Thereafter, the membranes were incubated with secondary antibody prepared by IRDye680-conjugated goat anti-mouse and IRDye800-conjugated goat anti-rabbit (1: 15000, LI-COR Biosciences, Lincoln, NE) for 60 min after washing. was collected for conducting ex vivo molecular/biochemical studies. == Results == Ethanol had no hemodynamic effects in OVX Sildenafil citrate rats, but reduced the left ventricular contractility index, dP/dtmax, and MAP after acute ER (PPT) or ER (DPN) activation. These responses were associated with increases in the phosphorylation of ERK1/2 and eNOS, and in ROS and MDA levels in the myocardium. GPER activation (G1) only unraveled a modest ethanol-evoked hypotension and elevation in myocardial ROS. PPT enhanced catalase, DPN reduced ALDH2, while G1 had no effect on the activity of either enzyme, and none of the agonists influenced ADH or CYP2E1 activities in the myocardium. Blood ethanol concentration (96. 0 mg/dL) was significantly reduced following ER (59. 8 mg/dL) or ER (62. 9 mg/dL), but not GPER (100. 3 mg/dL), activation in ethanol-treated OVX rats. == Conclusions == ER and ER play major roles in the E2-dependent myocardial dysfunction caused by ethanol by promoting combined accumulation of cardiotoxic (ROS and MDA) and cardio-depressant (NOS-derived NO) molecules in female myocardium. Keywords: Ovariectomized Rats, Estrogen Receptors, Ethanol, Myocardial Function, Oxidative Stress, Blood Pressure == INTRODUCTION == We documented the estrogen (E2)-dependence of ethanol-evoked myocardial dysfunction and hypotension because these effects are absent in ovariectomized (OVX) and male rats, and manifest in both preparations following E2administration (El-Mas and Abdel-Rahman, 2014, 2015). These findings, which are reminiscent of E2-dependent cardiovascular deficits following chronic ethanol administration (El-Mas and Abdel-Rahman, 2000), might be clinically relevant because ethanol reduces blood pressure (BP) in young, but not in older women, or men (Klatsky et al., 1990, Bae et al., Sildenafil citrate 2012). Our recent study identified a pivotal role for the estrogen receptor (ER) subtype ER in the E2-dependent myocardial dysfunction caused by ethanol (Yao and Abdel-Rahman, 2016). However , the use of selective ER blockade in proestrus rats (highest level of endogenous E2) in the latter study or the use of genetic ER knockout models (Sharma and Prossnitz, 2016) might confound data interpretation because a functional crosstalk exists between the 3 ERs (Shi et al., 2013). This limitation could be circumvented by adopting a regain of function approach to identify the ER subtype(s) required for restoring ethanol-evoked myocardial oxidative stress/dysfunction in conscious OVX rats. Such studies have become feasible following the availability of highly selective ER (PPT), ER (DPN) and G protein-coupled ER (GPER, G1) agonists (Mata et al., 2015). The elucidation of the ER subtype(s) implicated in a paradoxical transformation of E2into a pro-inflammatory hormone in the presence of conditions that promote cellular oxidative stress (White et al., 2010) is important for understanding the molecular mechanisms of the sex (E2)-dependent adverse hemodynamic effects of alcohol in females. All 3 ER subtypes, ER, ER and GPER, are distributed throughout the cardiovascular system (Meyer et al., 2006, Meyer et al., 2011), and confer favorable redox and functional cardiac effects under physiopathological conditions via both genomic and rapid signaling mechanisms (Weil et al., 2010, Lee et al., 2014). In OVX rats, acute E2administration enhances the activity of two cardiac enzymes, catalase and mitochondrial aldehyde dehydrogenase 2 (mitALDH2) (El-Mas and Abdel-Rahman, 2014), which serve antioxidant roles (Ren et al., 2007, Ma et al., 2010, Kandadi et al., 2012). Notably, these two enzymes Sildenafil citrate also catalyze ethanol oxidative metabolism (Soffia and Penna, 1987, Kinoshita et al., 2001), Gdnf and determine the level of the pro-oxidant metabolite acetaldehyde (Comporti et al., 2010). Therefore , identifying the ER(s) that activate(s) these two enzymes is critical for our understanding of paradoxical myocardial oxidative stress caused by combining E2and ethanol. Ethanol-evoked Sildenafil citrate accumulation of cardiotoxic molecules such as malondialdehyde (MDA) and reactive oxygen species (ROS) (El-Mas and Abdel-Rahman, 2014) is exacerbated in the presence of E2(El-Mas and Abdel-Rahman, 2014, Ibrahim et al., 2014). Further, E2is required for mediating the protein kinase B (Akt)/nitric oxide synthase (NOS)-dependent myocardial dysfunction caused by ethanol in female rats (El-Mas et al., 2009, 2012). Collectively, these cellular effects are expected to compromise the antioxidant/detoxifying effects of mitALDH2 (Ren et al., 2007, Ma et al., 2010, Kandadi et al., 2012), and ultimately result in ethanol-evoked myocardial oxidative stress/dysfunction. The main aim of the present study was to identify the ER subtype(s) implicated in the E2-dependent adverse biochemical and cardiac effects of ethanol. To achieve this goal, we conducted targeted ER, ER or GPER restoration of function studies, using the respective highly selective agonist (PPT, DPN or G1), in the absence or presence of ethanol in conscious OVX rats. Notably, in OVX rats, the activation of one or more ER subtype function, by the endogenous non-selective.