Published ahead of print on March 27, 2003, doi:10.1165/rcmb.2002-0197OC
© 2003 American Thoracic Society DOI: 10.1165/rcmb.2002-0197OC Involvement of Ca2+ Mobilization in Tachyphylaxis to ß-Adrenergic Receptors in TrachealisDivision of Respiratory Medicine, Department of Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan; and Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York Address correspondence to: Hiroaki Kume, M.D., Ph.D., Division of Respiratory Medicine, Department of Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan. E-mail: hkume{at}med.nagoya-u.ac.jp
We examined the mechanisms underlying tachyphylaxis to ß-adrenergic receptor agonists (ß-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca2+ concentration ([Ca2+]i) using fura-2–loaded guinea pig tracheas showed that the inhibitory effects of isoproterenol (ISO) on tension and increases in [Ca2+]i induced by methacholine exhibited marked tachyphylaxis with repeated exposure to ISO at intervals of 15 min. Similarly, the activation of single Ca2+-activated K+ (KCa) channels in on-cell patches by 1 µM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of ß-adrenergic receptor/KCa channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the voltage-dependent Ca2+ channel (VDCC) with verapamil, suggesting a surprising relationship between Ca2+ influx through VDCC and ß-adrenergic desensitization. Conversely, repeated exposure of 10 U/ml protein kinase A to inside-out patches did not result in desensitization of channel activation, and repeated exposure to 10 µM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca2+]i by methacholine, indicating that the mechanism of desensitization is mediated by the ß-adrenergic receptor/G protein complex. These results indicate that an uncoupling of ß-adrenergic receptor from KCa channels augments Ca2+ mobilization through VDCC and stimulates tachyphylaxis.
Abbreviations: intracellular Ca2+ concentration, [Ca2+]i • dimethyl sulfoxide, DMSO • ethyleneglycol-bis-(ß-aminoethyl ether)-N,N',-tetraacetic acid, EGTA • isoproterenol, ISO • Ca2+-activated K+, KCa • open probability, nPo • voltage-dependent Ca2+ channel, VDCC
Repeated or continuous exposure of smooth muscle tissue to ß-adrenergic receptor agonists (ß-agonists) results in a decline in agonist activity, referred to as tachyphylaxis or desensitization. In vitro, repeated exposure of guinea pig (1) and human (2) tracheal smooth muscle to ß-agonists results in the gradual attenuation of relaxation responses. Similarly, repeated inhalation of ß-agonists causes a reduction in bronchodilation by ß-agonists in anesthetized dogs in vivo (3), and clinical trials have demonstrated that regular administration of inhaled ß-agonists may cause not only a deterioration of asthma control and an exacerbation of airway hyperreactivity (4), but also may accelerate a decline in lung function in patients with asthma (5). Moreover, excessive inhalation of ß-agonists causes a decrease in the bronchodilator effects induced by ß-agonists, and in the protective effects of ß-agonists against bronchoconstriction induced by muscarinic agonists (6, 7). Desensitization to ß-adrenergic receptors may not only complicate asthma therapy, but may also play a role in the pathophysiology of bronchial asthma. Thus reduced responsiveness to ß-agonists is observed after continuous exposure to interleukin-1ß and tumor necrosis factor-  , proinflammatory cytokines involved in airway inflammation in the disease (8, 9) and continuous exposure to lysophosphatidylcholine, a lysophospholipid which is synthesized by phospholipase A2, causes desensitization of ß-adrenergic receptors in airway smooth muscle (10). Relaxation of airway smooth muscle is particularly dependent on stimulatory coupling between ß-adrenergic receptors and large conductance Ca2+-activated K+ (KCa) channels (11–13), as well as by other mechanisms (14). This stimulatory coupling is mediated by both cAMP-dependent (15, 16) and -independent (17) pathways. cAMP-independent pathways associated with the direct stimulation of KCa channels by the stimulatory GTP-binding (G) protein of adenylyl cyclase, GS, have recently been shown to be involved in ß-adrenergic actions (18). Here we examined the degree to which this stimulatory coupling is involved in the prominent tachyphylaxis observed in ß-adrenergic relaxation responses. Specifically, we sought to determine whether ß-adrenergic linkage to KCa channels and Ca2+ mobilization exhibited this phenomenon and whether adenylyl cyclase activity is involved in the tachyphylaxis to ß-agonists. Our findings indicate that ß-adrenergic stimulatory coupling to KCa channels displays marked tachyphylaxis and that the desensitization of this coupling likely underlies the progressive loss of functional muscle relaxation.
Tissue Preparation and Tension Records Methods were essentially similar to those described previously (19). Male guinea pigs (350–450 g) were killed and tracheas excised from the animal. The tracheal ring was opened by cutting longitudinally through the cartilaginous region, and the epithelium was dissected away. Muscle strips containing one cartilaginous ring were removed and placed vertically in a 1-ml organ bath to measure tension isometrically, and perfused with solution at a constant flow rate 2.0 ml/min throughout the experiments. The normal bath solution had the following composition (mM): 137 NaCl, 5.9 KHCO3, 2.4 CaCl2,1.2 MgCl2, and 11.8 glucose, bubbled with a gas mixture of 99% O2–1% CO2. For the Ca2+-free solution, 2.4 mM CaCl2 was replaced with 2.2 mM NaCl and 0.2 mM ethyleneglycol-bis- (ß-aminoethyl ether)-N,N',-tetraacetic acid (EGTA). Indomethacin (2 µM) was perfused throughout the experiments to abolish resting tone and passive tension was adjusted to 0.5 g after equilibrating the preparation in the normal bath solution for 60 min. One micromolar methacholine (MCh) was applied to the strips for 10 min at intervals of 20 min until the control response to 1 µM MCh was established, then the experiments were started. One micromolar MCh was repeatedly applied in the presence of 0.3 µM isoproterenol (ISO) at an interval of 30 min. ISO was applied 3 min before MCh. The relaxation observed following exposure to Ca2+-free solution, applied at the end of each experiment, was defined as complete relaxation (0% contraction). All experiments were performed at 37°C.
Measurement of Fura-2 Fluorescence
Single-Channel Records
cAMP Measurement
Materials
Analysis of Results
Desensitization of ß-Adrenergic Relaxation The trachealis strips were repeatedly contracted (every 30 min) with 1 µM MCh after pretreatment with 0.3 µM ISO (3 min before contraction). As shown in Figure 1A (upper trace), this protocol resulted in a gradual attenuation of relaxant effects from almost complete inhibition of contraction to only slight inhibition of control contractions. The values of percent contraction at the first and ninth application were 1.2 ± 0.6 and 72.9 ± 6.4% (n = 12), respectively (P < 0.01, Figure 1B). The desensitization of ß-adrenergic relaxation was markedly dependent on voltage-dependent calcium influx, as the same protocol in the presence of 3 µM verapamil prevented the loss of ISO efficacy (Figure 1A, middle trace). The value of percent contraction for MCh with ISO at the ninth application were decreased to 14.2 ± 5.6% (n = 12, P < 0.001, Figure 1B). Similar effects were observed with equimolar nifedipine. Conversely, the KCa channel inhibitor IbTX (30 nM) reduced the inhibitory effects of ISO and resulted in a rapid loss of relaxation efficacy after repeated exposure (Figure 1A, lower trace). The values of percent contraction for MCh with ISO at the first application were 31.4 ± 4.9%, and those values were increased to 100% at the sixth application (n = 12, Figure 1B). In control experiments repeated application of 1 µM MCh did not result in a significant attenuation of contractile responses. The values of percent contraction for MCh at the tenth applications of this agent was 96.8 ± 2.9% (n = 4, data not shown).
Stimulation of Adenylyl Cyclase and Desensitization We next examined the desensitization of direct adenylyl cyclase stimulation. Using 1 µM forskolin in place of ISO in protocols identical to those shown in Figure 1, the inhibitory effects of forskolin were not attenuated over time (Figure 2A, upper trace), as was observed with ISO. The values of percent contraction for MCh with forskolin at the first and ninth application were 66.9 ± 9.9 and 73.5 ± 6.6% (n = 12), respectively (not significant; Figure 2B). The same result was observed when the concentration of forskolin was increased 10-fold (10 µM) to achieve almost total inhibition of contraction; rather than demonstrating tachyphylaxis, the inhibitory effects of 10 µM forskolin on 1 µM MCh were gradually augmented after repeated exposure (Figure 2A, lower trace). The values of percent contraction with MCh with forskolin at the first and ninth application were 8.0 ± 3.9 and 1.3 ± 0.6% (n = 12), respectively (P < 0.01, Figure 2B). However, in the presence of 30 nM IbTX, the inhibitory effects of 10 µM forskolin on 1 µM MCh-induced contraction was gradually attenuated after repeated exposure to these agents. The values of percent contraction for MCh with forskolin were 43.6 ± 9.2%, and those values were increased to approximate 100% at the sixth application (n = 8, Figure 2B).
Desensitization of KCa Channel Stimulatory Coupling To examine the stimulatory coupling between ß-adrenergic receptors and KCa channels, we directly examined this relationship in on-cell patches. In separate experiments, KCa single channel currents were recorded in the cell-attached configuration during repeated exposure of ISO for 5 min every 15 min, and PKA was applied to inside-out patches in the same manner. Extracellular application of 1 µM ISO markedly augmented KCa channel activity and mean values of open probability (nPo) averaged over 5 min increased to 9.6 ± 3.1-fold (n = 6, Figure 3B). However, after repeated exposure to 1 µM ISO every 15 min, ISO channel stimulation was gradually attenuated with no change in unitary amplitude (Figure 3A). The values of fold stimulation of this channel by ISO at the third and sixth application were 5.3 ± 2.9- and 1.6 ± 1.1-fold (n = 6), respectively (P < 0.01, Figure 3B). This desensitization of stimulatory coupling was not due to channel rundown, as repeated application of 10 U/ml PKA to inside-out patches resulted in a sustained augmentation KCa channel activity (Figure 3C). Mean values of nPo increased 5.2 ± 1.8-fold (n = 12), and activation of channel open-state probability gradually increased with subsequent exposure to PKA. The values of fold stimulation of this channel by PKA at the third and sixth application were 6.9 ± 2.1- and 9.6 ± 2.0-fold (n = 12), respectively (P < 0.01, Figure 3D).
Desensitization of Inhibitory Effects on [Ca2+]i To examine the desensitization effect at the level of [Ca2+]i, we made simultaneous measurements of tension and F340/F380 in intact muscle segments. The fura-2 loaded tissues were repeatedly exposed to 1 µM MCh for 7 min in the presence of 0.3 µM ISO (1 min before MCh) every 15 min. As shown in Figure 4A, ISO markedly reduced MCh-induced contractions (upper trace) and increases in [Ca2+]i (lower trace), but this effect was gradually attenuated after repeated exposure to ISO. The values of percent contraction and F340/F380 at the first and third application were 4.9 ± 1.9 and 17.9 ± 5.8% (n = 4), and 27.5 ± 8.9 and 60.6 ± 9.6% (n = 4), respectively (P < 0.01, Figure 4B). Consistent with experiments examining contraction alone, verapamil (3 µM) prevented the tachyphylaxis at the level of contraction and [Ca2+]i (Figure 4C). The values of percent contraction and F340/F380 for MCh with ISO at the first and third application were 3.2 ± 1.6 and 5.1 ± 2.9% (n = 4), and 26.0 ± 6.9 and 29.3 ± 7.4% (n = 4), respectively (not significant, Figure 4D).
Similar to results at the level of contraction, the inhibitory effects of forskolin on [Ca2+]i did not display desensitization (Figure 5A). Application of 10 µM forskolin inhibited the MCh increase in force and [Ca2+]i without a loss of efficacy; the values of percent F340/F380 for MCh with forskolin were 54.2 ± 9.8 and 48.8 ± 8.6% (n = 4), respectively (not significant, Figure 5B). When the concentration of forskolin was lowered to 1 µM, the values of percent contraction and percent F340/F380 for MCh on forskolin were increased to 55.8 ± 10.8 and 60.9 ± 8.2% (n = 4), respectively (Figure 5C), and were not affected after repeated exposure to these agents. The values of percent contraction and percent F340/F380 at the third application of these agents were 70.1 ± 9.6 and 76.6 ± 6.9% (n = 4), respectively (not significant, Figure 5C).
Role of KCa Channel Suppression on Ca2+ Mobilization via VDCC Channels To further explore the relationship between KCa stimulation and VDCC activity, we examined contractile and [Ca2+]i responses in the presence of inhibitors of both channel types. When IbTX (30 nM) was applied to tissues precontracted by 1 µM MCh, an increase in contraction and [Ca2+]i was observed (Figure 6A). The percent contraction and F340/F380 were increased to 128.6 ± 8.6 and 133.7 ± 9.6% (n = 4), respectively (Figure 6B). Verapamil (0.1-10 µM) prevented this increase, however, in a concentration-dependent manner, limiting the increases to 116.8 ± 6.8 and 120.4 ± 7.9% (n = 4) for contraction and [Ca2+]i, respectively, at 0.3 µM (P < 0.05, Figures 6B and 6C) and to 96.1 ± 6.2 and 97.6 ± 2.9% (n = 4), respectively, at 3 µM (P < 0.01, Figures 6B and 6C). Verapamil alone (3 µM) only modestly inhibited contraction and F340/F380 induced by MCh. The values of percent contraction and F340/F380 for MCh with 3 µM verapamil were 94.1 ± 6.2 and 95.6 ± 3.9% (n = 4), respectively.
Concentration of Intracellular cAMP after Repeated Exposure to ISO and Forskolin Finally, we examined desensitization at the level of generation of intracellular cAMP. Exposure of tissues to 0.3 µM ISO for 10 min increased intracellular cAMP from 4.6 ± 3.2 to 12.5 ± 3.5 pmol/mg (n = 6, P < 0.01; Figure 7A). However, after repeated exposure to ISO for 10 min every 30 min, concentration of intracellular cAMP was markedly decreased to 5.7 ± 2.4 pmol/mg at the ninth application (n = 6, P < 0.05). Conversely, when tissues were repeatedly exposed to 10 µM forskolin in the same way, no desensitization was observed. Forskolin stimulated cAMP concentration to 288.9 ± 162.9 pmol/mg initially and to 477.8 ± 107.4 pmol/mg at the ninth application (n = 6, P < 0.05; Figure 7B).
In the present study, we demonstrate for the first time the involvement of Ca2+ mobilization regulated by KCa channels as an important mechanism underlying tachyphylaxis to ß-agonists in airway smooth muscle. ß-Adrenergic stimulatory coupling to KCa channels and inhibition of cholinergic force production is progressively lost with repeated application of ß-agonist, whereas forskolin-stimulated coupling does not desensitize (Figures 1, 2, 4, and 5). Consistent with a causal relationship between loss of KCa channel stimulatory activity and tachyphylaxis, pharmacologic inhibition of KCa channels augments the loss of relaxant activity with repeated ß-agonist exposure. Moreover, in the presence of verapamil, little loss of ß-agonist potency was observed. Because ß-agonist stimulatory coupling to KCa channels is an important regulator of VDCC activity during muscarinic contractions (Figure 6), this finding suggests that augmented Ca2+ influx through VDCC leads to rapid desensitization of ß-adrenergic receptors and tachyphylaxis. The effect of verapamil could not be explained by a progressive decrease in sarcoplasmic reticulum Ca2+, as repeated contractions in the presence of verapamil alone did not decrease force production (not shown). Thus, our results suggest that ß-adrenergic desensitization is highly sensitive to VDCC activity. A recent study in hippocampal neurons indicates interactions between the ß2-adrenergic receptor and the 1.2 subunit of the L-type Ca2+ channel (25). Although no evidence of interaction between the ß2-adrenergic receptor and smooth muscle VDCC has been reported, our findings suggest a surprising dependence of desensitization on VDCC activity. Thus, Ca2+ influx through VDCC appears to promote tachyphylaxis to ß-agonists. Muscarinic stimulation of airway smooth muscle produces a sustained, global rise in [Ca2+]i, which would be expected to markedly activate KCa channels and prevent tissue depolarization. However, muscarinic receptor stimulation also produces a potent and direct inhibition of KCa channel activity (21). ß-Adrenergic receptor stimulation opens KCa channels, resulting in competitive antagonism at the level of membrane potential (15, 26), likely explaining the marked dependence of relaxation on KCa activity (11–13). This competitive action can be seen in Figure 6, in which muscarinic increases in [Ca2+]i and tension are augmented by inhibition of KCa channels, an effect that occurs through stimulation of VDCC. Thus, VDCC activity appears to play an important role in ß-adrenergic relaxation in spite of the relatively modest effect of VDCC inhibitors alone in antagonizing muscarinic contraction. Our results indicate that VDCC regulates tachyphylaxis, rather than the VDCC being the key downstream target controlling contraction. Measurements of [Ca2+]i during repeated exposure to ß-agonist indicated that the ability of the agonist to inhibit cholinergic increases in [Ca2+]i also desensitized (Figure 4), providing further evidence of the functional importance of ß-adrenergic receptor/KCa channel stimulatory coupling in relaxation responses. Desensitization of stimulatory coupling was directly established in patch clamp experiments showing a progressive loss of stimulation of channel activity by ISO with repeated exposure (Figure 3), similar in magnitude and time-course to that observed at the levels of force and [Ca2+]i (Figure 4). Because the progressive reduction in KCa channel activity by ß-agonists was mimicked by outside-out patches (data not shown), membrane-delimited pathways may be involved in the phenomenon. Previous reports have demonstrated that an augmentation in KCa channel activity is a key component of the relaxant action and membrane hyperpolarization induced by ß-agonists (27). As shown in Figure 6, IbTX increased tension and [Ca2+]i induced by MCh, and verapamil suppressed the effects induced by IbTX, demonstrating that inhibition of KCa channel activity results in increased Ca2+ influx through VDCC, and augmented force production (28). We show that tachyphylaxis to ß-agonists also occurs at the level of [Ca2+]i as repeated exposure to ß-agonists resulted in gradual increase in [Ca2+]i (Figure 4), indicating a loss of efficacy in preventing Ca2+ mobilization. A substantial component of this increase in [Ca2+]i occurs through the activation of VDCC, as the overall increase in [Ca2+]i is partially inhibited by verapamil (Figure 4) and nifedipine (not shown). Strikingly, it is this component that appears to account for the ß-adrenergic desensitization, as inhibition of VDCC eliminates tachyphylaxis at the level of [Ca2+]i (Figure 4). Hence, Ca2+ mobilization through VDCC regulated by KCa channel activity leads to tachyphylaxis to ß-agonists. Stimulatory coupling between ß-adrenergic receptors and KCa channels is involved in functional relaxation, and desensitization of this coupling correlates extremely well with loss of ß-adrenergic efficacy. As shown in Figure 1, the first application of ISO is markedly less effective at reducing tension in the presence of IbTX, and this effect was also observed at the level of [Ca2+]i (data not shown). However, our data also point to KCa channel–independent mechanisms underlying relaxation induced by ß-agonists in airway smooth muscle, as previous reported (14, 29). Thus, although reduced in efficacy, ß-agonists relax tracheal smooth muscle in the presence of 30 nM IbTX (Figure 1B). Finally, we show that desensitization of ß-adrenergic stimulatory coupling is not associated with a loss of efficacy downstream of the receptor/G protein complex (Figures 2–5). The inhibitory effects of forskolin on contraction and [Ca2+]i were not attenuated with repeated exposure to this agent, suggesting that desensitization occurs at the level of ß-adrenergic receptor/adenylyl cyclase coupling. Moreover, cAMP generation was not attenuated following repeated exposure to forskolin (Figure 7), whereas adenylyl cyclase activity was significantly attenuated after repeated exposure to ISO. Thus, tachyphylaxis is considered to be associated with an uncoupling of the ß-adrenergic receptor from target molecules rather than a loss of efficacy of downstream signaling events, and is consistent with the well characterized phosphorylation and arrestin-mediated internalization of ß2-adrenergic receptors (30, 31). However, our data also demonstrate that forskolin relaxation is desensitized in the presence of IbTX (Figure 2B), revealing a postreceptor desensitization of adenylyl cyclase or downstream effectors that occurs with augmented Ca2+ influx. In summary, we have shown that a progressive loss of ß-adrenergic stimulatory coupling to KCa channels plays a major role in the loss of ß-adrenergic relaxation efficacy that occurs with tachyphylaxis. Loss of this stimulatory coupling is reflected in a parallel failure to effectively decrease [Ca2+]i. The mechanism underlying loss of coupling efficiency is upstream of the KCa channel and appears to result from receptor uncoupling. These results may provide a rationale for other strategies aimed at activating this system through nondesensitizing pathways. Received in original form September 30, 2002 Received in final form March 10, 2003
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Abstract
Introduction
2 x 2 x 10 mm) were cut by dissecting parallel to the longitudinal axis of the muscle and equilibrated in normal bath solution at 37° for 60 min, and then the experiments started. Two micromolars indomethacin was perfused throughout the experiments. At the end of each experiment, the strips were rapidly removed, blotted, and frozen in liquid nitrogen. Frozen strips were homogenized in cold 6% trichloroacetic acid at 2–8°C and the homogenate centrifuged at 2,000 x g for 15 min at 4°C. Precipitated protein was separated and used for the measurement of protein content (
