Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia

Abstract

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,β-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17β-estradiol (E2) at a low concentration (30 μg/ml) but not at high concentrations (50 and 150 μg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Keywords: airway hypersensitivity; estrogen; intermittent hypoxia; lung inflammation; lung vagal C fibers.

FIGURE 1

Ventilatory responses to the intravenous injection of three types of stimulants in three female rats after 14 days of exposure to room air (RA) or intermittent hypoxia (IH). (A) Responses of an intact rat exposed to RA; (B) responses of an intact rat exposed to IH; (C) responses of an ovariectomized (OVX) rat exposed to IH (OVX + IH). Sixteen hours after the last exposure, the animals’ responses to capsaicin (1.0 μg/kg), phenylbiguanide (PBG; 8.0 μg/kg), and α,β-methylene-ATP (α,β-meATP; 10 μg/kg) were measured in each rat. These chemicals are stimulants of lung vagal C fibers and were injected into the jugular vein as a bolus (0.1 ml volume) as indicated by the arrows. The tip of the injection catheter was inserted close to the right atrium. Approximately 15 min elapsed between any two injections. R, respiratory flow; VT, tidal volume; ABP, arterial blood pressure.

FIGURE 2

Role of lung vagal C fibers in IH-induced augmented apneic responses to stimulants in female rats. Groups of intact rats were exposed to RA or IH for 14 days, and OVX rats were exposed to RA (OVX + RA) or IH (OVX + IH) for the same duration. Each rat’s apneic responses to intravenous capsaicin (A), PBG (B), and α,β-meATP (C) were measured under control conditions, after perivagal capsaicin treatment (PCT; 250 μg/ml), and after vagotomy. Apneic duration indicates the longest expiratory duration (TE) during the first 20 s after stimulant injection. Baseline TE was calculated as the average over 10 consecutive breaths immediately before injection. The horizontal dashed lines indicate the apneic ratio of 100% (no response). ^∗p < 0.05 compared with responses of RA rats under the same experimental condition; ^#p < 0.05 compared with responses of IH rats; ^†p < 0.05 compared with control responses in the same group. Except for the RA group, which included rats in the four stages of the estrus cycle (n = 6/each stage; total number of RA rats: 24), all data from the other groups are presented as means ± SE of 10 rats. See the legend of Figure 1 for further explanation.

FIGURE 3

Responses of lung vagal C fibers to intravenous injection of three stimulants in three female rats exposed to RA or IH. (A) Responses of an intact rat exposed to RA; (B) responses of an intact rat exposed to IH; (C) responses of an OVX rat exposed to IH (OVX + IH). Afferent responses to capsaicin, PBG, and α,β-meATP were measured. These stimulants were injected into the jugular vein as a bolus (0.1 ml volume), as indicated by the arrows. Approximately 15 min elapsed between any two injections. AP, action potential; Ptr, tracheal pressure; ABP, arterial blood pressure. See the legend of Figure 1 for further explanation.

FIGURE 4

Sensitizing effect of IH on afferent responses of lung vagal C fibers to intravenous stimulants in female rats. Groups of intact rats were exposed to RA or IH for 14 days, and OVX rats were exposed to IH (OVX + IH) for the same duration. The afferent responses (fiber activity, FA) to intravenous capsaicin (A), PBG (B), and α,β-meATP (C) were measured. Baseline FA was calculated as the value averaged over 10-s interval before stimulation. The peak response was measured as the maximum averaged over 2-s interval after stimulation. Except for the RA group, which included rats in the four stages of the estrus cycle (n = 6/each stage; total number of RA rats: 24), all data from the other groups are presented as means ± SE of 10 fibers recorded from 10 rats. ^∗p < 0.05 compared with peak responses of RA rats; ^#p < 0.05 compared with peak responses of IH rats. See the legend of Figure 1 for further explanation.

FIGURE 5

Role of estrogen in the potentiating effect of OVX on apneic responses and afferent responses to stimulants in female rats exposed to IH. Apneic responses (A) and afferent responses (B) to intravenous capsaicin, PBG, and α,β-meATP were measured after 14 days of exposure to IH in OVX rats supplemented with vehicle (Veh + OVX + IH) or three different concentrations of 17β-estradiol (E2): 30, 50, and 150 μg/ml (30E2 + OVX + IH, 50E2 + OVX + IH, 150E2 + OVX + IH). ^∗p < 0.05 compared with Veh + OVX + IH rats; ^#p < 0.05 compared with control responses in the same group. Data in each group are presented as means ± SE of 10 rats. See legend of Figures 2 and 4 for further explanation.

FIGURE 6

Changes in total cell count and differential cell counts in bronchoalveolar lavage fluid (BALF) from female rats. Two groups of intact rats were exposed to RA or IH for 14 days. The four other groups consisted of OVX rats alone (OVX + IH) or under supplementation with three different concentrations of E2: 30, 50, and 150 μg/ml (30E2 + OVX + IH, 50E2 + OVX + IH, 150E2 + OVX + IH) and exposed to IH for 14 days. The indices measured were total protein concentration (A), total cell count (B), and differential cell count: macrophage (C), neutrophil (D), and lymphocyte (E) in BALF sampled from rats of the six study groups. ^∗p < 0.05 compared with responses of RA rats; ^#p < 0.05 compared with responses of IH rats; ^†p < 0.05 compared with OVX + IH rats. Data in each group are presented as means ± SE of five rats.

FIGURE 7

Expression of inflammatory cytokines and cyclooxygenase-2 (COX-2) and level of malondialdehyde (MDA) in lung samples and BALF from female rats. Two groups of intact rats were exposed to RA or IH for 14 days. The four other groups consisted of OVX rats (OVX + IH) or OVX rats receiving E2 replacement at the concentrations of 30, 50, and 150 μg/ml (30E2 + OVX + IH, 50E2 + OVX + IH, 150E2 + OVX + IH) and exposed to IH for 14 days. In (A), the levels of the protein expression of the p65 subunit of NF-κB in the nucleus of lung tissue samples were analyzed through Western blot analysis to assess NF-κB activation. The protein levels of interleukin-1β [IL-1β (B)] and COX-2 (C) in the lung tissues were measured. The BALF level of TNF-α (D) and the lung level of MDA (E), an indicator of oxidative stress, in these groups were analyzed through ELISA and an assay kit, respectively. These indices were measured and served as indications of lung inflammation. ^∗p < 0.05 compared with responses of RA rats; ^#p < 0.05 compared with responses of IH rats; ^†p < 0.05 compared with OVX + IH rats. Data in each group are presented as means ± SE of five rats.