Given the widespread use of mice as a model to study the pathogenesis of lung diseases and associated functional alterations, it is important to recognize factors that might influence experimental endpoints of interest. In this respect, the roles of sex and sex hormones are receiving increasing attention, particularly with regard to their effects on lung function. The present study was thus undertaken to examine the basis for the disparity in airway responsiveness to cholinergic stimulation that exists between naïve adult male and female mice (4
). Results indicate that the exaggerated airway responsiveness of male mice relative to that of female mice appears to be due to promotive effects of male sex hormones that are dependent on a reflex pathway mediated by the vagus nerves.
Differences in pulmonary anatomy exist between male and female mice, with females typically having proportionally smaller lungs and larger conducting airways than males (22
) and possessing smaller and more numerous alveoli (18
). Despite these dissimilarities, sex-dependent variation in baseline mechanical properties of the murine lung appears to be minimal (22
). On the basis of our previous observation of a sex difference in airway responsiveness to methacholine aerosol, we sought to determine if there is an inherent difference in the abilities of male and female airway smooth muscle to respond to direct cholinergic stimulation. Minimal sex differences in the contractility of isolated tracheal and bronchial ring segments to direct cholinergic stimulation were observed, however, suggesting that this is not the case. Numerous other studies have documented a lack of correlation between in vivo and ex vivo human airway responsiveness to cholinergic stimulation (1
), suggesting that the mechanism(s) responsible for in vivo responsiveness is (are) not necessarily present ex vivo. This was likely the case in the present study as well.
Parasympathetic innervation of the respiratory system is recognized as contributing significantly to the maintenance of airway tone in normal and diseased settings, and a significant role for parasympathetic reflex pathways in the bronchoconstrictive effects of numerous compounds (e.g., histamine, serotonin, and acetylcholine) has been demonstrated in a variety of species (reviewed in Ref. 3
). Specifically, bilateral vagotomy or cooling of the vagus nerves significantly reduces bronchoconstriction induced by cholinergic agonists in rats, sheep, and dogs (2
). However, we are unaware of reported effects of vagus nerve interruption on responsiveness of mice to cholinergic stimulation, and in particular to inhaled cholinergic agonists. We observed that bilateral vagotomy abolished the responsiveness of intact male mice to aerosolized methacholine, whereas it did not alter the responsiveness of intact female mice, suggesting that airway responsiveness to cholinergic stimulation in male mice is mediated almost entirely by vagal reflex pathways. The same may be true in female mice but may merely be undetectable in our assessments due to the relatively nominal innate responsiveness of females to begin with. The observation that castration of male mice eliminated airway responsiveness to a similar extent as did bilateral vagotomy suggested a potential association between male sex hormone effects and the vagal contribution to airway reactivity. This was confirmed when DHT administration was found to increase responsiveness of castrated male and intact female mice and was further corroborated when vagotomy was shown to nullify this effect. Of note, testosterone has also been shown to promote baroreceptor-mediated experimental bradycardia (7
), suggestive of a general promotive effect of male sex hormones on parasympathetic vagal activity. Our observations imply a novel role for male sex hormones in promoting airway responsiveness to cholinergic stimulation and underscore the importance of accounting for sex in the design and analysis of murine studies of lung function and disease.
To begin to address the underlying mechanistic basis for male sex hormone-mediated promotion of responsiveness to cholinergic stimulation, we determined pulmonary M2AchR expression levels in naïve male and female mice. The M2AchR mediates negative feedback inhibition of acetylcholine release from parasympathetic nerve terminals in the airways (9
), and its inhibition or absence promotes airway responsiveness to vagal stimulation (8
). Furthermore, we have observed that the airways of female estrogen receptor-α-deficient mice are male-like (i.e., more sensitive than those of wild-type females to methacholine aerosol) and that this phenotype is associated with decreased M2AchR expression and function (5
). No difference was observed in the amount of M2AchR protein in the lungs of naïve male and female mice, however, indicating that this mechanism was not likely contributing to the sex difference in airway reactivity observed here.
In addition to direct effects on specific receptors, it is thought that in most instances chemical mediator-induced bronchoconstriction results from indirect or subsequent effects on multiple receptors and pathways that mediate reflex responses including rapidly adapting stretch receptors, slowly adapting stretch receptors, and pulmonary and bronchial C fibers (reviewed in Ref. 6
). Furthermore, afferent nerves are subject to varying degrees of neuromodulation by many mediators and autocoids such that the electrical excitability of the nerves can be significantly altered (29
). The integration of pulmonary vagal afferent nerve inputs in the nucleus tractus solitarius provides another level of complexity in the processing of afferent information and in the subsequent regulation of efferent output to the lungs. Although we favor an enhanced excitatory (cholinergic) component in intact male mice as underlying the observed sex difference in airway responsiveness, an alternative explanation is that excitatory (cholinergic) and inhibitory (adrenergic and/or nonadrenergic) reflexes effectively cancel one another in female but not in male mice, implying an impaired or less effective inhibitory reflex in male mice. Parasympathetic nerve-mediated relaxations have been attributed in part to nitric oxide and/or S
), and there is published evidence for sex differences in S
-nitrosothiol metabolism in mice (14
), supporting the possibility of sex differences in this mechanism as underlying our observations. Whether male sex hormones exert regulatory effects on these and/or other processes involved in the control of airway tone and responsiveness to cholinergic stimulation remains to be determined and represents an important area of future investigation.
A recent report indicates a similar sex difference in airway responsiveness to methacholine in adult guinea pigs to that which we have observed in adult mice, namely an increased sensitivity of males compared with females (21
). In contrast to our observations in adult mice, however, healthy adult female humans are generally more sensitive to methacholine-induced airway constriction (measured as the decline in forced expiratory volume in 1 s, or FEV1
) than are healthy adult male humans (13
). Whereas female mouse airways are relatively larger than those of males, the opposite is true in humans, and these anatomical differences may contribute to the apparent species difference in cholinergic airway responsiveness between sexes. In humans, it has been demonstrated that the sex disparity in responsiveness can be accounted for by taking the relative differences in lung and airway size into account (25
). In our study, mice were ventilated based on body weight (and by correlation, lung size) such that tidal volume per unit body weight was equivalent for all mice examined. It is therefore difficult to directly compare our murine airway responsiveness data to those generated from human studies, and other potentially confounding factors must also be recognized. These include but are not limited to environmental factors (exposure to second hand smoke, ozone, etc.), which may affect human but not murine studies, and the innate methodological differences in human and murine lung function assessment protocols. Regardless, it is interesting to note that greater airway responsiveness appears to be associated with smaller caliber airways in both species.
In conclusion, we have demonstrated that male sex hormones promote murine airway responsiveness to cholinergic stimulation via a vagally mediated reflex pathway. This effect of male sex hormones represents the basis for the increased sensitivity of male vs. female mice to aerosolized methacholine and should be carefully considered when designing and interpreting murine studies of lung function that include cholinergic responsiveness as an experimental endpoint. Delineating the underlying basis for this effect of male sex hormones may reveal novel mechanisms underlying functional abnormalities associated with a variety of lung disease states.