Apelin-13 is a 13-amino acid polypeptide that participates in several physiological processes, particularly in cardiovascular regulation, fluid homeostasis, and energy metabolism (Galanth et al., 2012; Hu et al., 2022; Kleinz et al., 2005; Kleinz and Davenport, 2004). This compound derives from an endogenous pre-proapelin precursor of 77 amino acids. This precursor undergoes enzymatic hydrolysis to generate several active forms, including apelin-36 and apelin-13 (Kawamata et al., 2001). Among these forms, apelin-13 is the shortest length and exhibits the highest biological activity (Kawamata et al., 2001; Knauf et al., 2013; Masri et al., 2005). This particular form has been extensively used in vivo and in vitro experiments on myocardial glucose uptake (Xu et al., 2012).

Apelin-13 expresses in multiple peripheral tissues, such as the stomach, lung, heart, adipose tissue, and blood vessels, as well as within some areas of the central nervous system (CNS; O'Carroll et al., 2000; Reaux et al., 2001, Reaux et al., 2002), as the medial and lateral preoptic area (mPOA, lPOA), where numerous neuronal bodies with low-density but intense staining are observed (Reaux et al., 2002). A high density of apelin-13-positive nerve fibers is also present in the hypothalamus, which, together with the POA, regulates both appetitive and consummatory aspects of female sexual behavior in rodents (Kawamata et al., 2001; Napoli et al., 1972; Pfaff and Sakuma, 1979; Pfaus et al., 2015; Reaux et al., 2002). Additionally, apelin-13 is present in the hypothalamus-pituitary gland-gonads axis, which is an essential circuit in reproductive events for both females and males, regulating GnRH secretion (Tekin et al., 2017; Yang et al., 2019).

Apelin-13 exerts its effects on the reproductive system by several cellular mechanisms (Kurowska et al., 2018); however, the impact of apelin-13 on the activity of GnRH neurons remains unexplored. Nevertheless, the coexistence of apelin-13 and kisspeptin (Kiss) neuronal populations within the mediobasal hypothalamus suggests a plausible influence of apelin-13 on Kiss neuron activity, thereby potentially decreasing GnRH secretion and subsequently reducing luteinizing hormone (LH) release. Notably, Kiss and proopiomelanocortin (POMC) neurons arise from the same cell lineage (Sanz et al., 2015), and apelin-13 has been demonstrated to increase the electrical activity of approximately 50 % of POMC neurons (Lee et al., 2015). These findings collectively support the notion that apelin-13 might also modulate Kiss neurons. On the other hand, it has been reported that apelin-13 stimulates the activity of critical enzymes involved in steroidogenesis in granulosa and luteal cells of the ovary, such as aromatase and 3β-hydroxysteroid dehydrogenase, to synthesize estradiol and P (Shimizu et al., 2009). This ovarian effect of apelin-13 is thought to be mediated by the activation of intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways (enzymes involved in the regulation of female sexual behavior; Acosta-Martínez et al., 2006; Domínguez-Ordóñez et al., 2019; Etgen and Acosta-Martinez, 2003; González-Flores et al., 2009). These findings suggest that apelin-13 is crucial in the neuronal signaling pathways (Knauf et al., 2013; O'Donnell et al., 2007) for lordosis and other reproductive responses.

The APJ receptor belongs to the family of G protein-coupled receptors and is widely distributed in various human tissues (Habata et al., 1999). Activation of the APJ receptor by its ligand, apelin, can activate proteins such as Gαi2, Gαi3, Gα0, and Gαq (Bai et al., 2014). Recent research has shown that APJ plays a dual role in cardiac hypertrophy (Scimia et al., 2012). For example, Apelin-activated APJ elicits a protective response in cardiac hypertrophy through the Gαi pathway, while phosphorylation of various sites on the C-terminal end of the receptor facilitates β-arrestin-dependent signaling that induces cardiac hypertrophy (Chen et al., 2020). Thus, several active forms of apelin activate APJ receptor (a Gi protein-coupled receptor), which can inhibit adenylyl cyclase and cAMP formation (Habata et al., 1999), inhibiting protein kinase A (PKA). Therefore, activating this receptor induces Ca2+ release (Japp and Newby, 2008), which induces Ca2+/calmodulin kinase, and activates the nitric oxide (NO) pathway (Takata et al., 2011), causing endothelial vasodilatation. Moreover, PKA and nitric oxide (NO) have also been implicated in activating lordosis in EB-primed ovariectomized (OVX) rats (Domínguez-Ordóñez et al., 2019; García-Juárez et al., 2013; González-Flores et al., 2006; Mani et al., 2000; Mani et al., 1994).

NO is a gaseous neurotransmitter that diffuses throughout the body and is crucial in several reproduction-related brain functions. This molecule is produced from L-arginine by NO synthase (NOS), and its activity is induced by EB in the neurons of the ventromedial hypothalamus (VMH) in female rats (Garthwaite and Boulton, 1995). In intact cycling female rats, NO levels in the hypothalamus increase twofold between the afternoon and evening of proestrus (Lamar et al., 1999), coinciding with the GnRH/LH peak and the period of behavioral receptivity. In OVX female rats primed with EB plus P, inhibition of NOS or scavenging of NO attenuated lordosis, whereas an NO donor facilitated lordosis without P (Mani et al., 1994). These findings suggest that cGMP, synthesized after NO-activated soluble guanylyl cyclase (sGC), could be an essential cellular second messenger to stimulate lordosis in hormonally primed female rats. To test this idea, the 1H-[1,2,4]oxadiazolo[4,3-a]quinoxa-lin-1-one (ODQ), a selective inhibitor of NO-stimulated sGC, was used. ODQ infusion into the lateral or third ventricle suppressed lordosis in a dose-dependent manner in OVX female rats treated with EB plus P (Chu and Etgen, 1997).

The present study had a specific objective: to investigate whether the nitric oxide pathway (NO/cGMP/PKG pathway) is involved in the activation of female sexual behavior in OVX rats pre-treated with EB. This investigation was carried out through the administration of the following inhibitors: L-NAME (a nitric oxide synthase inhibitor), ODQ (a specific soluble guanylyl cyclase inhibitor), and KT5823 (a protein kinase G inhibitor). Our interest in conducting this study arises from our recent report, in which we demonstrated that the direct infusion of apelin-13 into the VMH induced dose-dependent lordosis in OVX rats primed with EB (García-Juárez et al., 2022). However, numerous questions remain regarding the signaling pathways involved in the modulation of this reproductive event by apelin-13.