Oxytocin regulates gonadal function such as labor-associated uterine contractions and milk discharge during lactation [1
], but the role of oxytocin in other tissues remains unexplored. Evidence shows that the incidence of asthma switches at puberty from male to female predominance, and in adult asthma the ratio is about 2:1 in favor of women [38
] and that asthma worsens in about 30% of pregnant women [9
]. Whether oxytocin plays a role in determining gender susceptibility to asthma remains unknown. Our present study demonstrates the existence of functional oxytocin receptors on HASMCs and their regulation by pro-inflammatory cytokines known to be involved in asthma pathogenesis. Further, we report that oxytocin induced force generation and contractile responses in isolated murine tracheal rings [32
] and lung slices [40
]. These data suggest that expression of oxytocin receptor (OXTR) on HASMCs can be differentially modulated by inflammatory cytokines in HASMCs, a mechanism that may contribute to the altered airway responsiveness observed in asthma assuming that such receptor changes would occur in vivo.
The study of the molecular mechanisms regulating the expression of oxytocin receptor remains complex [42
]. Most studies that attempt to elucidate the transcriptional regulation of oxytocin receptors have been performed in myometrial and uterine cells. Using these models, investigators found that OXTR expression was downregulated by IL-1β, IL-6 but not by TNFα [44
]. Others found that lysophospholipids increased translation of oxytocin receptor possibly as a consequence of increased mRNA stability [46
]. Our report is the first to demonstrate that expression of oxytocin receptor is increased in HASMCs treated with pro-inflammatory cytokines, either TNFα or IL-13. An increased gene transcription was detected by the real time PCR data demonstrating a rapid effect of these cytokines on oxytocin receptor expression. In addition, both TNFα and IL-13 up-regulated receptor expression at the protein level with a 7 fold and 3.5 fold increase over basal, respectively. Unexpectedly, IL-13, but not TNFα, enhanced oxytocin-evoked calcium responses in HASMCs. The mechanisms responsible for the differential effects of TNFα and IL13 to enhance oxytocin-evoked calcium responses remain unclear. This is surprising given evidence that IL-13 and TNFα comparably enhance calcium signals induced by bradykinin and acetylcholine [19
]. Our data does support the hypothesis that TNF and IL-13 have disparate effects on OXTR expression and that agonists with less efficacy at inducing calcium mobilization may be enhanced by increases in receptor expression rather than by processes that amplify downstream pro-contractile signaling pathways. Accordingly, we and others also reported that changes in bradykinin receptor expression in part explained the enhancing effects of cytokines (TNFα or IL-1β) on agonist-evoked calcium responses [49
]. In some but not all instances, modulation of receptor expression may not correlate with receptor function in ASM cells. In previous reports, we and others found that although calcium signals to acetycholine were significantly increased by TNFα, muscarinic receptor density were significantly reduced in TNFα-treated cells [51
]. Collectively, these data suggest that cytokine-induced changes in receptor function may be uncoupled from cell surface receptor density. Additionally, signaling pathways such as alterations in calcium sensitization could also contribute to the observed differences in cytokine-induced agonist responsiveness but such mechanisms require further study.
We show that oxytocin receptors are expressed on HASMCs and cytokines modulate receptor expression [48
]. In myometrium cells, TNFα enhanced oxytocin-induced calcium transients [20
]. Whether TNF effects were due to alterations in receptor expression was not addressed. These investigators, however, found that CD38 played a critical role in the enhanced oxytocin-induced calcium responses in myometrium cells. Interestingly, both TNFα and IL-13 also upregulated CD38 expression and function in HASMCs [48
]. Because IL-13, but not TNFα, enhanced oxytocin calcium responses in HASMCs, our findings possibly suggest that both CD38-dependent and independent pathways may contribute to cytokine-induced alterations in oxytocin responses.
Using two ex vivo
models to study airway responsiveness, namely, isolated tracheal rings [35
] and mouse lung slices [40
], we found that oxytocin induced ASM contraction and airway narrowing, suggesting that oxytocin serves as a bronchoconstrictor. Whether these responses, which are modest in magnitude compared to those induced by carbachol, are clinically relevant remains unknown. Our study, however, shows that these bronchoconstrictor responses induced by oxytocin were dramatically increased by IL13. However, we failed to detect any effects of IL-13 on oxytocin-evoked contractile responses (Amrani et al., unpublished observations). The reasons explaining the discordance observed between IL-13 effects on cultured cells and on ex vivo tissue remains unexplained but could reflect that IL-13 effects in complex tissue provides negative homeostatic effects that dampens IL13's ability to enhance oxytocin-induced bronchoconstriction. Alternatively, the diffusing barrier of the tissue thickness mitigates the ability of IL-13 and/or oxytocin to stimulate their cognate receptors. Accordingly, contractile responses to oxytocin could be modulated by effects on airway epithelial cells and/or vascular endothelium [16
] or by subsequent production of relevant factors such as nitric oxide [43
]. Further studies are needed to address whether IL-13 modulates oxytocin effects in other lung cells.
Given our observations that oxytocin induced airway smooth muscle contraction and that cytokines increased expression of the receptor, we characterized whether BAL fluid derived from healthy subjects as well as subjects with asthma had detectable oxytocin levels. Our studies revealed that detectable oxytocin levels were found in the BAL fluid in both cohorts; however, in stable asthma, there was no increase in oxytocin levels in BAL fluid. We postulate that the mechanism by which oxytocin may play a role in asthma in acute exacerbations concerns enhanced vascular permeability into tissue where the receptor number in ASM but not the ligand are markedly increased. To address this hypothesis, OXTR expression in ASM tissue derived from subjects with acute exacerbation would be required but such studies are beyond the scope of the current study.
In summary, our report provides the first evidence of a contractile role of oxytocin in the airways. Future studies will address the nature of the common transcription factors as well as signaling pathways by which both cytokines regulate the transcription of the oxytocin receptor gene. Since gender disparity in asthma is well recognized, studies could also address whether differential OXTR expression in the airways between men and women in part explains gender differences in asthma prevalence and morbidity or whether differential OXTR expression mediates asthma morbidity in pregnancy.