Acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress

Figures & data

Figure 1. Effects of restraint stress on exploration of the elevated plus-maze (EPM), plasma corticosterone concentrations and systolic blood pressure. (A) The rats were tested in the EPM and results represent the percent of open arm exploration [% entries (open bars) and time spent (hatched bars) in open arms]. Values are mean ± SEM of n = 9 for each group. (B) Plasma corticosterone was evaluated by radioimmunoassay in control (n = 7) and stressed rats (n = 8). (C) Systolic arterial pressure was evaluated by plethysmography before or 2 and 24 h after restraint (n = 8 for each group). *Compared to control group (p < 0.05, Student’s t-test).

Figure 2. Effects of restraint stress on the vascular reactivity of isolated aortas to phenylephrine, serotonin (5-HT), acetylcholine and sodium nitroprusside (SNP). The contraction induced by phenylephrine and 5-HT were determined in endothelium-intact (Endo+) and endothelium-denuded (Endo−) rat aortic rings. Relaxation to acetylcholine and SNP were obtained in Endo+ and Endo− aortic rings, respectively. Values are mean ± SEM of n = 6 for control and n = 9 for stressed rats. *Maximum effect elicited by the agonist (E_max) compared to control group Endo+ (p < 0.05, ANOVA followed by Bonferroni’s multiple comparison test).

Table 1. Effect of several inhibitors on the E_max (grams) and pD₂ (−log EC₅₀) values for contractile actions of phenylephrine on endothelium-intact rat aortic rings.

	Phenylephrine
	Control		Stressed
	Emax	pD2	Emax	pD2
None	1.1 ± 0.1 (7)	7.2 ± 0.1	1.5 ± 0.1 (7)*	7.2 ± 0.1
l-NAME (100 µM)	1.5 ± 0.1 (7)*	7.6 ± 0.1	1.5 ± 0.1 (7)*	7.6 ± 0.1
Tiron (1 mM)	1.1 ± 0.1 (8)	7.1 ± 0.1	0.9 ± 0.1 (7)†	7.1 ± 0.1
Indomethacin (10 µM)	1.1 ± 0.1 (5)	7.1 ± 0.1	0.9 ± 0.1 (7)†	7.4 ± 0.4
AH6809 (10 µM)	1.1 ± 0.1 (7)	7.5 ± 0.1	1.3 ± 0.1 (8)*	7.1 ± 0.1
SQ29548 (3 µM)	0.9 ± 0.1 (7)	7.1 ± 0.1	1.1 ± 0.1 (7)†	7.2 ± 0.2

Number within parentheses indicates the number of rats. Values are means ± S.E.M. l-NAME (NG-nitro-l-arginine methyl ester, non-selective NO synthase [NOS] inhibitor), Indomethacin (1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid, non-selective cyclooxygenase [COX] inhibitor), AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid, antagonist of prostaglandin F_2α [PGF_2α] receptors), SQ29548 (7-[3-[[2-(phenylcarbamoyl)hydrazinyl]methyl]-7-oxabicyclo[2.2.1]heptan-2-yl]hept-5-enoic acid, antagonist of PGH₂/thromboxane A₂ [TXA₂] receptors), tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate, superoxide anion scavenger []). E_max (maximum effect elicited by the agonist); pD₂ (negative logarithm of the molar concentration of agonist producing 50% of the maximal response).

*Compared to control group without inhibitor (p < 0.05, ANOVA followed by Bonferroni’s multiple comparison test).

†Compared to stressed group without inhibitor (p < 0.05, ANOVA followed by Bonferroni’s multiple comparison test).

Figure 3. Effects of restraint stress on prostaglandin (PG)F_2α and thromboxane (TX)B₂ concentrations in the rat aorta. Basal concentration of PGF_2α (A) and TXB₂ (B), a stable metabolite of TXA₂, were measured in the rat aorta by enzyme immunoassay. Results are mean ± SEM of n = 6 for control and n = 7 for stressed rats. *Compared to control group (p < 0.05, Student’s t test).

Figure 4. Effects of restraint stress on systemic and vascular oxidative stress and basal nitrate/nitrite (NOx) levels. (A) Visualization of reactive oxygen species (ROS) generation in aorta slices detected with fluorescent dye dihydroethidium (DHE); note stronger signal after stress. (B) Bar graphs represent lucigenin-chemiluminescence in aortic tissue, (C) plasma concentrations of thiobarbituric acid reactive substances (TBARS). (D) Basal plasma, and (E) aortic nitrate/nitrite (NOx). Results are mean ± SEM of n = 7 for control and n = 11 for stressed rats. *Compared to control group (p < 0.05, Student’s t test).

Figure 5. Effects of restraint stress on endothelial NOS (eNOS), inducible NOS (iNOS), cyclooxygenase (COX)-1 and COX-2 expression in the rat aorta. Upper panels, representative immunoblots for eNOS, iNOS, COX-1 and COX-2 protein expression. Lower panels, corresponding bar graphs show densitometric data for expression of (A) eNOS, (B) iNOS, (C) COX-1 and (D) COX-2. Results are mean ± SEM of n = 4 for control and n = 6 for stressed rats. *Compared to control group (p < 0.05, Student’s t-test).

Figure 6. Effects of restraint stress on antioxidant system and hydrogen peroxide (H₂O₂) concentration. Total antioxidant capacity was evaluated in the plasma (A). Superoxide dismutase (SOD, B) and catalase (CAT) activities (C) as well as H₂O₂ (D) and reduced glutathione (GSH) concentration (E) were determined in the aorta. Results are presented as mean ± SEM of n = 6 for control and n = 8 for stressed rats. *Compared to control group (p < 0.05, Student’s t test).