G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

J. K. L. Walker,1 R. R. Gainetdinov,2 D. S. Feldman,3 P. K. McFawn,4 M. G. Caron,2,5 R. J. Lefkowitz,1,5,6 R. T. Premont,1 and J. T. Fisher7

Departments of 1Medicine, 6Biochemistry, 2Cell Biology, 5Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710; 3Department of Medicine, Ohio State University, Columbus, Ohio 43210; 4School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley 6009, Western Australia, Australia; and 7Departments of Physiology, Medicine and Pediatrics, Queen's University, Kingston, Ontario, Canada K7L 3N6

Submitted 25 July 2003 ; accepted in final form 5 October 2003


    ABSTRACT
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 METHODS
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 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2 muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5-/-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5-/- and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from {beta}2-adrenergic receptor activation were diminished in GRK5-/- mice. These data suggest that M2 muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5-/- mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

transgenic mice; airway pressure time index; electric field stimulation; airway responsiveness


NUMEROUS STUDIES INDICATE that dysfunction of either pre- or postjunctional airway M2 muscarinic receptors is implicated in the pathophysiology of asthma. Evidence suggests that human asthmatics and animal models of asthma demonstrate prejunctional M2 receptor dysfunction (14, 15, 23). Other studies show that the activity of postjunctional M2 muscarinic receptors is exaggerated in sensitized tracheal tissue, resulting in diminished responsiveness to {beta}2-adrenergic receptor ({beta}2-AR) agonist-mediated relaxation (11, 19, 20, 43).

Five muscarinic cholinergic receptor subtypes (M1-M5) have been cloned, and four subtypes can be distinguished pharmacologically (21). Radioligand binding studies demonstrate differential distribution of M1-M3 muscarinic receptor subtypes in airways (18). In brief, M1 muscarinic receptors, located at the parasympathetic ganglion, are thought to filter the transmission of the neuronal signal. M3 muscarinic receptors mediate acetylcholine-induced contraction of airway smooth muscle (36) (Fig. 1). M2 muscarinic receptors, located both pre- and postjunctionally, effect dual regulation of airway smooth muscle tone (22). In a classic negative feedback loop, prejunctional M2 muscarinic receptors limit the amount of acetylcholine released from stimulated cholinergic nerves, thereby limiting the level of bronchoconstriction (12). In contrast, postjunctional M2 muscarinic receptors, through the activation of G{alpha}i, can enhance the overall level of bronchoconstriction by opposing G{alpha}s-mediated events, including activation of adenylyl cyclase or opening of large conductance calcium-activated potassium (KCa) channels.



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Fig. 1. M3 muscarinic receptors mediate acetylcholine (ACh)-induced contraction of airway smooth muscle (ASM). Prejunctional M2 muscarinic receptors limit the amount of ACh released from stimulated cholinergic nerves, thereby limiting the level of bronchoconstriction. In contrast, activation of postjunctional M2 muscarinic receptors can enhance the overall level of bronchoconstriction by opposing G{alpha}s-mediated ASM relaxation, as might occur through activation of {beta}2-adrenergic receptors ({beta}2-ARs), prostaglandin E2 receptors (PGE2), or through vasoactive intestinal polypeptide (VIP) receptors. GRK5, G protein-coupled receptor kinase 5.

 

Characteristic of G protein-coupled receptors (GPCRs), muscarinic receptor signaling is regulated by the coordination of two critical events. GPCR kinases (GRKs) phosphorylate the agonist-activated receptor, and this chemical modification promotes binding of an arrestin protein to the receptor. These events terminate further receptor activation of downstream G protein signaling while at the same time promoting arrestin-dependent signals.

We recently generated mice lacking the GRK5 gene (GRK5-/-) and showed that behavioral responses mediated by central muscarinic receptors were enhanced and prolonged in these mice when a muscarinic receptor agonist was administered (16). Because GRK5 is a ubiquitously expressed member of the GRK family, we hypothesized that in addition to regulating central muscarinic receptors, GRK5 might also regulate responses mediated by peripheral muscarinic receptors.

Because the mechanism of muscarinic receptor "dysfunction" as it relates to airway responsiveness is not well defined, it is important to determine whether GRK5 modulates muscarinic receptor-mediated airway responses. We measured tracheal pressure in GRK5-/- and littermate wild-type (WT) control mice during bilateral vagal stimulation or intravenous injection of the muscarinic receptor agonist methacholine (MCh). Because GRK5 mRNA is robustly expressed in the heart (34), where M2 muscarinic receptors mediate parasympathetic control over cardiac chronotropy, we also tested whether GRK5 regulates heart rate.


    METHODS
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Generation of GRK5-/- mice and genotyping. The murine GRK5 gene was deleted as described previously, and genotyping was performed using triplex PCR on DNA purified from tail tips (16). Heterozygous SVJ129xC57BL/6 mice were interbred to obtain the littermate WT and GRK5-deleted animals used in this study.

General. Mice were acclimated to an ambient temperature of 21-22°C and a photoperiod between 0600 and 2000. They were fed standard mouse chow and allowed free access to water. Experiments were carried out between 0800 and 1800. All experimental procedures conformed to the guidelines of the Canadian Council of Animal Care and were approved by the Queen's University Animal Care Committee and Duke University Institutional Animal Care and Use Committee.

In vivo animal preparation and experimental protocols. Animals were surgically prepared as previously reported (42). Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg) and mechanically ventilated through a steel cannula inserted into the trachea. A catheter in the jugular vein was used for intravenous administration of anesthetic and other drugs (injection volumes of 1 ml/kg). In respiratory protocols, mice were paralyzed with doxacurium chloride (0.25 mg/kg) to prevent respiratory efforts. Additional doses of doxacurium chloride were administered as necessary, and additional doses of anesthetic were administered at regular time intervals after paralysis. Mice were ventilated with 100% oxygen at a constant volume of 8-10 ml/kg and a frequency of 125 breaths/min. Measurement of airway pressure was made at a side port of the tracheal cannula connected to a Validyne differential pressure transducer. Electrocardiogram electrodes were placed subcutaneously for the measurement of heart rate. For studies involving vagal stimulation, the right and left branches of the vagus nerve were isolated from the carotid artery, placed on respective stimulating electrodes, and immersed in mineral oil. Electrocardiogram signals and tracheal pressure were acquired by computer and analyzed using peak detection software. Airway responses were denoted as airway pressure time index (APTI), which was calculated as the sum of the MCh-induced change in peak airway pressure integrated with respect to time. APTI was calculated every 10 s from the time of injection until the peak pressure plateaued (2 min). Others have validated this measure as a reasonable index of airway responsiveness (6, 10, 27). Average heart rate was calculated using the instantaneous heart rate derived from the electrocardiogram R-R interval.

Experimental Protocols

MCh injection. Increasing doses of MCh (25, 50, 100, and 150 µg/kg) were injected intravenously into anesthetized, paralyzed, and ventilated mice. Respiratory efforts were closely monitored to prevent their interference with accurate measurement of tracheal pressure. Mice were hyperinflated 1 min before each MCh injection to establish a constant volume history and respiratory mechanics. Each MCh injection was separated by a 5-min recovery period.

Vagal stimulation. A separate group of mice was surgically prepared for vagal stimulation by placing a length of nerve on platinum electrodes. A section of nerve proximal to the electrodes was crushed to prevent afferent nerve stimulation. Nerves were activated with a supramaximal stimulus (16 V, 2-ms duration) to ensure recruitment of all vagal efferents for a period of 10 s and at various stimulus frequencies (5, 10, 15, and 20 Hz). Bilateral vagal stimulation was randomized in the following order: 10, 15, 5, and 20 Hz, and a 5-min recovery period (between stimulations) was allowed. Mice were injected with propranolol (1 mg/kg) and doxacurium chloride (0.25 mg/kg) before the initiation of the vagal stimulation frequency response protocol.

In vitro tissue preparation and protocols. The trachea was immediately removed from mice (that had been killed) and placed in ice-cold oxygenated Krebs solution containing (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25.0 NaHCO3, 1.2 NaH2PO4, and 11 glucose, pH 7.35-7.40. Five to seven cartilage rings were cut, and smooth muscle was exposed. The tracheal preparation was mounted with small pins and surgical suture (4.0 silk) between platinum ring electrodes under an initial load of 0.4-0.6 g in an organ tissue bath (Radnoti, Monrovia, CA) containing Krebs solution. The Krebs solution was bubbled continuously with a mixture of 95% O2 and 5% CO2 and maintained at 37°C ± 1°C. Tracheal ring preparations were allowed to equilibrate for at least 60 min before being adjusted to optimal length. The isometric force of tracheal smooth muscle was measured using a calibrated force displacement transducer (Grass) and acquired using a computer data acquisition program (CODAS, DATAQ). A Grass S88 stimulator was used to produce electric field stimulation of tissues by passing current pulses between ring electrodes. In all protocols, pulses of 2-ms duration, 50 V, and 20 Hz, were applied for 10 s. These stimulus parameters were chosen based on preliminary studies that demonstrated maximal contractile tension with little or no direct effect on smooth muscle, as assessed by the ability of 1 µm of tetrodotoxin to abolish the response. For the KCl depolarization protocol, equimolar KCl was substituted for NaCl in the Krebs solution (32).

Protocols. Four protocols were conducted on each tracheal ring preparation with washout between protocols. The protocols, in order of occurrence, were as follows: 1) complete depolarization with 90 mM KCl; 2) cumulative concentration response to carbachol; 3) relaxation over 60 min after precontracting with carbachol to 70% of maximum; and 4) relaxation with increasing concentrations of isoproterenol after precontracting with carbachol to 70% of maximum. In a separate experiment, tissues were precontracted with 90 mM KCl and relaxed with increasing doses of isoproterenol. In a second group of mice, the following protocol was conducted: 1) complete depolarization with 90 mM KCl; 2) cumulative concentration response to carbachol; and 3) relaxation with increasing concentrations of isoproterenol after blocking M2 receptors with 0.3 µM methoctramine and precontracting with carbachol to 78% of maximum.

Western blots. Airway tissues including lung and trachea were removed and stored at -80°C until assayed for protein. Stored tissues analyzed for GRK5 protein were homogenized using liquid nitrogen and mortar and pestle. The homogenized tissue was solubilized in 800 µl of RIPA buffer (50 mM Tris·HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors) and centrifuged (100,000 g) for 30 min at 4°C. Solubilized proteins were separated using 10% SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted [monoclonal anti-GRK5 (A16/17)] (33), and visualized using autoradiography.

Statistical analyses. Statistical analyses were carried out using the computer software GraphPad Prism or SPSS. Differences between genotype, drug dose, and time after challenge were analyzed for MCh using multiway ANOVA in a general linear model with genotype, time, and dose as factors. Values are reported as means ± SE. To determine differences within a protocol for a mouse genotype, a paired two-tailed t-test was used unless more than two cases existed, in which case a one-way ANOVA for repeated measures was used, and differences were identified with Tukey's honest significant difference post hoc test. Results were considered significantly different if P <= 0.05.


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GRK5 expression. Figure 2 shows that GRK5 is indeed expressed in mouse lung and trachea and is, therefore, readily available to exert a physiological effect on GPCRs in those tissues. Notice that GRK5 remains measurable in tracheal tissue even after epithelial tissue has been denuded, suggesting that GRK5 is expressed in airway smooth muscle cells. Others have previously shown that GRK5 is expressed in the mouse heart (35), thus cardiac samples are not presented here.



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Fig. 2. GRK5 protein expression by Western blotting. Whole lung, trachea, and epithelium-denuded trachea from wild-type (WT) animals were subjected to immunoblotting using an anti-GRK5 antiserum (A16/17) (33). All WT tissues exhibited a 67-kDa band having migration consistent with that of control GRK5 protein. GRK5 control protein is loaded in the fourth lane from the left.

 

In vivo airway response to bilateral vagal stimulation and MCh. Control tracheal pressure was not different between WT (8.6 ± 0.1 cmH2O) and GRK5-/- (8.9 ± 0.3 cmH2O) mice at the start of an experiment. If GRK5 is important for the desensitization of prejunctional M2 muscarinic receptors, then stimulation of these receptors in GRK5-/- mice should result in excessive inhibition of junctional acetylcholine release. Thus one might observe decreased airway responsiveness to vagal stimulation in GRK5-/- mice. Both WT and GRK5-/- mice demonstrated a frequency-dependent increase in APTI during bilateral vagal stimulation. However, at stimulation frequencies of 5 and 15 Hz, average APTI values were significantly greater in GRK5-/- mice relative to WT mice (Fig. 3), a trend that was also apparent for the other stimulus frequencies. These results suggest that M2 muscarinic autoinhibitory receptors are not modulated by GRK5 but that airways of GRK5-/- mice are hyperresponsive to vagal stimulation. To assess whether the enhanced airway response to vagal stimulation in GRK5-/- mice was mediated by M3 muscarinic receptors on airway smooth muscle, we measured APTI during intravenous administration of MCh. Figure 4 shows that the airway responses to 25 and 50 µg/kg of MCh were not enhanced in GRK5-/- mice and that the effect of {beta}2-AR antagonism (propranolol) on the airway response to 50 µg/kg of MCh was not different between WT and GRK5-/- mice. These data suggest that GRK5 is not an important regulator of the M3 muscarinic receptors that mediate contraction of airway smooth muscle.



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Fig. 3. Airway response to increasing frequency of vagal stimulation is enhanced in GRK5-/- ({circ}) mice relative to WT ({blacksquare}) mice. Airway pressure time index (APTI) was calculated over a 30-s period beginning at the initiation of a 10-s vagus nerve stimulation at 5, 10, 15, or 20 Hz. Mice were anesthetized and paralyzed with doxacurium chloride. Data points are means ± SE for n = 10 WT and n = 12 GRK5-/-. *GRK5-/- significantly different from WT mice (P < 0.05) at 5 and 15 Hz.

 


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Fig. 4. Airway response to intravenous administration of methacholine (MCh). Response to 25 and 50 µg/kg of MCh is not different between WT (filled bars) and GRK5-/- (open bars) mice. Propranolol had no effect on the airway response to 50 µg/kg of MCh (50 pst-prop). Data points are means ± SE for n = 7 WT and n = 8 GRK5-/-.

 

In vitro tracheal smooth muscle contractile response to carbachol and isoproterenol. To further explore GRK5 regulation of airway muscarinic receptors, we assessed the in vitro response of tracheal smooth muscle to the muscarinic receptor agonist carbachol. The maximum absolute tracheal smooth muscle tension developed in response to 10 µM carbachol (maximal contraction) or 90 mM KCl was not different between WT and GRK5-/- mice (Table 1). The sensitivity of the contractile response to carbachol also was the same in WT and GRK5-/- mice (Fig. 5). These data provide additional support for the idea that GRK5 is not an important regulator of M3 muscarinic receptor-mediated contraction of airway smooth muscle.


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Table 1. Maximum contractile tension developed

 


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Fig. 5. Effect of carbachol on tracheal smooth muscle isometric force generation is not different between WT ({blacksquare}) and GRK5-/- ({circ}) mice. Force generated at 10 µM carbachol is defined as "maximum" force of contraction. All other values are expressed as a percentage of maximum force. Data points are means ± SE for n = 6 WT and n = 4 GRK5-/-. Nonlinear regression for sigmoidal dose response was calculated.

 

Having ruled out a regulatory effect of GRK5 on prejunctional M2 muscarinic receptors and postjunctional M3 muscarinic receptors, we focused on the possibility that GRK5 may desensitize M2 muscarinic receptors located on airway smooth muscle. If this were the case, then the physiological response mediated by these receptors (opposition of G{alpha}s-mediated relaxation) would be more effective in mice lacking the GRK5 protein (GRK5-/-). To test M2 muscarinic receptor-meditated inhibition of G{alpha}s-mediated relaxation, we precontracted tracheal smooth muscle with carbachol and then exposed it to increasing concentrations of isoproterenol, a {beta}2-AR agonist that causes G{alpha}s-mediated airway smooth muscle relaxation. Both WT (70 ± 4% of maximum) and GRK5-/- (68 ± 1% of maximum) mice were precontracted to similar tensions (Fig. 6A). However, tracheal smooth muscle from GRK5-/- mice was more resistant to the relaxation effects of isoproterenol (Fig. 6B). This observation was not a result of decreased responsiveness to isoproterenol alone (Fig. 7A) or inherent time-related differences in relaxation (Fig. 7B). When tissue was precontracted with KCl, rather than a muscarinic agonist, {beta}2-AR-mediated relaxation was not different in GRK5-/- mice relative to WT mice (Fig. 7A). Furthermore, time-series control data show that WT and GRK5-/- mice sustain their carbachol-induced contractions similarly over an interval equal to that of the isoproterenol relaxation challenge (Fig. 7B). Thus the relaxatory effect of isoproterenol was diminished in GRK5-/- mice precontracted with carbachol but not KCl. One mechanism to explain this observation is that {beta}2-AR-mediated relaxation is excessively opposed by enhanced activation of an M2 muscarinic receptor-mediated pathway in GRK5-/- mice. To test this hypothesis, we precontracted tracheal tissue with carbachol to 78% of maximum and relaxed it with isoproterenol in the presence of the selective M2 muscarinic receptor antagonist methoctramine. As shown in Fig. 7C, when M2 muscarinic receptors are pharmacologically inhibited, tracheal tissue relaxes similarly in GRK5-/- and WT mice. Together, the in vivo and in vitro airway data suggest that M2 receptors on airway smooth muscle cells are desensitized, at least in part, by GRK5, whereas M3 receptors on these same cells and M2 autoreceptors on the vagus nerves are not.



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Fig. 6. Effect of isoproterenol on tracheal smooth muscle precontracted with carbachol. A: tracheal tissue from WT (solid bar) and GRK5-/- (open bar) mice was precontracted to similar tensions. B: relaxation in response to log increases in isoproterenol concentration is inhibited in GRK5-/- ({circ}) mice relative to WT ({blacksquare}) mice. Data points are means ± SE for n = 6 WT and n = 4 GRK5-/-. Nonlinear regression for sigmoidal dose response was calculated. *Significant difference between WT and GRK5-/- (P < 0.05).

 


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Fig. 7. Effect of time and isoproterenol on the magnitude of tracheal tissue contraction. Tracheal tissue from WT ({blacksquare}) and GRK5-/- ({circ}) mice showed no difference in relaxation response to isoproterenol when precontracted with KCl (A) or when precontracted with carbachol and antagonized with methoctramine (C). Data points are means ± SE for n = 5 (A) and 4 (C) WT and n = 5 (A) and 4 (C) GRK5-/- mice, respectively. Nonlinear regression for sigmoidal dose response was calculated. B: time series control data for the isoproterenol relaxation protocols shows that the magnitude of the 70% maximum contraction is maintained similarly in WT and GRK5-/- mice. Data points are means ± SE for n = 4 WT and n = 4 GRK5-/-.

 

Heart rate response to bilateral vagal stimulation and MCh. We next tested the contribution that GRK5 may make to the regulation of cardiac chronotropy. Resting heart rate was not significantly different between anesthetized, intact WT (476 ± 11 beats/min, n = 6) and GRK5-/- (486 ± 17 beats/min, n = 6) mice. Propranolol caused a similar and significant decline in heart rate in WT (-18 ± 4 beats/min) and GRK5-/- (-26 ± 4 beats/min) mice.

To determine whether GRK5 regulates cardiac muscarinic receptor sensitivity, we measured heart rate during bilateral vagal nerve stimulation and before and after administration of the muscarinic receptor agonist MCh. At a stimulation frequency of 10 Hz, both WT and GRK5-/- mice demonstrated a significant bradycardia followed by heart rate recovery (Fig. 8A). Neither the heart rate decrease nor the heart rate recovery was different between genotypes. Intravenous injection of MCh produced a time- and dose-dependent reduction in heart rate in both WT and GRK5-/- mice (P < 0.001 all cases; Fig. 8B). This bradycardia was not different in GRK5-/- mice relative to WT mice. These data suggest that M2 muscarinic receptor-mediated bradycardia is not significantly modulated by GRK5.



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Fig. 8. Effect of GRK5 on activation of cardiac muscarinic receptors. Effect of 10 Hz bilateral vagal nerve stimulation (A) and 25 and 50 µg/kg doses of MCh (B) on heart rate in anesthetized, paralyzed WT (closed symbols) and GRK5-/- (open symbols) mice. Resting heart rate was not different between genotypes during control conditions. Genotype had no effect on bradycardic response to vagal stimulation or MCh injection. Heart rate responses to other stimulation frequencies or MCh doses were not different between WT and GRK5-/- mice. Means ± SE are shown. Two separate groups of mice were used. A: WT ({blacksquare}), n = 10; GRK5-/- ({circ}), n = 10. B: WT (25 µg/kg MCh {blacksquare}; 50 µg/kg MCh {blacktriangleup}), n = 6; GRK5-/- (25 µg/kg MCh {circ}; 50 µg/kg MCh {triangleup}), n = 6.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous work showed that GRK5-/- mice demonstrate pronounced central cholinergic supersensitivity and impaired M2 muscarinic receptor desensitization (16). Consistent with those findings, this study indicates that airway smooth muscle M2 muscarinic receptor function is exaggerated in GRK5-/- mice. However, the current study also shows that GRK5 is unimportant for regulating cardiac chronotropy through direct regulation of cardiac M2 muscarinic receptors.

Although M3 muscarinic receptors are the primary mediators of airway smooth muscle constriction, M2 receptors, expressed by airway smooth muscle of many species, including humans and mice, frequently outnumber M3 muscarinic receptors (13, 17, 29, 37). This relatively large concentration of M2 muscarinic receptors contributes to airway smooth muscle contraction predominantly through inhibition of relaxation (8, 30). However, in the absence of M3 muscarinic receptors, M2 muscarinic receptors can directly promote airway smooth muscle contraction (39). Our in vitro results show that when tracheal smooth muscle is precontracted with a muscarinic agonist, {beta}2-AR-mediated relaxation is significantly attenuated in GRK5-/- mice relative to WT mice and that this difference disappears when M2 muscarinic receptors are pharmacologically antagonized. Thus our data indicate that GRK5 is likely an important contributor to the desensitization of M2 muscarinic receptors on airway smooth muscle, thereby regulating the ability of these receptors to oppose airway smooth muscle relaxation. Our results are in contrast to a study by Matsui et al. (30), which showed that M2 muscarinic receptor activation does not oppose isoproterenol-induced relaxation of murine tracheal smooth muscle. However, this same study demonstrated that M2 muscarinic receptor activation inhibits forskolin-induced cAMP-mediated relaxation, thus supporting the existence of a pathway for M2 muscarinic receptor-mediated inhibition of tracheal smooth muscle relaxation. Methodological differences may underlie the inconsistency between our study and that of Matsui et al. We administered increasing doses of isoproterenol to submaximally contracted tissue to characterize relaxation, whereas Matsui et al. used a maximal dose of isoproterenol before contracting airway smooth muscle with increasing doses of oxotremorine-M. Because tissues were maximally relaxed in the Matsui study, the efficacy of M2 muscarinic receptors may have been masked.

There are a number of reports from other species that support the notion that M2 muscarinic receptors oppose {beta}2-AR-mediated relaxation. Through a pertussis toxin-sensitive mechanism, stimulation of airway smooth muscle M2 muscarinic receptors inhibits adenylyl cyclase activity (8, 30, 37) and significantly reduces the open-state probability of KCa channels (25, 26), two events promoted by {beta}2-AR stimulation (24).

Potassium ion channels exist in high density in airway smooth muscle membrane of many species, including mice (7, 25, 31). Activation (opening) of murine tracheal smooth muscle KCa channels results in relaxation (28). Thus stimulation of M2 muscarinic receptors opposes {beta}2-AR-mediated relaxation through regulation of second messenger systems or membrane ion channels.

M2 muscarinic receptor-mediated inhibition of {beta}2-AR-mediated relaxation may be of functional significance in humans. Severe asthmatics demonstrate reduced responsiveness to bronchodilators that act through {beta}2-ARs (3, 4). Some studies suggest that this impaired {beta}2-AR-mediated relaxation is due to increased cholinergic muscarinic antagonism of {beta}2-AR function rather than a primary defect in the adrenergic receptor itself (19, 43). Our data provide a potential mechanism by which the refractoriness to inhaled adrenergic bronchodilators of such patients may be explained. GRK5 may be reduced, or its function antagonized, in severe asthmatics, thereby enhancing the inhibitory activity of M2 muscarinic receptors. This may result in more effective opposition of {beta}2-AR-mediated bronchodilation. This hypothesis remains to be tested.

The enhanced airway response to vagal stimulation in GRK5-/- mice cannot be explained by enhanced activity of M3 muscarinic receptors on airway smooth muscle. Both our in vivo and in vitro experiments demonstrate that WT and GRK5-/- mice develop similar airway responses to increasing doses of muscarinic receptor agonists. We propose that the enhanced airway response to vagal stimulation in GRK5-/- mice is a result of unchecked stimulation of M2 muscarinic receptors that, in turn, excessively inhibits airway smooth muscle relaxation. Vagal stimulation activates both an airway smooth muscle cholinergic contractile pathway as well as a nonadrenergic, noncholinergic inhibitory pathway (40). Although the neurotransmitter for nonadrenergic, noncholinergic relaxation has not been completely resolved, nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are two major players (2, 44). One mechanism by which NO triggers airway smooth muscle relaxation is through activation of KCa channels (1, 9, 41). As much as 50% of the NO-mediated airway smooth muscle relaxation occurs through modification of KCa channels (5). Because M2 muscarinic receptors can inactivate KCa channels, it is plausible that nonadrenergic, noncholinergic-mediated airway smooth muscle relaxation, associated with vagal stimulation, may be excessively opposed in our GRK5-/- mice through M2 muscarinic receptor regulation of KCa channels. One can propose a similar scenario for M2 muscarinic receptor opposition of VIP-mediated nonadrenergic, noncholinergic airway smooth muscle relaxation.

VIP receptors activate adenylyl cyclase through G{alpha}s coupling (38). Because M2 muscarinic receptors couple to G{alpha}i, which inhibits adenylyl cyclase, VIP-mediated relaxation associated with vagal stimulation may be markedly offset by acetylcholine activation of M2 muscarinic receptors in the GRK5-/- mice. Thus it is plausible that vagally mediated relaxation of airway smooth muscle is excessively opposed in GRK5-/- mice and that this mechanism is responsible for the elevation of APTI during vagal stimulation.

This same trend, elevated APTI in GRK5-/- mice, should not be observed during intravenous MCh injection for a variety of reasons. First, nonadrenergic, noncholinergic relaxation does not occur during MCh infusion. Second, the inhibitory role of M2 muscarinic receptors is more pronounced at a low level of contraction (37). Our APTI data show much smaller airway responses to vagal stimulation than to MCh injections. Finally, intravenous administration of MCh induces transient hypotension and baroreflex release of norepinephrine (NE). Although one might expect NE-induced relaxation of airway smooth muscle to be opposed by activation of M2 muscarinic receptors, this NE has no effect on the resultant airway response, as evidenced by the lack of effect of propranolol on the APTI response to 50 µg/kg of MCh (Fig. 4).

Heart rate is primarily controlled by the opposing influences of the sympathetic and parasympathetic branches of the autonomic nervous system. Postganglionic cardiac parasympathetic fibers synapse onto cardiac pacemaker cells at the sinoatrial node where they exert an inhibitory effect on heart rate. Vagal release of acetylcholine induces bradycardia through activation of M2 muscarinic receptors expressed on these pacemaker cells. The cardiac chronotropic responses to vagal stimulation or MCh injection were not different between GRK5-/- and WT mice, indicating that M2 muscarinic receptors at the sinoatrial node are not appreciably desensitized by GRK5.

We showed that GRK5 is not important for desensitizing autoinhibitory M2 muscarinic receptors located prejunctionally on postganglionic airway nerves. Prejunctional muscarinic receptors exert an inhibitory influence on acetylcholine release, and if GRK5 were important for desensitizing these receptors, one would expect unchecked autoinhibition of acetylcholine release in GRK5-/- mice to result in a reduced airway response to vagal stimulation. Similarly, GRK5 is not important for regulating M3 muscarinic receptors in mouse airway smooth muscle. This result contrasts with our previous findings in GRK3-deficient mice that showed that the loss of GRK3 enhances airway responsiveness to MCh injection mediated by M3 muscarinic receptors (42). Together, our studies provide in vivo proof that GRKs selectively regulate individual muscarinic receptor subtypes.

This study provides insight into the functional role of GRK5 and contributes to the growing body of literature describing in vivo regulation of GPCR function by GRKs. Our results suggest that GRK5 regulates M2, but not M3, muscarinic receptors on airway smooth muscle. In addition, we show that M2 muscarinic receptors located prejunctionally on the vagus nerve or in cardiac pacemaker cells are not regulated by GRK5. These results, combined with our previous study (16), conclusively demonstrate not only that GRK5 selectively regulates individual GPCR subtypes (i.e., M2, but not M3, muscarinic receptors in airway smooth muscle), but that GRK5 regulation of a particular receptor subtype is tissue specific (i.e., GRK5 regulates central and airway smooth muscle M2 muscarinic receptors but not cardiac or vagal M2 muscarinic receptors). This paradigm of tissue and receptor specificity may be applicable to all GRKs.

Interestingly, severe asthmatics can become refractory to bronchodilators that act at {beta}2-ARs. Studies in animal airways show that excessive activation of postjunctional M2 muscarinic receptors results in impaired {beta}2-AR-induced relaxation. We speculate that, in asthma, reduced expression or diminished activity of GRK5 may result in excessive M2 muscarinic receptor-mediated inhibition of relaxation, thus making {beta}2-ARs appear refractory.


    ACKNOWLEDGMENTS
 
We thank Rachel McAdam and Humphrey Kendall for animal care and genotyping. Special thanks goes to Sandra Vincent and Barb Lawson for expert technical assistance.

R. J. Lefkowitz and M. G. Caron are investigators of the Howard Hughes Medical Institute.

GRANTS

This work was supported in part by the Canadian Institute of Health Research and the Ontario Thoracic Society (to J. T. Fisher), by the Sandler Program for Asthma Research (R. J. Lefkowitz), and National Institutes of Health Grant NS-19576 (to M. G. Caron), Grants HL-16037 and HL-70631 (to R. J. Lefkowitz), and Grant GM-59989 (to R. T. Premont). J. K. L. Walker was supported by the Canadian Lung Association, Sandler Program for Asthma Research, and the Veterans Administration at Durham. P. K. McFawn was supported by a Canadian Lung Association/Canadian Institute of Health Research/Glaxo Smith Kline Postdoctoral Fellowship. D. S. Feldman was supported by a Howard Hughes fellowship.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. K. L. Walker, Box 2629, Duke Univ. Medical Center, Durham, NC 27710 (walke082{at}mc.duke.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abderrahmane A, Salvail D, Dumoulin M, Garon J, Cadieux A, and Rousseau E. Direct activation of KCa channel in airway smooth muscle by nitric oxide: involvement of a nitrothiosylation mechanism? Am J Respir Cell Mol Biol 19: 485-497, 1998.[Abstract/Free Full Text]
  2. Aizawa H, Tanaka H, Sakai J, Takata S, Hara N, and Ito Y. L-NAME-sensitive and -insensitive nonadrenergic noncholinergic relaxation of cat airway in vivo and in vitro. Eur Respir J 10: 314-321, 1997.[Abstract/Free Full Text]
  3. Bai TR. {beta}2-adrenergic receptors in asthma: a current perspective. Lung 170: 125-141, 1992.[ISI][Medline]
  4. Bai TR, Mak JCW, and Barnes PJ. A comparison of {beta}-adrenergic receptors and in vitro relaxant responses to isoproterenol in asthmatic airway smooth muscle. Am J Respir Cell Mol Biol 6: 647-651, 1992.[ISI][Medline]
  5. Bialecki RA and Stinson-Fisher C. KCa channel antagonists reduce NO donor-mediated relaxation of vascular and tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L152-L159, 1995.[Abstract/Free Full Text]
  6. Drazen JM, Finn PW, and De Sanctis GT. Mouse models of airway responsiveness: physiological basis of observed outcomes and analysis of selected examples using these outcome indicators. Annu Rev Physiol 61: 593-625, 1999.[CrossRef][ISI][Medline]
  7. Edwards G and Weston AH. KATP-fact or artefact? New thoughts on the mode of action of the potassium channel openers. Cardiovasc Res 28: 735-737; discussion 741-735, 1994.[ISI][Medline]
  8. Eglen RM, Hegde SS, and Watson N. Muscarinic receptor subtypes and smooth muscle function. Pharmacol Rev 48: 531-565, 1996.[ISI][Medline]
  9. Ellis JL and Conanan ND. Effect of potassium channel blockers on relaxations to a nitric oxide donor and to nonadrenergic nerve stimulation in guinea pig trachea. J Pharmacol Exp Ther 271: 782-786, 1994.[Abstract]
  10. Ewart SL, Mitzner W, DiSilvestre DA, Meyers DA, and Levitt RC. Airway hyperresponsiveness to acetylcholine: segregation analysis and evidence for linkage to murine chromosome 6. Am J Respir Cell Mol Biol 14: 487-495, 1996.[Abstract]
  11. Fernandes LB, Fryer AD, and Hirshman CA. M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle. J Pharmacol Exp Ther 262: 119-126, 1992.[Abstract]
  12. Fryer AD, Adamko DJ, Yost BL, and Jacoby DB. Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung. Life Sci 64: 449-455, 1999.[CrossRef][ISI][Medline]
  13. Fryer AD and El-Fakahany EE. Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists. Life Sci 47: 611-618, 1990.[CrossRef][ISI][Medline]
  14. Fryer AD and Jacoby DB. Effect of inflammatory cell mediators on M2 muscarinic receptors in the lung. Life Sci 52: 529-536, 1993.[CrossRef][ISI][Medline]
  15. Fryer AD and Maclagan J. Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig. Br J Pharmacol 83: 973-978, 1984.[Abstract]
  16. Gainetdinov RR, Bohn LM, Walker JKL, Laporte SA, Macrae AD, Caron MG, Lefkowitz RJ, and Premont RT. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron 24: 1029-1036, 1999.[ISI][Medline]
  17. Haddad EB, Landry Y, and Gies JP. Muscarinic receptor subtypes in guinea-pig airways. Am J Physiol Lung Cell Mol Physiol 261: L327-L333, 1991.[Abstract/Free Full Text]
  18. Haddad EB, Mak JC, and Barnes PJ. Characterization of [3H]Ba 679 BR, a slowly dissociating muscarinic antagonist, in human lung: radioligand binding and autoradiographic mapping. Mol Pharmacol 45: 899-907, 1994.[Abstract]
  19. Hakonarson H, Herrick DJ, and Grunstein MM. Mechanism of impaired {beta}-adrenoceptor responsiveness in atopic sensitized airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 269: L645-L652, 1995.[Abstract/Free Full Text]
  20. Hakonarson H, Herrick DJ, Serrano PG, and Grunstein MM. Mechanism of cytokine-induced modulation of {beta}-adrenoceptor responsiveness in airway smooth muscle. J Clin Invest 97: 2593-2600, 1996.[Abstract/Free Full Text]
  21. Hulme EC, Birdsall NJ, and Buckley NJ. Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol 30: 633-673, 1990.[CrossRef][ISI][Medline]
  22. Jacoby DB and Fryer AD. Anticholinergic therapy for airway diseases. Life Sci 68: 2565-2572, 2001.[CrossRef][ISI][Medline]
  23. Jacoby DB and Fryer AD. Interaction of viral infections with muscarinic receptors. Clin Exp Allergy 29: 59-64, 1999.[CrossRef][ISI][Medline]
  24. Jones TR, Charette L, Garcia ML, and Kaczorowski GJ. Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca2+-activated K+ channel inhibitor. J Pharmacol Exp Ther 255: 697-706, 1990.[Abstract]
  25. Kotlikoff MI. Potassium channels in airway smooth muscle: a tale of two channels. Pharmacol Ther 58: 1-12, 1993.[CrossRef][ISI][Medline]
  26. Kume H and Kotlikoff MI. Muscarinic inhibition of single KCa channels in smooth muscle cells by a pertussis-sensitive G protein. Am J Physiol Cell Physiol 261: C1204-C1209, 1991.[Abstract/Free Full Text]
  27. Levitt RC and Mitzner W. Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice. FASEB J 2: 2605-2608, 1988.[Abstract/Free Full Text]
  28. Li L, Paakkari I, and Vapaatalo H. Effects of K+ channel inhibitors on the basal tone and KCl- or methacholine-induced contraction of mouse trachea. Eur J Pharmacol 346: 255-260, 1998.[CrossRef][ISI][Medline]
  29. Mak JCW, Baraniuk JN, and Barnes PJ. Localization of muscarinic receptor subtype mRNAs in human lung. Am J Respir Cell Mol Biol 7: 344-348, 1992.[ISI][Medline]
  30. Matsui M, Griffin MT, Shehnaz D, Taketo MM, and Ehlert FJ. Increased relaxant action of forskolin and isoproterenol against muscarinic agonist-induced contractions in smooth muscle from M2 receptor knockout mice. J Pharmacol Exp Ther 305: 106-113, 2003.[Abstract/Free Full Text]
  31. McCann JD and Welsh MJ. Calcium-activated potassium channels in canine airway smooth muscle. J Physiol 372: 113-127, 1986.[Abstract]
  32. Medina P, Noguera I, Aldasoro M, Vila JM, Flor B, and Lluch S. Enhancement by vasopressin of adrenergic responses in human mesenteric arteries. Am J Physiol Heart Circ Physiol 272: H1087-H1093, 1997.[Abstract/Free Full Text]
  33. Opperman M, Diverse-Pierluissi M, Drazen MH, Dyer SL, Freedman NJ, Peppel KC, and Lefkowitz RJ. Monoclonal antibodies reveal receptor specificity among G protein-coupled receptor kinases. Proc Natl Acad Sci USA 93: 7649-7654, 1996.[Abstract/Free Full Text]
  34. Premont RT, Koch WJ, Inglese J, and Lefkowitz RJ. Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases. J Biol Chem 269: 6832-6841, 1994.[Abstract/Free Full Text]
  35. Rockman HA, Choi DJ, Rahman NU, Akhter SA, Lefkowitz RJ, and Koch WJ. Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice. Proc Natl Acad Sci USA 93: 9954-9959, 1996.[Abstract/Free Full Text]
  36. Roffel AF, Elzinga CRS, and Zaagsma J. Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle. Pulm Pharmacol 3: 47-51, 1990.[Medline]
  37. Roux E, Molimard M, Savineau JP, and Marthan R. Muscarinic stimulation of airway smooth muscle cells. Gen Pharmacol 31: 349-356, 1998.[CrossRef][Medline]
  38. Shetzline MA, Walker JK, Valenzano KJ, and Premont RT. Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration. J Biol Chem 277: 25519-25526, 2002.[Abstract/Free Full Text]
  39. Stengel PW, Yamada M, Wess J, and Cohen ML. M3-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea. Am J Physiol Regul Integr Comp Physiol 282: R1443-R1449, 2002.[Abstract/Free Full Text]
  40. Undem BJ and Myers AC. Cholinergic and noncholinergic parasympathetic control of airway smooth muscle. In: Muscarinic Receptors in Airway Diseases, edited by Zaagsma J, Meurs H, and Roffel AF. Boston, MA: Birkhauser Verlag, 2001, p. 1-24.
  41. Vaali K, Li L, Paakkari I, and Vapaatalo H. Relaxing effects of NO donors on guinea pig trachea in vitro are mediated by calcium-sensitive potassium channels. J Pharmacol Exp Ther 286: 110-114, 1998.[Abstract/Free Full Text]
  42. Walker JKL, Peppel K, Lefkowitz RJ, Caron MG, and Fisher JT. Altered airway and cardiac responses in mice lacking G protein-coupled receptor kinase 3. Am J Physiol Regul Integr Comp Physiol 276: R1214-R1221, 1999.[Abstract/Free Full Text]
  43. Wills-Karp M and Gilmour MI. Increased cholinergic antagonism underlies impaired {beta}-adrenergic response in ovalbumin-sensitized guinea pigs. J Appl Physiol 4: 2729-2735, 1993.
  44. Yano S, Kiyota Y, Yamamoto M, and Watanabe K. Pharmacological features of non-adrenergic non-cholinergic (NANC) relaxation induced by electrical vagal stimulation in isolated mouse stomach. Jpn J Pharmacol 69: 9-15, 1995.[ISI][Medline]