Sex differences in endothelial function in porcine coronary arteries: a role for H2O2 and gap junctions?

Abstract

Background and purpose: Cardiovascular risk is higher in men and postmenopausal women compared with premenopausal women. This may be due to sex differences in endothelial function. Here, sex differences in endothelial function of porcine coronary arteries (PCAs) were investigated.

Experimental approach: Distal PCAs were studied under myographic conditions and after precontraction with U46619. Concentration-response curves to bradykinin were constructed in the presence of a range of inhibitors.

Key results: In male and female PCAs, bradykinin produced comparable vasorelaxant responses. Inhibition of NO and prostanoid synthesis produced greater inhibition in males compared with females. Removing H2 O2 with PEG-catalase reduced the maximum relaxation in the absence, but not the presence of L-NAME and indomethacin in females, and had no effect in males. Blocking gap junctions with 100 µM carbenoxolone or 18α-glycyrrhetinic acid further inhibited the endothelium-derived hyperpolarization (EDH)-mediated response in females but not in males. In female PCAs, the maximum EDH-mediated response was reduced by inhibiting SKCa with apamin and by inhibiting IKCa with TRAM-34, or with both. In male PCAs, at maximum bradykinin concentration, the EDH-mediated response was reduced in the presence of apamin but not TRAM-34. Western blot did not detect any differences in connexins 40 or 43 or in IKCa expression between male and female PCAs.

Conclusions and implications: H2 O2 mediated some part of endothelium-dependent vasorelaxation in female PCAs and EDH was more important in females, with differences in the contribution of gap junctions and IKCa channels. These findings may contribute to understanding vascular protection in premenopausal women.

Keywords: bradykinin; connexin (Cx); endothelium-derived hyperpolarization (EDH); gap junctions; hydrogen peroxide (H2O2); intermediate-conductance calcium-activated K+ channel (IKca); nitric oxide (NO); porcine coronary artery (PCA); sex differences; small-conductance calcium-activated K+ channel (SKca).

Figure 1

Log concentration-response curves for the vasorelaxant effects of bradykinin in the absence or presence of 300 µM L-NAME and 10 µM indomethacin in male and female PCAs (A). Data are expressed as a percentage change from U46619-induced tone and are mean ± SEM of 8–12 experiments. Original traces of recording showing the responses to increasing concentration of bradykinin (B,C). *P < 0.05, ****P < 0.0001; significantly different as indicated; one-way anova followed by Bonferroni's post hoc test.

Figure 2

Log concentration-response curves for the vasorelaxant effects of bradykinin in the absence or presence of 300 µM L-NAME, 10 µM indomethacin or 300 U mL⁻¹ PEG-catalase in female (A) and in male (B) PCAs. Data are expressed as a percentage change from U46619-induced tone and are mean ± SEM of 9–15 experiments. *P < 0.05; one-way anova followed by Bonferroni's post hoc test.

Figure 3

Log concentration-response curves for the vasorelaxant effects of bradykinin in the absence or presence of 300 µM L-NAME, 10 µM indomethacin or 100 µM carbenoxolone in female (A) and in male (B) PCAs. Data are expressed as a percentage change from U46619-induced tone and are mean ± SEM of 6–14 experiments. **P < 0.01, one-way anova followed by Bonferroni's post hoc test.

Figure 4

Log concentration-response curves for the vasorelaxant effects of bradykinin in the presence of 300 µM L-NAME, 10 µM indomethacin and 100 µM 18α-glycyrrhetinic acid (18α-GA) in female (A) and in male (B) PCAs. Data are expressed as a percentage change from U46619-induced tone and are mean ± S.E.M of six experiments. In female PCAs, presence of 18α-GA in L-NAME and indomethacin significantly shifted the bradykinin-induced vasorelaxation curve 2.2-fold to the right.

Figure 5

Log concentration-response curves for the vasorelaxant effects of bradykinin in the presence of 300 µM L-NAME, 10 µM indomethacin, 500 nM apamin and/or 10 µM TRAM-34 in female (A) and in male (B). Data are expressed as a percentage change from U46619-induced tone and are mean ± SEM of 5–11 experiments. *P < 0.05, **P < 0.01, ****P < 0.0001; one-way ANOVA followed by Bonferroni's post hoc test.

Figure 6

Log concentration-response curves for the vasorelaxant effects of NS309 in the presence of 300 µM L-NAME, 10 µM indomethacin and 500 nM apamin in female (A) and in male (B) PCAs or 10 µM TRAM-34 in male (C) or both apamin and TRAM-34 in male (D) and female (E) PCAs. *P < 0.05, two-tailed, paired Student's t-test. To examine the selectivity of NS309, log concentration-response curves for the vasorelaxant effects of NS309 in the presence of 300 µM L-NAME and 10 µM indomethacin precontracted with either U46619 or 60 mM KCl in female PCAs (F) was constructed where presence of 60 mM KCl significantly shifted the NS309-induced vasorelaxation 8.1-fold to the right (P < 0.001, two-tailed, paired Student's t-test). Data are expressed as a percentage change from U46619-induced tone and are mean ± SEM of five to ten experiments. Original traces of recording showing the responses to increasing concentration of NS309, a selective SK_Ca and IK_Ca channels activator (G).

Figure 7

Connexin 43 (43 kDa) and MLC (18 kDa) expression levels in 5 µg of female (F1–F5) and male PCAs (M1–M5) (A). Ratio of the expression levels of connexin 43 to MLC in male and female PCAs based on the intensities of their bands (B). Data are expressed in the ratio of Cx43 to MLC intensities bands and are mean ± SEM of five experiments.

Figure 8

Connexin 40 (40 kDa) and GAPDH (∼37 kDa) expression levels in 10 µg of female (F6–F10) and male PCAs (M6–M9) with 20 µg of pig kidney (PK) lysate as positive control (A). Ratio of the expression levels of connexin 40 to GAPDH in male and female PCAs based on the intensities of their bands (B). Data are expressed in the ratio of Cx40 to GAPDH intensities bands and are mean ± SEM of four to five experiments.

Figure 9

Connexin 37 (∼37 kDa) and β-actin (42 kDa) expression levels in 15 µg of female (F7–F9) and male PCAs (M6–M8) with 20 µg of pig (PK) and rat kidney (RK) lysate as positive controls.

Figure 10

IK_Ca (47.5 kDa) and GAPDH (∼37 kDa) expression levels in 15 µg of female (F6–F10) and male PCAs (M6–M10) with 10 µg of pig kidney (PK) lysate as positive control (A). Ratio of the expression levels of IK_Ca to GAPDH in male and female PCAs based on the intensities of their bands (B). Data are expressed in the ratio of IK_Ca to GAPDH intensities bands and are mean ± SEM of five experiments.

Figure 11

Hypothesized mechanism of action of sex differences in endothelial function underlying bradykinin-induced vasorelaxation in isolated PCAs from female (A) and male (B) pigs. Present study demonstrated a clear sex differences in endothelial function where only in PCAs from female pigs have greater EDH-mediated responses specifically the gap junction communication whereas endogenous H₂O₂ plays a role in the NO-mediated pathway in female pigs. Figure adapted from Shimokawa (2010).