in asthmatic patients . Therefore, mechanical stress is likely to have an impact on cellular function in normal and diseased lungs [101, 102]. Indeed, the development , contraction [104, 105], stiffness [106–108], proliferation , migration , organization of the actin cytoskeleton [106, 108, 111, 112] and protein synthesis [113, 114] of airway SMCs are influenced by the mechanical stress. These cellular responses of airway SMCs to mechanical strain are mediated via multiple intracellular pathways including RhoA, myosin light chain kinase, cyclooxygenase (COX), MAP-kinases (ERK1/2 and p38) and various transcription factors [104, 113, 115–118]. Although the involvement of stretch-activated channels is currently ill-defined, airway SMC contraction or IL-8 production induced by cyclic mechanical strain are significantly inhibited by the stretch-activated channel inhibitor Gd3+ [104, 113]. Therefore it is important to further characterize stretch-activated channels and their role in airway SMCs as they too may serve as drug targets for the treatment of airway disease [94, 119].
Although acute changes in membrane potential do not seem to be required to stimulate airway SMC contraction, it appears that [Ca2+]i and membrane potential are interconnected by Ca2+-dependent K+ channels and that this regulation is designed to operate over a longer time course to mediate changes in SMC phenotype.
The three major families of human Ca2+-activated K+ channels (KCa); the large conductance (KCa1.1), intermediate conductance (KCa3.1) and small conductance channels (3 varieties, KCa2.1, 2.2 and 2.3) , share the property of forming an open conformation in response to increased [Ca2+]i. Each family differs in their Ca2+-sensing mechanism and voltage sensitivity. While KCa1.1 channels bind Ca2+ directly at the C-terminus, KCa3.1 channels form a constitutive molecular partnerships with the Ca2+-binding protein calmodulin which results in a greater sensitivity to [Ca2+]i [120, 121]. In addition, the KCa1.1 channel, but not the KCa3.1 channel, is voltage-gated. These properties of high Ca2+-sensitivity and voltage-insensitivity allow the KCa3.1 channel to open during membrane hyper-polarization when it is likely that KCa1.1 is inactive. Such distinct functional properties suggest that these channels may operate during different conditions in the same cell, such as occur following a phenotypic alteration in airway SMC.
KCa channels also differ in their sensitivity to inhibitors allowing their different actions to be assessed pharmacologically; KCa1.1 channels are inhibited by TEA, iberiotoxin and charybdotoxin whereas KCa3.1 channels are inhibited by TRAM-34, clotrimazole, ICA17043 and charybdotoxin [120, 122].
A Ca2+-dependent and voltage-gated outward K+ current that is sensitive to charybdotoxin and iberiotoxin is widely found in isolated tracheal SMCs [123–125]. The properties of this current are consistent with the large conductance KCa channel (BK or KCa1.1); a channel commonly responsible for spontaneous transient outward currents (STOCS) that are associated with ‘Ca2+ sparks’ . In vascular SMCs, the Ca2+-activation of KCa1.1 channels leads to membrane hyper-polarization and the inhibition of voltage-gated L-type Ca2+ channels. This in turn, reduces the amount of Ca2+ influx, and thereby decreases SMC tone . The activation of the KCa1.1 channel by physiological (nitric oxide) or therapeutic bronchodilators (isoproterenol) is consistent with this paradigm, are activated by these agonists that appear to reduce Ca2+ by other although it is not clear how KCa mechanisms . However, the observations that voltage-gated Ca2+-channel inhibitors have no effect on agonist-induced Ca2+ signaling in airway SMCs , are ineffective bronchodilators  and that thedeletion of KCa1.1 channels in transgenic mice reduced airway contractility [22, 46, 128] all suggest that the contribution of membrane potential to regulation of [Ca2+]i in airway SMCs is minimal. Consequently, the role of KCa1.1 in airway SMC contraction remains controversial. Airway SMCs also contain Ca2+-activated Cl− channels  and these have been found to be responsible for spontaneous transient inward currents (STICS) . The exact role for Ca2+ activated Cl− currents in airway SMCs is also unclear, but these currents may enhance Ca2+ loading of the sarcoplasmic reticulum  and influence cell proliferation.
Airway SMCs are adaptable and may adopt a ‘synthetic’ or ‘proliferative’ phenotype in chronic asthma . SMC proliferation leads to hyperplasia in asthma and is a feature of isolated human airway SMCs in vitro . This entry into and progression through the cell-cycle requires a careful orchestration of the membrane potential with the [Ca2+]i  and this appears to be associated with distinct patterns of KCa channel expression.
Snetkov et al  demonstrated that KCa1.1 currents, found in isolated human bronchial SMCs, diminished with in vitro culture and suggested that a reduction in the expression of a major K+ channel correlated with a switch to a proliferative phenotype [133, 134]. Similarly, outwardly rectifying currents associated with KCa channels in proliferating human airway SMCs were relatively insensitive to iberiotoxin, implying a relatively small contribution by KCa1.1 . Under these conditions, few cells expressed currents typical of KCa 3.1 channels, but in response to TGF-β, the majority of cells increased the expression and activity of KCa3.1 channels. A loss of KCa1.1 channels and gain in KCa3.1 channels also occurs when vascular SMCs switch from a contractile to a proliferative phenotype [136, 137]. Thus, the switch from a contractile to a proliferative phenotype by SMCs in response to injury is associated with the replacement of relatively Ca2+-insensitive, voltage-gated KCa channels with voltage-insensitive, but more Ca2+-sensitive KCa channels that may facilitate the regulation of the cell-cycle.
The idea that the cell cycle is regulated in SMCs by KCa channels is attractive but, as yet, not well documented. On the other hand there is considerable evidence supporting the idea that a change in membrane potential serves as a critical determinant of cell cycle progression in other cells. A well-studied example is found with MCF-7 breast cancer cells in which membrane hyper-polarization, mediated by the activation of K+ channels, correlates with the progression of cells from the G0 phase into, and to the end of, the G1 phase [138, 139]. We review the data from MCF-7 breast cancer cells with the objective of gaining an insight to the mechanisms that may apply to SMCs expressing similar KCa channels. An arrest of the cell cycle can be induced by the inhibition of K+ channels but the particular K+ channel involved varies in different cell types. Ouadid et al.,  also investigating MCF-7 cells found that although the early phases of hyper-polarization (up to mid G1) were accompanied by increases in K+ channels related to the human ether-a-go-go related gene (Kv10), this channel density and mRNA expression fell later in G1 despite persistent membrane hyper-polarization. This sustained hyper-polarization was explained by the identification of a clotrimazole and TRAM-34 sensitive, Ca2+ activated-channel with the characteristics of K Ca3.1 . The mRNA and currents associated with KCa3.1 channel increased throughout G1 and were maximally elevated in early S-phase. KCa3.1 channels have also been implicated in proliferation of mesenchymal cells, vascular SMCs and transformed fibroblasts when stimulated by growth factors [141, 142]. Similarly, inactivation of KCa3.1 channels, with inhibitors such as TRAM-34 and clotrimazole, prevented human airway SMC proliferation , SMC re-growth in a model of post-angioplasty restenosis [137, 143] and cell proliferation induced by mitogens including serum, basic FGF and thrombin. By contrast, KCa1.1 and KCa2 inhibitors, such as iberiotoxin, TEA and apamin do not inhibit proliferation . These results indicate that a differential expression of K+ channels regulates membrane potential throughout the cell cycle and that expression of KCa3.1 channels is necessary to progress into the S-phase. Given the inhibitory effect of membrane hyper-polarization on voltage-gated Ca2+ channels, it is possible that the additional KCa3.1 channel activity provides the necessary electrochemical gradient to enhance Ca2+ entry, perhaps via another voltage-independent Ca2+ channel [145, 146].
KCa3.1 is expressed by proliferating airway SMCs rather than ‘normal’ contractile cells  and its expression is driven by the pathological mediator TGFβ that is increased in asthmatic airways. Its presence thus characterizes SMCs involved in disease progression making it an attractive therapeutic target in chronic asthma. In addition KCa3.1 channels are found in mesenchymal stem cells, epithelial cells and mast cells, all of which contribute to the pathology of chronic asthma [147, 148]. Inhibitors of KCa3.1 channels have proven to be safe during clinical trials for sickle cell disease and clinical trials with these inhibitors for the treatment of asthma are being developed .
In addition to their contractile and proliferative activities, airway SMCs have also been implicated as a target, as well as a source, of a variety of pro-inflammatory factors and extracellular matrix proteins. Indeed there is evidence that their proliferative and synthetic functions can overlap  and that growth factors such as TGF-β, not only induce proliferation, but also induce extracellular matrix deposition . Airway SMCs also respond to cytokines and produce the chemokines CXCL10 (IP-10), in response to interferon (IFN)-γ [151, 152], and IL-8 and eotaxin, in response to IL-4 and IL-13 . Moreover, pro-inflammatory cytokines such as TNF-α, IL-1β and IFN-γ induce the expression of multiple chemokines and adhesion molecules in airway SMCs [154–156]. Thus, airway SMCs can perpetuate the inflammation of asthmatic airways [157–159].
There is growing evidence that Ca2+ signaling is important in the regulation of the production of cytokines and chemokines in other cell types such as T cells . In human adipocytes, secretion of macrophage inhibitory factor (MIF), CD14, macrophage colony-stimulating factor, IL-6 and expression of monocyte chemoattractant protein-1 were all Ca2+-dependent . IL-1β production by murine peritoneal macrophages  and exercise-induced IL-6 expression by skeletal muscle cells  were also regulated by [Ca2+]i In human bronchial epithelial cells, particulate matter induced IL-1β and IL-8 mRNA expression with an elevation of [Ca2+]i . Additionally, perturbing Ca2+ homeostasis affects mRNA expression of heme oxygenase-1 in human endothelial cells  and nuclear factor κ B (NFκB) activation and reactive oxygen species production in monocytes .
In airway SMCs, thapsigargin, an inhibitor of SERCA [167–169], modulated the differential effect of TNF-α on the production of IL-6 and RANTES (regulated on activation, normal T cell expressed and secreted) . Thapsigargin also reduced TNF-α-induced expression of intercellular adhesion molecule 1 (ICAM-1) by suppressing the activation of NFκB . Moreover, nickel ions (Ni2+) which act as a broad Ca2+ channel blocker to reduce Ca2+ influx, abrogated CD40-induced NFκB activation in human airway SMCs . These findings suggest that the [Ca2+]i homeostasis is also involved in cytokine/chemokine production in airway SMCs. Furthermore, cytokine exposure affects airway SMC Ca2+ signaling by modulating of the sarcoplasmic reticulum Ca2+ stores .
One of the key proteins used to transduce increases in [Ca2+]i is calmodulin (CaM), a 148-amino acid protein that binds up to four Ca2+ ions. An important action of CaM is the activation of a family of Ca2+/CaM-dependant protein kinases including CaM kinases I, II, III, and IV, myosin light chain kinase, and phosphorylase kinase . In NIH 3T3 cells, an increase in [Ca2+]i induced by IFN-γ activates CaM kinase II, which in turn, phosphorylates signal transducers and activators of transcription-1 (STAT-1) on serine 727 for maximum transcription activation . In contrast, the inhibition of CaM kinases suppresses IFN-α induced STAT-1 tyrosine phosphorylation and the activation of macrophages . It would be of interest to determine if Ca2+ regulates airway SMC STAT-1 activation and cytokine production stimulated by the IFN family.
Airway SMCs possess a wide array of ion channels which reside in plasma membrane, including receptor- or store-operated channels that may be comprised of a variety of TRP channels, stretch-activated channels, voltage-gated channels and Ca2+-dependent K+ channels. In addition, SMCs have specialized receptor/channels that reside in the sarcoplasmic reticulum. The activity of these channels significantly contributes to changes in the [Ca2+]i, which, in turn, determines, in the short term, the magnitude of SMC contraction and, in the longer term, SMC proliferation and the production and secretion of pro-inflammatory factors. In an exaggerated state, these cell activities are believed to be collectively expressed as airway hyper-responsiveness. While changes in [Ca2+]i are common to each processes, the question is raised of how this single ion messenger mediates this diversity of responses. Although the nature of the Ca2+ signals associated with proliferation or secretion are uncharacterized, the Ca2+ waves and oscillations associated with SMC contraction clearly indicate that airway SMC Ca2+ signaling is spatially and temporally dynamic. The key implication of this is that pattern of the changes of the [Ca2+]i are also important. In this respect, the distribution and temporal expression of a variety of ion channels that alter [Ca2+]i provides the necessary plasticity to regulate different SMC functions. As a result, these ion channels form an attractive target for therapeutic approaches for asthma.
Yazan Alrashdan was supported by The Asthma Foundation New South Wales and the CRC for Asthma and Airways. Satoru Ito was supported by a Grant-in-Aid for Young Scientists (19689017) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Malcolm Shepherd acknowledges the support of Asthma UK. Michael Sanderson was supported by NIH Grants HL71930 and HL 087401. The authors would also like to thank Astra-Zeneca for sponsoring and the travel grants to attend the 6th International Young Investigators’ Symposium on Smooth Muscle in Sydney Australia, November, 2007.
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Jose F. Perez-Zoghbi, Assistant Professor, Dept. Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, 3601 4TH St. Lubbock, TX 79430, Phone: (806) 743 2522 ; Fax (806) 743 1512, Email: firstname.lastname@example.org.
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