Spontaneous airway hyperresponsiveness in estrogen receptor-α α deficient mice
Michelle A. Carey,1 Jeffrey W. Card,1 J. Alyce Bradbury,1 Michael P. Moorman,1 Najwa
Haykal-Coates,2 Stephen H. Gavett,2 Joan P. Graves,1 Vickie R. Walker,1 Gordon P. Flake,1
James W. Voltz,1 Daling Zhu,3 Elizabeth R. Jacobs,3 Azzeddine Dakhama,4 Gary L. Larsen,4
Joan E. Loader,4 Erwin W. Gelfand,4 Dori R. Germolec,1 Kenneth S. Korach1 and Darryl C.
1Division of Intramural Research, National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, North Carolina 27709; 2Experimental Toxicology
Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina 27711; 3Departments of Medicine
and Physiology, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee,
Wisconsin 53226; 4Division of Cell Biology, Department of Pediatrics, National Jewish Medical
and Research Center, Denver, Colorado 80206.
Address correspondence and reprint requests to:
Darryl C. Zeldin, M.D.
NIH/NIEHS, 111 T.W. Alexander Drive, Building 101, Room D236
Research Triangle Park, NC 27709
AJRCCM Articles in Press. Published on November 9, 2006 as doi:10.1164/rccm.200509-1493OC
Copyright (C) 2006 by the American Thoracic Society.
Funding: This research was supported by the Intramural Research Program of the NIEHS, NIH.
J.W. Card was supported by a Research Fellowship Award from the Davies Charitable
Foundation and by a Senior Research Training Fellowship from the American Lung Association
of North Carolina.
Running head: ERα and airway hyperresponsiveness
Word Count: 5,294
Descriptor number: 60
This article has an online data supplement which is accessible from this issues table of content
online at www.atsjournals.org
This paper has been reviewed and approved for release by the National Health and
Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. Approval
does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor
does mention of trade names or commercial products constitute endorsement or recommendation
Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic
evidence supports a role for female sex hormones in modulating lung function and airway
hyperresponsiveness in humans. Objectives: To examine the role of estrogen receptors in
modulating lung function and airway responsiveness using estrogen receptor deficient mice.
Methods: Lung function was assessed by a combination of whole body barometric
plethysmography, invasive measurement of airway resistance and isometric force measurements in
isolated bronchial rings. M2 muscarinic receptor expression was assessed by western blotting and
function was assessed by electrical field stimulation of tracheas in the presence/absence of
gallamine. Allergic airway disease was examined following ovalbumin sensitization and exposure.
Measurements and main results: Estrogen receptor-α knockout mice exhibit a variety of lung
function abnormalities and have enhanced airway responsiveness to inhaled methacholine and
serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor
expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway
responsiveness without increased inflammation following allergen sensitization and challenge.
Conclusions: These data suggest that estrogen receptor-α is a critical regulator of airway
hyperresponsiveness in mice.
Word count: 185
Keywords: lung function, asthma, hyperreactivity, M2 muscarinic receptor, estrogen receptor
A compelling body of evidence supports a role for female sex hormones in modulating lung
function, airway hyperresponsiveness and asthma in humans. Asthma prevalence rates are higher in
women than in men between the ages of puberty and menopause (1, 2). Menstrual cycle variations
in pulmonary function and airway hyperresponsiveness have been well documented (3, 4). Females
also appear to exhibit more severe airway hyperresponsiveness and more severe asthma than males
(5, 6). The effect of estrogens in asthma is highly controversial and the results of published studies
are contradictory. Both beneficial and detrimental effects have been reported (7, 8). For example,
long-term and/or high doses of postmenopausal estrogen therapy have been reported to increase
subsequent risk of asthma (7). In contrast, another study reported that supplemental estrogens could
be used as steroid-sparing agents in asthmatic women (8).
Airway hyperresponsiveness is one of the main features of asthma and is also a major risk
factor for accelerated lung function decline, and hence the development of chronic obstructive
pulmonary disease. The dominant autonomic control of airway smooth muscle in the lungs is
provided by the parasympathetic nervous system (9, 10). The parasympathetic nerves release
acetylcholine which stimulates muscarinic M3 receptors on the smooth muscle to cause contraction.
Concurrently, acetylcholine also stimulates M2 muscarinic receptors located on the nerves to limit
further acetylcholine release (9). Loss of M2 receptor function increases acetylcholine release and
potentiates vagally mediated bronchoconstriction (11). There is substantial evidence that loss of M2
muscarinic receptor expression and/or function on parasympathetic nerves is responsible for the
development of airway hyperresponsiveness (9). Indeed, M2 muscarinic receptors are dysfunctional
in asthmatics (12, 13) and in animal models of allergic airway disease (14, 15).
Estrogens mediate both transcriptional and non-genomic effects via alpha (α) or beta (β)
estrogen receptors (ERs). Both nuclear receptors are expressed in the lung with ERβ being more
abundant than ERα (16), but their functions in this organ are largely unknown. Mice lacking either
ERα (αERKO) or ERβ (βERKO) have been developed using gene targeting strategies (17, 18). The
objective of the present study was to examine the role of ERs in modulating lung function and
airway hyperresponsiveness. Our results show that ERα is a critical regulator of airway
hyperresponsiveness and that αERKO mice have reduced M2 muscarinic receptor expression and
function in the lung. Hence ERs could represent a novel therapeutic target for asthma and other
diseases associated with reactive airways. Parts of this work have been published in abstract form
Methods (Word count: 376)
All procedures were performed under an approved animal study protocol in accordance with
the NIH Guide for the Care and Use of Laboratory Animals. Mice (αERKO, βERKO and wild type
littermate controls on a pure C57BL/6 background, 12-16 weeks of age) were obtained from Taconic
Farms. Further details about animals and treatments are provided in the online supplement.
Whole body barometric plethysmography
Basal lung function was measured in unrestrained mice using whole body barometric
plethysmography (Buxco Electronics). Greater detail about these measurements is provided in the
Invasive analysis of lung function was performed on anesthetized mice using the Flexivent
system (Scireq). Further detail is provided in the online supplement.
Acetylcholine release assay
Acetylcholine (ACh) release was determined by spectrofluorometry using the Amplex® Red
acetylcholine/acetylcholinesterase assay kit following the manufacturer’s instructions (Molecular
Probes). This assay is described in more detail in the online supplement.
Lungs homogenates were analyzed by western blotting for M2 muscarinic receptor
expression which was then normalized to actin expression by analyzing the M2 muscarinic
receptor/actin band density ratio in each sample. Details are provided in the online supplement.
Assessment of M2 muscarinic receptor function by electrical field stimulation
Airway responsiveness to electrical field stimulation in presence or absence of the M2
muscarinic receptor antagonist gallamine was assessed ex vivo as previously described (20), with
some modifications which are described in detail in the online supplement.
Isometric force measurements in isolated bronchial rings
The tension response in isolated bronchial rings to incrementally increasing concentrations of
carbachol (10-7 M to 10-3 M) was examined. Details are provided in the online supplement.
Allergic airway disease model
Allergic airway disease was induced as previously described (21). Invasive lung function
measurements were performed as described in the “Respiratory Mechanics” section.
Bronchoalveolar lavage was performed and various endpoints examined as described in detail in the
Values for all measurements are expressed as mean ± SEM. ANOVA was used to determine
the levels of difference between all groups. Comparisons for all pairs were performed by unpaired
two-tailed Student’s t test. Statistics were performed using GraphPad Prism (version 4) statistical
software (GraphPad Software Inc., San Diego, CA) and Microsoft Excel 2002 software.
Significance levels were set at a p value of 0.05.
Baseline lung function and airway hyperresponsiveness
Whole body barometric plethysmography
Whole body barometric plethysmography was used to non-invasively assess baseline lung
function in αERKO and βERKO mice. Breathing frequency was significantly reduced in male and
female αERKO mice relative to wild type controls (Table 1). Male wild type mice were found to
have a significantly higher tidal volume than female wild type mice; however, this pattern was
reversed in αERKO mice (Table 1). Similarly, minute ventilation, peak inspiratory flow and peak
expiratory flow were higher in male versus female wild type but not in αERKO mice (Table 1). In
contrast, disruption of ERβ had no influence on gender differences in tidal volume, minute
ventilation, peak inspiratory flow and peak expiratory flow (Table 1). However, breathing frequency
was significantly lower and peak inspiratory flow was significantly higher in female βERKO mice
relative to female wild type mice. Moreover, tidal volume was higher in both male and female
βERKO mice relative to their gender-matched wild type controls (Table 1). Together, these data
suggest that both ERα and ERβ play a role in the regulation of breathing with ERα having the more
αERKO females exhibited substantially enhanced bronchial responsiveness to inhaled
methacholine compared to wild type females (Figure 1A). Similarly, male αERKO mice were
hyperresponsive to methacholine compared to their male wild type counterparts, although the
differences were less pronounced than in females (Figure 1B). In contrast, there were no significant
differences in methacholine responsiveness between male or female βERKO mice and their gender-
matched wild type controls (Figure 1, A and B). As Penh is not a universally accepted measure of
bronchoconstriction in mice (22), we also examined airway hyperresponsiveness using invasive
Invasive measurement of lung function and airway responsiveness
We focused our attention on the female αERKO mice with the more robust lung phenotype.
Lung function and methacholine responsiveness were measured in anesthetized, intubated and
mechanically ventilated mice. Under basal conditions, there were no significant differences between
αERKO and wild type females with respect to total elastance (E), Newtonian resistance (Rn) or
tissue elastance (H) (Table 2). However, total respiratory resistance (R), tissue resistance (G) and
hysteresivity (η) were significantly reduced in αERKO female mice compared to wild type females
(Table 2). The reductions in total respiratory resistance and in tissue resistance at baseline in the
αERKO females are consistent with the reduced baseline Penh as assessed by barometric
plethysmography (Table 1). Consistent with the non-invasive barometric plethysmography results,
invasive measurement of lung function confirmed that αERKO females exhibit hyperresponsiveness
to inhaled methacholine (Figure 2). Specifically, PC200R, PC50E and PC200G were significantly
reduced in αERKO female mice relative to wild type females indicating that the lung periphery
plays a role in the enhanced bronchoconstriction to inhaled methacholine in the αERKO mice.
αERKO females also tended to have reduced PC200Rn and reduced PC50H, although these
differences were not statistically significant. Together, these data confirm, via an alternative
method, that lack of ERα leads to basal lung function abnormalities and hyperresponsiveness to
Role of circulating estrogen in the α αERKO phenotype
Female αERKO mice have elevated circulating levels of estrogen and androgen (23). To
address the possibility that altered sex hormone levels in the female αERKO mice were responsible
for the methacholine hyperresponsiveness, wild type and αERKO female mice were ovariectomized
and lung function was assessed 3 weeks later using whole body barometric plethysmography.
Ovariectomy reduced absolute responsiveness to methacholine in the αERKO mice (compare
αERKO mice in Figure 1A to αERKO ovariectomized mice in Figure 3). However, removal of the
ovaries failed to completely abolish differences in methacholine responsiveness between αERKO
and wild type female mice as ovariectomized αERKO mice were still hyperresponsive relative to
ovariectomized wild type mice (Figure 3). Interestingly, ovariectomy did not alter the response of
wild type mice to methacholine. Ovariectomy abolished most of the differences between wild type
and αERKO mice with respect to basal lung function parameters (Table E1, online supplement).
Following ovariectomy, breathing frequency in αERKO mice was still reduced relative wild type
mice, but this did not reach statistical significance (p = 0.06) (Table E1, online supplement).
We next investigated the role of estrogen in modulating airway hyperresponsiveness using
invasive measurements of lung function. Similar to the whole body plethysmography experiments,
we examined airway hyperresponsiveness in ovariectomized mice. In addition, to determine
whether high levels of estradiol alone could recapitulate the hyperresponsive phenotype in wild type
mice, we administered estradiol using implantable, sustained-release pellets, which have been shown
to produce circulating, steady-state estradiol levels comparable to those found in αERKO female
mice. In wild type mice, neither ovariectomy nor estradiol treatment had any effect on airway
responsiveness to methacholine (Figure 4A). In contrast, ovariectomy reduced responsiveness to
methacholine in αERKO mice as evidenced by increased values for PC200R, PC200G and PC50H
(Figure 4B). Estradiol treatment had no potentiating effect on airway hyperresponsiveness in
αERKO mice. Collectively, these data suggest that the high circulating levels of estrogen in the
αERKO mice may contribute to the hyperresponsive phenotype; however, the data also suggest that
high circulating estrogen levels alone are not sufficient for the phenotype to occur – the ERα must
also be absent.
Role of nerve and muscle in airway hyperresponsiveness
Acetylcholine release following electrical field stimulation of tracheas
In order to assess the involvement of neural pathways in the hyperresponsive phenotype of
αERKO mice, we measured release of acetylcholine from isolated tracheas stimulated by electrical
field stimulation. As shown in Figure 5, there was enhanced release of acetylcholine from tracheas
of αERKO female mice relative to wild type controls. As acetylcholine release is controlled by
prejunctional inhibitory M2 muscarinic autoreceptors (9), the increased release of acetylcholine
strongly suggests that these receptors are dysfunctional in the αERKO mice.
M2 muscarinic autoreceptor function
We next investigated the function of the M2 muscarinic receptors. Tracheas were subjected
to electrical field stimulation in the presence or absence of the specific M2 muscarinic receptor
antagonist, gallamine. Electrical field stimulation of tracheas from both genotypes resulted in
frequency-dependent contractile responses. As predicted, pre-treatment with gallamine potentiated
the contractile response in tissues from wild type mice (Figure 6A) demonstrating the presence of
functional muscarinic M2 muscarinic receptors. In contrast, pre-treatment of αERKO mouse tissues
with gallamine had no effect on contractile response to electrical field stimulation (Figure 6B). The
lack of effect of gallamine on contractile response in αERKO mouse tissues indicates that αERKO
mice have dysfunctional M2 muscarinic receptors. One possible mechanism for M2 muscarinic
receptor dysfunction is downregulation of M2 receptor expression (9). To examine this possibility,
we measured protein levels of this receptor in lung homogenates using a specific M2 muscarinic
receptor antibody. We found significantly reduced M2 muscarinic receptor expression in lungs from
αERKO female mice relative to wild type controls (Figure 7A). Densitometric analyses normalized
to actin expression revealed an approximate 50% reduction in M2 muscarinic receptor expression in
αERKO females (Figure 7B). Together, these data suggest that dysfunctional M2 muscarinic
receptors may contribute to the hyperresponsive phenotype in αERKO mice.
Assessment of airway smooth muscle responsiveness.
In order to assess the involvement of airway smooth muscle in the hyperresponsive
phenotype of αERKO mice, isometric force measurements using isolated bronchial ring preparations
were employed. Carbachol induced a significantly greater increase in isometric tension in female
αERKO compared to female wild type bronchial rings ex vivo (Figure 8). These data suggest that
the hyperresponsive phenotype observed in αERKO female mice may be due, at least in part, to
alterations in airway smooth muscle contractile function.
Airway responsiveness to serotonin
In order to determine whether the hyperresponsive phenotype of αERKO mice was specific
to cholinergic agonists, we examined responsivity to serotonin. Similar to the results for
methacholine, αERKO mice were hyperresponsive to inhaled serotonin with significantly reduced
PC200R, PC50E, PC200Rn, PC200G and PC50H relative to wild type mice (Figure 9). These data
suggest that αERKO mice are also hyperresponsive to other bronchoconstrictors.
Role of ERα α in allergic airway disease
Airway responsiveness following allergen challenge
Our next objective was to assess the role of ERα in a clinically relevant lung disease model.
Airway responsiveness to methacholine was assessed in wild type and αERKO mice in an
established model of allergic airway disease involving initial sensitization and subsequent exposure
to ovalbumin. Disruption of ERα had profound effects on the degree of airway hyperresponsiveness
following allergen challenge (Figure 10). Thus, compared to allergic wild type mice and non-
allergic mice of both genotypes, allergic αERKO mice exhibited significantly reduced PC200R,
PC50E, PC200Rn, PC200G and PC50H. It should be noted that, in contrast to naïve αERKO mice,
allergic αERKO mice exhibited enhanced responsiveness to methacholine in the central airways as
evidenced by significantly reduced PC200Rn. These results indicate that absence of ERα leads to
greatly enhanced airway responsiveness following allergen challenge.
Inflammation and cytokine release following allergen challenge
Allergen sensitization and exposure resulted in an influx of inflammatory cells into the
airways. Interestingly, there were no differences between allergic wild type and allergic αERKO
mice with respect to numbers of total cells, eosinophils, lymphocytes and macrophages recovered in
the bronchoalveolar lavage (BAL) fluid (Figure 11A). There was a small reduction in the number of
neutrophils in the airways of allergic αERKO relative to allergic wild type mice. There were no
differences between allergic wild type and allergic αERKO mice with respect to tissue inflammation
as assessed histologically (Figure E1, online supplement). There were no differences between
allergic wild type and allergic αERKO mice in BAL fluid levels of IL-4, IL-5, IL-12 or TNF-
α (Figure 11B). BAL fluid levels of total protein, a marker of alveolar epithelial permeability, were
also similar in allergic wild type and αERKO mice (Figure 11C). These results suggest that lack of
ERα does not appreciably alter the inflammatory response in the allergic airway despite having
profound effects on the development of allergen-induced airway hyperresponsiveness.
The physiological roles of ERs in the lung are largely unknown, hence we examined lung
function and airway hyperresponsiveness in ER deficient mice. The results described herein
implicate a major role for ERα in modulating lung function and airway hyperresponsiveness, and
describe a potential mechanism by which ERα mediates airway responsiveness.
There is considerable evidence supporting a role for sex hormones in the neural control of
breathing (24). Breathing disorders such as obstructive sleep apnea have been linked to sex hormone
levels (24). There is an increase in sleep disordered breathing after menopause which can be
alleviated by hormone replacement therapy (25, 26). Respiratory rhythm is generated by medullary
neurons in the brainstem (27), a site where ERα has been shown to be abundantly expressed (27-29).
Interestingly, we found a marked reduction in breathing frequency in male and female αERKO mice
relative to wild type controls. Male wild type mice have a significantly higher tidal volume than
female wild type mice; however, this pattern is reversed in αERKO mice. Similarly, minute
ventilation, peak inspiratory flow, and peak expiratory flow are higher in male versus female wild
type but not αERKO mice. Together, these data indicate that functional disruption of ERα leads to
changes in a variety of respiratory parameters and suggest that this nuclear receptor may be a critical
regulator of breathing and respiratory rhythmogenesis in mice.
ERβ disruption has no influence on gender differences in tidal volume, minute ventilation,
peak inspiratory flow and peak expiratory flow. However, breathing frequency is significantly lower
and peak inspiratory flow is significantly higher in female βERKO relative to female wild type mice.
Tidal volume is higher in both male and female βERKO mice relative to their respective wild type
controls. Consistent with this observation, Massaro and Massaro recently reported that βERKO
mice have a higher body mass-specific lung volume relative to wild type mice (30). These data
suggest that ERβ does play a role in the regulation of breathing, albeit a much less dominant role
Airway hyperresponsiveness to cholinergic stimuli is a cardinal feature of asthma and a
major risk factor for accelerated decline of lung function and development of chronic obstructive
pulmonary disease (COPD) in humans (31, 32). The exact mechanism(s) underlying the
development of airway hyperresponsiveness in chronic lung diseases such as asthma remains
unknown. Several studies of risk factors associated with airway hyperresponsiveness have reported
higher responsiveness in females compared to males (32-34) suggesting the involvement of sex
hormones in the pathogenesis. Herein we demonstrate that in the absence of immunologic
stimulation, αERKO female mice exhibit substantially enhanced airway responsiveness to inhaled
methacholine compared to wild type females, suggesting that ERα is a critical regulator of this
Traditionally, airway hyperresponsiveness has been presumed to mainly involve the central
airways and not the periphery. However, physiologic and pathologic evidence has emerged in recent
years to support the role of the lung parenchyma and distal airways in the pathogenesis of airway
hyperresponsiveness (35-38). Airway hyperresponsiveness is influenced by properties of the central
airways and the surrounding pulmonary parenchyma which is tethered to the airways, and by
interactions between these two compartments (39). The exact location and precise mechanism for
changes in tissue resistance are controversial, but many hypotheses have been proposed including
contraction of parenchymal interstitial cells, contraction of smooth muscle cells within alveolar ducts
and changes in the architecture of the alveoli and alveolar ducts (40-42). It has also been suggested
that parenchymal changes could be secondary to airway narrowing, either by direct interaction
between the airways and parenchyma or indirectly by altering lung volume (39). Invasive
measurement of lung function in the αERKO mice at baseline revealed hyperresponsiveness
primarily in the periphery. Following allergen challenge, there was marked hyperresponsiveness in
both the central and peripheral airways. Interestingly, Massaro and Massaro recently reported that
estrogen receptors are required for the formation of a full complement of alveoli in female mice (30).
Thus, it is possible that structural abnormalities related to formation and size of alveoli may play a
role in the abnormal hyperresponsive phenotype observed in these mice. Alternatively, the
parenchymal defect in the αERKO mice could be secondary to airway narrowing.
The reduced airway responsiveness to methacholine following ovariectomy of the αERKO
mice suggests that ovarian products may play a role in the hyperresponsive phenotype. However,
the lack of an effect of estrogen supplementation or ovariectomy on airway responsiveness in wild
type mice suggests that estrogen alone is not the only culprit. One possible explanation for these
findings may be that the absence of ERα allows ERβ to predominate in this model. ERα and ERβ
have distinct expression patterns, with some organs having a predominance of one receptor over the
other and other tissues having comparable expression of both receptors (16, 43, 44). Studies with
αERKO and βERΚΟ mice have revealed that these receptors have both overlapping and unique (and
sometimes opposite) roles in mediating estrogen dependent action in vivo. Both receptors are
expressed in the lung with ERβ levels being higher than ERα (16). Studies have revealed that there
is a complex interplay between ERα and ERβ in the regulation and autoregulation of their respective
promoters (44) and that some of the biological functions of one receptor may be dependent on the
presence of the other receptor (16, 44, 45). In the present study, estrogen may produce different
effects via ERβ and the observed hyperresponsive phenotype in αERKO mice may be due to an
altered estrogen response rather than an absent one, as has been postulated with other phenotypes
displayed by these mice (46-48). An alternative possibility could be that the hyperresponsive
phenotype is driven by an ovarian product other than estrogen such as an androgen. In this regard,
the ovaries of the αERKO mice produce 17β-hydroxysteroid dehydrogenase type III, an enzyme
normally found only in testes, that converts androstenedione to testosterone. Indeed, αERKO
females have elevated plasma levels of androgens, similar to those seen in wild type males (23).
Estrogen has been shown to modulate the density of muscarinic receptors in vivo in
extrapulmonary tissues (49, 50), but there are no reports of estrogen modulation of muscarinic
receptor expression or function in the lung. Importantly, expression of the M2 muscarinic receptor
is markedly reduced in αERKO female mice relative to wild type controls. Consistent with this
finding, tracheas from αERKO female mice release more acetylcholine in response to electrical field
stimulation than tracheas from wild type controls. Furthermore, the lack of effect of gallamine, a
selective M2 muscarinic receptor antagonist, on the contractile response of αERKO tracheas to
electrical field stimulation conclusively demonstrates M2 muscarinic receptor dysfunction in these
mice. Together, these data indicate that one potential mechanism for airway hyperresponsiveness in
αERKO female mice could be downregulation of M2 muscarinic receptor expression and function
leading to increased acetylcholine in the neuromuscular junction and resulting in enhanced
bronchoconstriction following cholinergic agonist stimulation.
In light of our finding that αERKO mice are also hyperresponsive to inhaled serotonin, it is
possible that a reflex mechanism may be contributing to the generalized airway hyperresponsiveness
in these mice. It is generally assumed that the response to methacholine reflects only a direct effect
of the agonist on airway smooth muscle. However, studies suggest that a substantial component of
the airway smooth muscle response to cholinergic agonists depends on a vagally mediated reflex
(51). For example, administering cholinergic agonists increases the firing of sensory nerves in the
lung and vagotomy decreases the bronchoconstrictive response to methacholine (52, 53). Studies
suggest that in the mouse, the respiratory response to serotonin also involves a vagal reflex (54).
Blockade of muscarinic M2 receptors with the subtype selective antagonist gallamine potentiates
vagally induced bronchoconstriction by increasing acetylcholine release (55). In addition,
dysfunctional muscarinic M2 receptors have been shown to enhance reflex bronchoconstriction (56).
Thus, if reflex bronchoconstriction is contributing to the enhanced airway hyperresponsiveness to
methacholine and serotonin, then dysfunctional M2 receptors could be playing a major role by
leading to enhanced acetylcholine release following vagal stimulation. The in vitro response to
carbachol may reflect an additional smooth muscle defect and this may or may not contribute to the
airway hyperresponsiveness observed in the intact animal. The combined defects at the level of
smooth muscle and nerve may explain why the hyperresponsiveness is so prominent in the αERKO
While deletion of ERα has minimal effects on airway inflammation following allergen
challenge, it has a profound effect on the development of allergen-induced airway
hyperresponsiveness. Indeed, the differences in airway responsiveness between wild type and
αERKO mice are even more pronounced following exposure to ovalbumin. Although airway
inflammation is frequently correlated with airway hyperresponsiveness to methacholine, and
treatment of inflammation often improves hyperresponsiveness, dissociation between airway
inflammation and hyperresponsiveness has been observed in other murine models of allergic airway
disease (57, 58). Of note, allergen challenge induced minimal changes in airway responsiveness in
wild type mice in our study. This is not surprising since other investigators have shown that the
C57BL/6 strain is one of the least responsive strains in terms of the development of airway
responsiveness following ovalbumin sensitization and exposure (59, 60). Indeed, the fact that
allergen challenge induced such a profound degree of airway hyperresponsiveness in αERKO mice
on a C57BL/6 background indicates that ERα is a potent regulator of this process.
While targeted disruption of specific genes in mice offers a powerful tool to investigators,
caution must be exercised in extrapolating from these studies to physiological effects in humans.
Knockout of a gene in mice may produce different effects than inactivation of the same gene in
humans. In addition, targeted disruption in mice may lead to compensatory changes in expression of
other genes which may hamper interpretation of the results. Nevertheless, it is of interest to
speculate on the potential clinical significance of our findings. There are several polymorphic sites
in the human ERα locus and some of these polymorphisms have been associated with diseases such
as cancer and osteoporosis (61, 62). Interestingly, in some instances, the phenotype associated with
the polymorphism is also dependent on high levels of estrogen (62). The role of hormone
replacement therapy in the treatment and prevention of cardiovascular disease has been highly
controversial due to conflicting findings. It has been suggested that variation in estrogen effects on
the cardiovascular system may be related, at least in part, to common variants in the ERα gene
which can significantly alter the way a person responds to endogenous and exogenous estrogen (63).
It is possible that the conflicting human data on estrogen effects in the lung could also be due to
genetic variability in ERα. Indeed, genetic variability in ERα with resultant effects on estrogen
sensitivity could underlie the greater incidence and severity of asthma and airway
hyperresponsiveness in females between the ages of puberty and menopause when circulating
estrogen levels are high, and could also underlie the phenomenon of premenstrual asthma as
estrogen levels fluctuate during the menstrual cycle. Of interest, the relevance of our findings to
humans is supported by a recent publication by Dijkstra and co-workers who found that variations in
ERα were associated with airway hyperresponsiveness and more rapid lung function decline in
In conclusion, our data suggest that in the mouse, lack of ERα leads to airway
hyperresponsiveness via defects at the level of airway smooth muscle and nerves, and may involve
regulation of M2 muscarinic receptor expression and function. Our data also suggest that the
αERKO mouse may prove to be a useful model to study the mechanisms of sex hormone modulation
of airway responsiveness in humans.
The authors gratefully acknowledge Drs. Anton Jetten, William Schrader and Steven
Kleeberger for helpful suggestions during preparation of this manuscript. They thank Laura Miller
DeGraff for assistance with pellet implantations and surgeries. They also thank Sandy Ward for help
with cell differentials.
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Figure 1. Airway responsiveness to inhaled methacholine in wild type, αERKO and βERKO
mice. Responsiveness to inhaled methacholine was measured using whole body barometric
plethysmography in awake unrestrained female (A) and male (B) mice. Penh, an index of
bronchoconstriction, was measured at baseline and after sequential delivery of increasing
concentrations of methacholine (6.25 – 100 mg/ml). Results are reported as % increase in Penh over
baseline values. Data represent means ± SEM of at least eight mice per group; *p<0.05 vs. wild
type; ^ p<0.05 vs. βERKO.
Figure 2. Invasive measurement of airway responsiveness in female wild type and αERKO
mice. Following acquisition of baseline data, airway responsiveness to inhaled methacholine (0 – 25
mg/ml) was assessed using the forced oscillation technique in anesthetized, intubated and
mechanically ventilated mice. Linear interpolation was used to determine the provocative
concentration of methacholine aerosol at which a 200% increase (PC200) over baseline values was
observed for R, Rn and G and at which a 50% increase (PC50) over baseline values was observed
for E and H. Data represent the means ± SEM of at least six mice per group; *p<0.05 vs. wild type.
Figure 3. Airway responsiveness to inhaled methacholine in ovariectomized wild type and
αERKO female mice. Responsiveness to inhaled methacholine was measured using whole body
barometric plethysmography in awake unrestrained female mice. Penh, an index of
bronchoconstriction, was measured at baseline and after sequential delivery of increasing
concentrations of methacholine (6.25 – 100 mg/ml). Results are reported as % increase in Penh over
baseline values. Data represent means ± SEM of eight mice per group; *p<0.05 vs. wild type ovex.
Figure 4. Invasive measurement of airway responsiveness to methacholine in female wild
type (A) and αERKO (B) mice following ovariectomy (ovex) or implantation of sustained-release
pellets containing estradiol. Control animals were sham ovariectomized or received placebo pellets
and combined into one control group. Following acquisition of baseline data, airway responsiveness
to inhaled methacholine (0 – 25 mg/ml) was assessed using the forced oscillation technique in
anesthetized, intubated and mechanically ventilated mice. Linear interpolation was used to
determine the provocative concentration of methacholine aerosol at which a 200% increase (PC200)
over baseline values was observed for R, Rn and G and at which a 50% increase (PC50) over
baseline values was observed for E and H. Data represent the means ± SEM of at least six mice per
group; *p<0.05 vs. αERKO sham/placebo.
Figure 5. Acetylcholine release from wild type and αERKO tracheas in response to
electrical field stimulation. Data represent the means ± SEM of at least four mice per group;
*p<0.05 vs. wild type.
Figure 6. M2 muscarinic receptor function in wild type and αERKO tracheas. The ex vivo
response of tracheal smooth muscle from (A) wild type female and (B) αERKO female mice to
electrical field stimulation in the presence and absence of the M2 muscarinic receptor antagonist
gallamine. Data represent the means ± SEM of at least eight mice per group; *p<0.05 vs. wild type.
Figure 7. Pulmonary M2 muscarinic receptor expression in female wild type and αERKO
mice. (A) M2 muscarinic receptor and actin expression by immunoblotting in lungs from female
wild type and αERKO mice. (B) Density of M2 muscarinic receptor expression was normalized to
actin expression in each sample. Data represent the means ± SEM of four mice per group; *p<0.05
vs. wild type.
Figure 8. Isometric force measurements in isolated bronchial rings from female wild type
and αERKO mice. The tension response of rings to incrementally increasing concentrations of
carbachol (10-7 M – 10-3 M) was examined. Data represent the means ± SEM of at least eight mice
per group; *p<0.05 vs. wild type.
Figure 9. Invasive measurement of airway responsiveness to inhaled serotonin in female
wild type and αERKO mice. Following acquisition of baseline data, airway responsiveness to
inhaled serotonin (0 – 20 mg/ml) was assessed using the forced oscillation technique in anesthetized,
intubated and mechanically ventilated mice. Linear interpolation was used to determine the
provocative concentration of serotonin aerosol at which a 200% increase (PC200) over baseline
values was observed for R, Rn and G and at which a 50% increase (PC50) over baseline values was
observed for E and H. Data represent the means ± SEM of at least ten mice per group; *p<0.05 vs.
Figure 10. Airway responsiveness to inhaled methacholine following ovalbumin
sensitization and exposure in female wild type and αERKO mice. Following acquisition of baseline
data, airway responsiveness to inhaled methacholine (0 to 25 mg/ml) was assessed using the forced
oscillation technique in anesthetized, intubated and mechanically ventilated mice. Linear
interpolation was used to determine the provocative concentration of methacholine aerosol at which
a 200% increase (PC200) over baseline values was observed for R, Rn and G and at which a 50%
increase (PC50) over baseline values was observed for E and H. Data represent the means ± SEM of
at least four non-allergic and twelve allergic mice per group; *p<0.05 vs. wild type vehicle ; ^p<0.05
vs. αERKO vehicle; #p<0.05 vs. wild type ovalbumin. OVA, ovalbumin.
Figure 11. BAL fluid cells (A), cytokines (B) and protein (C) following ovalbumin
sensitization and exposure in female wild type and αERKO mice. BAL fluid was collected 24 hours
following the fifth daily ovalbumin aerosol challenge. Data represent the means ± SEM of at least
three non-allergic and twelve allergic mice per group; * p<0.05 vs. wild type ovalbumin. OVA,
Table 1. Basal lung function in wild type, αERKO and βERKO mice
Number of animals
44 59 20 20 22 38
Body weight (g) 22.0±0.2 27.2±0.3* 23.9±0.6 26.9±0.4 23.1±0.4 28.9±0.2
0.56±0.01 0.56±0.02 0.50±0.02 0.50±0.02 0.56±0.02 0.56±0.01
507±11 501±11 468±6* 447±13^ 471±12* 495±11
252±7 277±6* 275±9* 256±8^ 293±13* 311±10^
127±5 138±5 128±4 113±6^ 137±8 150±6
Peak inspiratory flow
8.6±0.3 9.9±0.3* 10.0±0.3* 8.8±0.4^ 9.6±0.4* 10.4±0.4
Peak expiratory flow
6.0±0.3 6.6±0.3 6.0±0.2 5.3±0.2^ 6.5±0.3 7.1±0.3
Respiratory parameters were measured by non-invasive whole body barometric plethysmography in
wild type, αERKO and βERKO mice. Data represent means ± SEM;* p < 0.05 vs. wild type
females; ^ p < 0.05 vs. wild type males
Table 2. Basal lung function in wild type and αERKO mice
Number of animals 9
R (cm H2O.s/ml)
E (cm H2O/ml)
Rn (cm H2O.s/ml)
G (cm H2O/ml)
H (cm H2O.s/ml)
η ( η (G/H)
Respiratory parameters were assessed by invasive measurement
of lung function in wild type and αERKO female mice. Data
represent means ± SEM;* p < 0.05 vs. wild type.
Spontaneous airway hyperresponsiveness in estrogen receptor-α α deficient mice
Michelle A. Carey, Jeffrey W. Card, J. Alyce Bradbury, Michael P. Moorman, Najwa Haykal-
Coates, Stephen H. Gavett, Joan P. Graves, Vickie R. Walker, Gordon P. Flake, James W. Voltz,
Daling Zhu, Elizabeth R. Jacobs, Azzeddine Dakhama, Gary L. Larsen, Joan E. Loader, Erwin
W. Gelfand, Dori R. Germolec, Kenneth S. Korach and Darryl C. Zeldin.
Online Data Supplement
Mice. All animals were maintained in plastic cages under a 12-h light, 12-h dark
schedule in a temperature-controlled room (21-22ºC) and fed NIH 31 mouse chow and fresh
water ad libitum. In ovariectomy studies, ovaries were removed three weeks prior to
experimentation to allow the levels of endogenous ovarian steroids to decrease. Control mice
were sham ovariectomized. In studies involving estradiol treatment, pellets (0.05 mg
estradiol/21 day release) purchased from Innovative Research of America were implanted
subcutaneously in the shoulder region. Similarly, control mice were implanted with placebo
pellets. Experiments involving pellet implanted mice were performed 2 weeks post-
implantation. This particular pellet was chosen because it produces circulating levels of estradiol
in wild type mice which mimic levels found in αERKO mice (1-3).
Whole body barometric plethysmography. Basal lung function was measured in
unrestrained mice using whole body barometric plethysmography (Buxco Electronics). From
each chamber, a pressure signal was generated from the pressure difference between the main
chamber containing the unrestrained mouse and a reference chamber. Signals were analyzed
using BioSystem XA software (Buxco Electronics) to derive whole body flow parameters
including respiratory frequency, tidal volume, minute ventilation, peak inspiratory flow, peak
expiratory flow and Penh (enhanced pause). Penh is a unitless parameter that strongly correlates
with lung resistance (4). Signals were recorded every 6 seconds for 60 minutes and the values
were averaged. Airway responsiveness to inhaled methacholine aerosol was then assessed.
After measurement of baseline Penh for 10 minutes, changes in Penh in response to increasing
doses of methacholine (0, 6.25, 12.5, 25, 50 and 100 mg/ml) were measured. Penh was recorded
every 6 seconds during the 3 minute aerosol delivery and for 5 minutes following each aerosol
delivery. Penh values were averaged over the 5 minute response time only and are expressed as
% increase in Penh over Penh response to saline aerosol.
Respiratory Mechanics. Invasive measurements of lung function were performed using
the FlexiVent system (Scireq) and the forced oscillation technique (5). This FlexiVent system is
a platform that integrates a computer-controlled small animal ventilator with measurements of
respiratory mechanics. Using this system, two models were employed to assess respiratory
mechanics: 1) the linear first-order single compartment model, and 2) the constant phase model.
The single compartment model which is the standard model of respiratory mechanics, was
employed to determine total respiratory resistance (R) and elastance (E). The constant phase
model is an advanced model of respiratory mechanics that offers parametric distinction between
central and peripheral respiratory mechanics and which was introduced by Hantos et al. (6). This
model was employed to determine Newtonian resistance (Rn, a measure of central airways
resistance), tissue damping (G, a measure of resistance in the peripheral airways and
parenchyma), and tissue elastance (H, a measure of elastance in the peripheral airways and
parenchyma). This model has been employed by numerous investigators (7-10).
Mice were anesthetized with urethane (1.5 g/kg i.p.). Once surgical anesthesia had been
established, a tracheostomy was performed and a 19 gauge stainless steel cannula was inserted
into the trachea. Animals were then paralyzed with pancuronium bromide (0.8 mg/kg i.p.) and
placed on a 37°C heating pad, and the cannula was connected to the computer-controlled
flexivent ventilator. Ventilation was maintained at a rate of 150 breaths/minute and a tidal
volume of 7.5 ml/kg, with a positive end-expiratory pressure of 3 cm H2O. All data points were
determined by the FlexiVent software (version 4.0) by using multiple linear regression to fit each
data point to the single compartment or the constant phase model, as appropriate. Baseline values
for each mouse were obtained by applying a 2 s perturbation at a frequency of 2.5 Hz followed
by an 8 s pseudo-random perturbation consisting of waveforms of mutually prime frequencies
(0.5 to 19.6 Hz) a total of 3 times at 30 s intervals; these maneuvers generated data that were fit
to the single compartment and constant phase models, respectively. The averages of these
measurements for each mouse served as its baseline values. Following acquisition of baseline
data, airway responsiveness to aerosolized methacholine (0 to 25 mg/ml saline; delivered by
ultrasonic nebulizer) or serotonin (0 to 20 mg/ml saline) was assessed. Aerosols were delivered
for 10 seconds without altering the ventilatory pattern, after which the 8 second forced
oscillation perturbation was applied every 30 seconds for 5 minutes. Peak responses during each
5 minute period were determined for R, E, Rn, G and H. Linear interpolation was used to
determine the provocative concentration of methacholine aerosol at which a 200% increase
(PC200) over baseline values was observed for R, Rn and G and at which a 50% increase (PC50)
over baseline values was observed for E and H. Brief occlusion of the expiratory tube was
performed prior to each aerosol administration in order to reset the volume history. Only derived
values with a coefficient of determination of 0.9 or greater were used.
Isometric force measurements in isolated bronchial rings. Mice were anesthetized
with methoxyflurane followed by pentobarbital sodium (0.5-1.0 mg i.p.). The hearts and lungs
were removed en bloc, and the left and right mainstem bronchial rings (~1-2 mm in diameter)
were dissected free in ice-cold phosphate buffered saline solution (11). Rings were mounted on
two tungsten wires, one connected to a fixed holder and the other to a force displacement
transducer (Model FT03, Gould Electronics) for continuous measurement of isometric tension.
The rings were immersed in pH-adjusted, oxygenated physiologic salt solution at 37°C. Tension
data were relayed from transducers to a signal amplifier (600 series eight channel amplifier,
5 Download full-text
Gould Electronics). Data were acquired and analyzed using CODAS software (DataQ
Instruments, Inc.). Rings were loaded with 0.1 g passive tension and then equilibrated for an
additional 30 minutes before the studies began. The tension response of rings to incrementally
increasing concentrations of carbachol (10-7 M to 10-3 M) was examined.
Immunoblotting. Lungs were homogenized in Tris Buffer (100 mM; pH 8.5) and
protein concentrations of the homogenates were determined using the Bio Rad Assay reagent
(Bio-Rad). Aliquots containing equivalent amounts of protein were prepared and boiled for 5
minutes in sample loading buffer containing sodium dodecyl sulfate (SDS). Twenty µg of
protein from each sample was subjected to electrophoresis on SDS-PAGE gels and transferred to
nitrocellulose membranes. Membranes were blocked for one hour at room temperature in
TBSTM (Tris buffered saline containing 0.1% Tween and 10% non-fat milk protein) and
incubated with rabbit anti-mAChR M2 (Santa Cruz Biotechnology) diluted 1:100 in TBSTM at
4ºC overnight. After removing primary antibody with several washes of TBST (Tris buffered
saline with 0.1% Tween), the membrane was incubated with bovine anti-rabbit IgG conjugated to
horseradish peroxidase (Santa Cruz Biotechnology) for 30 minutes. The membrane was washed
as before and the antibody-antigen complexes were visualized using a chemiluminescence
detection system (SuperSignal West Pico Chemiluminescent Substrate, Pierce). The blot was
stripped using Restore Western Blot Stripping Buffer (Pierce) and re-probed with goat anti-actin,
an antibody reactive with a broad range of actin isoforms (Santa Cruz Biotechnology) and
developed as before. Density of staining was assessed with the image analysis program Scion
Image for windows (Scion Corporation) and M2 muscarinic receptor expression was normalized
to actin expression by analyzing the M2 receptor/actin band density ratio in each sample.