Chlorine gas exposure causes systemic endothelial dysfunction by inhibiting endothelial nitric oxide synthase-dependent signaling

Abstract

Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

Figure 1.

Chlorine gas (Cl₂) exposure inhibits aortic vasodilation in response to acetylcholine. Rats were exposed to air (open squares) or Cl₂ (400 ppm, 30 min). Aorta were isolated at 6 (closed triangles), 24 (closed circles), or 48 hours (open circles) thereafter, and vasoconstriction and vasodilation responses to phenylephrine (PE) (A) or acetycholine (Ach) (B), respectively, were determined. Data are means ± SEM for cumulative dose-dependent changes in tension. *P < 0.05 by two-way repeated measures ANOVA with Bonferroni post-test for 24 and 48 hours after Cl₂ exposure relative to control (n = 3–12). (C and D) EC₅₀ values for PE-dependent vasoconstriction and Ach-dependent vasodilation, respectively, in aorta isolated from rats 24 hours after exposure to different doses of Cl₂ (0–400 ppm) for 30 minutes. Data show mean ± SEM. *P < 0.05 by one-way ANOVA with Bonferroni post-test relative to control (n = 5–11).

Figure 2.

Cl₂ exposure does not affect Mahma/NONOate (MNO)- or sodium nitroprusside (SNP)-induced vasodilation of aorta. Rats were exposed to air (open squares) or Cl₂ (400 ppm, 30 min). Aorta were isolated at 6 (closed triangles), 24 (closed circles), or 48 hours (open circles) thereafter, and vasodilation responses to the NO donors MNO (A) or SNP (B) were determined. Data show mean ± SEM. No significant differences were observed by two-way repeated measures ANOVA with Bonferroni post-test (n = 3–8).

Figure 3.

Effects of Cl₂ on aortic endothelial nitric oxide synthase (eNOS) expression eNOS mRNA (A) or protein (B and C) were determined in aorta isolated from rats 24 hours after exposure to air or Cl₂ (400 ppm, 30 min). eNOS protein levels were determined by immunoflouresence (B) or by Western blotting (C; inset shows representative Western blots). Data show mean ± SEM (n = 3–4). **P < 0.05, *P < 0.03, and ^#P < 0.001 relative to control. (D) Plasma nitrite concentrations in 24 hours after air or Cl₂ exposure. Data show mean ± SEM (n = 5–7). ^#P < 0.05 relative to control.

Figure 4.

Cl₂ decreases eNOS protein expression. Representative immunofluoresence images for eNOS (A and B) or von Willebrand factor (vWF) (C and D) staining from aorta collected 24 hours after exposure to air (A and C) or Cl₂ (400 ppm, 30 min) (B and D). Red = eNOS or vWF as indicated; blue = Hoechst staining for nuclei.

Figure 5.

Effects of Cl₂ on blood pressure. Rats were acclimatized to tail-cuff blood pressure measurement protocols and then exposed to air (open squares) or Cl₂ (closed squares; 400 ppm, 30 min), and blood pressure measured again at 24 and 48 hours thereafter. −48 h and −24 h indicate measurements during acclimatization and indicate stable blood pressures before Cl₂ exposure. Time 0 indicates measurements (30–60 min before Cl₂ exposure). No significant differences between mean arterial blood pressure (MAP) as a function of time in air or Cl₂ groups (by one-way ANOVA) or between groups (by two-way repeated measures ANOVA) were observed. Data are mean ± SEM (n = 2–4).

Figure 6.

Role of systemic inducible nitric oxide synthase (iNOS) induction in post-Cl₂–induced changes in MAP. (A) Rats were exposed to air or Cl₂ (400 ppm, 30 min), and 24 hours thereafter neuronal nitric oxide synthase (nNOS) or iNOS mRNA expression from aorta or circulating leukocytes was determined. mRNA levels are expressed relative to air controls and after normalization to GAPDH. Right-hand y axis is for leukocyte mRNA levels. Data are mean ± SEM (n = 2–3). *P < 0.001 by t test relative to respective air control. (B and C) Representative immunofluoresence images of iNOS (red) staining in aorta from control or 24 hours after Cl₂ exposure, respectively. (D) Rats were exposed to air or Cl₂ (400 ppm, 30 min). MAP was measured 24 hours thereafter, and saline or 1400W (10 mg/kg) was added by intraperitoneal injection. After a further 18 to 24 hours, MAP was measured. Data show changes in MAP (post 1400W – pre 1400W administration) and are mean ± SEM (n = 2–4). *P < 0.05 relative to air + 1400W or chlorine + saline by one-way ANOVA with Tukey post-test. (E and F) Effects of 1400W (10 μM) or L-NMMA (100 μM) on PE-induced vasoconstriction or Ach-induced vasodilation of aorta isolated from control of Cl₂–exposed rats, respectively. Data show mean ± SEM (n = 3–5). *P < 0.05 for control versus control + L-NMMA or Cl₂ versus Cl₂ + L-NMMA by two-way ANOVA with Bonferroni post-test.