INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

G PROTEIN-COUPLED receptors (GPCRs) are membrane proteins characterized by seven transmembrane spanning domains that mediate the actions of many extracellular signals. GPCRs interact with heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) that modulate a variety of second messenger systems or ionic conductances to effect physiological responses (28). The signaling event produced by agonist activation of GPCRs is rapidly attenuated by phosphorylation of the receptor and its subsequent uncoupling from the signal transduction mechanism. There is substantial biochemical evidence implicating G protein-coupled receptor kinases (GRKs) in the phosphorylation and acute desensitization of agonist-activated GPCRs (24). There are six members in the GRK family; the relative importance of each GRK, in each tissue, is governed by a number of factors and is, at present, incompletely defined. Nevertheless, there is evidence to indicate that these kinases can play an integral role in vivo. Jaber et al. (17) showed that GRK2 is critical for normal cardiac development in mice. Furthermore, Koch et al. (19) demonstrated that cardiac-specific overexpression of GRK2 reduces agonist-induced myocardial contractility and adenylyl cyclase activity and reduces functional coupling of -adrenergic receptors. In the same study, mice expressing a GRK2 inhibitor displayed enhanced cardiac contractility. Recently, a primary role for GRK3 desensitization of odorant receptors was demonstrated (23).

Contractile and relaxant agonists exert their effects on airway smooth muscle through GPCR binding; dysfunction of these receptors has been implicated in asthma, an inflammatory disease characterized by hyperresponsiveness to bronchoconstrictors and hyporesponsiveness to bronchodilators (3). Dysfunction of M₃ muscarinic ACh receptors (mAChRs) located on airway smooth muscle cells may play a role in the pathophysiology of asthma. In the A/J mouse, which is a genetic model of airway hyperreactivity, heightened muscarinic receptor affinity and coupling is believed to be responsible for the exaggerated airway response to acetylcholine (11). Severe asthmatics also display a decreased responsiveness to ₂-adrenergic receptor (₂-AR) agonists, and, although the mechanism for this functional hyporesponsiveness is unknown, there is some evidence to suggest that uncoupling of the receptor from its signal transduction mechanism is involved (1). Thus regulation of GPCRs (specifically, muscarinic and adrenergic) may play an important role in the pathogenesis of asthma.

₂- And ₂-ARs are phosphorylated by GRKs in vitro (reviewed in Ref. 24). Because of technical difficulties, many GPCRs have not yet been tested for the ability to undergo phosphorylation by GRKs. However, tissue culture experiments have demonstrated that GRK2 and GRK3 phosphorylate and desensitize human M₂ and human M3 mAChRs (6). We hypothesized that GRK2 and GRK3 may be modulators of these muscarinic receptors in vivo and may therefore play an important role in desensitizing agonist-activated muscarinic receptors on airway smooth muscle cells. Unfortunately, selective GRK antagonists do not, as yet, exist. However, transgenic mice having a disruption of the GRK2 or GRK3 gene represent a powerful model in which to explore the in vivo role of GRKs in the modulation of airway reactivity.

To test our hypothesis, airway and heart rate (HR) responses to intravenous administration of methacholine (MCh) were assessed in GRK2 +/, GRK3 /, and wild-type mice. Due to the embryonic lethality of the GRK2 homozygote (/) genotype, only GRK2 heterozygote (+/) mice were available for study (17). MCh was used as a direct agonist of muscarinic receptors, presumably located on airway smooth muscle cells and cardiac pacemaker cells, to cause airway narrowing and bradycardia, respectively. Airway responses were estimated as the time-integrated change in peak airway pressure, denoted as airway pressure time index (APTI) (20).

Our results demonstrate that in the absence of the GRK3 gene mice display an enhanced cholinergic airway responsiveness that is accompanied by an altered HR recovery from MCh-induced bradycardia. Further studies suggest that the changes in the HR recovery of the GRK3 / mice are due to an effect on the baroreceptor reflex rather than an adaptation of cardiac muscarinic receptor signaling pathways. Mice lacking one copy of GRK2 (GRK2 +/) showed no significant differences in either respiratory or cardiac parameters evaluated.

These results are consistent with a role for GRK3 in desensitizing the airway response to MCh and in regulating the chronotropic component of the baroreceptor reflex in mice.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Genotyping: Southern blot analysis. Mouse tail DNA was isolated after overnight digestion with proteinase K in 1% SDS at 65°C. Approximately 10 µg of DNA was digested overnight with EcoR I, separated on a 0.8% agarose gel, and transferred to Nytran membranes. The 5' probe was a 189-bp EcoR I-Hind III fragment located 5' to the targeting vector. The 3' probe was a 200-bp DNA fragment that was PCR amplified with primers 5'-TATAGTGCACACCAGCTC-3' and 5'-CACTGAGGTGGCTGAGAG-3'. All DNAs used as probes were gel purified and labeled with [³²P]dCTP using Stratgene's random primer labeling kit. Prehybridization and hybridization were done at 55°C in 4× SSC, 25% formamide, 1% SDS, 50 µg/ml tRNA, and 10% dextran sulfate. After overnight hybridization, the filters were washed initially in 2× SSC, 1% SDS at 65°C. The final wash was in 0.2× SSC, 0.2% SDS at 70°C. The filters were exposed to X-R Kodak film for up to 3 days (23).

General. GRK3 /, GRK2 +/, and wild-type mice, bred from a background of C57B and 129SvJ strains, 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 from 0800 to 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.

Animal experimentation. Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg) diluted 50% with saline. Once an appropriate plane of anesthesia was achieved, mice were surgically prepared with a tracheotomy, into which a specially fabricated stainless steel cannula was inserted, and mechanical ventilation was initiated. Access to the abdominal vena cava was obtained through a longitudinal abdominal incision, and a catheter containing 5% heparin saline (Hepalean, Organon Teknika) was inserted and fixed in place with cyanoacrylate (26). The abdominal incision was closed, and the catheter, which is designed to minimize dead space (<10 µl), was used for intravenous administration of anesthetic and other drugs (injection volumes of 1 ml/kg). In respiratory protocols, mice were paralyzed with pancuronium bromide (1 mg/kg) to prevent respiratory efforts. Additional doses of pancuronium were administered as necessary, and additional doses of anesthetic were administered at regular time intervals after paralysis. The mice were ventilated by a small animal ventilator (1 ml maximum volume; Harvard Apparatus) with 100% oxygen at a constant volume of 8-10 ml/kg and a frequency of 130 breaths/min. These ventilator settings result in a peak airway pressure of 6-8 cmH₂O and were previously shown to provide normal arterial blood gases (20). Measurement of airway pressure (P_aw) 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 HR. P_aw and electrocardiogram signals were acquired by a computer data acquisition package (CODAS DATAQ, Akron, OH) at a sampling frequency of 1,000 samples · s¹ · channel¹. Acquired data was analyzed using peak detection software and data were imported to a spreadsheet for calculation of HR and APTI.

A second group of mice was used to study cardiovascular parameters. For studies involving vagal stimulation, the right vagus nerve was isolated from the carotid artery and prepared for electrical stimulation. Activation of vagal efferents was achieved by placing the nerve on a stimulating electrode immersed in mineral oil or covered in low-melting-point wax. The proximal end of the vagus was crushed, and the nerve was 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 (10, 15, and 20 pulses/s).

Experimental protocols. Increasing doses of MCh (5, 10, 25, 50, 100, and 250 µg/kg) were injected intravenously into anesthetized, paralyzed (pancuronium bromide, 0.4 mg/kg), and ventilated mice. Respiratory efforts were closely monitored to prevent their interference with accurate measurement of tracheal pressure. Mice were hyperinflated 2 min before each MCh injection to establish a constant volume history and respiratory mechanics. Each MCh injection was separated by a 7- to 10-min period. Tracheal pressure and HR data were acquired immediately before, during, and after MCh injection. APTI was calculated as the time-integrated change in peak airway pressure (see Measurements/data analysis). This airway reactivity protocol was conducted on five GRK2 +/ mice and five age-matched wild-type controls, as well as seven GRK3 / mice and three age-matched wild-type controls. Measurements from each of the wild-type controls, which did not differ significantly, were pooled for graphical and statistical analyses.

To assess HR responses, a second group of mice was used, and multiple consecutive protocols were employed due to the limited number of mice available. The protocols were designed to build on the prior pharmacological blockade of each protocol. An MCh injection protocol, similar to that used with the first group of animals, was performed in six GRK3 / and eight age-matched wild-type mice, which were not under the influence of pancuronium. Mice were injected with a 10- µg/kg and, after recovery, a 25-µg/kg dose of MCh. To assess the baroreflex response, a 500-µg/kg bolus injection of the vasodilator sodium nitroprusside (SNP) was administered to induce a transient hypotension. All injections were separated by a 7- to 10-min recovery period. HR data were acquired for 30 s before each injection and 2 min after.

After the injection protocols, the right vagus nerve was isolated and a stimulus frequency versus HR response protocol was conducted. The vagus nerve was stimulated for 10 s at 15, 10, and 20 pulses/s (in that order) with a 5-min recovery between stimulations. HR data were acquired for 30 s before and 1 min after stimulation.

Subsequently, cardiac M₂ receptors were again activated by a 10 pulse/s vagal nerve stimulation 2 min before and 2 min after a 1 mg/kg injection of propranolol (specific -AR antagonist). The postpropranolol stimulation served as a control for a gallamine cumulative dose-response protocol; thus -ARs were blocked during the gallamine-vagal stimulation protocol. The cumulative dose of gallamine was increased in half log increments (from 0.01 to 10 mg/kg), and successive gallamine injections were followed every 2 min by 10 pulses/s vagal nerve stimulation. HR data were acquired 30 s before, during, and 30 s after vagal nerve stimulation.

Measurements/data analysis. Airway responses were estimated as the time-integrated change in peak airway pressure, denoted as APTI (20). The change in P_aw was calculated as the difference between the breath-by-breath P_aw and the average P_aw measured 15 s before injection. The APTI value is the sum of the differences measured for 80 s after MCh injection.

Nonlinear regression analysis was conducted on raw data from the MCh protocol for each mouse using an equation of the form y = a · e^kx. The dose of MCh required to elicit an airway response of magnitude 40 or 70 cmH₂O/s was calculated using the results from the curve fitting procedure. These values were chosen because they represent 50 and 85% of the average APTI response to 250 µg/kg MCh in wild-type mice (82.4 ± 12.5 cmH₂O/s).

The time to HR recovery was calculated as the difference between the time at which HR reached a minimum and the time at which HR returned to within 90% of preinjection control HR. The percent change in HR was calculated on the basis of the preinjection HR and the minimum HR.

Control HR was calculated as the average of 5 s of data acquired just before injection of any agonist or antagonist. HR was measured for 60 s after MCh or SNP injections. At a given time period after injection, for example 30 s, HR was calculated as an average of 2 s of data, in this case between 29 and 31 s postinjection. Two minutes after injection of propranolol, 5 s of data was averaged to calculate postpropranolol HR.

For vagal stimulation protocols, 5 s of data before stimulation was averaged for control HR. HR during the entire 10-s stimulation period was averaged to obtain "stimulation" HR value. During vagal stimulation (stimulus frequency to HR response protocol), the decrease in HR was calculated for 2-s intervals from 0 to 10 s of stimulation; as well, the recovery HR was averaged over 2-s intervals from 0 to 10 s after the cessation of vagal stimulation, and this value was compared with control HR.

Statistical analysis. Statistical analyses were carried out using the computer software package SYSTAT for Windows. Values presented are means ± SE. To determine differences between or among mouse genotypes, a two-way ANOVA was used and differences were identified using a contrast matrix. 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 a Tukey honest significant difference post hoc test. Results were considered significantly different if P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway response to MCh. To assess airway reactivity, increasing doses of MCh were used to initiate muscarinic receptor-mediated bronchoconstriction. Paralysis of respiratory muscles was imperative to prevent confounding pressure changes associated with respiratory efforts. GRK3 / mice demonstrated a significant enhancement in the airway response at both the 100- and 250-µg/kg doses of MCh compared with wild-type and GRK2 +/ mice (Fig. 1A). The calculated dose of MCh required to reach an airway response that was 50 and 85% of the wild-type maximal response was less in the GRK3 / phenotype (Fig. 1B). All mouse genotypes displayed a dose-dependent increase in airway response to MCh. However, as hypothesized, GRK3 / mice displayed a more pronounced airway response to high doses of MCh, suggesting that GRK3 contributes to desensitization of the muscarinic receptor-mediated airway response.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Airway response to increasing doses of methacholine (MCh) (A) and calculated dose of MCh required to elicit an arbitrary airway pressure time index (APTI) response (B). A shows APTI at 5, 10, 25, 50, 100, and 250 µg/kg MCh injection for wild-type (), G protein-coupled receptor kinase 2 (GRK2) +/ (), and G protein-coupled receptor kinase 3 (GRK3) / () mice. Data points are means ± SE. B shows calculated dose of MCh required to elicit an APTI response 50 and 85% of wild-type average maximal response (to 250 µg/kg MCh). Doses were calculated for individual mice using nonlinear regression analysis and an equation in the form y = a · ekx. Values presented are means ± SE for wild-type (filled bars), GRK2 +/ (open bars), and GRK3 / (hatched bars) mice.  GRK3 / significantly different from wild-type and GRK2 +/ mice (P  0.05).  GRK3 / significantly different from wild-type mice (P  0.05). Mice were anesthetized and paralyzed with pancuronium bromide.

HR response to MCh. Initial analysis of HR data from the airway study (where mice were paralyzed by pancuronium) suggested that GRK3 / mice have an altered HR response to MCh. However, pancuronium is an antagonist of M₂ muscarinic receptors (16) and thereby reduces the bradycardia associated with MCh exposure. To avoid the potential confounding effect of pancuronium, we evaluated the HR response to MCh in eight wild-type and 6 GRK3 / age- and litter-matched mice in which pancuronium was omitted. In this protocol, a lower dose of MCh was required to achieve levels of bradycardia similar to those of the airway study.

HR responses in age- and litter-matched controls. To confirm the HR recovery findings of the airway study, 10- and 25-µg/kg doses of MCh were injected into age- and litter-matched GRK3 / and wild-type mice that were not under the influence of pancuronium. The HR response to 10 µg/kg MCh was not different between GRK3 / and wild-type mice (data not shown). However, the time required for recovery of HR (to 90% of control value) after 25 µg/kg MCh injection was significantly less in GRK3 / mice (10.1 ± 2.5 s) compared with that of wild-type mice (18.8 ± 2.7 s) (Fig. 2A). Minimum HRs were not different between genotypes (237 ± 18 in GRK / vs. 250 ± 8 beats/min for wild-type mice). Interestingly, MCh-induced bradycardia was followed by HRs which, in GRK3 / mice, consistently exceeded their control HR (Fig. 2B). In contrast, HR of the wild-type mice returned only to their control values (Fig. 2B). Note that before each MCh injection control HRs were not different between wild-type (394 ± 38 beats/min) or GRK3 / mice (HR 367 ± 18 beats/min). These data corroborate the findings of the airway study and imply that MCh-induced bradycardia was reversed by an additional process that caused a relative tachycardia.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Recovery of heart rate (HR) after injection of 25 µg/kg MCh in anesthetized, but not paralyzed, GRK3 / mice and their age- and litter-matched wild types. A: means ± SE of time required to return to 90% of control HR from minimum HR. After MCh injection, GRK3 / mice (hatched bar) required significantly less time to return to control HR than wild-type mice (filled bar) (P  0.05). B: means ± SE of percent change from respective control HR for GRK3 / mice (hatched bar) and wild-type mice (filled bar) after 25 µg/kg MCh injection. GRK3 / mice consistently exceeded control HR while recovering from MCh injection, whereas wild-type mice returned HR only to control values.  Significant difference between genotypes (P  0.05).

HR baroreflex responses to SNP. To assess whether baroreflexes contribute to the observed HR recovery differences, SNP was administered to induce a substantial, but transient, hypotension. Through a non-GPCR mechanism, SNP liberates nitric oxide, causing rapid, transient relaxation of vascular smooth muscle cells and concomitant hypotension (13). GRK3 / mice displayed a consistently greater increase in HR than wild-type mice after SNP injection, and the percent difference from the control value reached statistical significance (Fig. 3). Although blood pressure was not measured (limited by the number of knockout mice available for the study) the administered dose of 500 µg/kg SNP elicited a HR change consistent with initiation of a baroreflex response and this was true in both GRK3 / and wild-type mice (Fig. 3). This 500 µg/kg dose of SNP is 50-fold the dose that, when administered to rats, induces a 25-mmHg decrease in mean arterial pressure (MAP) and an immediate baroreflex increase in HR (27). Furthermore, Ma et al. (21) showed that in anesthetized Webster mice, MAP dropped from 87 to 40 mmHg with a 1,333-µg/kg injection of SNP. The mean resting HR for the GRK3 / mice (362 ± 22 beats/min) tended to be lower than the wild-type mice (410 ± 36 beats/min), although this did not reach statistical significance.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   HR response to injection of vasodilator sodium nitroprusside (SNP) in anesthetized, but not paralyzed, GRK3 / mice and their age- and litter-matched wild types. Percent increase in HR from respective control HRs for GRK3 / mice () and wild-type mice () at 10, 15, 30, 45, and 60 s post-SNP injection.  Significantly greater increase in HR in GRK3 / mice compared with wild-type mice (P  0.05).

HR responses to vagal stimulation. After the injection protocols, the right vagus nerve was isolated and a stimulus-frequency versus HR-response protocol was conducted. Vagal nerve stimulation provides an opportunity to selectively stimulate cardiac M₂ receptors and thus to determine if their altered regulation contributes to the HR recovery differences identified in GRK3 / mice. During vagal stimulation, GRK3 / and wild-type mice decreased HR similarly, on both a relative and an absolute basis (Fig. 4A). The time course of the recovery of HR was also comparable for GRK3 / and wild-type mice (Fig. 4B). These data suggest that regulation of cardiac M₂ receptors is not different in GRK3 / mice compared with wild-type mice.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   HR response to vagal stimulation in anesthetized, but not paralyzed, GRK3 / mice and their age- and litter-matched wild types. A: control HR (measured just before stimulation) and minimum HR (calculated over entire 10-s stimulation period) as means ± SE for 10, 15, and 20 pulses/s stimulation frequencies. HR decreased significantly and similarly in GRK3 / () and wild-type mice () at each stimulation frequency. * Significant difference from control within genotypes (P  0.05). B: recovery of HR after cessation of vagal stimulation was not different between GRK3 / (hatched bars) and wild-type mice (filled bars) at 2 or 10 s of recovery. C: percent inhibition of vagal stimulation-induced bradycardia with half-log increments in cumulative dose of gallamine from 0.01 to 10.0 mg/kg. Means ± SE are shown for GRK3 / () and wild-type mice (), and there is no difference between them. For the gallamine protocol, all mice are -adrenergic receptor blocked.

To further rule out any potential differences in cardiac M₂ receptors, the HR response to vagal stimulation was assessed in the presence of increasing doses of the selective M₂ antagonist gallamine. The gallamine dose response was performed in the presence of -adrenergic blockade with propranolol (see below). Gallamine caused a dose-dependent blockade of the decrease in HR associated with vagal stimulation, and this was similar for GRK3 / and wild-type mice (Fig. 4C).

HR responses to -AR block. To determine if -ARs contributed to the differences in the HR recovery observed in GRK3 / mice, a 1 mg/kg injection of the -AR antagonist propranolol was administered. Resting HR decreased significantly in wild-type but not GRK3 / mice after propranolol injection (Fig. 5), suggestive of tonic -adrenergic input in the former, but not in the latter, group. Resting HR was almost identical in the GRK3 / (426 ± 25 beats/min) and wild-type (415 ± 20 beats/min) mice after -adrenergic block (Fig. 5). During -AR block, vagal stimulation caused similar HR decreases and HR recovery in GRK3 / and wild-type mice (data not shown). Thus -AR regulation does not seem to contribute to the enhanced HR recovery observed in GRK3 / mice.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   HR response to -adrenergic receptor blockade with propranolol in anesthetized, but not paralyzed, GRK3 / mice and their age- and litter-matched wild types. Control HR (con) before and after (post) -adrenergic receptor blockade with propranolol is shown. Vagus nerve was crushed proximal to electrode stimulation site. After propranolol injection, HR significantly decreased from control in wild-type mice (filled bars) but not GRK3 / mice (hatched bars). * Significant difference from control within a genotype (P  0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

GRK3 / mice display an enhanced airway response to MCh injection, suggesting that GRK3 may be an important kinase for quenching airway responsiveness mediated by muscarinic receptors in vivo. Although the airway response to MCh in GRK2 +/ mice was not different from wild-type mice, it is difficult to speculate on the potential role of GRK2 in desensitization of airway reactivity because it is not possible to study homozygote GRK2 knockout mice. MCh is known to activate M₃ receptors to cause airway smooth muscle contraction in many species (reviewed in Ref. 2). Our results are consistent with, but not proof of, a role for GRK3 in the phosphorylation and thereby desensitization of M₃ receptors on airway/lung smooth muscle cells of mice. The absence of GRK3 in the knockout mouse may diminish the desensitization of muscarinic receptors leading to an enhanced bronchoconstrictor response. This speculation is supported by biochemical evidence suggesting that GRK3 is expressed in the lung.

Benovic et al. (4) showed GRK3 mRNA to be present in bovine lung. Although mRNA for GRK3 is typically present at lower levels than GRK2 (4), the physiological relevance of the kinase depends not only on its abundance, but on its distribution, ability to recognize a receptor as a substrate, ability to translocate to the membrane-bound receptor, and the ability of the receptor substrate to activate the kinase (25). Although GRK3 mRNA transcripts could not be detected in primary cultures of human airway smooth muscle cells (22), these cells are known to dedifferentiate and change phenotype after several passages (12, 14). Preliminary Western studies from our laboratory (unpublished data) demonstrate the presence of GRK3 in murine lung homogenate, and these results support its potential physiological role revealed in our studies.

Airway responses were estimated as the time-integrated change in peak airway pressure (denoted as APTI), a measure that others have validated for its ability to provide a reasonable index of airway responsiveness (11, 20) as assessed by the more specific mechanical variables of resistance and compliance. This is especially true for small species at high respiratory frequencies in which tracheal pressure closely parallels changes in lung or respiratory resistance (29). Our use of the term airway responsiveness reflects the ubiquitous use of this term with respect to a decrement in lung function (e.g., spirometry, resistance, or APTI) in response to smooth muscle contractile agonists. It does not indicate the anatomic site of action of agonist or of GRK3. Indeed, on the basis of our data it is not possible to identify the location of action of MCh and we are unaware of evidence as to the distribution of muscarinic receptor subtypes within the murine lung. Despite some controversy regarding airway versus parenchymal sites of action, there is substantial evidence in other species to indicate that M₃ muscarinic receptors mediate contraction of airway smooth muscle and lead to increased airway resistance or airway narrowing (2, 5). Muscarinic receptor subtypes have been characterized in murine tracheal smooth muscle, and M₃ receptors have been implicated as the primary subtype leading to contractile signal transduction (10). If one speculates that receptor distribution in mouse airways is similar to other mammals, then it is likely that M₃ muscarinic receptors located on airway smooth muscle cells contribute to the bronchoconstrictor response to MCh in mice.

Deburrman et al. (6) showed that both the M₂ and M₃ subtypes of the muscarinic receptor are phosphorylated and desensitized by GRK3. Thus GRK3 modulation of M₂ receptors could also modulate airway responsiveness (10). M₂ receptors couple to G_i to inhibit adenylate cyclase, and thus activation of M₂ receptors on airway smooth muscle could inhibit any tonic G_s-coupled relaxation (i.e., ₂-AR) (8, 18). It is unclear whether GRK3 modulates M₂ and/or M₃ receptors of murine airways. However, given the magnitude of the enhanced airway response in GRK3 / mice, it is unlikely that our results could be totally explained by loss of M₂ receptor inhibition of bronchodilation or by loss of M₂ autoinhibitory receptors, which limit further release of ACh from vagal postganglionic efferents (10). Interestingly, regulation of muscarinic receptors was shown to be involved in airway hyperresponsiveness in the A/J mouse (11).

Given the pharmacological properties of MCh, it is reasonable to assume that the airway response observed in this study was mediated primarily through muscarinic receptors. On this basis and the basis of the enhanced response of the APTI in GRK3 / mice, we speculate that GRK3 modulates muscarinic receptors in murine airways to dampen lung responsiveness.

HR responses. MCh presumably acts at cardiac M₂ mAChRs to decrease HR and at vascular M₃ mAChRs to cause vasodilatation. If GRK3 was crucial for phosphorylating and thus desensitizing cardiac M₂ mAChRs in vivo, then one might expect that during MCh injection, GRK3 / mice would have a more pronounced decrease in HR and a longer recovery period compared with wild-type mice. There was no evidence to support these assumptions. In fact, HR in GRK3 / mice significantly exceeded control HR after MCh injection, whereas this HR "overshoot" did not occur in wild-type mice. These results suggest at least two mechanistic possibilities. First, GRK3 may be important for desensitizing the baroreflex response to the vasodilatation-induced decrease in MAP subsequent to MCh injection. The baroreflex response is mediated through multiple central nervous system structures, some of which activate adrenergic neurons in this reflex pathway (15). Second, cardiac M₂ mAChRs or their signal transduction mechanisms may have adapted during development in the GRK3 / mice.

The increase in HR after SNP administration is indicative of the baroreflex loop. GRK3 / mice displayed a significantly greater percentage increase in HR compared with wild-type mice after SNP administration. This may reflect an altered regulation of the baroreflex control loop in the GRK3 / mice. However, because blood pressure was not measured, it is not possible to differentiate between an altered blood pressure response to SNP versus a change in the baroreceptor reflex loop gain. Interestingly, the mechanism responsible for this difference between the wild-type and GRK3 / mice response to SNP is also true for the baroreceptor response to MCh-induced hypotension. Although suggestive, it remains to be determined what role GRK3 / plays in the quenching of the cardiac component of the baroreceptor reflex.

Although ₁-ARs mediate the effects of cardiac sympathetic efferents, it is unlikely, given our results with propranolol, that GRK3 regulates cardiac -AR activity. If cardiac -ARs were desensitized by GRK3 then one would expect a greater resting HR in the GRK3 / mice compared with the wild-type mice; however, an opposite trend was observed.

We rejected the possibility that altered regulation of cardiac M₂ mAChRs contributed to the distinct HR response observed in GRK3 / mice on the basis of the vagal stimulation (i.e., selective activation of cardiac M₂ receptors) and gallamine dose-response protocols. These protocols demonstrated that the HR response to vagal nerve stimulation and the inhibitory action of the M₂ antagonist gallamine on decrease in HR were similar for GRK3 / and wild-type mice. Because the vagus nerve was crushed cranial to the stimulation site, vasodilatation and baroreflex responses were prevented from reflexively affecting HR.

Of interest, although not statistically significant, was the tendency for GRK3 / mice to have a lower resting HR. This difference was emphasized by the lack of response to propranolol in the GRK3 / mice. These data suggest that the tonic sympathetic outflow from central cardiorespiratory centers may be diminished in GRK3 / mice. This is in contrast to that observed for wild-type mice or reported for other strains where -block has a reasonably strong influence on resting HR (7).

In summary, these data provide evidence of a physiological role for GRK3 in the modulation of airway reactivity and cardiac baroreflex chronotropy. GRK3 / mice display enhanced airway responsiveness to intravenous MCh and a potentiated baroreflex chronotropic response to hypotension.

Perspectives