Sex differences in progesterone-induced relaxation in the coronary bed from normotensive rats

in Journal of Molecular Endocrinology
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  • 1 Department of Physiological Sciences, Health Sciences Center, Federal University of Espirito Santo, Vitoria, Espirito Santo, Brazil
  • 2 Department of Physiology and Biophysics, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Correspondence should be addressed to R L dos Santos: rogerlyrio@hotmail.com

Progesterone seems to play a role in cardiovascular physiology since its receptors are expressed on endothelial cells from both sexes of mammals. However, little is known about its role on the coronary circulation. Thus, this study aims to evaluate the effect of acute administration of progesterone on the coronary bed and the endothelial pathways involved in this action in normotensive rats of both sexes. A dose–response curve of progesterone (1–50 μmol/L) in isolated hearts using the Langendorff preparation was performed. Baseline coronary perfusion pressure (CPP) was determined, and the vasoactive effect of progesterone was evaluated before and after infusion with Nω-nitro-L-arginine methyl ester (L-NAME), indomethacin, catalase, and Tiron. The analysis of nitric oxide (NO) and superoxide anion (O2 · ) was performed by DAF-2DA and DHE, respectively. Female group showed higher CPP. Nevertheless, progesterone promoted a similar relaxing response in both sexes. The use of L-NAME increased vasodilatory response in both sexes. When indomethacin was used, only the males showed a reduced relaxing response, and in the combined inhibition with L-NAME, indomethacin, and catalase, or with the use of Tiron, only the females presented reduced responses. NO and O2 ·− production has increased in female group, while the male group has increased only NO production. Our results suggest that progesterone is able to modulate vascular reactivity in coronary vascular bed with a vasodilatory response in both sexes. These effects seem to be, at least in part, mediated by different endothelial pathways, involving NO and EDH pathways in females and NO and prostanoids pathways in males.

Abstract

Progesterone seems to play a role in cardiovascular physiology since its receptors are expressed on endothelial cells from both sexes of mammals. However, little is known about its role on the coronary circulation. Thus, this study aims to evaluate the effect of acute administration of progesterone on the coronary bed and the endothelial pathways involved in this action in normotensive rats of both sexes. A dose–response curve of progesterone (1–50 μmol/L) in isolated hearts using the Langendorff preparation was performed. Baseline coronary perfusion pressure (CPP) was determined, and the vasoactive effect of progesterone was evaluated before and after infusion with Nω-nitro-L-arginine methyl ester (L-NAME), indomethacin, catalase, and Tiron. The analysis of nitric oxide (NO) and superoxide anion (O2 · ) was performed by DAF-2DA and DHE, respectively. Female group showed higher CPP. Nevertheless, progesterone promoted a similar relaxing response in both sexes. The use of L-NAME increased vasodilatory response in both sexes. When indomethacin was used, only the males showed a reduced relaxing response, and in the combined inhibition with L-NAME, indomethacin, and catalase, or with the use of Tiron, only the females presented reduced responses. NO and O2 ·− production has increased in female group, while the male group has increased only NO production. Our results suggest that progesterone is able to modulate vascular reactivity in coronary vascular bed with a vasodilatory response in both sexes. These effects seem to be, at least in part, mediated by different endothelial pathways, involving NO and EDH pathways in females and NO and prostanoids pathways in males.

Introduction

Progesterone plays an important role in physiological processes in reproductive organs (Morel et al. 2016). However, it is already known that it also acts on non-reproductive organs, such as the nervous system (Rossetti et al. 2016), bone (Xiu et al. 2016), and cardiovascular system (Pang et al. 2015). Evidence shows that the female sex hormones, estrogen and progesterone, exert a cardioprotective effect (Orshal & Khalil 2004), considering that the risk of the development of cardiovascular disease, such as hypertension and coronary artery disease, in postmenopausal women and men is higher than in premenopausal women (Benjamin et al. 2018). Furthermore, endothelial dysfunction appears to be the common point between risk factors for the development of cardiovascular diseases (Hadi et al. 2005), and the sex difference seems to be a key element on the modulation of endothelial function and may lead to an increase in cardiovascular risk (Mcculloch & Randall 1998, Rosano et al. 2017).

The endothelium can release vasodilatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and endothelium-dependent hyperpolarizing (EDH), or vasoconstriction factors, such as thromboxane A2 (TXA2) and endothelin-1 (ET-1) that participate in the modulation of vascular tone (Orshal & Khalil 2004). It is already known that sex hormones can interact with the endothelium through nuclear and extranuclear receptors and/or second messengers, thus stimulating the release of vasoactive factors (Mendelsohn & Karas 2005). The literature already demonstrates the presence of progesterone receptors in endothelial cells (Vázquez et al. 1999, Orshal & Khalil 2004) both in women and men (Ingegno et al. 1988), suggesting that this hormone may play a physiological role in the maintenance of vascular processes (Thompson & Khalil 2003). However, little is known about the effects of progesterone on the vascular bed (Selles et al. 2001).

In animal and human models, it has been demonstrated that progesterone, through extranuclear (non-classical) actions, can act in the vessel, stimulating the release of the endothelium-derived relaxing factors (Cutini et al. 2009, Pang et al. 2015). In the rat aorta (Selles et al. 2001), porcine and ovine arteries (Ross et al. 2008), and human endothelial cells (Simoncini et al. 2007) progesterone caused an increase in the activity of endothelial NO synthase (eNOS), which is associated with the production of vasodilator endothelial factors, such as NO (Selles et al. 2001, Simoncini et al. 2007, Ross et al. 2008) and EDH (Matoba et al. 2000), and the decrease of vasoconstriction factors such as prostaglandin F2α (PGF2α) (Crews & Khalil 1999). Progesterone can also interact with vascular smooth muscle, through a rapid decrease in the influx of Ca2+ in rats, rabbits, pigs, and humans (Murphy & Khalil 1999, Minshall et al. 2002, Cairrão et al. 2012) , which suggests that this hormone exerts a vascular protective effect.

To our knowledge, there are few studies evaluating the action of progesterone in the coronary vascular bed and on how it interacts in the different sexes. Therefore, to elucidate the mechanisms involved in the action of progesterone in both male and female may provide a basis for better understanding its actions, especially in the coronary circulation. Our hypothesis is that progesterone has a vasodilatory and protective role in the coronary bed. Thus, our objective was to evaluate the acute action of progesterone in order to clarify its effects and provide the possible mechanisms involved in its action in the coronary vascular bed in rats from different sexes.

Materials and methods

Experimental animals

For this study we used adult Wistar rats (Rattus norvegicus albinus) of both sexes aged between 10 and 12 weeks old. The animals were divided into two groups, females and males, which were provided by the animal facility of the Health Sciences Center of the Federal University of Espirito Santo. The animals were maintained in group-housed and under controlled conditions of temperature (22–24°C) and humidity (40–60%), with a 12-h light-dark cycle with water and food ad libitum (standard diet for housed animal, Purina Labina, SP, Brazil). All procedures were conducted in accordance with recommendations in the Brazilian Guidelines for the Care and Use of animals for the Scientific Purpose and Didactics and the Guidelines Euthanasia Practice (CONCEA-MCT 2016) and approved by the Institutional Ethics Committee for the Use of Animals from the Federal University of Espirito Santo under protocol no. 64/2017.

Vaginal smears

The estrous cycle of female rats was monitored by vaginal smears. The vaginal epithelial cells were collected daily between 08:00 and 09:00 h and were examined according to the protocol of Marcondes et al. (2002). The male group was submitted to the same daily handling to reproduce the possible stress suffered by the females during the collection. Although there was no difference in the progesterone response between the phases of the estrous cycle (see the results), the estrogenic hormones, such as 17β-estradiol, can exert rapid effects on the vessel (Santos et al. 2004). To avoid any effects caused by estrogens, we chose to perform the experimental protocols only with the females that were in diestrus I phase with basal concentration of female hormones (Goldman et al. 2007).

Vascular reactivity: studies in isolated hearts

The isolated hearts study was performed as previously described (Debortoli et al. 2017). The animals were anesthetized with ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The thoracic cavity was opened, and the heart was dissected from its connections and immediately transferred to the apparatus for perfusion of the isolated heart. The aorta was cannulated, at the level of its curvature, and then the Langendorff retrograde perfusion technique (Hugo Sachs Electronics, March-Hugstetten, Germany) started. The isolated hearts were perfused with a modified Krebs solution containing NaCl, 120 mmol/L; CaCl2H2O, 1.25 mmol/L; KCl, 5.4 mmol/L; MgSO4.7H2O, 2.5 mmol/L; NaH2PO4.H2O, 2.0 mmol/L; NaHCO3, 27.0 mmol/L; Na2SO4, 1.2 mmol/L; EDTA, 0.03 mmol/L, and glucose 11.0 mmol/L, continuously heated at 37°C by a water bath and saturated with a carbogenic mixture (95% O2 and 5% CO2) in a saturation chamber to maintain a stable pH of 7.4. Coronary flow was maintained constant at 10 mL/min using a roller pump (Hugo Sachs, Germany). The isovolumetric pressure of the left ventricle was maintained through a latex balloon at the end of a steel cannula that was inserted into the left ventricle and connected to a pressure transducer (AD Instruments MLT0380/A Reusable BP Transducer). The balloon was pressurized with the aid of a glass syringe to maintain a preload of 10 mmHg. The baseline coronary perfusion pressure (CPP) was measured with a pressure transducer (AD Instruments MLT0380/A Reusable BP Transducer) that was immediately connected in close proximity to the aortic perfusion cannula by which the coronary artery bed was perfused and connected to a digital data acquisition system (PowerLab System, AD Instruments, Bella Vista, New South Wales, Australia). Because the flow rate was maintained constant, the changes in CPP were directly related to changes in vascular resistance.

After 40 min of stabilization, baseline CPP was determined, and a dose–response curve of progesterone was administered, in bolus, at increasing concentrations (1–50 μmol/L) before and after perfusion with Nω-nitro-L-arginine metal ester (100 μmol/L – L-NAME, a non-selective inhibitor of the enzyme nitric oxide synthase – NOS), indomethacin (2.8 µmol/L, a non-selective inhibitor of the enzyme cyclooxygenase – COX), catalase (1 000 units/mL, enzyme that specifically decomposes H2O2), and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate (Tiron – 1 mmol/L, O2 ·− scavenger). The progesterone curve concentrations were established in agreement with in vitro previous studies (Crews & Khalil 1999). There is a possibility that chronic exposure of coronary vascular bed to physiological levels of sexual hormones in vivo may have the same effect as the supraphysiological levels in vitro (Nakano et al. 1998, Selles et al. 2002). All inhibitors were perfused for at least 20 min and then the dose–response curve of progesterone was repeated. The relaxing response was expressed as the percentage of relaxation and calculated by the following equation:

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Dissection of the coronary arteries

The dissection of coronary arteries was assessed as previously described (Debortoli et al. 2017). The animals were anesthetized with ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The thoracic cavity was opened, the heart was dissected from its connections and maintained in cold modified Krebs solution containing: NaCl, 120 mmol/L; CaCl2H2O, 1.25 mmol/L; KCl, 5.4 mmol/L; MgSO4.7H2O, 2.5 mmol/L; NaH2PO4.H2O, 2.0 mmol/L; NaHCO3, 27.0 mmol/L; Na2SO4, 1.2 mmol/L; EDTA, 0.03 mmol/L and glucose 11.0 mmol/L, during the dissection procedure. The anterior descending branch of the left and septal coronary arteries were isolated, with the aid of a stereomicroscope (BEL Photonics, SZ-B LED, São Paulo, Brazil), free of surrounding ventricular muscle tissue and then frozen in cryoprotectant liquid (Tissue Tek – OCT) at −80°C until its use.

Vascular NO and O2·− levels

The analysis of NO and O2 ·− levels was assessed as previously described (Silva et al. 2016). The coronary arteries of the females (n = 4) and males (n = 4) were cut into 10 µm cross-sections and placed on slides. The slides were then incubated with 4,5-diaminofluorescein diacetate (DAF-2DA, Sigma-Aldrich, 10 µmol/L) and dihydroethidium (DHE; Invitrogen; 5 µmol/L) to detect NO and O2 ·−, respectively. The incubation protocols were performed according to Silva et al. (2016), with modifications. The production rates of this substance were evaluated at four distinct time-points, by performing the following steps on separate slides: (i.) investigation of baseline production; (ii.) negative control of baseline production with L-NAME (300 µmol/L) and Tiron (10 µmol/L); (iii.) analysis of NO and O2 ·− production stimulated by progesterone (50 µmol/L); and (iv.) measurement of the production of these substances after stimulation with progesterone combined with the use of L-NAME and Tiron. This last analysis was used as a control to verify that the fluorescence observed was due to the specific production of NO and O2 ·−. For all substances (DAF-2DA, DHE, progesterone, L-NAME, and Tiron), slides were incubated for 30 min at 37°C in the dark. The excitation wavelengths used were 515 and 500 nm for the analysis of DAF-2DA and 518 and 480 nm for the analysis of DHE. The images were obtained using a 40× lens on a Zeiss Axio Imager fluorescence microscope with an ApoTome module and were analyzed using ImageJ software, version 1.48.

Statistical analysis

Data analysis was performed by the GraphPad Prism 6 (GraphPad Software) and all data are expressed as mean ±  s.d. Data normality was evaluated by Shapiro–Wilk test. After data normality was confirmed, the comparisons of CPP among groups were performed through the unpaired Student’s t-test, and the vasodilator effect of progesterone was evaluated through two-way ANOVA followed by Bonferroni post hoc test. The fluorescence microscopy analysis was evaluated through one-way ANOVA followed by Tukey’s post hoc test. The significance level of P < 0.05 was established.

Results

Vascular reactivity of the coronary bed

A difference in the baseline CPP between female and male groups was observed. Female group had a higher CPP when compared to male group (female (F) = 87.39 ± 14.8 mmHg; male (M) = 69.04 ± 14.1 mmHg). Despite this sexual difference in baseline CPP, the progesterone-induced vasodilation (Fig. 1A) was similar in both groups (F = 10.8 ± 3.6%; M = 10.2 ± 5.1%). To know whether hormonal changes along the estrous cycle of rats can modify the progesterone-mediated vasodilatory response, we evaluated female rats at different stages of the estrous cycle. Thus, the results showed us that the variation of the estrous cycle neither changes CPP (proestrus: 93.61 ± 20.0 mmHg; estrus: 94.13 ± 22.2 mmHg; diestrus I: 86.26 ± 19.0 mmHg; diestrus II: 98.54 ± 23.3 mmHg) nor vasodilatation response stimulated by progesterone (Fig. 1B – proestrus: 11.0 ± 1.7%; estrus: 12.4 ± 3.4%; diestrus I: 12.7 ± 5.7%; diestrus II: 12.9 ± 6.6%).

Figure 1
Figure 1

Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) between the female (n = 10) and male (n = 9) groups (A) and different stages of the estrous cycle (B: Proestrus, n = 6; Estrus, n = 5; Diestrus I, n = 7; and Diestrus II, n = 5). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

We analyzed the possible endothelial pathways by which this hormone can act, that is, NO, prostanoids (e.g. PGI2), and EDH. Thus, at first, we verified the participation of NO in this action. After inhibition of NOS, both female and male groups had increased the vasodilator response, when compared to their respective controls (Fig. 2A: F = 10.8 ± 3.6% to 23.5 ± 8.6%; Fig. 2B: M = 10.2 ± 5.1% to 26.6 ± 6.7%). After inhibition of prostanoids synthesis, only the male group had reduced response (Fig. 2C: F = 10.8 ± 3.6% to 11.5 ± 6.6%; Fig. 2D: M = 10.2 ± 5.1% to 4.3 ± 2.5%).

Figure 2
Figure 2

Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with L-NAME (A: Female, n = 8; B: Male, n = 9) and indomethacin (Indo, C: Female, n = 8; D: Male, n = 8). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

In order to identify the participation of the EDH in the response evoked by progesterone, a progesterone curve was made in the presence of L-NAME plus indomethacin. We observed that the combined inhibition potentiated the vasodilator response of progesterone. However, in the male group, although the response was potentiated, this was lower than that observed with the individual L-NAME inhibition (Fig. 3A: F = 10.8 ± 3.6% to 19.6 ± 8.4%; Fig. 3B: M = 10.2 ± 5.1% to 17.9 ± 5.6%).

Figure 3
Figure 3

Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with L-NAME + indomethacin (L-NAME + Indo, A: Female, n = 8; B: Male n = 8) and L-NAME + indomethacin + catalase (L-NAME + Indo + Cat, C: Female, n = 8; D: Male, n = 7). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

Aiming to analyze the participation of H2O2 in EDH, the progesterone curve was performed after inhibition with L-NAME, indomethacin, and catalase. The vasodilator response to progesterone was lower in the female group, whereas in the male group there were no differences (Fig. 3C: F = 10.8 ± 3.6% to 6.7 ± 2.2%; Fig. 3D: M = 10.2 ± 5.1% to 17.0 ± 5.9%).

Since the inhibition of NO synthesis potentiates the vasodilator effect of progesterone, we suggest that the NO formation pathway could generate metabolites (such as, O2 ·−) that could act for inhibiting the progesterone-induced response. Thus, we verified the response to progesterone after inhibition with Tiron, an O2 ·− scavenger. In the female group, after the addition of Tiron to the perfusion solution, the progesterone-induced relaxation was reduced, whereas in the male group there were no differences (Fig. 4A: F = 10.8 ± 3.6% to 1.9 ± 3.2%; Fig. 4B: M = 10.2 ± 5.1% to 6.9 ± 3.9%).

Figure 4
Figure 4

Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with Tiron (A: Female, n = 7; B: Male, n = 7). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

Vascular NO and O2·− levels

Under baseline conditions (without stimulation), the production of fluorescence emitted from the oxidation of DAF-2DA by NO in coronary arteries was higher in the male group when compared to the female group (Fig. 5). These results were maintained after stimulation with progesterone. Although there was such a difference between the sexes, both groups had increased fluorescence after progesterone stimulation condition. In addition, in the presence of L-NAME, the fluorescence levels were decreased in all groups.

Figure 5
Figure 5

Fluorescence microscopy analysis emitted by DAF-2DA in coronary arteries of female (n = 4) and male (n = 4) in the absence or presence of stimulation by progesterone, with quantification of the fluorescence produced. Scale bar = 20 μm. The values were expressed as mean ± s.d. ****P < 0.0001 compared to the same group under baseline condition. aaaaP < 0.0001 compared female group in same conditions. ####P < 0.0001 compared same conditions without L-NAME. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0171.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

Under baseline conditions (without stimulation), the production of fluorescence emitted from the oxidation of DHE by O2 ·− in coronary arteries was higher in the male group when compared to the female group (Fig. 6). After stimulation with progesterone, the fluorescence levels were increased in females compared to baseline condition, whereas in male group, there was no differences. In addition, in the presence of Tiron the fluorescence levels were decreased in all groups.

Figure 6
Figure 6

Fluorescence microscopy analysis emitted by DHE in coronary arteries of female (n = 4) and male (n = 4) in the absence or presence of stimulation by progesterone, with quantification of the fluorescence produced. Scale bar = 20 μm. The values were expressed as mean ± s.d. ****P < 0.0001 compared to the same group under baseline condition. aaaaP < 0.0001 compared female group in same conditions. ####P < 0.0001 compared same conditions without Tiron. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0171.

Citation: Journal of Molecular Endocrinology 64, 2; 10.1530/JME-19-0171

Discussion

The main finding of this study was that progesterone could promote a relaxing response in the coronary bed of normotensive rats of both sexes and this effect was similar between females and males. Although this response was similar between female and male groups, the mediators involved in this response appear to be different.

Our first observation was the existence of a difference in baseline CPP between females and males, corroborating previous studies (Moysés et al. 2001, Santos et al. 2016, Debortoli et al. 2017). The mechanisms by which coronary vascular tone of normotensive female rats is higher than male rats is not yet fully elucidated. Experiments with gonadectomized rats of both sexes demonstrated that after castration the CPP falls in females, but does not change in males. After treatment with estrogen, the CPP was restored in females and increased in males with similar values to those observed in non-gonadectomized females (Moysés et al. 2001). Moreover, Santos et al. demonstrated that castration had no effect on baseline CPP of hearts from male rats and reduced baseline CPP of hearts from female group, suggesting a role for female sex hormones in the maintenance of coronary tone. However, further studies are needed to determine if this elevation of CPP would play a role in the cardioprotective effects of estrogens observed in females, as well as the possible pathways involved in this response. Perhaps a higher CPP allows greater vasodilation when the supply of oxygen and/or nutrients becomes reduced (Santos et al. 2016). According to Figueroa-Valverde et al., the estrogen infusion significantly increases CPP and vascular resistance in isolated hearts due to the influx of calcium by activation of the L-type calcium channels via an extra-nuclear mechanism which could justify the higher tonus in the coronary circulation of female rats (Figueroa-Valverde et al. 2011).

Although the basal CPP of female rats was higher than the male group, when we analyzed the vascular reactivity, we observed a similar vasodilatory concentration-dependent effect of progesterone (1–50 µmol/L) in both sexes. It is known that sex hormones can modulate coronary vascular reactivity and this effect can be observed either by the action of estrogen (Santos et al. 2016) or by the action of testosterone in rat coronary arteries (Rouver et al. 2015). A growing body of evidence has indicated that progesterone also exerts an important physiological effect, characterized by rapid action, (extra-nuclear effect) which regulates vasodilation and vascular tone (Minshall et al. 2002, Ramírez-Rosas et al. 2014), although we recognize that a limitation of the study was the non-quantification of the progesterone receptors. Our results demonstrate that progesterone can also act in the modulation of the resistance of the coronary arteries in both sexes, corroborating other studies in which the administration of progesterone caused vasodilation in both females (Molinari et al. 2001, Minshall et al. 2002) and males (Cairrão et al. 2012).

It has been demonstrated that females may have physiological responses that can differ according to the phase of the estrous cycle (Kaur et al. 2018). Notwithstanding, to minimize the effects of hormonal variation, studies have aimed to demonstrate the effects of progesterone on cell culture or using male animals (Berger et al. 1992, Crews & Khalil 1999, Barbagallo et al. 2001, Cairrão et al. 2012). On the other hand, our study showed that this hormonal variation in females at different stages of the estrous cycle was not able to alter progesterone-mediated vasodilation.

Although the vasodilatation response seems to be similar between sexes, we analyzed the possible pathways involved in this response in the coronary bed from rats in order to know whether the pathways involved in this response are different in each group. After inhibition of NO synthesis, progesterone had a potentiated vasodilator effect in all groups. Other studies have shown that progesterone causes an acute increase in the activity of the enzyme that synthesizes NO, (i.e. eNOS), which was associated with the NO production (Rupnow et al. 2001, Selles et al. 2001, 2002, Simoncini et al. 2004, Pang et al. 2015). Our results suggest that the NO formation pathway can modulate the progesterone-induced relaxation, since after progesterone stimulation the NO levels were increased in both females and males, but it was higher in males. Curiously, the involvement of NO in relaxation by progesterone is established of negative way, as after inhibition of the NO production the vasodilatory response induced by progesterone increased. A possible explanation for this result involves the production of O2 ·−. The O2 ·− can act as a vasoconstrictor directly resulting in Ca2+ sensitization and arterial constriction (Knock et al. 2009) or indirectly decreasing the bioavailability of NO (Zou et al. 2002). An important source of O2 ·− production is NOS itself during the formation of NO (Stuehr et al. 2001, Morikawa et al. 2003). Furthermore, there are other sources of O2 ·−, for example, the enzymes COX (Tang & Vanhoutte 2009) and NOX (Tarafdar et al. 2018). Once it has been formed, the O2 ·− can interact with NO to form peroxynitrite (ONOO) (Zou et al. 2002) or may suffer the action of antioxidant enzymes, such as superoxide dismutase (SOD), that act in the formation of hydrogen peroxide (H2O2) (Faraci & Didion 2004). However, ONOO formation is about three to four times faster than the formation of H2O2 (Kerr et al. 1999).

Once the ONOO is formed by oxidation of NO by O2 ·−, it can act by inhibiting the synthesis of endothelium-derived relaxing factors, since it may decrease substrates required for NO formation (i.e. tetrahydrobiopterin, BH4), which in turn results in an increased O2 ·− synthesis instead of NO production by NOS (Laursen et al. 2001, Satoh et al. 2005). Besides, ONOO is able to inhibit the PGI2 synthesis even at low concentrations (0.01 µmol/L) (Zou & Ullrich 1996), and it also impairs EDH-mediated vasodilation reducing K+ channel activity (Liu et al. 2002). Therefore, after the inhibition of NOS, with consequent decrease of this ONOO formation, the vasodilatory response to progesterone would be increased. Thus, we believe that progesterone may stimulate the synthesis of NO, which in turn could form ONOO, that can act as a vasoconstrictor impairing the production of NO and/or PGI2, by NOS and COX respectively, also impairing the EDH. Thus, in the absence of NO (for ONOO formation), the impairment of vasodilatation promoted by this pathway could be reversed.

To confirm the involvement of the O2 ·− in the progesterone-induced response, it would be interesting to perform the evaluation analysis of O2 ·− production. Indeed, after progesterone stimulation the levels of O2 ·− production increased only in the female group as showed by the fluorescence intensity. Since the female group had lower production of O2 ·− and NO, which are necessary for ONOO formation, but showed increased relaxation response after inhibition of the NO pathway similar to the male group, we suggest that another factor (vasodilator) could be evidenced in females.

Since ONOO can impair the production of PGI2, we also assessed the involvement of the prostanoids formation pathway in the relaxing response of the progesterone in the coronary bed. Our data demonstrated that the inhibition of this pathway decreased the vasodilatory progesterone response only in males, whereas in the female group the same did not occur. It seems that prostacyclin has a relevant role on the progesterone-induced relaxation in male group coronary vascular bed since this effect was blocked by indomethacin. On the other hand, indomethacin had no effect in female group coronary vascular bed. Moreover, in double-knockout mice for eNOS and COX (called EDH mice), male mice became hypertensive, whereas female mice remained normotensive with greater endothelium-dependent vasorelaxation (Scotland et al. 2005). Another study demonstrated that progesterone can act positively by modulating COX activity, thus increasing the bioavailability of vasodilatory prostanoids in aortic rings of young females (Cutini et al. 2014). However, it is known that the effects of progesterone may be different depending on the type of vessel (Santos et al. 2014).

In addition to the NO and prostanoids formation pathways, we investigated whether there was participation of EDH in progesterone vasodilator effect. It is accepted that the nature of EDH factors varies depending upon the vascular beds and species examined (Shimokawa & Godo 2016). In 2001, Campbell and Harder considered that a factor capable of promoting EDH must exert an endothelium-derived relaxation response that is not inhibited after inhibition of NOS and COX (Campbell & Harder 2001). When we performed conjugated inhibition of NO and prostanoids formation, the vasodilator response was potentiated in females, similar to that found with individual inhibition with L-NAME, confirming that the COX metabolites do not seem to be involved in the vasodilator response in this group. These findings suggest that EDH occurs independently of the participation of NO and prostanoids in this response. In males, on the other hand, albeit the response has been potentiated, and this response was different from that observed in the individual inhibition with L-NAME. We suggest that the reduction observed in the conjugated inhibition (NO + prostanoids) may be due to an important role of COX metabolites in vasodilatation induced by progesterone.

Studies have described that H2O2 can promote EDH in rat and human mesenteric arteries, murine, and coronary arteries from mice (Matoba et al. 2000, 2002, 2003, Wheal et al. 2012). However, the H2O2 effect may vary among vascular beds, species, and experimental conditions (Gluais et al. 2005, Lucchesi et al. 2005). Considering that H2O2 can mediate the EDH, we added a H2O2 degradation catalyst (catalase) and the progesterone response was reduced only in females. Thus, we suggest that vasodilation induced by progesterone, in female group, has the participation, at least in part, of H2O2.

Knowing that the formation of H2O2 can occur by the spontaneous dismutation of O2 ·− through SOD (Faraci & Didion 2004), we performed the progesterone curve in the presence of Tiron, an O2 ·− scavenger. The vasodilatation response was reduced in the female whereas there was no difference in the male group. Thus, we suggest that O2 ·− would have been catalyzed to H2O2, contributing to the vasodilator response in females. Indeed, in other experimental models, H2O2 appears to be involved in relaxation in coronary arteries (Liu et al. 2003, Matoba et al. 2003). Wassman et al. demonstrated that in thoracic aorta from female gonadectomized rats, progesterone promoted the synthesis of O2 ·− and this synthesis, in part, occurred by NAD phosphate (NADPH) oxidase (NOX) (Wassmann et al. 2005). In this context, SOD would act in the formation of H2O2, which would act as vasodilator. Moreover, we suggest that progesterone-stimulated O2 ·− production may have contributed to ONOO formation in both sexes.

Conclusions

In conclusion, the progesterone promotes vasodilatation response in the coronary vascular bed of both sexes in a concentration-dependent manner. This response appears to be performed through different endothelial mediators in each sex involving NO and H2O2 in female and NO and prostanoids in male group. In addition, the participation of other factors in the relaxing response to progesterone cannot be disregarded. Notwithstanding, to better elucidate the mechanisms involved in the action of the progesterone, additional studies that could further contribute to the understanding of the action of progesterone on the coronary circulation are required.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by CAPES.

Author contribution statement

J A Soares Giesen: Conception and design of the study, or acquisition of data, or analysis and interpretation of data; Drafting the article or revising it critically for important intellectual content; Final approval of the version to be submitted; W do Nascimento Rouver: Conception and design of the study, or acquisition of data, or analysis and interpretation of data; Drafting the article or revising it critically for important intellectual content; Final approval of the version to be submitted; E Damasceno Costa: Conception and design of the study, or acquisition of data, or analysis and interpretation of data; V Soares Lemos: Final approval of the version to be submitted; R Lyrio dos Santos: Drafting the article or revising it critically for important intellectual content and final approval of the version to be submitted.

Acknowledgments

The authors would like to thank Erick Roberto Gonçalves Claudio, Juan Carlos Arapa Diaz, and Tagana Rosa da Cunha for the assistance with the helpful suggestions regarding other issues with the manuscript.

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    Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) between the female (n = 10) and male (n = 9) groups (A) and different stages of the estrous cycle (B: Proestrus, n = 6; Estrus, n = 5; Diestrus I, n = 7; and Diestrus II, n = 5). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test.

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    Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with L-NAME (A: Female, n = 8; B: Male, n = 9) and indomethacin (Indo, C: Female, n = 8; D: Male, n = 8). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

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    Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with L-NAME + indomethacin (L-NAME + Indo, A: Female, n = 8; B: Male n = 8) and L-NAME + indomethacin + catalase (L-NAME + Indo + Cat, C: Female, n = 8; D: Male, n = 7). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

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    Vasodilator response to increasing concentrations of progesterone (1–50 μmol/L) before and after inhibition with Tiron (A: Female, n = 7; B: Male, n = 7). The values were expressed as mean ± s.d. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the control group.

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    Fluorescence microscopy analysis emitted by DAF-2DA in coronary arteries of female (n = 4) and male (n = 4) in the absence or presence of stimulation by progesterone, with quantification of the fluorescence produced. Scale bar = 20 μm. The values were expressed as mean ± s.d. ****P < 0.0001 compared to the same group under baseline condition. aaaaP < 0.0001 compared female group in same conditions. ####P < 0.0001 compared same conditions without L-NAME. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0171.

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    Fluorescence microscopy analysis emitted by DHE in coronary arteries of female (n = 4) and male (n = 4) in the absence or presence of stimulation by progesterone, with quantification of the fluorescence produced. Scale bar = 20 μm. The values were expressed as mean ± s.d. ****P < 0.0001 compared to the same group under baseline condition. aaaaP < 0.0001 compared female group in same conditions. ####P < 0.0001 compared same conditions without Tiron. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0171.

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