Ca2+ influx triggered by depletion of sarcoplasmic reticulum (SR) Ca2+ stores [mediated via store-operated Ca2+ channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca2+ was depleted by either 5 μM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca2+, subsequent introduction of extracellular Ca2+ further elevated [Ca2+]i. SOCC was insensitive to 1 μM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM–1 mM La3+ or Ni2+ inhibited SOCC. Exposure to ACh increased Ca2+ influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca2+ release by 20 μM xestospongin D inhibited SOCC, whereas ACh-induced IP3 production by 5 μM U-73122 had no effect. Inhibition of Ca2+ release through ryanodine receptors (RyR) by 100 μM ryanodine also prevented Ca2+ influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca2+ influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca2+ depletion. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx via SOCC in porcine ASM cells involves SR Ca2+ release through both IP3 and RyR channels. Additional regulation of Ca2+ influx by agonist may be related to a receptor-operated, noncapacitative mechanism.
- sarcoplasmic reticulum
- calcium release-activated calcium channel
- inositol trisphosphate
regulation of intracellular calcium concentration ([Ca +]i) in airway smooth muscle (ASM) with agonist stimulation is mediated by both extracellular Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum (SR) through both inositol 1,4,5-trisphosphate (IP3) receptor (5) and ryanodine receptor (RyR) (22) channels. Previous studies have shown that the [Ca2+]i response to agonist stimulation is biphasic, with the initial, transient rise in [Ca2+]i representing predominantly SR Ca2+ release (3) and the latter sustained, steady-state [Ca2+]i response representing predominantly Ca2+ influx (34). In ASM cells, Ca2+ influx may occur through both voltage-gated (26, 59) and receptor-gated channels (35). In addition to these influx pathways, there is now significant data from different cell types demonstrating that controlled Ca2+ influx also occurs in response to depletion of SR Ca2+, thus allowing for replenishment of intracellular Ca2+ stores [capacitative Ca2+ entry, Ca2+ release-activated channels, or store-operated Ca2+ channels (SOCC)] (40, 45–48). Initial studies in parotid acinar cells and vascular smooth muscle have demonstrated that extremely rapid refilling of SR stores following depletion is dependent on the presence of extracellular Ca2+ concentration ([Ca2+]o) but does not occur through either L-type or receptor-operated channels (reviewed in Ref. 40). Expression of the transient receptor potential channel (TRPC) genes have been demonstrated to be crucial for store-operated Ca2+ influx in mammalian cells, suggesting that TRPC-encoded proteins are the putative SOCC responsible for the observed Ca2+ influx (33, 49, 52, 63).
There is now considerable evidence for SOCC in vascular (1, 8, 13, 24, 28, 36, 40, 41, 54, 56, 57) and other smooth muscle types (1, 8, 24, 36, 41, 54, 56, 57). It has also been suggested that Ca2+ influx mediated via SOCC is involved in actual smooth muscle contraction (29). The role of SOCC in ASM had been suggested by experiments in guinea pigs, based on comparisons of contractions induced by agonists vs. inhibitors of the SR Ca2+ ATPase (SERCA) (24). Furthermore, SERCA inhibitors have been shown to increase [Ca2+]i in human bronchioles and bovine ASM, suggesting the involvement of SOCC (52). However, characterization of SOCC in ASM is relatively lacking. Given previous findings that both Ca2+ influx and SR Ca2+ release play important roles in [Ca2+]i regulation, identification and characterization of SOCC in ASM are important.
Previous studies suggested that SOCC is restricted to the replenishment of IP3-sensitive SR Ca2+ stores (17, 40). Some studies have demonstrated that elevation of IP3 (either by agonist stimulation or by direct introduction into the cytoplasm) triggers SOCC (46). However, other experiments using SERCA inhibitors such as thapsigargin directly demonstrated that SR depletion triggered SOCC with no further potentiation by additional application of IP3 (48). Certainly, IP3 plays an integral part in the [Ca2+]i response of ASM to agonists (5, 22). Accordingly, we hypothesize that SOCC in ASM is modulated by SR Ca2+ release via IP3 receptor channels.
We have previously shown that RyR channels are involved in the [Ca2+]i response of porcine ASM to agonist stimulation (22, 39, 42) and that the novel second messenger cyclic ADP ribose mediates agonist-induced Ca2+ release via RyR channels (43). Recent studies in tissues other than ASM have suggested that Ca2+ release through RyR channels may also trigger SOCC (10, 55, 57). We hypothesize that SR Ca2+ release via RyR channels is also a trigger for SOCC in ASM.
Porcine tracheae were obtained from a local abattoir. Single ASM cells were dissociated from the smooth muscle layer as previously described (22, 42). Briefly, the smooth muscle layer was excised, freed of endothelium, and minced in Hanks' balanced salt solution (HBSS) with 10 mM HEPES (pH 7.4, Life Technologies). The tissue was then incubated for 2 h with 20 U/ml papain and 2,000 U/ml DNase (Worthington Biochemical) and subsequently for 1 h at 37°C with 1 mg/ml type IV collagenase (Worthington Biochemical). The sample was gently triturated, centrifuged, and resuspended in minimum essential medium with 10% fetal calf serum. For [Ca2+]i imaging, isolated cells were plated on collagen-coated glass coverslips and incubated for 1 h in 5% CO2 at 37°C. Trypan blue exclusion was used to verify cell viability (>90% of all cells).
Identification of Transient Receptor Potential Isoforms
ASM cells in suspension were processed for microsomal enrichment and separation of plasma membrane and intracellular membranes. Freshly isolated cells (2×107 cells) were extracted (50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 10 μM leupeptin, and 100 μM PMSF) at 4°C and homogenized with a Polytron homogenizer, and the extract was centrifuged at 750 g for 10 min at 4°C. The resulting supernatant was centrifuged at 50,000 g for 30 min at 4°C, and the pellet was resuspended in extraction buffer and stored at –80°C. Protein concentrations were determined using a Lowry assay.
Western blot analyses of candidate transient receptor potential (TRP) proteins (TRPC family) were performed using microsome samples (40 μg of protein). The samples were separated by 9% SDS-PAGE, transferred to nitrocellulose, and blocked 3–4 h with Tris buffer (2 0 mM Tris·HCl, pH 8.0, 500 mM NaCl, and 0.1% Tween 20) and 5% skimmed milk powder. The membranes were then incubated overnight at 4°C with rabbit anti-TRPC1 (1 μg/ml in Tris buffer, Chemicon International), goat anti-TRPC3 (which recognized TRPC3, TRPC6, and TRPC7), and anti-TRPC4 antibodies (1 μg/ml in Tris buffer, Santa Cruz Biotechnology). After three washes, peroxidase-conjugated anti-rabbit or anti-goat IgG antibody (1:2,000 in Tris buffer) was applied for 2 h at room temperature and finally washed. Proteins were detected with a peroxidase staining kit (Vector Labs). Appropriate control antigens and rat brain extracts (positive control) were also used.
Real-Time Confocal Imaging
Cells were loaded with 5 μM fluo 3-AM (Molecular Probes) for 30–45 min at 37°C in HBSS and washed in HBSS, and the coverslip was mounted on an open slide chamber (RC-25F, Warner Instruments) and perfused at 2–3 ml/min at room temperature.
Details of the confocal imaging technique have also been previously published (22, 42). An Odyssey XL real-time confocal system (Noran Instruments) with an Ar-Kr laser (488-nm line) and a Nikon ×40/1.3 oil-immersion objective lens was used to visualize the cells (640 × 480 pixels, 0.063 μm2/pixel, 1 μM optical section thickness). Measurements were made across the whole cell. Based on previous studies (22, 42), a fixed combination of laser intensity (20% maximum) and photomultiplier gain (45% maximum) was used to ensure pixel intensities between 25 and 255 gray levels. At these settings, intermittent laser exposure at 3 mW (<5 min) caused <1% photo-bleaching. Empirical calibration of fluo 3 fluorescence levels for [Ca2+]i based on exposure to known Ca2+ concentrations was performed as described previously (42).
Establishment of SOCC by passive SR depletion. Previous studies on SOCC have employed passive SR depletion with cyclopiazonic acid (CPA) to inhibit SERCA (40, 47). This approach presumably depletes Ca2+ from both IP3- and ryanodine-sensitive SR stores. ASM cells were initially perfused with HBSS for 1–2 min, and baseline Ca2+ levels were measured. [Ca2+]o was then removed with zero-Ca2+ HBSS (5 mM EGTA). The cells were then rapidly exposed to 1 μM CPA in zero-Ca2+ HBSS. Continued SR Ca2+ leak resulted in elevation of [Ca2+]i levels. When Ca2+ reached a plateau level or was starting to trend downward, 2.5 mM [Ca2+]o was rapidly reintroduced (in the continued presence of CPA), and the [Ca2+]i response was recorded.
The effect of various pharmacological manipulations on SOCC was investigated. Details of these protocols are provided in the results section, to avoid repetition.
Establishment of SOCC by caffeine depletion of ryanodine-sensitive SR Ca2+. It is well recognized that caffeine largely depletes ryanodine-sensitive Ca2+ stores. Although some studies have indicated caffeine effects on IP3-sensitive stores (16, 32), these studies have found inhibition rather than activation of such stores by caffeine. Therefore, caffeine is likely a suitable agent for selective depletion of ryanodine-sensitive Ca2+ stores and for the assessment of any interactions between Ca2+ release via RyR channels and SOCC.
ASM cells were initially perfused with HBSS for 1–2 min and baseline Ca2+ levels were measured. [Ca2+]o was then removed with zero-Ca2+ HBSS (5 mM EGTA). The cells were then rapidly exposed to 5 mM caffeine in zero-Ca2+ HBSS, and the peak of the [Ca2+]i response was measured. The cells were then washed with HBSS for 15 min to replenish the SR. [Ca2+]o was removed again, and the cells were reexposed to caffeine. In the continued presence of caffeine, 2.5 mM [Ca2+]o was rapidly reintroduced, and the [Ca2+]i response was recorded. As with the CPA protocols, the effect of various pharmacological manipulations on SOCC was investigated.
For each ASM cell, comparisons before and after exposure to a drug or with a manipulation were made by paired Student's t-test. Multiple comparisons were performed with repeated-measures ANOVA and post hoc testing using Bonferroni and Scheffé's tests. Because it was not possible to apply all experimental protocols to the same cells, results were replicated in at least three to five cells obtained from each of five animals (with paired comparisons within cells and independent testing across cells). Statistical significance was tested at the P < 0.05 level. Values are reported as means ± SE.
Identification of TRP Isoforms
Western blots of ASM showed positive reactivity with anti-TRPC1, anti-TRPC3, and anti-TRPC4 antibodies, indicating the predominant expression of TRPC1, TRPC4, and one or all of TRPC3, TRPC6, and TRPC7 (Fig. 1, n = 5 tracheae). Using gel densitometry on blots where the protein levels were known and controlled, we estimated the relative intensities of the these blots. The most intense staining was for TRPC3.
Establishment of SOCC. Baseline [Ca2+]i levels of ASM cells were 85 ± 4 nM (n = 149). There was no significant change in [Ca2+]i levels with removal of extracellular Ca2+. In the absence of extracellular Ca2+, exposure of ASM cells to 1 μM CPA resulted in elevation of [Ca2+]i levels that eventually reached a plateau (n = 89). Under these conditions, rapid reintroduction of [Ca2+]o resulted in a further, sustained elevation of [Ca2+]i (Fig. 2). The plateau of the response to CPA ranged from 250 to 450 nM. The plateau of the response to [Ca2+]o ranged from 340 to 660 nM (P < 0.05 compared with the first plateau), representing a rise in [Ca2+]i of ∼90–250 nM using CPA-induced depletion of SR Ca2+ stores.
Exposure to 5 mM caffeine in the absence of [Ca2+]o resulted in the well-recognized transient [Ca2+]i response (Fig. 2). After a wash in HBSS, the peak of the second [Ca2+]i response to caffeine was not significantly different from the peak of the first response. In the continued presence of caffeine, reintroduction of 2.5 mM [Ca2+]o resulted in a relatively sustained elevation in [Ca2+]i, demonstrating Ca2+ influx (Fig. 2, n = 138). The plateau of the response to 2.5 mM [Ca2+]o ranged from 150 to 400 nM, whereas the actual amplitude of the response (measured from the Ca2+ level just before activation) ranged from 75 to 138 nM (Fig. 2). Compared with a peak caffeine response ranging from 600 to 950 nM, the influx response ranged from 18 to 28% of the caffeine peak (Fig. 2C). Compared with the Ca2+ influx observed with CPA (see above), the influx observed with reintroduction of [Ca2+]o following caffeine exposure was significantly smaller (P < 0.05).
Sensitivity to nifedipine. To distinguish SOCC-mediated Ca2+ influx from that via L-type Ca2+ channels, we conducted experiments in a separate set of cells using 1 μM nifedipine. In these cells, the [Ca2+]i response to rapid reintroduction of [Ca2+]o in the presence of CPA (n = 25) or caffeine (n = 14) was determined as above. After a wash in HBSS, ASM cells were exposed to 1 μM nifedipine, [Ca2+]o was removed, and, in the continued presence of nifedipine, cells were reexposed to CPA (or caffeine) with both zero-Ca2+ and regular HBSS, thus inhibiting L-type Ca2+ channels through most of the protocol. The presence of nifedipine during rapid reintroduction of [Ca2+]o did not significantly influence the amplitude of the [Ca2+]i response to Ca2+ influx for both CPA and caffeine (Fig. 3, A and B), indicating that the observed Ca2+ influx in both instances was not mediated through L-type Ca2+ channels.
Role of membrane potential. A potential confounding factor in SOCC-mediated Ca2+ influx is the membrane potential, which will alter the driving forces for Ca2+ entry. Accordingly, separate experiments were performed to determine the role of membrane potential. A control response to [Ca2+]o was recorded in the presence of nifedipine (now present in all further experiments) and CPA (n = 15) or caffeine (n = 14). After a HBSS wash, ASM cells were reexposed to nifedipine, [Ca2+]o was removed, and 10, 40, or 60 mM KCl was added in the continued presence of nifedipine. Under these conditions, cells were reexposed to CPA or caffeine, and [Ca2+]o was rapidly reintroduced. The presence of 10 and 40 mM KCl did not significantly influence the amplitude of the [Ca2+]i response to CPA or caffeine (Fig. 3C). However, in the presence of 60 mM KCl, the [Ca2+]i response upon reintroduction of [Ca2+]o was ∼25% smaller than that in its absence for both CPA and caffeine (P < 0.05, Fig. 3C).
Sensitivity to Ni2+ and La3+. Previous studies in other tissues have found SOCC-mediated Ca2+ influx to be inhibited by Ni2+ and La3+. In a separate group of cells, following a control activation of SOCC with CPA (n = 16) or caffeine (n = 21) and HBSS wash, ASM cells were exposed to nifedipine, 10 mM KCl, and NiCl2 (1 μM or 1 mM) in HBSS. [Ca2+]o was then removed, and, in the continued presence of nifedipine, KCl, and Ni2+, cells were reexposed to CPA or caffeine. [Ca2+]o was then rapidly reintroduced. In a subset of experiments (n = 11), Ni2+ was added to the medium only after the [Ca2+]i response to CPA had reached near plateau or the [Ca2+]i response to caffeine had decreased to near baseline values. In the presence of both 1 μM and 1 mM Ni2+, reintroduction of [Ca2+]o following CPA or caffeine exposure did not result in any significant increase in [Ca2+]i (Fig. 4, P < 0.05 compared with control), indicating that SOCC-mediated Ca2+ influx in ASM cells is Ni2+ sensitive. The timing of introduction of Ni2+ did not significantly affect the results.
The CPA (n = 8)- and caffeine-based (n = 11) protocols used to test Ni2+ sensitivity were repeated by using 1 μM and 1 mM LaCl3 instead of Ni2+. As with the Ni2+ experiments, SOCC activation was significantly inhibited by both concentrations of LaCl3 (Fig. 4).
Sensitivity to SKF-96365. Previous studies in other tissues have used inhibition of Ca2+ influx by SKF-96365 as adjunct evidence of SOCC-mediated Ca2+ influx. However, this compound is also reported to inhibit receptor-operated Ca2+ influx. Regardless, in a separate group of cells, following a control SOCC activation with CPA (n = 25) or caffeine (n = 18) and HBSS wash, ASM cells were exposed to nifedipine and KCl (as in above protocols) and to 10 μM SKF-96365 in HBSS. The SOCC protocol was then repeated in the continued presence of these agents. SKF-96365 inhibited SOCC-mediated Ca2+ influx with both CPA and caffeine protocols (Fig. 4B, P < 0.05 compared with controls). However, it must also be noted in ∼15–20% of the cells, SKF had no effect on SOCC-mediated Ca2+ influx.
Effect of ACh. After a control activation of SOCC and a wash, ASM cells were exposed to CPA or caffeine in zero-[Ca2+]o and then to 10 nM, 100 nM, or 1 μM ACh with nifedipine and KCl in the continued presence of CPA or caffeine (n = 15 for each ACh concentration for either CPA or caffeine). Under these circumstances, [Ca2+]o was rapidly reintroduced. The goal of these protocols was to ensure that SR Ca2+ release occurred before activation of muscarinic receptors by ACh. In the case of CPA, SR Ca2+ depletion before ACh was passive and occurred through both IP3 and RyR channels. In the case of caffeine, release through RyR channels certainly occurred before ACh, whereas release via the IP3 pathway occurred only after ACh exposure. Although only a CPA protocol would seem appropriate for examination of SOCC, the above protocols were conducted for comparison with other protocols listed below. Compared with controls (no ACh), preexposure of ASM cells to either 100 nM or 1 μM (but not 10 nM) ACh resulted in significant enhancement of Ca2+ influx with reintroduction of [Ca2+]o (P < 0.05, Fig. 5).
A potential confounding factor in the above protocols examining ACh interactions with SOCC is the possible contribution of noncapacitative muscarinic receptor-operated Ca2+ influx (20), which is likely activated on exposure to ACh. Indeed, it may be difficult to distinguish between capacitative (i.e., SOCC) vs. noncapacitative Ca2+ influx (29). Accordingly, in separate experiments (n = 14), where a control SOCC activation and a Ca2+ influx response to ACh following caffeine depletion was confirmed, ASM cells were thoroughly washed to replenish SR Ca2+ stores and then exposed to 20 μM xestospongin D [XeD, a cell-permeant IP3 receptor blocker (12)] and 50 μM ryanodine for at least 15 min. This ensured the simultaneous inhibition of both IP3 receptor and RyR channels, while maintaining the SR in a state of repletion. In the additional presence of nifedipine, KCl, and [Ca2+]o, the cells were exposed to 1 μM ACh. Under these circumstances, ACh produced a significant elevation in [Ca2+]i, indicating the additional presence of receptor-operated, noncapacitative Ca2+ influx. However, the amplitude of the Ca2+ influx under these conditions was 36.7 ± 3.5% of that induced by ACh (upon reintroduction of [Ca2+]o) when SR Ca2+ stores were depleted by caffeine.
Role of IP3. The caffeine protocol used to evaluate the influence of ACh on SOCC was modified. After a control activation of SOCC in the absence and presence of 1 μM ACh with intervening washes, [Ca2+]o was removed, and the cells were exposed to 5 μM U-73122 or 20 μM XeD for 15 min to inhibit PLC- or IP3-induced SR Ca2+ release (n = 8, respectively). The SOCC was then activated by [Ca2+]o in the presence of caffeine, ACh, and U-73122 or XeD. Inhibition of ACh-induced IP3 production by U-73122 did not significantly influence SOCC-mediated Ca2+ influx compared with 1 μM ACh alone (Fig. 6). However, inhibition of IP3-induced SR Ca2+ release by XeD significantly decreased Ca2+ influx (P < 0.05 compared with control and ACh, Fig. 6). It should be noted that the above protocols were not performed with CPA, since inhibition of IP3 receptors would not necessarily prevent depletion of IP3-sensitive SR Ca2+ stores, especially if Ca2+ release via IP3 and RyR channels occurs from a common pool.
Role of RyR. The caffeine protocol used to establish SOCC in ASM cells (see above) essentially demonstrates that depletion of caffeine/ryanodine-sensitive SR Ca2+ stores also results in SOCC activation. Lack of caffeine-induced Ca2+ release via IP3 receptor channels was verified when the presence of 20 μM XeD had no significant effect on SOCC-mediated Ca2+ influx in the caffeine protocol (Fig. 7). To further characterize this feature, we first performed a control SOCC activation in a separate set of ASM cells, and following a wash, the cells were preexposed to 100 μM ryanodine to inhibit RyR channels. In the presence of ryanodine, reintroduction of [Ca2+]o did not result in SOCC-mediated Ca2+ influx following exposure to caffeine (P < 0.05 compared with control, Fig. 7). Furthermore, ryanodine prevented SOCC-mediated Ca2+ influx even in the presence of 1 μM ACh. It should be noted that in the presence of ryanodine, there was only a minimal [Ca2+]i response to caffeine (∼10% of control).
In certain respects, the characteristics of SOCC in ASM are similar to those observed in vascular (13, 28, 40) and other smooth muscle types (56, 58) in that Ca2+ influx mediated via this mechanism is insensitive to nifedipine and involves IP3-mediated SR Ca2+ release. However, the present study found that in addition to passive SR depletion or depletion of IP3-sensitive stores, SOCC in ASM occurs in response to depletion of ryanodine/caffeine-sensitive stores. Overall, these results indicate that SOCC in ASM occurs in response to generalized SR Ca2+ depletion (thus representing a store-operated mechanism) as well as during agonist stimulation.
Ca2+ Influx in ASM
In ASM, Ca2+ influx occurs through both voltage-gated (26, 59) and receptor-gated channels (2, 21, 27, 35). For example, in cultured human ASM cells, the steady-state [Ca2+]i response to ACh stimulation has been attributed to Ca2+ influx through receptor-gated channels (35), whereas in porcine and canine ASM cells, voltage-gated channels are involved (9, 23).
Initial studies in parotid acinar cells and vascular smooth muscle demonstrated that extremely rapid SR refilling following depletion is dependent on [Ca2+]o but not L-type or receptor-operated channels (reviewed in Ref. 40). In vascular smooth muscle, SR Ca2+ depletion by thapsigargin (another SERCA inhibitor) (53) activates Ca2+ influx independently of IP3 generation and is insensitive to nicardipine (61). Studies in a variety of preparations have demonstrated that SERCA inhibitors increase Ca2+ influx and/or smooth muscle tone (1, 6, 8, 11, 14, 37). In this regard, it is interesting that CPA appears to cause a slower, more gradual elevation in [Ca2+]i, whereas thapsigargin (at least in vascular smooth muscle) causes a biphasic response. Sensitivity of SERCA to these inhibitors, as well as relative activity of other Ca2+ efflux mechanisms in different cell types, may underlie this difference.
Although [Ca2+]o is necessary for maintenance of muscle tone or [Ca2+]i, sensitivity to Ca2+ channel blockers is not consistent, such that nifedipine completely inhibits influx in rat aorta but not pulmonary artery. These data suggested that both voltage-gated Ca2+ channels as well as SOCC contribute to smooth muscle Ca2+ regulation and tone. The contribution of either mechanism likely differs across preparations such that tonic muscles including blood vessels (13, 28, 40), gastric fundus (41), and pulmonary artery (8) depend on a larger contribution from SOCC. One previous study used measurement of Ca2+-activated Cl- currents to suggest the existence of SOCC in ASM cells (24); however, definitive experiments and characterization of SOCC in ASM have only been recently reported in the guinea pig ASM (20).
Characteristics of SOCC in ASM
Unlike L-type Ca2+ channels, SOCC does not appear to be gated by changes in membrane potential (40), although once activated, SOCC has a current-voltage relationship showing large currents at negative voltages and a reversal potential at ∼50 mV, beyond which there is no ionic flow. (18, 40). Because the present study did not use electrophysiological techniques to examine SOCC, these properties were only indirectly addressed. The lack of nifedipine sensitivity of the [Ca2+]i response to [Ca2+]o in either the CPA or the caffeine protocol indicates that SOCC rather than voltage-gated channels are involved in this response. This is further demonstrated by persistence of Ca2+ influx when membrane potential was held at different levels using KCl. Indeed, KCl-induced depolarization resulted in less influx. If such influx had been mediated via voltage-gated channels, more positive membrane potentials should increase Ca2+ influx, whereas SOCC-mediated influx should be smaller due to decreased driving force for Ca2+ (40). Regardless, while demonstrating SOCC, these data do not rule out the already established role for L-type Ca2+ channels in ASM (50, 62). The relative contribution of voltage-gated channels vs. SOCC likely depends on whether a specific agonist causes membrane depolarization and/or only SR Ca2+ release.
As with voltage-gated Ca2+ channels, SOCC have been shown to be blocked by several divalent cations in the mM range, including Ni2+ (20, 28, 36, 55, 57). We also found that the [Ca2+]i response to [Ca2+]o was inhibited by both low and high concentrations of Ni2+. Although inhibition by Ni2+ in isolation does not provide convincing evidence of SOCC, the facts that Ni2+ inhibition occurred at membrane potentials where voltage-gated channels were likely inactivated and that even low Ni2+ concentrations were effective provide stronger evidence for inhibition of SOCC. In this regard, La3+ inhibition of Ca2+ influx is further evidence of SOCC and is consistent with findings in anococcygeus smooth muscle (57) as well as pulmonary artery (28).
IP3-Induced SR Ca2+ Release and SOCC in ASM
Previous studies suggested that SOCC is restricted to the replenishment of IP3-sensitive SR Ca2+ stores (17, 40). Some studies have demonstrated that elevation of IP3 triggers SOCC (46). In one theory of SOCC regulation (48), IP3 receptors directly interact with plasma membrane SOCC such that decreased SR Ca2+ induces conformational change in the IP3 receptor leading to protein-protein interaction and activation of Ca2+ influx. Alternatively, proteins that mediate SOCC may be colocalized with IP3 receptor channels (essentially by proximity of the SR and plasma membranes). For example, Trp3 and IP3 receptor colocalize within caveolae in cortical neurons (24a). The mechanism by which SOCC-IP3 receptor interactions take place in ASM is not known.
Certainly, IP3 plays an integral part in the [Ca2+]i response of ASM to agonists (5, 22). The present study demonstrates that IP3 is also involved in SOCC activation in porcine ASM. In this regard, the lack of effect of PLC inhibition on SOCC is consistent with the idea that IP3 itself is nonessential for SOCC (48) and that SR Ca2+ release via IP3 receptor channels is involved. In this regard, the specificity of XeD for IP3 receptors has been previously demonstrated, and thus our findings of SOCC inhibition by XeD are consistent with a role for IP3 receptors. Some studies have used 2-aminoethyldiphenyl borate (2-APB) to examine the role of IP3 receptor channels in SOCC. However, several recent studies have indicated that not all of the observed interactions between 2-APB and SOCC are actually mediated via IP3 receptor channels. For example, in rat hepatocytes, where 2-APB did not inhibit IP3-induced SR Ca2+ release, inhibition of SOCC involves binding of this compound to the SOCC protein itself or a regulatory protein (15). In other cell types where IP3 receptors were knocked out, inhibition of SOCC by 2-APB was identical in knockout vs. wild-type cells (25, 44). Whether SOCC occurs through mechanisms independent of IP3 receptors is not known and was not determined in the present study.
Caffeine-Induced SR Ca2+ Release and SOCC in ASM
Although the role of SR Ca2+ release through IP3 receptor channels appears to be more definite, interactions between RyR channels and SOCC are less clearly defined. Recent studies in different tissues, including skeletal muscle where RyR channels are key to Ca2+ regulation, have demonstrated SOCC activation in response to depletion of caffeine-sensitive SR Ca2+ stores and subsequent introduction of [Ca2+]o (30, 51). In the present study, the fact that SOCC was activated following SR depletion by caffeine is certainly indicative of a role for RyR channels. Most studies employ low mM caffeine to “specifically” release Ca2+ through RyR channels. In addition to regulating Ca2+ release via RyR channels, caffeine may also inhibit IP3-induced SR Ca2+ release (16, 32). However, in the present study, if caffeine did inhibit IP3-induced SR Ca2+ release, this would only further strengthen the role for RyR channels in SOCC activation. Another issue with the use of caffeine is its known inhibitory effect on phosphodiesterases, resulting in increased cAMP and cGMP (7), which may themselves alter Ca2+ influx. However, a previous study in neurons demonstrated that SOCC does not occur in the presence of a cAMP phosphodiesterase inhibitor more potent than caffeine (55). Furthermore, studies in mouse anococcygeus smooth muscle (57) as well as rat pulmonary artery (36) have found that low concentrations of ryanodine (which activate RyR channels) stimulate SOCC, whereas higher ryanodine concentrations (which inhibit RyR channels) prevent subsequent SOCC activation. Altogether, these data strongly support a role for Ca2+ release through RyR channels in SOCC activation.
Agonist Stimulation and SOCC in ASM
Whether agonist stimulation modulates SOCC is not clear. A confounding issue is the presence of other mechanisms of Ca2+ influx that are neither voltage gated nor mediated by SOCC, such as agonist-induced noncapacitative Ca2+ influx via G protein-coupled receptor-operated mechanisms (4, 20, 29). Whether such a mechanism is relevant to ASM is not clear (20). In the present study, selective activation of the noncapacitative component by preventing SR Ca2+ depletion (by blocking both IP3 receptor and RyR channels) resulted in significantly less enhancement of Ca2+ influx by ACh compared with when the SR was depleted, suggesting the existence of both SOCC and noncapacitative Ca2+ influx in ASM. The relative roles of these influx mechanisms in [Ca2+]i regulation is likely species and agonist dependent.
TRP Expression in ASM
There is now increasing evidence that members of the TRP family are the proteins mediating SOCC, and several mammalian TRP genes have been identified (31, 45). Electrophysiological studies of cells overexpressing certain TRP genes (the TRPC family) have shown increased Ca2+ influx following depletion of intracellular stores (45). The present study found the expression of several TRPC isoforms in porcine ASM. Although the antibodies used in the present study did not identify the expression of each of the individual TRPC isoforms, the data at least suggest a correlation between TRPC expression and the presence of SOCC in porcine ASM and are generally consistent with previous studies based on RT-PCR of isolated RNA from smooth muscle (28, 60). A very recent study by Ong et al. (38) found TRPC1, TRPC3, and TRPC6 proteins in guinea pig ASM, although the authors did not specifically examine whether these proteins mediate SOCC in that tissue. TRPC1 has been reported to be involved in SOCC (60), whereas TRP6 appears to be involved in α-adrenoceptor-activated Ca2+ influx in vascular smooth muscle (19). Some recent studies have also examined the effect of altered TRP isoform expression on SOCC and found that decreased TRPC3 expression results in decreased SOCC-mediated Ca2+ influx (31). Therefore, it is possible that SOCC in porcine ASM is mediated via TRPC proteins.
The authors thank Larry Hunter, Kathy Street, and Thomas Keller for superb technical assistance.
This work was supported by National Institute of General Medical Sciences Grant GM-56686 (G. C. Sieck).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society