Airway smooth muscle relaxation results from a reduction in the frequency of Ca2+ oscillations induced by a cAMP-mediated inhibition of the IP3 receptor
© Bai and Sanderson. 2006
Received: 05 December 2005
Accepted: 23 February 2006
Published: 23 February 2006
It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca2+ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca2+ oscillations.
The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca2+ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.
All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca2+ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca2+ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca2+ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca2+ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca2+ available to stimulate contraction because an elevation of intracellular Ca2+ concentration induced by exposure to a Ca2+ ionophore (ionomycin) or by photolysis of caged-Ca2+ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca2+-activated K+ channels, which would be expected to increase Ca2+ influx and contraction. By contrast, the photolysis of caged-IP3 in the presence of agonist, to further elevate the intracellular IP3 concentration, reversed the slowing of the frequency of the Ca2+ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP3R to IP3 was reduced by FSK and this was supported by the reduced ability of IP3 to release Ca2+ in SMCs in the presence of FSK.
These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca2+ oscillation frequency by reducing internal Ca2+ release through IP3 receptors.
A major symptom of asthma is an excessive contraction of airway smooth muscle cells (SMCs) which results in airway hyper-reactivity. To alleviate this acute and chronic airway constriction, β-adrenergic agonists, that relax SMCs, are commonly administered . Yet, despite their efficacy and wide-spread application, the signaling pathways underlying the relaxing effect of β-agonists are not fully understood.
It is generally accepted that stimulation of β-adrenergic receptors activates adenylyl cyclase (AC), via receptor associated G proteins, to increase cAMP levels to mediate SMC relaxation . The role of cAMP in relaxation has been confirmed with other compounds that increase intracellular cAMP concentration ([cAMP]i), such as forskolin (FSK) that directly activates AC, theophylline that inhibits phosphodiesterase and 8-bromo-cAMP that is an analog of cAMP [3–5]. The traditional mechanism for cAMP action is via the stimulation of protein kinase A (PKA) to phosphorylate a variety of target proteins to induce airway SMCs relaxation. Alternatively, cAMP may act independently of PKA by interacting with exchange proteins (EPACs) . EPACs have been found to activate PCLε to enhance IP3 induced Ca2+ release in non-smooth muscle cells . However, before the specific details of cAMP-mediated signaling can be explored, it is essential to initially identify the fundamental action of cAMP on the mechanisms that regulate SMC contraction.
Airway SMC contraction is determined by the balance between phosphorylation and de-phosphorylation of the regulatory light chain of myosin (rMLC). Phosphorylation of rMLC is induced by Ca2+-calmodulin activated myosin light-chain kinase (MLCK). De-phosphorylation of rMLC is believed to be mediated by myosin light chain phosphatase (MLCP) . As a result, SMC relaxation may be the result of different cellular pathways that culminate in either or both a reduction in MLCK activity and an increase in MLCP activity.
Perhaps the most direct method of relaxation is a reversal of the Ca2+ response that stimulates MLCK activity and contraction. Ca2+ signaling in airway SMCs is frequently induced by agonists such as ACh, 5-HT and ATP and consists of Ca2+ oscillations [9–11]. In our recent studies with lung slices, we found that the frequency of these Ca2+ oscillations correlated with the contractile state of the airways, a relationship that indicates the SMC tone is regulated in a frequency-modulated manner [12–14].
In previous studies with isolated tracheal SMCs, cAMP was found to modulate the frequency of Ca2+ oscillations [15, 16]. However, its effect on airway contraction or the mechanism of action was not determined. In other studies with different SMC types, it has been suggested that cAMP interacts with intracellular Ca2+ signaling pathways at multiple sites . These include a decrease in Ca2+ influx [3, 15, 16, 18], particularly by the activation of large conductance Ca2+-activated K+ channels (BKCa) to hyperpolarize the membrane [19–21], an increase in Ca2+ efflux  or uptake by the sarcoplasmic reticulum [23, 24], or an inhibition of agonist-induced IP3 formation [5, 25]. All of these effects of cAMP would be expected to decrease the available intracellular Ca2+ concentration ([Ca2+]i). However, it is unknown how any of these effects would influence the frequency of Ca2+ oscillations of SMCs.
To explore the hypothesis that cAMP induces intrapulmonary airway relaxation by down-regulating Ca2+ oscillations, we examined the effect of isoproterenol (ISO) and other cAMP elevating agents (FSK and 8-bromo-cAMP) on methacholine (MCh)-induced contractility and Ca2+ signaling of airway SMCs in murine lung slices. The importance of using lung slices, instead of cultured or isolated cells, is that Ca2+ signaling and airway contractility can be measured simultaneously. We found that cAMP induced relaxation of MCh-contracted airways and reduced the frequency of the Ca2+ oscillations. In addition, we confirmed that cAMP did not affect Ca2+ influx or the refilling of depleted Ca2+ stores. By contrast, we found that an elevation of intracellular IP3 concentration ([IP3]i) reversed the effect of FSK by increasing the frequency of the Ca2+ oscillations to induce contraction. In addition, we also show that cAMP inhibits the Ca2+ release via the IP3 receptor. From these data, we suggest that airway relaxation induced by cAMP is achieved by decreasing the Ca2+ oscillation frequency of airway SMCs by inhibiting IP3-induced Ca2+ mobilization.
Materials and methods
Cell culture reagents were obtained from GIBCO/Invitrogen Corp. Oregon Green 488 BAPTA-1 AM and O-nitrophenyl EGTA AM (NP-EGTA AM) were obtained from Molecular Probes (Eugene, OR). Caged-iso-Ins(1,4,5)P3/PM was obtained from Alexis Biochemicals (San Diego, CA) and dissolved in DMSO, aliquoted and frozen at -80°C. Other reagents were obtained from Sigma-Aldrich or Calbiochem. Hanks' balanced salt solution was supplemented with 20 mM HEPES buffer (sHBSS) and adjusted to pH 7.4.
The preparation of lung slices has been previously described in detail . Briefly, male Balb/C inbred strain mice (Charles River Breeding Labs, Needham, MA, 7–10 weeks old) were killed by intra-peritoneal injection of 0.3 ml of pentobarbital sodium (Nembutal) as approved by the IACUC of the University of Massachusetts Medical School. The trachea was exposed and cannulated. After the thoracic cavity was opened, the lung lobes were slowly inflated with a warm agarose solution (2%, 37°C, ~1.3 ml). Subsequently ~0.2 ml of air was injected to flush the agarose out of the airway into the distal alveoli. The lungs were cooled at 4°C to gel the agarose. A single lung lobe was removed and sectioned with a vibratome (model EMS-4000; Electron Microscope Sciences, EMS) into slices of ~140 μm thick starting at the lung periphery. Airways were identified by their lining of epithelial cells with beating cilia. Serial sections were collected and transferred to culture solution containing DMEM supplemented with antibiotics and anti-mycotics and NaHCO3. Slices were maintained in culture media at 37°C and 10% CO2 for up to 3 days.
Measurement of airway contraction and relaxation
Lung slices were observed in a custom-made perfusion chamber constructed from 2 cover-glasses sealed with thin strips of silicone grease (Valve lubricant and sealant, Dow Corning, Midland, MI) on a Plexiglas support. The slices were held in place with a small sheet of nylon mesh (CMN-300-B, Small Parts Inc, Miami Lakes, FL) with a small hole to view the selected airway and avoid the mesh from influencing the contraction of the airway. A gravity-driven perfusion system with a multi-tube manifold with a single output (Warner Instruments, Inc.) was used, in conjunction with valves (LFVA, Lee Company, CT) under TTL control, to exchange solutions in the chamber. The chamber volume was about 100 μl. The perfusion rate was 800 μl/min.
The lung slices were observed with an inverted microscope (IX71, Olympus) with a 10× objective and a zoom adapter. Phase-contrast images were recorded using a CCD camera (model LCL-902C, Watec America Corp, USA), a camera interface ("Picolo", Eurosys Inc., Belgium) and image acquisition software ("Video Savant"; IO industries, Inc, London, Ontario, Canada). Digital images were recorded in time-lapse (0.5 Hz) and analyzed with "NIH /Scion Image" software (Scion Corp.). The lumen area of the airway was measured by summing the number of pixels below a selected threshold grey level with respect to time. All experiments were performed at room temperature (RT, 20 ± 2°C).
Measurements of [Ca2+]i
Slices were loaded for 30 minutes at 30°C with 20 μM Oregon Green 488 BAPTA-1 AM in sHBSS containing 0.1% pluronic (Pluronic F-127; Calbiochem) and 100 μM sulfobromophthalein. Lung slices were de-esterified for another 30 minutes at 30°C in sHBSS containing 100 μM sulfobromophthalein before being placed in the perfusion chamber as described above.
Fluorescence imaging was performed using a custom-built video-rate confocal or 2-photon scanning laser microscope. Either a 488 nm beam from a diode laser or an 800 nm beam from a Ti-sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA) pumped with a 5 W 525 nm diode laser (Millennia, Spectra-Physics) was scanned across the specimen with two oscillating mirrors (X- and Y-scan) through an inverted microscope (Nikon DIAPHOT 200 for confocal microscopy; Olympus IX71FVSF-2 for 2-photon microscopy). For confocal microscopy, the emitted fluorescence (>510 nm) was separated from the excitation light by a dichroic mirror, a long pass filter and a confocal aperture . For 2-photon microscopy, the emitted fluorescence was separated with a dichroic mirror (670uvdclp, Chroma Technology Corp, Rockingham, VT) and a long pass filter (E700SP or E600SP-2P, Chroma Technology Corp) positioned immediately below the objective. Emitted fluorescence was detected with a photomultiplier tube (PMT, R5929, Hamamatsu USA, Bridgewater, NJ). Grayscale images were recorded at 1, 15 or 30 Hz with a frame-grabber (Raven; Bit Flow, Inc). Changes in fluorescence intensity were obtained, frame-by-frame, from selected regions of interest (ROI, ~5 × 5 pixel). A line-scan analysis was made by extracting a row of pixels from each frame and aligning them in a time sequence.
Flash photolysis of caged-Ca2+ or caged-IP3
Flash photolysis of caged-Ca2+ (NP-EGTA) or caged-IP3 was used to experimentally increase the [Ca2+]i or [IP3]i. Slices were initially loaded with Oregon Green 488 BAPTA-1 AM as described above. Subsequently, slices were incubated, at RT, with either 1 μM NP-EGTA AM (for 15 min) or 2 μM Caged-iso-Ins(1,4,5)P3/PM (for 1 hr) in sHBSS containing 0.1% pluronic and 100 μM sulfobromophthalein followed by de-esterification for 30 min in the sHBSS containing 100 μM sulfobromophthalein. The details of the flash photolysis setup have been previously described . Briefly, a flash of UV light was produced from a mercury arc lamp with a mechanical shutter and focused into the microscope with a biconvex lens (focal distance 200 mm) through a bandpass filter (330 nm). The flash duration was determined by electronic control and the image acquisition software. The intensity of the flash was regulated by a neutral density filter (Optical Density, OD 0.5 ~ 1.0, transmission = log [1/OD]). The size of the area illuminated was adjusted with an iris diaphragm and measured by illuminating and monitoring the emitted fluorescence from a thin uniform layer of fura-2 salt solution (1 μM) between 2 cover glasses.
Results are expressed as means ± S.E. Concentration-response curves of MCh, ISO and FSK were fitted using a non-linear interactive fitting program (Microcal Origin 7.0, Microcal Software Inc, MA, USA). Statistical significance was determined by using paired student's t-test.
The relaxing effect of ISO on airways contracted by MCh
Because MCh can stimulate the M2 receptor and this could possibly lead to the inhibition of adenylyl cyclase (AC) to enhance the contractile response, we examined the effects of the M2 receptor antagonist, methoctramine (0.2 μM) on ISO-induced relaxation. In the presence of the M2 antagonist, airway contraction induced by MCh (200 nM) was decreased by about ~5%. However, the transient relaxation induced by ISO (10 μM) was unchanged in the presence of methoctramine. A similar transient relaxation was also observed in response to ISO (10 μM), when the airway was pre-contracted with another agonist, serotonin (5-HT, 1 μM).
Ca2+ events underlying airway contraction and relaxation
In response to 200 nM MCh, the airway SMCs demonstrated an initial increase of intracellular Ca2+ which was followed by the onset of Ca2+ oscillations (Figs. 3B and 3C). These Ca2+ oscillations were superimposed on a slightly elevated baseline. The frequency of the Ca2+ oscillations increased to a stable rate within 1 minute. Upon exposure to 1 μM ISO, the frequency of the Ca2+ oscillations slowed, although the baseline of the Ca2+ signal did not substantially change. In the continued exposure to ISO with MCh and after about 150 seconds, the frequency of Ca2+ oscillations began to increase and approached a new steady rate; this rate was less than the initial rate induced by MCh alone.
The frequency of the Ca2+ oscillations was determined from the period of each oscillation (time interval between 2 adjacent peaks of the Ca2+ signal). As a result a correlation between the dynamic changes in the frequency of the Ca2+ oscillations with the changes in the cross-sectional area of the airway could be made (Fig. 3D). In response to MCh, the airway contracted to a smaller stable size and this correlated with the initiation and increase in the frequency of the Ca2+ oscillations to a stable rate. Similarly, the ISO-induced decrease in the frequency of the Ca2+ oscillations correlated with the ISO-induced initial relaxation of the airway. The subsequent re-contraction of the airway correlated with the late increase in the frequency of the Ca2+ oscillations.
The effect of ISO on the frequency of the Ca2+ oscillations was expressed as the ratio of the frequency of the Ca2+ oscillations after ISO exposure (60 to 90 seconds) to the frequency of the Ca2+ oscillations before ISO exposure. In the range from 1 nM to 10 μM, ISO induced a concentration-dependent decrease in the frequency ratio from 0.93 ± 0.03 (n = 5 slices from 3 mice) to 0.56 ± 0.03 (n = 6 slices from 4 mice). The decline in the frequency ratio was inversely proportional to the change in airway size (Fig. 3E).
Relaxation of airways by repeated exposure to ISO
Relaxing effect of FSK and 8-bromo-cAMP on contracted airways
Recordings of intracellular Ca2+ in airway SMCs revealed that both FSK and 8-bromo-cAMP reduced the frequency of the Ca2+ oscillations initiated by MCh (Fig. 5B and 5E). In contrast to the effects of ISO, the frequency of the Ca2+ oscillations declined throughout the experiment. In range from 10 nM to 10 μM, FSK induced a concentration-dependent decrease in the frequency of the Ca2+ oscillations (Fig. 5C). The frequency of the Ca2+ oscillations after 5 minutes of FSK exposure, expressed as the ratio to the Ca2+ oscillation frequency induced by MCh exposure, decreased from 0.95 ± 0.03 to 0.42 ± 0.04 (n = 4 slices from 3 mice) as the FSK concentration increased. The changes in frequency of the Ca2+ oscillations induced by FSK or 8-bromo-cAMP correlated with a progressive relaxation of the airway (Fig. 5A and 5D).
A decrease in Ca2+ oscillation frequency correlates with airway relaxation
cAMP does not affect caffeine-induced Ca2+ release
Supplementation of intracellular Ca2+ does not block the relaxing effect of cAMP
Because a reduction of [Ca2+]i, brought about by decreasing Ca2+ influx and/or increasing Ca2+ efflux or reuptake, has been proposed as a mechanism by which cAMP relaxed airway SMCs, we examined the effect of elevating [Ca2+]i during the response of SMCs to cAMP. The [Ca2+]i was increased with 3 different agents: 1) NP-EGTA, a caged-Ca2+ compound, which upon UV photolysis releases Ca2+, 2) ionomycin, a Ca2+ selective ionophore, and 3) iberiotoxin (IbTX), a specific inhibitor of BKCa channels, which enhances Ca2+ influx by blocking K+ efflux to induce hyper-polarization.
In summary, we used three different methods to increase the [Ca2+]i during FSK- induced relaxation but the frequency of the Ca2+ oscillation could not be increased to reverse the FSK effect. Moreover, the increase of [Ca2+]i induced by ionomycin inhibited the Ca2+ oscillations to induce further relaxation. These data suggest that a reduction in the availability of Ca2+ is not the cause of cAMP-induced slowing of Ca2+ oscillations.
cAMP exerts a relaxant effect by inhibiting IP3-induced Ca2+ release
Because Ca2+ release through the IP3 receptor of internal Ca2+ stores is a major mechanism contributing to the formation of Ca2+ oscillations, we investigated whether cAMP inhibited IP3-induced Ca2+ release. When airway SMCs loaded with caged-IP3 were illuminated with UV light for about 1 s, several Ca2+ oscillations were observed to propagate as Ca2+ waves from the illuminated zone through the rest of the SMC (data not shown). When the intensity and exposure time of UV light was optimized, repetitive UV flashes could induce similar repetitive Ca2+ waves in the same SMC.
CICR through the ryanodine receptor is not required for Ca2+ oscillations
To examine the Ca2+ conditions required for the induction of CICR, we released Ca2+ by flash photolysis of caged Ca2+. When a restricted area within a single SMC was exposed to UV light (Fig 13C), only a brief localized increase in [Ca2+]i was detected (Fig. 13D). The increase in Ca2+, as determined by the fluorescence change, was similar to that induced by a Ca2+ oscillation, but this Ca2+ change did not spread to the rest of the cell. By contrast, when the same airway SMCs was treated with sHBSS containing 50 mM K+ for ~3 minutes, a second identical UV flash induced a larger Ca2+ release in the zone of illumination and induced a Ca2+ wave that propagated throughout the cell (Fig 13D, 5 airways from 3 mice). Treatment with high K+ was used because it has previously been reported that high K+ initiates membrane-depolarization that results in excessive Ca2+ influx, overfilling of internal Ca2+ store and sensitization of the RyR . These results indicate that CICR through RyR only occurs in airway SMCs under certain circumstances or "stressed conditions" and implies that RyRs do not play a significant role in the normal maintenance of agonist-induced Ca2+ oscillations.
In this study, thin lung slices were used to explore the changes in intracellular Ca2+ that occur in airway SMCs during relaxation induced by ISO and other cAMP elevating compounds. The airway SMCs were initially contracted with MCh, an analogue of ACh, which is more slowly metabolized and widely used in clinical lung function tests. We found that MCh induced both a concentration-dependent contraction of the airways and increase in the frequency of Ca2+ oscillations within airway SMCs. These results are similar to our previous findings obtained with ACh in lung slices [12, 14]. Upon exposure to ISO, the MCh-contracted airways initially displayed a transient relaxation but subsequently re-contracted. The ability of ISO to relax the airway was dependent both on the ISO concentration and the MCh concentration used to contract the airway. If the airways were contracted with high concentration of MCh (1 μM), ISO, even at high concentrations (i.e.10 μM) was ineffective at relaxing the airway. Only at low MCh concentrations was ISO able to induce substantial relaxation. A similar relaxation and subsequent re-contraction in response to ISO (10 nM ~10 μM) was also observed in MCh-contracted (1 μM) rat lung slices (data not shown). These results suggest that the relaxing action of ISO is relatively weak in comparison to the contractile action of MCh and that the airway becomes de-sensitized to ISO-induced relaxation to allow re-contraction.
In addition to being activated by β-adrenoceptor associated G-stimulatory proteins (Gs), AC can also be inhibited by receptor-coupled G-inhibitory proteins (Gi). Therefore, a potential mechanism for the down-regulation of the ISO response is the inhibition of AC by activation of M2 muscarinic receptors and their associated Gi by MCh [28, 29]. However, in the presence of the M2 receptor antagonist, methoctramine, MCh only induced a slightly smaller airway contraction (about 95%), but more importantly, ISO induced a similar transient relaxation. This transient response to a high concentration of ISO also persisted across a wide range of MCh concentrations, which would be expected to differentially activate the M2 receptors. Moreover, a similar transient response to ISO was also observed in the airways contracted by 5-HT, an agonist with no effect on the AC. In other studies [30, 31], the pretreatment of cultured SMCs or bronchial smooth muscle strips with ISO (30 min to 24 hours) reduced subsequent ISO-mediated cAMP generation and relaxation. Similarly, we found that repetitive exposure of the airway to ISO (1 μM, 10 min to 2 hours) significantly decreased the relaxation response (by 30 to 50%). These results indicate that the M2 receptor has a minimal effect on AC activity at mid-range MCh concentrations and that the down-regulation of the ISO response is mediated by rapid de-sensitization and slow re-sensitization at the β-adrenoceptor. Desensitization of the β-adrenoceptor can be mediated by phosphorylation of the receptor by cAMP-independent β-adrenoceptor kinase or other G protein-coupled receptor kinases or by receptor internalization – a process that takes a longer time to reverse (hours) [2, 32].
Because β-adrenoceptor agonists primarily relax SMCs by increasing [cAMP]i, an implication of receptor desensitization is that the [cAMP]i within the SMCs initially increases but subsequently declines. The idea that increases in [cAMP]i correlate with relaxation was addressed by circumventing the β-adrenergic receptor by either activating adenylyl cyclase with FSK or by increasing the [cAMP]i directly with 8-bromo-cAMP. A major characteristic of FSK or 8-bromo-cAMP-induced relaxation was that the relaxation was sustained and that re-contraction never occurred. This response is consistent with the idea that the [cAMP]i must be maintained or increased if relaxation is to be sustained and that some form of desensitization of β-adrenoceptor occurs allowing re-contraction.
The correlation of increases in [cAMP]i with airway relaxation has been reported in mice , rats [23, 34], guinea pigs , rabbits [36, 37], dogs , cows [4, 24, 38], and humans [24, 37]. However, these studies were all conducted on isolated airway muscle strips or tracheal rings by measuring changes in isometric tension. Consequently, to explore the cellular mechanisms by which ISO and other agents acting via cAMP to relax SMCs, we examined their effects on intracellular Ca2+. As previously described, MCh, as well as ACh, initiates Ca2+ oscillations in SMCs and the frequency of these Ca2+ oscillations determines the extent of the SMC contraction [9, 11, 12, 14]. During the initial response to ISO, the frequency of the MCh-induced Ca2+ oscillations was quickly reduced and this was accompanied by fast relaxation of the airway. However, after a short time, the Ca2+ oscillation frequency began to slowly increase and this was accompanied by the slow re-contraction of the airway. A similar slowing of the frequency of the Ca2+ oscillations and airway relaxation was induced by FSK and 8-bromo-cAMP. But, in contrast to ISO, the frequency of the Ca2+ oscillations continued to slow to lower frequencies; a result that correlates with the greater airway relaxation. Although the relationship to contraction could not be measured, Salbutamol, a β2-adrenoceptor agonist, has also been found to slow and reduce the baseline of ACh-induced Ca2+ oscillation in dissociated porcine tracheal SMCs . In our studies, we found that the baseline of the Ca2+ oscillations was relatively stable. Consequently, we hypothesized, especially in view of the fact that the peak [Ca2+]i of the Ca2+ oscillations is substantially high, that increases in [cAMP]i induce airway relaxation by reducing the frequency of Ca2+ oscillations in SMCs rather than by lowering the baseline of global [Ca2+]i.
We [12–14] and others [10, 39] have shown that agonist-induced Ca2+ oscillations in airway SMCs are generated by cycles of Ca2+ release and uptake by the sarcoplasmic reticulum (SR). Because extracellular Ca2+ is required to replenish internal stores, it has been proposed that cAMP might exert its action on Ca2+ oscillations by 1) the inhibition of Ca2+ replenishment of the internal stores by inhibiting Ca2+ pumps and/or 2) blocking the Ca2+ influx through voltage-dependent Ca2+ channels (VOC) or store-operated Ca2+ channels (SOCC), or 3) by inhibiting the Ca2+ release via IP3 and/or ryanodine receptors.
To test the first hypothesis, that the internal Ca2+ stores were depleted, we investigated the effects of caffeine on cAMP-treated lung slices. We found that elevated [cAMP]i did not significantly affect the Ca2+ signaling or contractile response induced by repetitive exposures to caffeine. These results demonstrate that the Ca2+ capacity of the internal store was fully restored to normal in the presence of cAMP and imply that Ca2+ loading of the SR by Ca2+ pumps was not significantly altered.
To investigate the second possibility that cAMP decreased Ca2+ influx to limit the Ca2+ available to replenish internal stores and maintain the ongoing Ca2+ oscillation, we used flash photolysis of caged-Ca2+ or exposure to ionomycin, a Ca2+ ionophore, to directly increase the available Ca2+. We found that while a transient global increase in [Ca2+]i resulted from the photolysis of caged-Ca2+, this had little effect on the slow frequency of the Ca2+ oscillations in the presence of FSK. Furthermore, the application of ionomycin initially inhibited the FSK-reduced Ca2+ oscillation to relax the airway. However, the gradual increase in [Ca2+]i induced by continued exposure to ionomycin subsequently induced re-contraction of the airway. Because in both experiments, an increase in the frequency of the Ca2+ oscillations was not observed, these results indicate that a lowered Ca2+ influx is not a limiting factor. It is possible that ionomycin might have emptied the Ca2+ of SR to inhibit the Ca2+ oscillations; however considering the higher concentration of Ca2+ in the extracellular environment it is unlikely that the internal store would have been emptied before an influx of Ca2+ had occurred.
Another mechanism inhibiting SMC Ca2+ influx appears to involve membrane hyper-polarization resulting from cAMP-activation of BKCa channels to down-regulate voltage-dependent Ca2+ influx [19–21]. Specific blockers of the BKCa channels such as IbTX or charybdotoxin have been shown to antagonize cAMP-induced relaxation in isolated trachea or bronchi from guinea-pigs , pig , horse . However, in keeping with the effects of ionomycin, we found that in lung slices treated with MCh and FSK, IbTX further inhibited the Ca2+ oscillations to enhance airway relaxation. This effect suggests IbTX (and ionomycin) may actually inhibit Ca2+ oscillation by enhancing Ca2+ influx. The failure to observe a significant elevation of baseline Ca2+ suggests either that the influx of Ca2+ is sufficiently low and quickly sequestered into internal stores or that an alternative, non-specific action of IbTX must be considered. The failure of IbTX to induce re-contraction is consistent with the fact that voltage-dependent Ca2+ influx does not appear to play a prominent role in maintaining the agonist-induced Ca2+ oscillations in airway SMCs and that airway contraction and Ca2+signaling induced by membrane depolarization (with KCl) were completely different to that induced by agonists . Furthermore, there are conflicting reports regarding the contribution of BKCa channels based on differences amongst species and regions of the airway as well as experimental conditions [43, 44]. Consequently, it appears unlikely that the BKCa channel is a major mechanism mediating the effects of cAMP in mouse airway SMCs. Taken together, these data led us to believe that decreased [Ca2+]i is not the primary cause of the modulation of the frequency of Ca2+ oscillations.
Therefore, we examined the third idea that cAMP altered the Ca2+ release from SR. Although the RyR has an important role in CICR in many other cell types , Ca2+ oscillations and airway contraction induced by MCh were minimally affected in the short term by the presence of ryanodine. This suggests that the Ca2+ oscillations do not require RyRs and therefore it is unlikely that any effects of cAMP on RyR will be significant. The slight decline in the Ca2+ oscillation frequency may be explained by an increased leak of Ca2+ through RyR locked in the open state by ryanodine. However, this process must be occurring independently of the Ca2+ oscillations otherwise the Ca2+ oscillations would quickly stop as a result of empty Ca2+ stores. These results are also consistent with the fact that the release of caged-Ca2+ only initiated CICR when the cell was previously depolarized with KCl to overload the internal Ca2+ stores and sensitize the RyR.
The IP3R is another important Ca2+ release channel of the SR and we found that the slow frequency of Ca2+ oscillations induced by FSK could be increased when the [IP3]i was increased by flash photolysis. This response was accompanied by the re-contraction of airway SMCs. The idea that the sensitivity of the IP3R to IP3 is down-regulated by FSK is further supported by the finding that photolysis of caged IP3 induced a propagating Ca2+ wave in the absence, but not in the presence of FSK. A similar effect of cAMP on Ca2+ release has also been reported in other types of SMCs or non-excitable cells [46–49]. The underlying mechanism may involve cAMP-dependent PKA to phosphorylate the IP3R. However, the outcome of phosphorylation differs with the preparation and either an enhancement or inhibition of Ca2+ release can result [46–49]. Alternatively, cAMP may influence the IP3R via EPACs.
Although the molecular details mediating the action of cAMP will require further investigation, we propose that the cAMP has an inhibitory effect on the IP3R and reduces the open probability of the receptor. Moreover, cAMP may also change the Ca2+ sensitivity of the IP3R. Normally, the open probability of the IP3R is a bell-shaped function related to the [Ca2+]i; the receptor is closed by high Ca2+ concentrations at a constant IP3 concentration . With a decrease in [IP3]i, the bell-shaped response curve is shifted to the left, with the result that the IP3R closes at a lower [Ca2+]i [51, 52]. In view of this change in sensitivity, we hypothesize that the inhibition of IP3R by cAMP results in a similar leftward and downward shift of the bell-shaped curve. This idea is consistent with our observation that in the presence of cAMP, an increase in Ca2+ influx, induced by IbTX or ionomycin, leads to the closure of the IP3R to mediate the cessation of Ca2+ oscillations and relaxation of the airway SMCs.
An additional mechanism that would augment the inhibitory action of cAMP on the IP3R is a reduction in the production of IP3. This idea is based on the evidence that G protein-activated PLCβ activity can be inhibited by PKA-induced phosphorylation [4, 5, 53, 54]. This requires further investigation but is beyond the scope of this study.
An alternative mechanism for cAMP-induced SMC relaxation is the desensitization of contractile apparatus to Ca2+ [8, 24, 40, 55]. Phosphorylation of MLCK can lead to decreased affinity of MLCK for Ca2+/calmodulin while phosphorylation of Rho-kinase or protein kinase C can increase the activity of MLCP. We have also found that cAMP can down-regulate the Ca2+-sensitivity of agonist-induced contraction in lung slices . However, it is not yet clear if such changes occur in parallel with the changes in Ca2+ oscillation frequency.
In summary, we conclude that the frequency of Ca2+ oscillations controls the contractile state of airway SMCs and that compounds that elevate cAMP slow the frequency of Ca2+ oscillations within SMCs to proportionally induce airway relaxation. Furthermore, cAMP appears to regulate the Ca2+ oscillation frequency by inhibiting Ca2+ release through IP3R rather than influencing Ca2+ influx or the refilling of internal stores. In addition, we find that, under normal conditions, the RyR is not essential for Ca2+ oscillations of airway SMCs.
This work was supported by the NIH grant HL 71930 to MJS
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