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=The Role of Nitric Oxide in Sexual Behavior and Health in Women=

Abstract
Nitric oxide (NO) is a naturally occurring, gaseous molecule located in the central nervous system that helps regulate sexual behavior by interacting with several different neurotransmitters and hormones. Studies have been completed on various species, including rats, sheep, guinea pigs, and humans. NO stimulates the release of luteinizing hormone-releasing hormone, which onsets sexual behavior. NO also works with dopamine, oxytocin, and progesterone to make reproduction successful. It has been shown that in terms of sexual function, testosterone and estrogen (the gonadal hormones) help regulate NO. Cyclic guanosine monophosphate is also a regulator of NO synthesis, since it is required to initiate the binding of the substrate, L-arginine, to the enzyme, NO synthase. NO may also interact with norepinephrine to generate sexual behavior in females; NO and norepinephrine are correlated with the onset of sexual behavior, although it is still quite unclear as to the cause and effect relationship between the two. More research studying nitric oxide’s function in sexual behavior in females will determine the mechanisms that include nitric oxide and result in sexual function or dysfunction, which may help diagnose and treat women with certain sexual dysfunctions and possibly even fertility.

Introduction
Nitric oxide is a naturally occurring free radical in the human body. It functions in regulating sexual behavior, contributing to platelet coagulation (wound healing in general), modifying smooth muscle, developing cells (especially oocytes), and apoptosis [9, 19]. Nitric oxide also plays an important role in neurotransmission and in the regulation of neurotoxicity [9, 12, 19]. Nitric oxide is important to study with respect to sexual function in females because it closely interacts with other hormones and neurotransmitters to cause display of sexual behavior. Understanding the function of nitric oxide may help to advance the diagnosis and treatment of sexual dysfunction and infertility in women [5, 19]. Nitric oxide (NO) is a gaseous and found in the nervous system of various species [1], where it can easily diffuse through cellular membranes [19]. Recent studies focus on NO pathways in rats, mice, guinea pigs, cats, monkeys, pigs, ewes, and humans [1]. NO was first thought to be simply a local hormone. However, with the advancement in the study of NO, it is now believed to be a neurotransmitter that regulates other neurotransmitters and sexual behavior and reproduction in both males and females [2, 18]. NO is naturally synthesized by the de-amination of L-arginine (Arg) to an intermediate, NOHA (N-hydroxy-L-arginine). NOHA is then oxidized to NO and citrulline [4, 17]. In order for NO to be synthesized, a catalyst must be present: nitric oxide synthase (NOS). There are three isoforms of NOS: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) [13]. eNOS and nNOS use calcium-dependent calmodulin (CaM) to initiate and regulate NO synthesis. iNOS is derived from macrophages and is calcium independent [1, 13]. All three isoforms of NOS contain iron-centered heme and require NADPH as a cofactor, as well as FAD, FMN, and tetrahyrdobiopterin (THB) for the donation of electrons [1, 17]. Molecular oxygen is also required for the formation of NO [1]. To initiate the activation of eNOS and nNOS, CaM binds to the enzyme, which leads to the transfer of electrons from NADPH to heme. This first transfer of electrons results in a ferrous heme-NO complex [16, 17]. The reduction of the heme-NO complex to release NO is the rate-limiting step in the synthesis of NO [17] and requires the donation of one electron from THB and another electron from NADPH via FMN [16]. For the activation of iNOS, inflammatory cytokines and lipopolysaccharides bind to the enzyme complex [19]. NO synthesis in the endothelial cells, neuronal cells, and macrophages is regulated by NO and the ferrous-heme complex via a negative feedback loop. An increase in NO inhibits the ferrous-heme complex from releasing any NO that is bound to it. In endothelial and neuronal cells only, the NO bound to the ferrous-heme complex prevents the complex from binding to CaM, which inhibits the initial step [16, 17]. In macrophages, the ferrous-heme complex inhibits the binding of inflammatory cytokines and lipopolysaccharides to iNOS [19]. Inhibitors of NO synthesis are useful for determining what effects NO has on physiological function. Two unnatural inhibitors used in recent studies are Ng-momomethyl-L-arginine (L-NMMA) and nitro-L-arginine methyl ester (L-NAME). Both of these inhibitors have been shown to block NO synthesis in vitro and in vivo [7, 10, 12, 19]. These inhibitors have also been shown to attenuate the sexual response of lordosis, the arching of the spine, in female rats [10]. Also, in estrogen primed rats, lordosis was completely blocked with the addition of L-NMMA [12]. NO donors have the opposite effect of the inhibitors: they amplify NO synthesis instead of blocking it. The two most common NO donors used in recent studies are sodium nitroprusside (SNP) and S-nitroso-L-acetyl-penicillamin (SNAP) [7, 19]. During meiosis of oocytes, the addition of SNP has been shown to increase the maturation rate. However, conflicting studies have shown that the increase in maturation rate may lead to developmentally incompetent oocytes [19]. Like NO, the inhibitors and donors are active throughout the nervous system in various species. Their activity can help researchers determine the effect nitric oxide has on neurotransmitters, especially the gonadal hormones, which may help to determine how sexual behavior is influenced by nitric oxide. However, nitric oxide is not the only molecule involved in the sexual behavior and function of women. Nitric oxide works with other neurotransmitters and hormones to regulate sexual behavior [5].

Cyclic Guanosine Monophosphate
Cyclic guanosine monophosphate (cGMP) is a second messenger used to stimulate the release of luteinizing hormone, which prompts the onset of ovulation [1]. cGMP is generated through NO. NO activates guanlyl cyclase, the enzyme used to synthesize cGMP [14]. Increasing the amount of cGMP, results in increased calcium. This increase in calcium results in an increase in NO produced from endothelial and neuronal cells (since eNOS and nNOS need calcium to initiate NO synthesis), resulting in positive feedback. cGMP and NO will continue to increase until another hormone is introduced to inhibit the cycle [15, 19]. It is hypothesized that the NO-cGMP pathway described above allows hormones and neurotransmitters, especially the sex hormones and its derivatives [10], to be released, which results in an increase in sexual behavior [14]. Increasing the amount of cGMP in rats has been shown to increase the lordosis response [10]. Inhibitors of this pathway include NOS inhibitors and cGMP inhibitors. When both types of these inhibitors are introduced into female rats, lordosis behavior is lost [13] and sexual functioning is diminished [3].

Progesterone
Progesterone is a hormone that plays an important part in female sexual behavior and reproduction [5]. Injections of progesterone result in intense sexual behavior depending on the amount, indicating that progesterone determines the intensity of sexual behavior [10]. Sexual behavior that was induced by the addition of progesterone has been shown to be inhibited by inhibitors of NO. This indicates that NO influences progesterone and its effects on sexual behavior and function [5]. Studies have shown that an increase in NO results in the activation of progesterone receptors, and these progesterone receptors regulate lordosis behavior in guinea pigs [7]. This finding has also been reported for ewes [8]. In other studies, it has been proven that neurons with progesterone receptors also have NOS, indicating a direct relationship between progesterone and NO. However, it is highly doubted that progesterone has a role in NO’s regulation of lordosis. When a NOS inhibitor was injected into female rats before the injection of progesterone, lordosis was blocked. This implies that NO may have a regulatory role over progesterone. However, the exact role of neurons with progesterone receptors and NOS is unknown as is the relationship between progesterone and NO[7].

Testosterone
Testosterone is a gonad hormone most known for triggering the onset of the secondary sex characteristics in males during puberty. However, testosterone is also important in females and is responsible for the engorgement of the clitoris and for the sex drive. In females with higher than normal levels of testosterone, male sexual behavior that does not require male sex organs can be observed [20]. It is also known that testosterone promotes other neurotransmitters to be released, especially dopamine [11, 20]. From recent studies, it is now thought that testosterone promotes the release of dopamine by upregulating NOS, which in turn promotes the release of dopamine [11]. Studies are still being done to determine the exact pathways in which testosterone promotes dopamine and the role of nitric oxide.

Dopamine
Dopamine is a neurotransmitter responsible for male sexual behavior and is released before and during sexual intercourse. Sexual behavior in females may also be regulated by dopamine, but norepinephrine is the dominant regulator [11]. Dopamine is required for initiation of sexual behavior in both sexes [20]. It allows pathways for other neurotransmitters to become open and for sexual arousal and intercourse to occur [11]. The relationship between dopamine and nitric oxide is unclear and still being studied. However, it has been shown that when a NOS inhibitor is introduced to various animals, dopamine release was decreased, and sexual activity did not occur. This indicates that NO is a regulator of dopamine. When combined with testosterone, it has been shown that the presence of testosterone increases NOS activation, which increases NO synthesis. NO elevates the release of dopamine, which elevates sexual motivation, reflexes of the sex organs, and the probability of the act of intercourse [11, 20].

Norepinephrine
The interaction between NO and norepinephrine is probably the most unclear. Norepinephrine (NE) is in the class of catecholamine and acts as a hormone and a neurotransmitter. NE is responsible for sexual behavior and function in females, much like dopamine in males [2]. It has been shown that NO regulates the release of NE from neurons in the hippocampus and the cortex parts of the brain, which then releases the luteinizing hormone-releasing hormone (LHRH) [2, 15]. However, there are conflicting studies showing that NO does not regulate the release of NE. This may be explained by the different methods that were used in each experiment. Other studies have concluded that NE stimulates the release of NO [1]. Given these conflicting studies, it is hypothesized that NO and NE also partake in a feedback loop that regulates sexual behavior. However, the exact mechanism is still unclear [1, 3, 15]. More research needs to be completed to determine a standard operating procedure for these studies and to determine the effect NO has on NE release from neurons [2].

Luteinizing hormone-releasing hormone
Luteinizing hormone-releasing hormone, also called gonadotropin-releasing hormone (GnRH), functions in signaling the luteinizing hormone (LH) to be released from the pituitary gland. The release of LH results in the onset of ovulation [19]. Speculation about the relationship between NO and LHRH started upon the discovery that neurons containing NOS are in close vicinity to neurons that release LHRH [12]. NO has been shown to activate the release of LHRH in rats and LH in cows [19]. In the central nervous system of the woman, it has been demonstrated that NO regulates the release of LHRH, shown by the use of inhibitors. When NO inhibitors were introduced, particularly L-NMMA, NO synthesis was blocked, and LHRH was repressed [15]. NO donors were also added to determine the validity of the role of NO in LHRH release. When SNP was introduced, LHRH levels increased [1]. LHRH is known to promote the release of LH and also stimulate sexual behavior. Through the regulation of LHRH, NO regulates sexual behavior in females [1, 12, 15].

Multiple Pathways
The exact roles of dopamine, progesterone, NO, and NE in the pathway to sexual behavior are still undetermined. Studies showed that the presence of NO increased the presence of dopamine, which then lead to sexual behavior [11, 18], and then, dopamine leads to the activation of progesterone [20]. The activated progesterone then increases NADPH which increases NO synthesis, creating a feedback loop [8]. Activated progesterone also induces LHRH release, leading to sexual behavior [15]. However, it is still unknown if both progesterone and NO induce LHRH release, or if LHRH is release by one and regulated by the other [5].

Oxytocin
Oxytocin is one of the most abundant peptides in the body. It is required for the proper function of the mammary glands and for contractions during child birth. It has also been shown that oxytocin plays an important part in sexual behavior and maternal instincts. Injecting oxytocin into female rats resulted in lordosis [15]. It has been suggested that NO may modulate the release of oxytocin [1], since neurons containing NOS are the same neurons that contain oxytocin [15] and NO; as well as oxytocin levels increase during pregnancy [10]. Experiments have demonstrated that when L-NMMA is introduced into a female, the amount of LHRH released by the promotion of oxytocin decreased. This indicates that NO may be a modulator of oxytocin. It also indicates that there is a more complicated dependence between NO, oxytocin, and LHRH [1, 12, 15]. However, there have been conflicting experiments reported. Some studies have shown that the presence of oxytocin stimulates and enhances NOS to synthesize NO [15, 19]. Similar studies have found that NO regulates oxytocin release [1]. It is possible that oxytocin promotes the synthesis of NO and that the increase in NO production blocks further release of oxytocin, as a feedback loop. However, enough information has simply not been observed to prove this hypothesis valid [1, 15, 19].

Estradiol
Estradiol is a classification of three sex hormones. Estradiol functions in regulating the development of the sex organs in females of different species. In humans, the predominant estradiol is estrogen, although the other two types may be present. Estrogen is responsible for the secondary female sex characteristics that develop in girls during puberty. Estrogen also regulates vaginal lubrication and the thickening of the uterine wall during the menstrual cycle and pregnancy [18]. During the preparation for sexual intercourse (foreplay), estrogen functions in priming the neural connections and releasing vaginal lubrication [10]. Estrogen is also known for regulating dopamine, which also plays an important role in sexual behavior. Increasing estrogen levels results in an increase in dopamine levels in the limbic system of the brain, the area that is linked to the pleasure center. Likewise, decreasing estrogen leads to a decrease in sexual dopamine release [8, 11]. The relationship between NO and dopamine has already been described, but what is the relationship between estrogen and NO? It has been shown that in order to enhance estrogen caused sexual behavior (i.e. increased vaginal lubrication) NO levels can be increased to achieve the desired affect [10, 20]. It has also been shown that NOS acts upon neurons with estrogen receptors [20]. Not only does NOS act upon neurons with estrogen receptors, NOS is found within neurons that contain estrogen receptors [8]. Studies with estrogen receptors have also showed that when estrogen was introduced to neurons in the hypothalamus region of the brain, the number of NADPH cells increased, which lead to an increase in NO. This implies that estrogen may be a regulator of NO [8]. It has also been determined that NO regulates ovulation and the movement of oocytes during menstruation; however, the exact process is unknown, as well as which neurotransmitters have an effect. When NOS was removed from mice, ovulation ceased, indicating that NO is essential for reproduction. It was thought that NOS containing neurons in the brain remain constant in the expression of NOS during the menstrual cycle. This would imply that NO is irrelevant to reproduction. However, newer studies have focused on NOS containing neurons in the limbic system portion of the brain, which feeds into the brain’s pleasure center. These studies have reported that throughout the menstrual cycle and various estrogen levels, NOS levels significantly fluctuate. However, the overall NOS levels throughout the entire brain remain constant. Therefore, it is hypothesized that as NOS increases in the limbic system during the menstrual cycle, it decreases in other areas of the brain (i.e. the ventrolateral section of the ventromedial nucleus). In support of this hypothesis, the same study showed that there are two different types of NOS containing neurons: neurons related to the germ cells (sex cells) and neurons related to the somatic cells [5, 18].

Regulation of Nitric Oxide
Most of the analyzed studies considered interactions between NO and only one or two different neurotransmitters. However, sexual behavior in females and males arises from all of the neurotransmitters working together. NO is an important regulator of molecules that contribute to sexual behavior, but NO needs to be regulated as well. Recent studies have shown that the gonadal hormones regulate NO. When testosterone was increased in neurons, NOS activity was increased [11, 20]. Studies with estrogen have shown that an increase in estrogen results in an increase in NADPH cells. Since NADPH is a cofactor of NOS, NO synthesis is increased [8]. Previously, it was thought that estrogen, along with NE, regulated LHRH release, and thus, regulated LH [10]. However, now it is known that estrogen acts through NO to regulate the release of LH [1, 10, 12, 15]. NE, like estrogen, can stimulate NO release which releases LHRH [1]. Another regulator of NO is cGMP. Reduced cGMP, reduces the rate of NO synthesis. cGMP inhibitors prevent the release of LH because no cGMP is available to initiate NO synthesis [11]. An increase in estrogen results in an increase in calcium. Since cGMP is calcium dependent, this increase in calcium leads to an increase in NO synthesis, which activates LHRH, [12] and leads to sexual behavior [19].

Conclusion
Due to the vast amount of studies preformed, it is known that nitric oxide plays an integral part as a neurotransmitter in regulating sexual behavior. However, the mechanism of the specific interactions between NO and all the other hormones and neurotransmitters as a functioning system is still unknown. The relationship of NO with individual hormones and neurotransmitters is known, although the mechanisms and exact reactions are still undetermined or still hypothetical [1]. More comprehensive studies need to be completed in order to determine the exact role that nitric oxide plays as a neurotransmitter, and how it interacts with other molecules that are known to function in sexual behavior. In order to determine the proper function of nitric oxide in sexual behavior along with the pathways and reactions that result in the behavior, more information is required [5, 19].