Hydrogen peroxide affects contractile activity and anti-oxidant enzymes in rat uterus

Abstract

Background and purpose: The effects of hydrogen peroxide (H(2)O(2)) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca(2+))-induced] and the effect of such treatment on anti-oxidative enzyme activities.

Experimental approach: Uteri were isolated from virgin Wistar rats and suspended in an organ bath. Uteri were allowed to contract spontaneously or in the presence of Ca(2+) (6 mM) and treated with H(2)O(2) (2 microM-3 mM) over 2 h. Anti-oxidative enzyme activities (manganese superoxide dismutase-MnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT, glutathione peroxidase-GSHPx and glutathione reductase-GR) in H(2)O(2)-treated uteri were compared with those in uteri immediately frozen after isolation or undergoing spontaneous or Ca(2+)-induced contractions, without treatment with H(2)O(2). The effect of inhibitors (propranolol, methylene blue, L-NAME, tetraethylamonium, glibenclamide and 4-aminopyridine) on H(2)O(2)-mediated relaxation was explored.

Key results: H(2)O(2) caused concentration-dependent relaxation of both spontaneous and Ca(2+)-induced uterine contractions. After H(2)O(2) treatment, GSHPx and MnSOD activities were increased, while CuZnSOD and GR (In Ca(2+)-induced rat uteri) were decreased. N(omega)-nitro-L-arginine methyl ester antagonized the effect of H(2)O(2) on Ca(2+)-induced contractions. H(2)O(2)-induced relaxation was not affected by propranolol, potentiated by methylene blue and antagonized by tetraethylamonium, 4-aminopyridine and glibenclamide, with the last compound being the least effective.

Conclusions and implications: H(2)O(2) induced dose-dependent relaxation of isolated rat uteri mainly via changes in voltage-dependent potassium channels. Decreasing generation of reactive oxygen species by stimulation of anti-oxidative pathways may lead to new approaches to the management of dysfunctional uteri.

Figure 1

(A) A representative original trace of spontaneous uterine contractions treated with H₂O₂ (2, 20, 200, 400 µM and 3 mM). (B) A representative original trace of Ca²⁺-induced uterine contractions treated with H₂O₂ (2, 20, 200, 400 µM and 3 mM). (C) Contractile activity of spontaneous and Ca²⁺-induced rat uteri treated with H₂O₂ (2, 20, 200, 400 µM and 3 mM). Contractile activity was expressed as the relative ratio between mean height peak of untreated control and treated uteri. Data are expressed as mean ± SEM (n= 8). Data were analysed by two-way anova (factors: type of contractions and H₂O₂ dose), and showed significant dose effect (F= 380, P < 0.001) and non-significant type of contractions effect (F= 0.7). (D) Linear fit and regression analysis of contractile activity of spontaneous and Ca²⁺-induced rat uteri treated with H₂O₂ (2, 20, 200, 400 µM and 3 mM). Data are presented as individual points. H₂O₂ dose effect was significant for both types of contractions (P < 0.0001). There were no statistical differences between slopes and correlation coefficients of spontaneous (R=−0.88 ± 0.18) and Ca²⁺-induced (R=−0.90 ± 0.16) linear fitted lines (F-test).

Figure 2

Anti-oxidant enzyme activities in spontaneously active rat uteri. Enzyme activities were determined in untreated rat uteri immediately frozen after dissection (control, C0h, n= 8), in untreated ispontaneously active rat uteri incubated for the equivalent experimental time (2 h at 37°C) without the addition of H₂O₂ (C2h, n= 10) and spontaneously active rat uteri incubated for 2 h at 37°C treated with increasing concentration of H₂O₂ (n= 7). Data are expressed as mean ± SEM. The groups were compared by one-way anova (P < 0.05 was considered as significant) followed by honestly significant difference post hoc test for unequal n (n-number of samples). Probability levels are presented to denote the individual differences. Concentrations of H₂O₂: 2, 20, 200, 400 µM, 3 mM. CAT, catalase; CuZnSOD, copper-zinc superoxide dismutase; GR, glutathione reductase; GSHPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase.

Figure 3

Anti-oxidant enzyme activities in Ca²⁺-activated rat uteri. Enzyme activities were determined in untreated isolated rat uteri immediately frozen after dissection (control, C0h, n= 8), in untreated isolated Ca²⁺-activated rat uteri incubated for the equivalent experimental time (2 h at 37°C) without the addition of H₂O₂ (C2h, n= 10) and isolated Ca²⁺-activated rat uteri incubated for 2 h at 37°C treated with increasing concentration of H₂O₂, (n= 7). Data are expressed as mean ± SEM. The groups were compared by one-way anova (P < 0.05 was considered as significant) followed by the honestly significant difference post hoc test for unequal n (n-number of samples). Probability levels are presented to denote the individual differences. Concentrations of H₂O₂: 2, 20, 200, 400 µM and 3 mM. CAT, catalase; CuZnSOD, copper-zinc superoxide dismutase; GR, glutathione reductase; GSHPx, glutathione peroxidase; MnSOD, manganese superoxide dismutase.

Figure 4

(A) Effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on spontaneous contractile activity of rat uteri in the presence of N^ω-nitro-L-arginine methyl ester (L-NAME) 10 µM, propranolol 1 µM and methylene blue (MB) 0.4 µM. Contractile activity was expressed as the relative ratio between mean height peak of untreated control and treated uteri. Data are expressed as mean ± SEM (n= 8); Results were compared by two-way anova (factors: treatment and H₂O₂ concentration). There were significant H₂O₂ dose (F= 187.2, P < 0.001) and treatment effects (F= 2.9, P < 0.05). Post hoc comparison showed that MB treatment was significantly different compared with the other treatments (P < 0.05). (B) Linear fit of data presented in Figure 4A. Regression analysis of effects of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on spontaneous contractile activity of rat uteri in the presence of L-NAME (10 µM), propranolol (1 µM0 and MB (0.4 µM) showed significant effect of H₂O₂ dose (P < 0.0001). There was no difference between correlation coefficients of treatments: H₂O₂ (−0.88 ± 0.18), L-NAME (−0.80 ± 0.25), propranolol (−0.78 ± 0.25) and MB (−0.89 ± 0.17) (mean ± SD).

Figure 5

(A) Effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on Ca²⁺-induced contractile activity of rat uteri in the presence of N^ω-nitro-L-arginine methyl ester (L-NAME) (10 µM), propranolol (1 µM0) and MB (0.4 µM). Contractile activity was expressed as the relative ratio between mean height peak of untreated control and treated uteri. Data are expressed as mean ± SEM (n= 8); Results were compared by two-way anova (factors: treatment and H₂O₂ concentration). There were significant H₂O₂ dose (F= 278.2, P < 0.001) and treatment effects (F= 10.4, P < 0.001). Post hoc comparison showed that MB treatment was significantly different compared with H₂O₂ treatment (P < 0.001). (B) Linear fit of data presented in Figure 5A. Regression analysis of effects of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on Ca²⁺-induced contractile activity of rat uteri in the presence of L-NAME (10 µM), propranolol (1 µM) and MB (0.4 µM) showed significant effect of H₂O₂ dose (P < 0.0001). There were no differences between correlation coefficients of treatments: H₂O₂ (−0.90 ± 0.16), L-NAME (−0.78 ± 0.22), propranolol (−0.87 ± 0.17) and MB (−0.90 ± 0.17) (mean ± SD). F-test showed that at the 0.05 significance level, the fitted lines were not statistically different.

Figure 6

(A) Effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on spontaneous contractile activity of rat uteri in the presence of tetraethylamonium (TEA) (6 mM), glibenclamide (6 µM) and 4-aminopyridine (4-AP) (1 mM). Contractile activity was expressed as the relative ratio between mean height peak of untreated control and treated uteri. Data are expressed as mean ± SEM (n= 8); results were compared by two-way anova (factors: treatment and H₂O₂ concentration). There were significant H₂O₂ dose (F= 207, P < 0.001) and treatment effects (F= 17.1, P < 0.001). Post hoc comparison showed that all the treatments significantly altered H₂O₂ response (P < 0.001). (B) Linear fit of data presented in Figure 6A. Regression analysis of effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on spontaneous contractile activity of rat uteri in the presence of TEA (6 mM), glibenclamide (6 µM) and 4-AP (1 mM) showed a significant effect of H₂O₂ dose (P < 0.0001). Correlation coefficient of 4-AP treatment (−0.72 ± 0.18) was different from the others: H₂O₂ (−0.88 ± 0.18), glibenclamide (−0.91 ± 0.15) and TEA (−0.87 ± 0.12) (mean ± SD). F-test analysis revealed that linear fit of 4-AP and TEA data were statistically significant compared with H₂O₂ (both P < 0.001).

Figure 7

(A) Effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on Ca²⁺-induced contractile activity of rat uteri in the presence of tetraethylamonium (TEA) (6 mM), glibenclamide (6 µM) and 4-aminopyridine (4-AP) (1 mM). Contractile activity was expressed as the relative ratio between mean height peak of untreated control and treated uteri. Data are expressed as mean ± SEM (n= 8); There were significant H₂O₂ dose (F= 146.2, P < 0.001) and treatment effects (F= 16.1, P < 0.001). Post hoc comparison showed that TEA and 4-AP pretreatments were significantly different compared with H₂O₂ only treatment (P < 0.001). Glibenclamide treatment had effect, but at the concentrations above 200 µM (P < 0.01). (B) Linear fit of data presented in Figure 7A. Regression analysis of effect of H₂O₂ (2, 20, 200, 400 µM and 3 mM) on Ca²⁺-induced contractile activity of rat uteri in the presence of TEA (6 mM), glibenclamide (6 µM), 4-AP (1 mM) showed significant effect of H₂O₂ dose (P < 0.0001). Correlation coefficient of 4-AP treatment (−0.71 ± 0.16) was different from the others: H₂O₂ (−0.90 ± 0.16), glibenclamide (−0.83 ± 0.21) and TEA (−0.86 ± 0.12) (mean ± SD). F-test analysis revealed that the linear fit of 4-AP data was statistically significant compared with H₂O₂ (P < 0.05).