Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFalpha and IL-13

Abstract

Background: During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFalpha, cytokines known to be important in asthma.

Method: Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.

Results: PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFalpha) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFalpha had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.

Conclusion: Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.

Figure 1

Real time PCR (TaqMan^®) analysis showing the quantity of oxytocin receptor normalized to 18 S and relative to untreated in primary human airway smooth muscle cells from normal donors when treated with IL-13 or IL-13R130Q (A) and TNFα (B) for 6 hr and 18 hr. * Significant difference between treated and untreated conditions (p < 0.05), but not between the two treated conditions; **Significant difference between treated and untreated conditions (p < 0.05), as well as between the two treated conditions.

Figure 2

(A) Representative flow-cytometric analysis of oxytocin receptor expression in basal and HASM cells exposed to 50 ng/ml IL-13 or 10 ng/ml TNFα for 24 hr. (B) Graphical representation of the data as percentage increase in mean fluorescence intensity over basal. *P < 0.05 as compared with untreated cells, n = 9.

Figure 3

Effect of IL-13 on oxytocin-evoked cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). IL-13 (50 ng/ml) was pre-incubated for 24 hr before cells were exposed to 100 nM oxytocin. (A) Typical Ca²⁺ traces from cells incubated in the absence or presence of IL- 13. (B) Graphical representation of values for the Ca²⁺ peak phase from cells incubated in the absence or presence of IL-13. Results are expressed as the net increase in [Ca²⁺]_i over basal (unstimulated) levels. Values are means ± SE of 3 separate experiments and are significantly different from oxytocin only control (P < 0.01). TNFα did not have a modulatory effect on oxytocin-induced calcium response in these cells.

Figure 4

Representative trace showing the contractile response induced by oxytocin in isolated murine tracheal rings. Similar responses were observed in eight different tracheal rings. The insert shows the contractile responses expressed as means ± SEM from 8 different tracheal rings stimulated with 1 μM oxytocin or 10 μM carbachol (p < 0.05 compared to basal values, statistical significance using ANOVA).

Figure 5

Effect of oxytocin (OT) on murine intra-pulmonary airways with respect to a sub-maximal dose of carbachol (CCh). (A) Representational images show a murine airway at baseline (0% contraction), following 1.0 μM OT (20.3%) and 1.0 μM CCh (65.0%). (B) Maximum contraction of individual airways following OT and CCh. Mean ± SEM values of the data shown.