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q signaling
Department of Biochemistry and Molecular Biology, 1Division of Pulmonary Medicine, 2Division of Hematology and Oncology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259; 3Division of Biology, California Institute of Technology, Pasadena, California 91125; and 4Pharmacology Department, University of Texas Southwestern, Dallas, Texas 75390-9041
Submitted 25 September 2002 ; accepted in final form 24 February 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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q-containing G proteins are proposed to be a
prominent signaling pathway, and the availability of knockout mice deficient
of this subunit has allowed for an investigation of its potential role in
airway function. Airway responses in Gq-deficient mice
(activities assessed by both tracheal tension and in vivo lung function
measurements) were attenuated relative to wild-type controls. Moreover,
ovalbumin sensitization/aerosol challenge of Gq-deficient
mice also failed to elicit an allergen-induced increase in airway reactivity
to methacholine. These findings indicate that cholinergic receptor-mediated
responses are dependent on Gq-mediated signaling events and
identify Gq as a potential target of
preventative/intervening therapies for lung dysfunction. G protein; gene knockout mice
The Gq family includes four members identified by their
-subunits as Gq, G11, G14, and
G15/16 (40). These
proteins are functionally similar, yet multiple members of the Gq
family are usually coexpressed in the same cells
(40). This has led to several
investigations aimed at examining whether the various members of the
Gq family, most notably Gq and G11, display
distinct functional coupling to receptors or whether they are interchangeable
with multiple receptors. For example, Gq and G11 have
comparable abilities to activate phospholipase C in transfected cell systems
(21,
28), and Xu et al.
(43) have demonstrated that
Gq and G11 promiscuously couple M3 receptors to mediate
identical calcium signaling responses in pancreatic and submandibular gland
cells. Furthermore, knockout mice deficient for Gq and
G11 exhibit functional redundancy in craniofacial development and
cardiomyocyte proliferation in the fetus
(31) and cardiomyocyte
hypertrophy in adults (38).
Alternatively, distinct functions of Gq and G11
following activation of 1-adrenoreceptors have been
demonstrated in rat portal vein myocytes
(25), and the involvement of
Gq, but not G11, has been shown in G protein-deficient
fibroblasts in response to Pateurella multocida bacterial toxin
(44).
Gene knockout mice deficient in the -subunit of Gq
(
mice) were
utilized to determine the role of this G protein in airway responsiveness. The
data show that mice deficient in Gq are hyporesponsive to
cholinergic receptor activation. These responses are much greater in whole
animal studies than in isolated trachea, suggesting that effects of the
Gq deletion may not be smooth muscle autonomous but instead
affect multiple cell types contributing to airway responsiveness in vivo.
Furthermore, mice lacking Gq were unable to develop airway
hyperresponsiveness (AHR) in an allergen-induced model of increased airway
reactivity. Together, these data demonstrate an important role for
Gq in the development of airway resistance and AHR.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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q
(30) knockout mice has been
previously described. Mice were maintained on a 129/Sv x C57BL/6
crossbred background. All procedures were conducted on mice 10–14 wk of
age that were maintained in microisolator cages housed in a specific
pathogen-free animal facility. The sentinel cages within this animal colony
were negative for viral antibodies and the presence of known mouse pathogens.
Protocols and studies involving animals were conducted in accordance with
National Institutes of Health and Mayo Clinic Foundation guidelines.
Western blot analysis. Protein was extracted from tracheal
homogenates including epithelium, smooth muscle, and cartilage and subjected
to SDS-PAGE (10% polyacrylamide). Rabbit antisera specific for
Gq, G11, and Gq/11 have
been described in Hepler et al.
(16). We detected proteins
(
42 kDa) using the ECL Chemiluminescence Western Blotting Detection Kit
(Amersham, Piscataway, NJ) with a donkey anti-rabbit IgG, horseradish
peroxidase-linked secondary antibody (Amersham).
Tissue preparation and measurement of isometric tension. Measurement of mouse tracheal contractility was performed as previously described (18). Briefly, mice (20–30 g) were euthanized with 500 mg/kg pentobarbital sodium, and distal segments of trachea containing six cartilaginous rings (containing epithelium and cartilage) were rapidly removed and placed, at room temperature, in modified Krebs-Henseleit (KH) solution [(in mM) 116 NaCl, 4.7 KCl, 2.5 CaCl, 0.5 MgCl2, 25 NaHCO3/NaH2PO4, and 11 glucose] that was continuously oxygenated (95% O2-5% CO2) to maintain a pH of 7.4. No differences were observed in tissue wet weight between the two groups. Excess tissue was subsequently removed, and the tracheas were mounted longitudinally on stainless steel rings in a 30-ml water-jacketed organ bath (model 159920; Radnotti, Monrovia, CA) containing continuously oxygenated modified KH maintained at 37°C with a thermocirculator (model 50–1932; Harvard Apparatus, Holliston, MA). We allowed the mounted tracheas to equilibrate for 30 min before suspending them upright on glass hooks under a tension of 0.8 g. Stable tension was achieved after approximately five cycles of 15-min incubations, washings, and readjustments of tension to 0.8 g. Changes in force were measured isometrically with a force-displacement transducer (Harvard Apparatus) and collected with a BIOPAC data-acquisition unit and BIOPAC Acqknowledge software (BIOPAC, Santa Barbara, CA). Once a stable tension was established, the tracheal smooth muscle was depolarized with KCl in KH solution at a final concentration of 50 mM. Hyperosmotic KCl exposure was maintained until the isometric force generated by the tracheal contraction reached a plateau. The tracheas were then washed for two cycles to achieve a stable resting tension before methacholine (MCh) dose-response measurements. Increasing doses of MCh (10-9 M–10-4 M) were applied, allowing the isometric forces to plateau. Active tension (i.e., tension generated above resting tension) is reported as a percentage of the maximal tension obtained with 10-4 M MCh.
Measurements of lung mechanics. Respiratory system mechanics were assessed in mice according to the method of Gomes et al. (11). Mice were anesthetized with pentobarbital sodium (50 mg/kg ip, Nembutal; Abbott, Chicago, IL). Tracheas were surgically accessed through a ventral midline incision and connected with a small animal ventilator (SAV, FlexiVent; SCIREQ, Montreal, Quebec, Canada) via an 18-gauge needle. Mice were subsequently paralyzed with doxacurium chloride (0.5 mg/kg) (Nuromax; Catalytica, Greenville, NC) and ventilated at a frequency of 150 breaths/min and at a volume of 6 ml/kg. The mice expired passively through the expiratory valve of the ventilator against a positive end-expiratory pressure of 3 cmH2O. MCh aerosol was generated with an in-line nebulizer (model 5500D; De-Vilbiss, Somerset, PA) and administered directly through the ventilator. We determined the resistive properties of the lungs at constant volume using forced oscillations by the SAV. The oscillations consisted of applying a small-amplitude volume perturbation at 3 Hz to the airway opening. Measurements of piston volume displacement and cylinder pressure were used to calculate the impedance of the respiratory system from which respiratory system resistance values were derived.
Whole body plethysmography. We estimated total pulmonary airflow in unrestrained conscious mice with a whole body plethysmograph (Buxco Electronics, Troy, NY). Pressure differences between a chamber containing the mice and a reference chamber were used to extrapolate minute volume, tidal volume, breathing frequency, and enhanced paused (Penh). Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice during the respiratory cycle. Penh is described by the equation Penh = (PEP/PIP) x Pause; where PEP is the peak expiratory pressure, PIP is the peak inspiratory pressure, and Pause is a component of expiration time. This parameter is dependent on the breathing pattern of the mice and was also shown to correlate with airway resistance as measured by traditional invasive techniques on ventilated mice (15).
Lung histology. We obtained lung tissue for histological analysis
by instilling 1 ml of 10% neutral-buffered formalin (30 cmH2O
constant pressure) through a cannula inserted into the trachea. After
instillation of fixative, the trachea was ligated, and the excised lung was
immersed in formalin for 24 h (at 4°C). Two nonconsecutive parasaggital
sections (5 µm) were obtained from paraffin-embedded tissue, stained with
hematoxylin and eosin, and analyzed by bright-field microscopy (n = 5
mice per group).
Antigen sensitization and challenge. Mice (6–8 wk) were sensitized by an intraperitoneal injection (100 µl) of 20 µg of chicken ovalbumin (OVA; Sigma, St. Louis, MO) emulsified in 2 mg of Imject Alum [Al(OH)3/Mg(OH)2; Pierce, Rockfield, IL] on days 0 and 14. Mice were subsequently challenged with an aerosol generated from 1% OVA in saline or saline alone for 20 min by ultrasonic nebulization (DeVilbiss) on days 24, 25, and 26. Assessments of airway responsiveness were made on day 28.
Statistical analysis. Data presented are the means ± SE. Statistical analysis was performed on parametric data using t-tests with differences between means considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mice are hyporesponsive to MCh ex
vivo. Isometric force generation assessments of tracheal smooth muscle in
response to 50 mM KCl (i.e., depolarized maximal contraction) showed that no
intrinsic differences exist between wild-type and
mice regarding the
capacity of these tissues to constrict
(Table 1). However, MCh
dose-response curves revealed that the tracheas isolated from
mice were
hyporeactive relative to wild type (Table
1 and Fig.
1A). This response was similar when plotted either as
percent maximum response to MCh or as a percentage of maximum KCl response.
Despite this shift in responsiveness, the tension generated in response to
high doses of MCh by
tracheas reached a
plateau similar to wild-type tracheas
(Table 1). Western blot
analysis of the protein from the tracheal preparations confirmed that the
knockout mice did not express Gq. Moreover, these data
demonstrated that the expression level of another subfamily member,
G11, was significant in the trachea of wild-type and
mice and was
unaffected by the loss of Gq
(Fig. 1B).
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mice are hyporesponsive to
cholinergic stimuli in vivo. The consequences of altered smooth muscle
responses were further examined in vivo by invasive measurements of airway
resistance and whole body plethysmography. The lung mechanics of the
mice differed
significantly from wild-type littermates in response to MCh administration
(Fig. 2). Lung resistance
induced by MCh was minimally affected in
mice by even the
highest doses of MCh (50–100 mg/ml). Similar airway responses to inhaled
MCh and serotonin (an agonist that acts presynaptically to induce
acetylcholine release) were observed in these mice by whole body
plethysmography.
mice exhibited minimal reactivity to MCh regardless of the dose applied
(Fig. 3A) and no
significant reactivity to serotonin even at doses that resulted in maximum
reactivity of wild-type mice (Fig.
3B).
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Airway morphology of mice is
indistinguishable from wild-type mice. Microscopic examination of the
lungs from wild-type and
mice revealed no
obvious differences between the two groups
(Fig. 4, A and
B).
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mice fail to exhibit airway
reactivity to MCh in a model of allergen-induced AHR. Airway reactivity
of OVA-sensitized/aerosol-challenged Gq-deficient mice
following provocation with MCh was examined by whole body plethysmography to
determine whether Gq-mediated signaling is also required for
the development of allergen-mediated AHR. In contrast to OVA
sensitization/aerosol challenge of wild-type mice, which leads to a
significant increase in airway obstruction (i.e., Penh levels) relative to
control mice challenged with saline only,
mice challenged
with OVA did not demonstrate an increase in Penh compared with a
control group
challenged with saline only (Fig.
5). It is noteworthy that in addition to this failure to develop
AHR to MCh, neither group of Gq mice (i.e., saline or OVA
treated) had a significant dose-dependent response to MCh relative to
saline-treated control wild-type animals.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mice differed
quantitatively and qualitatively from the effects observed in whole animal
experiments. Specifically, the effects of MCh on tracheal rings appear to be
less pronounced compared with the in vivo assessments. The reasons for this
discrepancy are unclear; however, several possible mechanisms may account for
the observed differences: 1) the resistance to airflow is inversely
proportional to the fourth power of the radius of the airway
(26). Therefore, small changes
observed in airway smooth muscle shortening (tracheal tension) will be
magnified in the in vivo measurements, because radius is the most important
determinant of the resistance to airflow (e.g., decreasing the radius of an
airway by half will increase the resistance to flow 16-fold). 2) The
differences observed between these two assessments of smooth muscle
contraction may reflect differences in the contributions of different airway
generations and differences in Gq-coupled receptor
distribution along these airways. The airways that primarily contribute to the
pressure changes measured in the in vivo experiments are thought to be the
midsized and smaller-diameter intrapulmonary airways (i.e., not the trachea)
(24). This hypothesis is
supported by observed differences in muscarinic receptor density and
distribution observed throughout the airways. Emala and colleagues
(10) have shown that M2
receptors are the predominant receptor in the trachea and that they
continually diminish in density in the bronchi and peripheral lung, whereas
the M3 receptor is present at [1/10] the density of M2, but the relative
density of the M3 increases continually (compared to the M2 receptor) in the
bronchi and peripheral lung. 3) It is possible that other cell types
besides smooth muscle may represent a significant component of the signaling
process involved in airway smooth muscle contraction. Airway responsiveness in
response to a cholinergic receptor such as MCh is a complex phenotype
influenced by several cell types in the airways, including smooth muscle,
neurons, epithelial cells, and endothelial cells
(1,
2,
5). M1 muscarinic receptors are
coupled to the Gq family
(6) and are located on
postganglionic neurons that contribute to bronchoconstriction by positively
regulating depolarization and cholinergic neurotransmission
(20). However, specific
inhibition of M1 receptor activation with pirenzipine does not block the
effects of inhaled MCh (20),
suggesting that this mechanism is not responsible for the different effects
observed in the in vitro and in vivo experiments. Additionally, epithelial
cells and endothelial cells respond to M3 receptor stimulation by generating
nitric oxide, which may play an important role in bronchodilation
(2). It is more difficult to
speculate on how Gq deletion in these cells would lead to a
hyporesponsive phenotype in naive mice, but, without additional data, the
contribution of these cells to the observed phenotype cannot be excluded.
The involvement of nonairway smooth muscle cell types in the regulation of
airway responsiveness may be particularly relevant to the loss of AHR in
sensitized/challenged
mice. AHR
development in this model is incompletely understood, but there are data to
support the involvement of Th2 inflammatory responses, including the
production of cytokines and leukotrienes and the infiltration of the pulmonary
parenchyma by activated T lymphocytes and eosinophils
(22,
41). The relative importance
of the recruitment and activation of these leukocytes is difficult to assess,
as the magnitude of the leukocyte infiltrate is a poor prognostic indicator
for the development of AHR (3,
17,
36). Nonetheless, we have
previously reported that OVA-treated
mice exhibit an
67% reduction in airway inflammation and a concurrent decrease in
pulmonary eosinophilia without a decrease in Th2 cytokine production or T cell
activation (4), suggesting that
the loss of lung eosinophils may contribute to the failure of OVA-treated
mice to develop
AHR. This inflammation-dependent contribution, however, is likely to be
modulatory and not directly responsible for the failure of these mice to react
following MCh provocation, as saline control
mice (i.e.,
animals with little to no pulmonary eosinophil infiltrate) also failed to
display significant dose-dependent reactions to this cholinergic receptor
agonist.
It is noteworthy that any interpretations of these data are necessarily
complicated by the fact that these signals do not function in isolation. That
is, several G proteins are expressed in each of the potentially relevant cell
types, and a degree of promiscuity exists regarding G protein receptor
coupling. For example, signaling through M3 and thromboxane A2
activates both Gq and G12 to mediate distinct effects in
response to the same agonist
(19,
33). Also, evidence for
simultaneous coupling to G proteins with opposing effects, Gi and
Gs, has been clearly demonstrated for the
2-adrenergic receptor
(7) and
2-adrenergic receptor
(42) in mammalian hearts. The
2-adrenergic receptor has also been shown to similarly
activate two members of the Gi family, Gi2 and
Gi3 (42). Moreover,
tissues are constantly exposed to and stimulated by agonists that signal
through other G proteins and other signaling mechanisms (e.g., tyrosine
kinases).
Seemingly conflicting data exist in the literature concerning the function
of G proteins studied by various techniques and in various cell types. It has
been suggested that a functional overlap exists between Gq and
G11 on the basis of experiments in double knockout mice
(31). These studies
demonstrate that homozygous deficiency at both alleles results in embryonic
lethality, that expression of a just single copy of the four alleles results
in death within 1 h after birth, and that at least two alleles of the four
contributed by these two genes are required for survival after birth. The
conclusion from these data was that Gq and G11 can
functionally compensate for each other during development
(31). Subsequently, mice with
conditionally inactivated Gq on a
G11-deficient background have been used to demonstrate that
these two proteins are critical in the development of myocardial hypertrophy
in adults (38). Evidence for
promiscuous coupling of Gq and G11 to effector proteins
that activate calcium release has been shown in pancreatic and submandibular
gland cells in vitro (43),
whereas evidence for distinct roles for Gq and G11 has
been shown in fibroblasts
(44), neurons
(14), and portal vein myocytes
(25). Interestingly, Western
blot analyses demonstrate that a genetic deficiency in Gq
has no significant effect on the expression levels of the other G protein
(G11). These observations suggest that the results presented
can be attributed to the loss of Gq signaling and are not a
simple consequence of differential G protein expression/usage in airway smooth
muscle, although potential effects of G protein deletion on the complex
balance of integrated signaling that occurs in any cell type cannot be ruled
out.
Mouse models of human airway disease are increasingly and successfully used in laboratory investigations because of the nominal costs associated with small animals, the similarity of the genomes, and the ease of genetic manipulation. However, there are some limitations to their use and the interpretations of the data derived from experiments on mice. Although the structural components are similar between the two species, obvious differences between human and mouse airways in size and morphology may contribute to differences in airway reactivity to various agonists. Also, differences in the reactivity to certain agonists likely arise from differences in receptor expression and utilization. For example, serotonin may have direct and indirect effects on airway smooth muscle responsiveness in some species but affects only neural pathways in the mouse (23). This effect is also seen in the present study, as serotonin has potent effects on Penh (with neural component) but does not affect the response of mouse trachea ex vivo (without neural component).
The mechanisms leading to the development of airway dysfunction in
pulmonary diseases are likely to be combinatorial, involving alterations in
receptor function, smooth muscle electrophysiology, airway
morphology/geometry, airway caliber, airway inflammation, and epithelial
damage. The loss of allergen-induced AHR in
mice suggests that
the intracellular signaling pathways mediated by Gq are
required in one or more of these mechanisms. Furthermore, the demonstration of
an effect on airway reactivity in naive
mice implies that
Gq signaling directly in airway smooth muscle (e.g.,
muscarinic receptor signaling) is likely to be critical. The significance of
these findings to human health resides in the identification of
Gq as a potential target that mediates bronchodilation in
airway diseases by regulating both baseline responsiveness and
inflammatory-mediated contraction of parasympathetic neuromuscular activity in
the airways. Airway inflammation increases the level of bronchomotor tone in
both asthma (34) and COPD
(12) patients, each of which,
as the data presented here would predict, benefits from the administration of
anticholinergic agents. However, currently available anticholinergic agents
inhibit all three muscarinic receptors that have a role in airway function
(i.e., M1, M2, and M3). Strategies targeting Gq thus
represent novel therapeutic modalities to regulate airway reactivity.
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ACKNOWLEDGMENTS |
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This study was supported by the Mayo Clinic Foundation, National Institutes of Health Grants HL-60793–01S (to J. J. Lee) and GM-61395 (to T. M. Wilkie), and National Heart, Lung, and Blood Institute National Research Service Award HL-10361 (to M. T. Borchers).
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FOOTNOTES |
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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.
Present address for M. T. Borchers: Dept. of Environmental Health, Univ. of Cincinnati Medical Center, Cincinnati, OH 45267 (E-mail: michael.borchers{at}uc.edu).
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REFERENCES |
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