Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca²⁺ release in smooth muscle

Abstract

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.

Ca²⁺ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) in response to membrane depolarization drives the mechanical force and other cellular responses in skeletal and cardiac muscle cells. The underlying molecular processes in these 2 types of muscle cells differ greatly. In skeletal muscle cells, membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) in the plasma membrane, which can open RyR1 via their physical interaction with plasmalemmal VDCCs without an extracellular Ca²⁺ influx, inducing massive Ca²⁺ release from the SR. This process is termed voltage-induced Ca²⁺ release (VICR). In cardiac myocytes, activation of VDCCs by depolarization results in a small amount of extracellular Ca²⁺ influx. This Ca²⁺ influx subsequently causes the opening of RyR2, and then induces large Ca²⁺ release from the SR, a process of Ca²⁺-induced Ca²⁺ release (CICR) (1).

All 3 subtypes of RyRs (RyR1, RyR2, and RyR3) are expressed in smooth muscle cells (SMCs). However, there is no electrophysiological study showing skeletal VICR in SMCs. Cardiac-like CICR has been observed in bladder myocytes; however, unlike in cardiac cells, VDCCs and RyRs in these SMCs are “loosely coupled,” by which activation of VDCCs following depolarization produces non-obligate Ca²⁺ release from the SR; as such, sufficient Ca²⁺ influx through VDCCs is required to activate CICR (2, 3). In contrast, voltage depolarization does not evoke Ca²⁺ release in SMCs from numerous tissues including the trachea (4).

We here report that long voltage or chemical depolarization induces local Ca²⁺ release from the SR in airway SMCs without the involvement of VDCCs and associated extracellular Ca²⁺ influx. This form of local Ca²⁺ release results from a direct activation of native M₃ muscarinic receptors (M₃Rs) in the absence of exogenous agonists. Moreover, depolarization-evoked excitation of M₃Rs turns on their downstream G protein−phospholipase C (PLC)-IP₃ signaling axis, leading to initial Ca²⁺ release through IP₃Rs and then further Ca²⁺ release via RyRs due to a local IP₃R/RyR interaction-mediated CICR mechanism. The GPCR- and IP₃R-dependent, depolarization-induced local Ca²⁺ release is specifically mediated by RyR2, but not RyR1 and RyR3. This M₃R-dependent, depolarization-induced Ca²⁺ release evokes airway muscle contraction, showing an important physiological significance in SMCs.

Results

Membrane Depolarization Induces Ca²⁺ Release Through Ryanodine Receptors in SMCs.

Voltage depolarization from a holding potential of −60 mV to various potentials between −40 mV and 30 mV for 250 ms generated VDCC currents that were selectively blocked by the channel blocker nifedipine (10 μM) for 5 min (Fig. 1A). As examples shown in Fig. 1B, voltage depolarization in such duration did not induce detectable, instantaneous Ca²⁺ release in 12–16 cells tested. Long depolarization for 5 s also did not evoke Ca²⁺ release in the initial first second. After that, however, local Ca²⁺ release was evident, leading to a large increase in the frequency of Ca²⁺ sparks (Fig. 1C). The mean frequency of Ca²⁺ sparks was increased from 0.061 ± 0.011 to 0.107 ± 0.014 sparks/s/μm (n = 14, P < 0.05). Thus, there is a latency of >1 s for depolarization-induced Ca²⁺ release. We also observed that the amplitude of Ca²⁺ sparks was reduced from 0.829 ± 0.062 to 0.659 ± 0.028 ΔF/F₀ (P < 0.05). On the other hand, there were no changes in the Ca²⁺ spark rise time (31.9 ± 1.7 vs. 29.7 ± 0.9 ms), full duration at half maximal amplitude (29.7 ± 2.2 ms vs. 27.6 ± 1.3 ms), decay time (129.5 ± 15.1 vs. 116.3 ± 7.4 ms), and full width at half maximal amplitude (5.2 ± 0.4 vs. 4.9 ± 0.2 μm). As an example shown in Fig. 1D, application of ryanodine (100 μM) for 8 min to specifically inhibit RyRs abolished spontaneous Ca²⁺ sparks and subsequent depolarization-evoked Ca²⁺ sparks as well in 5 cells tested. In agreement with prominent Ca²⁺ release, voltage depolarization for 5 s caused significant contraction, determined by cell shortening, in isolated cells (Fig. 1E).

Fig. 1.

Voltage depolarization for a long, but not short duration induces local Ca²⁺ release in airway SMCs. (A) Original recordings (left) show VDCC currents generated by voltage depolarization from a holding potential of −60 mV to 10 mV for 250 ms in a cell before and after application of nifedipine (10 μM) for 5 min. The cell was voltage-clamped using nystatin-perforated patch clamp technique. Graph (right) illustrates the current-voltage curves for Ca²⁺ currents obtained from 6 different cells. (B) Voltage depolarization from −60 mV to −20, 0, and 20 mV for 250 ms did not induce detectable Ca²⁺ release in 12, 14, and 16 cells, respectively. (C) Voltage depolarization from −60 mV to −20 mV for over 1 s significantly increased the frequency of Ca²⁺ sparks, and decreased the amplitude of Ca²⁺ sparks. *, P < 0.05 compared with −60 mV. (D) Application of ryanodine (100 μM) for 8 min to specifically block RyRs abolished spontaneous and depolarization-induced local Ca²⁺ release. (E) Voltage depolarization from −60 mV to −20 mV for 5 s caused significant cell contraction, determined as the difference in cell length before and after voltage depolarization under perforated patch-clamp conditions.

Similar to voltage depolarization, chemical depolarization by elevating extracellular K⁺ from 5 to 60 mM for 1 min also caused local Ca²⁺ release in the absence of exogenous agonists, resulting in an increase in Ca²⁺ spark frequency and a decrease in Ca²⁺ spark amplitude without affecting other spatiotemporal characteristics (Fig. 2A). Chemical depolarization failed to induce local Ca²⁺ release in cells that were voltage-clamped at −60 mV (Fig. 2A) or pretreated with ryanodine (100 μM) for 8 min (Fig. 2B).

Fig. 2.

Chemical depolarization with high K⁺ exposure for 1 min also induces local Ca²⁺ release in airway SMCs. (A) Application of high K⁺ (60 mM) for 1 min significantly increased the frequency of Ca²⁺ sparks, and reduced the amplitude of Ca²⁺ sparks in cells that were not voltage-clamped; however, high K⁺ had no effect in cells voltage-clamped at −60 mV using the perforated patch clamp technique. (B) Application of ryanodine (Rya, 100 μM) prevented high K⁺-induced local Ca²⁺ release.

Membrane Depolarization-Induced Ca²⁺ Release Can Be Independent of Voltage-Dependent Ca²⁺ Channels and Associated Extracellular Ca²⁺ Influx in SMCs.

The selective VDCC blocker nifedipine (10 μM) or D600 (100 μM) for 5 min did not prevent the effect of chemical depolarization on the frequency and amplitude of Ca²⁺ sparks (Fig. 3A). Removal of extracellular Ca²⁺ (equilibration with nominally Ca²⁺-free plus 0.25 mM EGTA for 30 s) did not block the effect of chemical depolarization, either. This effect was not due to a significant Ca²⁺ contamination, as the free Ca²⁺ concentration in the recording chamber was found to be 1.87 ± 0.02 nM (n = 6). Moreover, chemical depolarization was able to induce significant contraction in isolated airway smooth muscle cells and tissues after treatment with nifedipine or removal of extracellular Ca²⁺ (Fig. 3B). Taken together, these data reveal that VDCCs and associated extracellular Ca²⁺ influx are not required for RyR-mediated local Ca²⁺ release and attendant contraction in SMCs.

Fig. 3.

Membrane depolarization-induced local Ca²⁺ release is independent of VDCCs and associated extracellular Ca²⁺ influx in airway SMCs. (A) Application of nifedipine (Nif, 10 μM) for 5 min to inhibit VDCCs or incubation with nominally Ca²⁺-free plus 0.25 mM EGTA bath solution (0 Ca²⁺) for 30 s to remove extracellular Ca²⁺ did not prevent chemical depolarization-evoked local Ca²⁺ release. (B) High K⁺ exposure was able to cause significant shortening in isolated airway SMCs (left) and muscle contraction (tension) in tracheal rings (normalized to tissue weight; right) after treatment with nifedipine (10 μM) for 5 min and incubation with nominally Ca²⁺-free plus 0.25 mM EGTA bath solution for 30 s.

Direct Activation of G Protein-Coupled Receptors and Their Downstream Signaling Axis Are an Initial, Indispensible Event for Depolarization-Induced Local Ca²⁺ Release in SMCs.

The long latency for depolarization-induced Ca²⁺ release in airway SMCs is similar to that for agonist-induced Ca²⁺ release, which is generally considered to represent the time course of the GPCR-dependent biochemical cascade reactions in SMCs (6). It has also been reported that membrane depolarization causes charge movement (gating current) of heterologously expressed GPCRs, M₁Rs and M₂Rs, in Xenopus oocytes (7). Thus, we wondered whether membrane depolarization might result in a direct activation of native GPCRs and their downstream signaling axis to trigger RyR-mediated local Ca²⁺ release in the absence of exogenous agonists. Our results indicate that targeted gene deletion of M₃Rs, functionally important GPCRs expressed in SMCs, could prevented depolarization causing an increase in the frequency of Ca²⁺ sparks and a reduction in the amplitude of Ca²⁺ sparks (Fig. 4A). On the contrary, M₂R and M₁R gene deletion did not significantly affect chemical depolarization-triggered local Ca²⁺ release (Fig. 4 B and C).

Fig. 4.

Membrane depolarization-induced local Ca²⁺ release is a direct activation of G protein-coupled, M₃ muscarinic receptors in airway SMCs. (A) Depolarization with high K⁺ increased Ca²⁺ spark frequency and decreased Ca²⁺ spark amplitude in M₃R^+/+, but not in M₃R^−/− cells. (B) Effect of high K⁺ on Ca²⁺ sparks were comparable in M₂R^−/− and M₂R^+/+ cells. (C) M₁R gene deletion did not affect chemical depolarization-induced local Ca²⁺ release, either. (D) Treatment with acetylcholine esterase (85 U/mL) for 10 min had no effect on the activity of spontaneous and high K⁺-evoked Ca²⁺ sparks, but fully blocked acetylcholine (3 and 10 nM)-evoked response. (E) M₃R, but not M₂R, gene deletion prevented high K⁺-induced muscle contraction in isolated tracheal rings.

Membrane depolarization can significantly enhance muscarinic agonist-induced Ca²⁺ release by affecting multiple signaling cascade sites in several types of cells (8), and muscarinic receptors provide a predominant neural control of Ca²⁺ signaling in airway SMCs (9). Thus, to exclude the potential involvement of the endogenous muscarinic agonist acetylcholine, we examined the effect of acetylcholine esterase, an endogenous enzyme known to rapidly metabolize acetylcholine, on depolarization-induced Ca²⁺ release. Treatment with this enzyme (85 U/mL) for 10 min, as used in lung slice preparations (10), did not affect the activity of spontaneous and depolarization-induced Ca²⁺ sparks in isolated cells (Fig. 4D). We also found that application of acetylcholine at 1 nM had no effect on Ca²⁺ sparks, but at 3 and 10 nM significantly increased the activity of Ca²⁺ sparks (Fig. 4D). The acetylcholine (3 and 10 nM)-evoked responses were completely blocked by pretreatment with acetylcholine esterase (85 U/mL) for 10 min. These results suggest that the muscarinic agonist acetylcholine is unlikely to be required for depolarization-induced Ca²⁺ release in single isolated airway SMCs.

Chemical depolarization with high K⁺ could result in large muscle contraction in M₃R^+/+ tracheal rings, but had not effect in M₃R^−/− tracheal rings (Fig. 4E). In contrast, high K⁺ caused similar muscle contraction in M₂R^+/+ and M₂R^−/− tracheal rings. Therefore, M₃Rs are the important sites of the primary voltage sensor to mediate the initial phase of depolarization-induced local Ca²⁺ release in SMCs.

In line with the critical role of M₃Rs as the loci of the primary voltage sensor, chemical depolarization significantly increased the activity of Gq proteins, determined using a newly-developed, europium-labeled GTP binding assay, in M₃R^+/+, but not in M₃R^−/− mouse airway smooth muscle tissues (Fig. 5A).

Fig. 5.

Gq protein/PLC/IP₃/IP₃R signaling axis is involved in depolarization-induced local Ca²⁺ release in airway SMCs. (A) high K⁺ exposure significantly increased the activity of Gqα proteins in M₃R^+/+, but not in M₃R^−/− mouse airway smooth muscle tissues. The activity of Gqα proteins was determined by measuring europium (Eu)-derived fluorescence, which is expressed as arbitrary units (a.u.), with an Eu-labeled GTP binding assay. (B) Intracellular dialysis of GDP-β-S to specifically inhibit G proteins prevented the effect of voltage depolarization on Ca²⁺ sparks. Cells were voltage-clamped at −60 mV using the classic patch clamp technique. Ca²⁺ sparks were recorded after dialysis of GDP-β-S (1 mM) for 5 min. (C) Dialysis of anti-Gqα antibodies (Gqα Ab), but not anti-Giα1 antibodies (Giα1 Ab) (4 μg/mL) for 5 min abolished voltage depolarization-induced local Ca²⁺ release. (D) Application of the selective PLC inhibitor U73122 (2.5 μM), but not its inactive analog U73433 (2.5 μM) for 8 min blocked high K⁺-induced increase in Ca²⁺ spark frequency and reduction in Ca²⁺ spark amplitudes. (E) Application of the IP₃R antagonist 2-APB (100 μM) for 8 min completely inhibit the effect of high K⁺ on Ca²⁺ sparks. (F) High K⁺ increased in IP₃ production (level) in tracheal muscle tissues. Treatment with U73122 (2.5 μM) for 8 min blocked high K⁺-induced IP₃ production, but incubation with nominally Ca²⁺-free plus 0.25 mM EGTA bath solution for 30 s had no effect.

Consistent with depolarization-induced activation of Gq proteins, intracellular dialysis of GDP-β-s (1 mM) for 5 min to specifically inhibit G proteins prevented voltage depolarization-elicited Ca²⁺ release (Fig. 5B). Furthermore, dialysis of specific anti-Gqα antibodies (4 μg/mL) for 5 min to block Gq protein abolished the effect of voltage depolarization as well (Fig. 5C). However, dialysis of anti-Giα1 antibodies (4 μg/mL) had no effect (Fig. 5C).

The selective PLC inhibitor U73122 (2.5 μM) for 8 min fully blocked the effect of depolarization on the frequency and amplitude of Ca²⁺ sparks (Fig. 5D). In contrast, the structurally similar, inactive analog U73433 (2.5 μM) was without effect.

Similar to our recent report (5), application of 2-aminoethoxydiphenyl-borate (2-APB, 100 μM) for 8 min to block IP₃Rs could significantly inhibit spontaneous Ca²⁺ sparks in airway SMCs. However, in the continued presence of 2-APB, chemical depolarization failed to affect either Ca²⁺ spark frequency or amplitude (Fig. 5E). Bath application of the IP₃R antagonist xestospongin-C (10 μM) for 8 min and intracellular dialysis of the selective IP₃R blocker heparin (5 mg/mL) for 5 min prevented high K⁺ from inducing local Ca²⁺ release as well.

We also found that chemical depolarization significantly increased IP₃ production, measured using a [H³]IP₃ radioreceptor assay, in tracheal muscle tissues. The increased production of IP₃ by chemical depolarization was blocked by treatment with U73122 (2.5 μM) for 8 min, but not by elimination of extracellular Ca²⁺ (Fig. 5F). Collectively, depolarization-induced, RyR-mediated local Ca²⁺ release results from the initial Ca²⁺ release from IP₃Rs due to a local CICR process through the GPCR-dependent PLC−IP₃ signaling pathway in SMCs.

Ryanodine Receptor 2, But Not Ryanodine Receptor 1 and 3 Is Responsible for Membrane Depolarization-induced Local Ca²⁺ Release in SMCs.

RyR2 is known to mediate CICR in cardiac cells; thus, we examined whether these Ca²⁺ release channels might also underlie depolarization-induced local CICR due to the IP₃R/RyR interaction in SMCs using RyR2^+/− mice [RyR2^−/− mice die at early embryonic stage (11)]. Compared with RyR2^+/+ cells, spontaneous Ca²⁺ spark frequency and amplitude were both reduced in RyR2^+/− cells (Fig. 6A). Moreover, depolarization-induced local Ca²⁺ release was almost prevented in RyR2^+/− cells. After high K⁺ exposure, the frequency of Ca²⁺ sparks was increased by 20.4% (from 0.054 ± 0.005 to 0.065 ± 0.006 sparks/s/μm) in RyR2^+/− cells (n = 36), and 109.2% (from 0.076 ± 0.010 to 0.159 ± 0.014 sparks/s/μm) in RyR2^+/+ cells (n = 23). In addition, the effect of chemical depolarization with high K⁺ on the amplitude of Ca²⁺ sparks was blocked in RyR2^+/− cells as well.

Fig. 6.

RyR2 mediates depolarization-induced local Ca²⁺ release in airway SMCs. (A) Depolarization by high K⁺ exposure almost failed to affect Ca²⁺ sparks in RyR2^+/− cells; the frequency and amplitude of spontaneous Ca²⁺ spark were significantly lower in RyR2^+/− cells as well. (B) RyR1 gene deletion did not affect depolarization-induced Ca²⁺ sparks, although it inhibited spontaneous Ca²⁺ sparks. (C) RyR3 gene deletion neither inhibited spontaneous nor high K⁺-induced Ca²⁺ sparks. (D) Intracellular dialysis of IP₃ (1 μM) for 5 min significantly increased Ca²⁺ spark frequency and decreased Ca²⁺ spark amplitude in RyR2^+/+, but not in RyR2^+/− cells.

We next examined the role of RyR1 in depolarization-induced local Ca²⁺ release using RyR1^−/− and RyR1^+/+ mice at embryonic day 17, as RyR1^−/− mice die at or just before birth (12). We found that the frequency and amplitude of spontaneous Ca²⁺ sparks were decreased in RyR1^−/− cells, relative to RyR1^+/+ cells. However, depolarization with high K⁺ caused a similar increase in the activity of Ca²⁺ sparks and reduction in the amplitude of Ca²⁺ sparks in airway SMCs from embryonic RyR1^−/− and RyR1^+/+ mice (Fig. 6B).

RyR3 gene deletion did not affect either spontaneous or depolarization-induced local Ca²⁺ release (Fig. 6C). Following high K⁺ exposure, the mean increase in the frequency of Ca²⁺ sparks and the mean reduction in the amplitude of Ca²⁺ sparks were both comparable in RyR3^−/− and RyR3^+/+ cells.

Finally, we examined and compared IP₃R-dependent local Ca²⁺ release in RyR2^+/+ and RyR2^+/− cells. Intracellular dialysis of IP₃ to directly activate IP₃Rs resulted in a large increase in the frequency of Ca²⁺ sparks and decrease in the amplitude of Ca²⁺ sparks in RyR2^+/+cells. In contrast, the effect of dialysis of IP₃ was strongly inhibited in RyR2^+/− cells (Fig. 6D). These findings provide compelling evidence supporting the importance of RyR2 in depolarization-induced local Ca²⁺ release as a result of the activation of IP₃Rs in SMCs.

Discussion

As membrane depolarization normally leads to an obligatory Ca²⁺ release through RyRs within a few milliseconds, the so-called instantaneous Ca²⁺ release, in cardiac and skeletal myocytes (1), we wondered whether an analogous Ca²⁺ release mechanism could exist in SMCs. Unlike in cardiac and skeletal myocytes, voltage depolarization for 250 ms does not induce instantaneous Ca²⁺ release in freshly isolated airway myocytes. After voltage depolarization for over one second, however, prominent local Ca²⁺ release occurs, manifesting itself in a great increase in the frequency of RyR-mediated Ca²⁺ sparks and significant contraction. Long chemical depolarization with high K⁺ exposure has similar effects. Both long voltage and chemical depolarization can decrease the amplitude of Ca²⁺ sparks. This effect is likely to be secondary to a reduction in local Ca²⁺ content in the release sites due to the increased activity of Ca²⁺ sparks (5). In agreement with our results, long voltage and chemical depolarization also induce Ca²⁺ release in basilar artery myocytes (13). Voltage and chemical depolarization-evoked local Ca²⁺ release may result from the opening of RyRs, as they are prevented by specific block of RyRs with ryanodine.

VDCCs are loosely coupled to RyRs in bladder SMCs, by which sufficient extracellular Ca²⁺ influx through VDCCs following depolarization is required to activate RyRs leading to SR Ca²⁺ release (2). However, depolarization-evoked Ca²⁺ release can be independent of extracellular Ca²⁺ influx in basilar artery SMCs (13). Accordingly, we sought to test whether VDCCs, extracellular Ca²⁺ influx or both would be involved in depolarization-triggered Ca²⁺ release in airway myocytes. Our data reveal that chemical depolarization causes a significant increase in the activity of Ca²⁺ sparks and associated contraction in the presence of the specific VDCC blocker nifedipine or D600. Similarly, depolarization-induced local Ca²⁺ release and contraction occur under extracellular Ca²⁺-free conditions. These data demonstrate a form of depolarization-induced Ca²⁺ release and contraction in SMCs, which are independent of VDCCs and the associated Ca²⁺ influx.

The long latency for depolarization-induced Ca²⁺ release in airway SMCs suggests the involvement of GPCR-dependent biochemical cascade reactions. In addition, membrane depolarization causes charge movement (gating currents) in heterologously expressed M₁Rs and M₂Rs in Xenopus oocytes (7). As a consequence, we hypothesized that the form of VDCC- and Ca²⁺ influx-independent, depolarization-induced Ca²⁺ release could result from a direct activation of native GPCRs in the absence of exogenous agonists. In support of this hypothesis, we have discovered that targeted gene deletion of M₃Rs, functionally important GPCRs in airway, bladder, gastric, intestinal, and other SMCs (9), completely inhibits depolarization-induced local Ca²⁺ release in airway SMCs. In contrast, neither M₂R nor M₁R gene deletion produces an effect. Furthermore, depolarization-induced muscle contraction is abolished in M₃R^−/−, but not in M₂R^−/− airway tissues. These findings are consistent with the well-known fact that M₃Rs, but not M₂Rs and M₁Rs, normally provide predominant neuronal control of Ca²⁺ and contractile responses in SMCs. Billups et al. have shown that voltage depolarization significantly increases the muscarinic agonist oxotremorine-evoked Ca²⁺ release by augmenting IP₃ production in neurons (14). It is also known that membrane depolarization can affect other multiple GPCR-dependent signaling cascade sites to enhance muscarinic agonist-induced Ca²⁺ release in a variety of cells (8). Presumably, if the muscarinic agonist acetylcholine could be endogenously present, even at a low concentration, membrane depolarization might amplify the role of endogenous acetylcholine, causing significant Ca²⁺ release. However, our data do not favor this assumption, because acetylcholine esterase which rapidly degrades acetylcholine (10) has no effect on spontaneous and depolarization-induced local Ca²⁺ release in isolated cells. Supportively, application of exogenous acetylcholine (3 or 10 nM) leads to a large increase in the activity of Ca²⁺ sparks in cells untreated, but not treated with acetylcholine esterase. Therefore, M₃Rs are the important loci of the primary voltage sensor in airway SMCs, similar to expressed M₁Rs and M₂Rs in Xenopus oocytes (7). However, depolarization-induced activation of native M₃Rs results in Ca²⁺ release in airway SMCs (in this study), whereas depolarization-induced charge movement in expressed M₁Rs and M₂Rs, respectively, enhances and reduces agonist binding in Xenopus oocytes (7).

Since native M₃Rs serve as the primary voltage sensor, we expect that target gene deletion of these receptors can prevent depolarization-induced activation of their downstream signaling molecules. In agreement with this view, M₃R gene deletion fully blocks depolarization-caused increase in the activity of Gqα proteins in airway smooth muscle tissues. Specific inhibition of the activation of Gq proteins by intracellular dialysis of GDP-β-s or anti-Gqα antibodies, PLC with U73122 and IP₃Rs with 2-APB, xestospongin-C, and heparin all prevent depolarization causing local Ca²⁺ release. Dialysis of GDP-β-s or application of U73122 fully blocks depolarization-evoked Ca²⁺ release as well in basilar artery SMCs (13). Moreover, our studies also disclose that membrane depolarization increases IP₃ production in airway muscle tissues; the increased production of IP₃ production is completely inhibited by U73122, but not affected by elimination of extracellular Ca²⁺. These data provide convincing evidence that membrane depolarization induces RyR-mediated local Ca²⁺ release due to the GPCR signaling-dependent activation of IP₃Rs in SMCs.

We further examined which subtype of RyRs would locally interact with IP₃Rs to mediate depolarization-triggered local Ca²⁺ release using receptor gene deletion mice. The frequency and amplitude of spontaneous Ca²⁺ sparks are both decreased in RyR2^+/− airway SMCs. Similar inhibition of Ca²⁺ sparks has been observed in RyR2^+/− bladder SMCs (15). More importantly, depolarization-evoked local Ca²⁺ release is almost totally blocked in RyR2^+/− cells. Local Ca²⁺ release (Ca²⁺ spark) is due to the instantaneous opening of functionally or structurally congregated RyRs; as such, the decreased expression of RyR1 by heterozygous gene deletion may lead to the inability of the remaining channels to assemble the functional or structural constitution necessary to generate a Ca²⁺ spark. This may explain the failure of depolarization to induce local Ca²⁺ release in RyR2^+/− cells. Consistent with our findings, depolarization-induced local Ca²⁺ release is reduced as well in RyR2^+/− bladder myocytes (15) and RyR2 gene knocked-down portal vein SMCs (16). RyR1 gene deletion greatly inhibits spontaneous Ca²⁺ release in airway SMCs. However, depolarization-induced local Ca²⁺ release is comparable in RyR1^+/+ and RyR1^−/− cells. RyR3 gene deletion neither affects spontaneous nor depolarization-evoked local Ca²⁺ release. Consonant with our data, suppression of RyR3 gene expression has no effect on spontaneous and/or depolarization-induced Ca²⁺ sparks in rat portal vein myocytes (16) and mouse cerebral artery myocytes (17). Thus, depolarization-evoked local Ca²⁺ release via the GPCR/PLC/IP₃/IP₃R signaling axis may be specifically mediated by RyR2, but not RyR1 and RyR3 in SMCs.

Direct activation of IP₃Rs by intracellular dialysis of IP₃ results in a large increase in the activity of Ca²⁺ sparks in RyR2^+/+, but not in RyR2^+/− cells. These results provide additional evidence for the specific role of RyR2 in depolarization-induced, GPCR/PLC/IP₃/IP₃R-dependent local Ca²⁺ release in SMCs. The basal activity of IP₃Rs significantly enhances spontaneous Ca²⁺ sparks in airway (5) and portal vein myocytes (18), downregulates Ca²⁺ sparks in vas deferens myocytes (19), and has no effect in pulmonary artery SMCs (20). Despite the effects of IP₃Rs on Ca²⁺ release in different types of SMCs, stimulation of IP₃Rs by neurotransmitters, hormones and growth factors can activate adjacent RyRs leading to further Ca²⁺ release through RyRs in airway and other SMCs (21–26). Collectively, our findings suggest that RyR2 may also be essential for agonist-induced, IP₃R-mediated global Ca²⁺ release in SMCs.

Experimental Procedures

Measurement of Ca²⁺ Sparks.

Single mouse airway SMCs from Swiss Webster, M₁R^−/−, M₂R^−/−, M₃R^−/−, RyR1^−/−, RyR2^+/−, RyR3^−/−, and corresponding control (wild-type) mice were obtained using a 2-step enzymatic digestion method (5, 27). M₁R^−/−, M₂R^−/−, M₃R^−/−, RyR1^−/−, RyR2^+/−, and RyR3^−/− mice were generated and maintained, as reported previously (11, 12, 28–31).

Ca²⁺ sparks were measured using a LSM-510 laser scanning confocal microscope (Carl Zeiss) in the line-scan mode (5, 27). Freshly isolated cells were incubated in physiological saline solution (PSS) containing 2.5 μM fluo-4/AM. The composition of PSS was (mM): 125 NaCl, 5 KCl, 2.0 CaCl₂, 1 MgSO₄, 10 glucose, and 10 Hepes (pH 7.4). Spatiotemporal characteristics of Ca²⁺ sparks were analyzed using the Physiology Analysis software (Carl Zeiss) and Interactive Data Language software (Research Systems).

Patch Clamp Recordings.

Cells were loaded with fluo-4/AM and voltage clamped using the nystatin-perforated or classic patch clamp technique with the EPC-9 patch clamp system (HEKA) (5, 27). The intracellular solution contained (mM): 125 KCl, 1 MgSO₄, 10 HEPS, 0.5 CaCl₂, and 0.85 EGTA (pH 7.4). Fluo-4 free acid (50 μM) was included in the patch pipette solution to avoid a potential reduction in fluorescent intensity due to dialysis effect. To record VDCC currents, the pipette solution was composed of (mM): 130 CsCl, 1 MgCl₂, 2 CaCl₂, 3 EGTA, and 10 Hepes (pH 7.4), and bath solution contained (mM): 130 TEACl, 1 MgCl₂, 2 CaCl₂, 10 Hepes, and 10 Glucose (pH 7.4).

Measurement of Cell Contraction.

Single cell contraction was measured by assessing cell length shortening (32). Transmitted-light x-y images were taken using Zeiss LSM510 confocal microscope. Cell length in each image was determined at its longest axis.

Muscle contraction in isolated tracheal rings was measured using the organ bath technique (25). Tracheal rings were removed off the connective tissue and epithelia, and then mounted in 2-ml organ bath chambers containing PSS at 37 °C. Muscle tension was determined in the presence of nifedipine (10 μM) and Y27632 (10 μM) to prevent VDCC-mediated Ca²⁺ influx and RhoA kinase-linked Ca²⁺ sensitivity.

Measurement of IP₃ Production.

Mouse tracheal tissues were dissected free of the epithelium, cartilage and connective tissue, and then homogenized in ice-cold, 1 M trichloroacetic acid (TCA). After centrifugation at 1,000 × g for 10 min at 4 °C, the supernatant was incubated at room temperature for 15 min. TCA was removed from the supernatant by adding 1-ml 1,1,2-trichlorotrifluoroethane and tri-n-octlyamine (3:1), followed by vigorous shaking and centrifugation at 2,000 × g for 1 min. The upper phase of the reaction buffer was used to assay IP₃ production using an [H³]IP₃ radioreceptor assay kit (PerkinElmer). The radioactivity of the reaction samples was determined in an LS6500 β-counter (Beckman Instruments). IP₃ concentrations were calculated from a standard curve that was generated by plotting the percentage binding (bound/free ratio) as a function of the logarithm of known IP₃ concentrations.

Measurement of Gqα-protein Activity.

Gqα-protein activity was determined using DELFIA GTP Binding Kit (Perkin-Elmer), according to the manufacturer's instruction. Isolated mouse tracheas were homogenized and centrifuged to obtain cell membrane fractions. Samples were incubated with specific anti-Gqα antibodies with protein-A beads for 3 h at room temperature and then centrifuged 3 times at 2,000 × g for 3 min to collect the beads. Proteins were eluted from the beads using eluting buffer containing 1% SDS, incubated in reaction buffer containing 2.5 mM MgCl₂, 0.5 μM GDP, 50 mM NaCl, and 15 μg/mL saponin for 30 min, and then added with 1 μM GTP-europium reagent followed by incubation for 30 min. After 3 washes on manifold vacuum, europium-derived fluorescence was measured at 615 nm emission with 340 nm excitation on a FlexStation-III microplate reader (Molecular Devices).

Reagents.

2-APB, U73122, U73433 and xestospongin-C were purchased from Calbiochem; ryanodine, D600, heparin, GDP-β-s, and Y27632 from Sigma; fluo-4/AM and fluo-4 free acid from Invitrogen; nifedipine from Tocris; and anti-Gqα and anti-Giα1 antibodies from Santa Cruz Biotechnology. All experiments were conducted at room temperature (≈22 °C), unless indicated otherwise.

Data Analysis.

Data are presented as mean ± SEM. Statistical comparisons between groups were performed with Student's t test or 1-way ANOVA with an appropriate posthoc test. Differences at P < 0.05 were considered statistically significant.