Possible role of nitric oxide (NO) in the regulation of gender related differences in stress induced anxiogenesis in rats

Highlights

• Restraint stress induced greater anxiety in males than females.

• Brain NO was suppressed and ADMA enhanced more in males.

• L-arginine attenuated and L-NAME aggravated such anxiogenesis.

• Formestane aggravated stress induced behavioral and biochemical changes in females.

• Gender differences in such anxiety involve estrogen–NO interactions.

Abstract

Gender related differences in stress induced neurobehavioral disorders have been reported although the mechanisms involved are not yet clear. The present study investigated the role of nitric oxide, an important biomodulator in the sex related differences in stress induced anxiety like behavior in rats. Restraint stress (RS for 1 h) was used as the experimental stressor and the effects of NO modulators were assessed in the elevated plus maze (EPM) test in both male and female rats. No metabolites (NOx) and asymmetric dimethyl arginine (ADMA) were measured in brain homogenates of these rats for corroborative purposes. RS induced anxiogenesis in both male and female rats and such changes were greater in males as compared to females. The behavioral alterations were associated with enhanced levels of ADMA and reductions in levels of NOx in brain homogenates – the effects being greater in intensity in males as compared to females. Pretreatment with NO precursor L-arginine (500 mg/kg) reversed the RS induced behavioral and biochemical changes, while NO synthase inhibitor L-NAME (50 mg/kg) had opposite effects. Additionally, Formestane (50 mg/kg), an estrogen synthesis blocker, aggravated stress induced anxiogenesis with a corresponding increase in ADMA and decrease in NOx levels in the females. To our knowledge, this is the first report indicating the involvement of ADMA, an endogenous nitric oxide synthase inhibitor in stress induced neurobehavioral changes. Furthermore, it is also evident that nitric oxide and its interactions with estrogens play a crucial modulatory role in the differential anxiogenic response to stress among males and females.

Introduction

Stress can be considered as a state of threatened ‘homeostasis’ and ability to cope with stressful insults is crucial determinant of health and disease [1]. Both animal and human studies have shown that exposure to stress may lead to morphological and functional changes in the brain and can lead to development of behavioral disorders associated with cognitive impairment, depression, fear and anxiety [2], [3]. There have also been reports showing that there are distinct gender related differences in stress-responses and in susceptibility to stress-associated disorders [4], [5], but the mechanisms involved are not yet clearly understood.

Nitric oxide signaling system plays important role in anxiety modulation [6] and NO synthase (NOS) is present in regions involved with anxiety such as hypothalamus, amygdala and hippocampus [7]. Besides, central nitrergic mechanisms are also known to play crucial role in both stress susceptibility and adaptation [8], [9], [10]. Since both ROS and reactive nitrogen species (RNS) may be simultaneously generated during various pathophysiological states, the balance between two moieties may play a more vital role in the initiation and progression of various disease states [11], [12]. We have earlier demonstrated that reactive oxygen species (ROS) are involved in the differential anxiogenic response to stress among males and females [13], [14].

Asymmetric dimethylarginine (ADMA) is a major endogenous inhibitor of NOS and plays a key role in regulation of NO synthesis [15]. Elevated ADMA levels are associated with increased risk of stroke and ischemia attacks and is also implicated in the pathophysiology of cognitive impairment [16], [17]. Oxidative stress has been shown to decrease the activity of ADMA degrading enzymes leading to increased ADMA concentrations [18]. Thus this endogenous inhibitor of NO synthase plays an important role in maintaining the cellular ROS–RNS balance. However, to the best of our knowledge, the role of ADMA during stress induced neurobehavioral responses has not yet been studied.

Estrogens are known to influence a wide variety of functions in the mammalian brain ranging from regulation of various aspects of neurotransmitter function and modulation of behavior to the stimulation of differentiation and plasticity of distinct neuronal populations and circuits [19]. There is substantial evidence suggesting that estrogen can interact with stress systems to produce unique stress effects in females [20]. Studies performed in both peripheral and brain tissues have also demonstrated that estrogen influences the NO system [21], [22]. In vivo estrogen treatment increased NOS activity in cerebral blood vessels [23] and McNeill et al. [24] have suggested increased NO production may contribute to the neuroprotective effects of estrogen. However, there is paucity of information whether estrogen–nitric oxide interactions play any role in sex related differences in stress induced anxiogenesis.

Therefore, in the present study, we tested the hypothesis that exposure to stress will differentially alter ADMA and thereby NO levels in males and females and such alterations would be associated to sex related differences in stress induced anxiety like behavior. In addition, we conducted studies to determine how pharmacological blockade of estrogen synthesis/receptors affects stress induced anxiety like behavior as well as nitrosative and oxidative stress markers in females so as to gain a better insight into the regulatory role of NO in the gender related differences in stress induced neurobehavioral changes.