The Adaptation Mechanism of Nitric Oxide
The Adaptation Mechanism of Nitric Oxide
1. Introduction
Stress is a state of threatened homeostasis, which initiates a state of physiological manifestations to antagonize the influence of stressors. Limbic-Hypothalamus-Pituitary-Adrenal Axis (LHPA) regulates the stressful condition. Amongst the various stress manifestations anxiety is one, which can be easily marked in experimental animals. The consequence of repeated exposure to stress is habituation or adaptation leading to gradual inhibition of primary responses. Various excitatory neurotransmitters activate the corticotropin releasing factor (CRF) increasing anxiety. To down regulate the activation of stress manifestation various neurotransmitters and neurohormones play an active role in developing adaptation. The studies carried on neuronal mechanism of stress manifestations include characteristic feed forward and feed back pathways involving several hormones and neurotransmitters but interlinking relationship between neurotransmitters have not been classified.
The elevated plus maze is a validated animal model of anxiety that is based on the natural aversion of rodents to open spaces and is a sensitive model to the effects of both anxiolytic and anxiogenic agents. Animal placed in the centre tends to move to the closed arm due to fear or anxiety. On repeated exposure to the instrument there is a gradual decline in the time spent in closed arm. Luteinizing hormone-releasing hormone (LHRH) and nitric oxide (NO), the neurohormone and neuromodulator, apart from various peripheral and central activities influence behavioral activities. The agonistic analog
of LHRH was found to exert sedation, anxiolysis, neuroleptic and analgesic activities (Kadar et al.,
1990). Nitric oxide mediates anxiolytic effect (Caton et al., 1994). Nitric oxide has been found to be a genetical factor in certain rats providing a resistance to stressful condition and involved in adaptive process (Pshennikova et al., 2001, Gulati et al., 2007). The exhaustive study reveals that nitric oxide negatively influences LHPA axis. Nitric oxide also inhibits CRF release and conversely activates the release of LHRH. The behavioral activity of CRF is opposed by LHRH (Telegdy et al., 1990). L-arginine, nitric oxide precursor and N (G)-nitro-L-arginine methyl ester (L-NAME), nitric oxide synthase inhibitor were used as nitric oxide modulators. To observe the effect of LHRH, the third generation LHRH antagonist, Antide([N-AC-D-Nal(2)',pCl-DPhe2,o-Pa1(3)3,Lys(Nic)5,o-Lys(Nic)6,Lys(iPR)~,o-Ala'o] GnRH), was used. Antide has a high affinity but with minimal histamine release side effects. It carries many amino acid substitutions with respect to the structure of the native GnRH decapeptide. Antide elicit unusual long-lasting inhibitory effects on gonadotropin secretion.
The involvement of stress during the exposure of the animal to the elevated plus maze is uncertain. Thus, to establish a certain stressful situation and farther adaptation the audiogenic stressor was applied before exposing the subjects to elevated plus maze. Therefore, the aim of the present study was to investigate the participation of nitric oxide in adaptation mechanism through LHRH in stress induced subjects.
2. Materials and methods
- Animals
Male swiss albino mice (obtained from CPCSEA approved animal house Nagpur University, Nagpur, India, 20-25g) were housed in a cage under a 12-hr/12-hr light/dark cycle (lights on at 07:00, lights off at 19:00) at a constant temperature of 23 1oC. Food and water were available ad libitum for 1 week before the experiments. Mice were handled 1 day before the test. This study was performed according to the guidance of Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and Institutional Animal Ethics Committee (IAEC).
- Drugs and administration
L-Arginine (Loba Chemie, India), L-NAME (Sigma-Aldrich Lab) and Antide (Sigma Laboratories, USA) were freshly prepared in 0.9% saline. Mice were divided into 10 groups of 8 mice each. Five of the groups were introduced to audiogenic stress and the other five groups were kept as control. Each of the five groups of control and stressed separately were administered with saline (10ml/kg, i.p.), L-Arginine (300mg/10ml/kg, i.p.), L-NAME (40mg/10ml/kg, 100mg/10ml/kg i.p.) and Antide (1g/mouse, s.c.), respectively.
- Elevated Plus Maze test
The experiment was carried out as per reported method (Lister, 1987). Mice from each group were individually placed in a four arm maze made of plexiglass consisting of two opposite closed arms (16 cm long x 5 cm wide x12 cm high) and two opposite open arms (16cm x 5 cm) elevated to a height of 25cm above the surface (Sharma and Kulkarni, 1991). The arms extended from a central platform 5 x 5 cm. Mice were placed in the center of the maze facing the open arm for a test period of 300 seconds. The total time spent by an animal in the open arm is considered as the index for anxiolytic behavior. The apparatus was cleaned with wetted towels after each test.
- Audiogenic Stress
The stress was applied to the animals with the help of buzzer with decibel range from 85-90(Armario et al., 1984). The five groups of animals stressed were exposed to the buzzer in a sound attenuated closed chamber. The stressor was applied for a period of 6 h. The buzzer was switched on for 10 seconds after every 2 minutes. The stressor was daily applied for a period of 14 days before subjection to elevated plus maze.
- Assessment of Adaptation
All behavioral tests were carried out in a dimly lit room. The control mice and stressed mice were subjected to the elevated plus maze individually after receiving the respective dose of individual drug, half an hour prior to the test. The stressed group received the treatment after audiogenic stress exposure. The time spent on the open arm by the animals is taken as the negative index of anxiety and the development of adaptation.
2.6. Statistical Analysis
Data for the Elevated plus maze was analyzed by one-way and two-way analysis of variance (ANOVA). Significance was set at P<0.05. Data are represented as mean S.E.M.
3. Results
3.1. Development of adaptation
The effect of 14 days exposure of control to elevated plus maze and exposure to audiogenic stress and then effect of elevated plus maze on stressed animals is shown in Fig. 1. There is a gradual increase in time spent in open arm upto 10th day and on 11th day onwards the animals remained in the open arm for a consistent period with the development of adaptation. The adaptation developed in stressed animal lesser than control.
3.2. Effect of Nitric oxide on adaptation
The open arm stay of mice after L-NAME treatment at two different doses on control and stressed group is shown in Table 1 and Fig. 2. In control group the consistent stay of animals in open arm or adaptation is inhibited by L-NAME dose dependently. The L-NAME treatment also showed a dose dependent inhibition in adaptation process on audiogenic stressed mice.
The L-Arginine treatment on control and stressed animals has been shown in Table 1 and Fig. 2. It was observed that L-Arginine alone did not have influence on adaptation but it activated adaptation in stressed mice.
3.3. Effect of Luteinizing Hormone Releasing Hormone (LHRH)
The effect of LHRH antagonist on adaptation is shown in Table 2 and Fig. 3. It was observed that LHRH antagonist impaired the process of adaptation on both control and audiogenic stressed mice.
4. Discussion
Several physiological changes have been identified when an individual is subjected to a stressful stimulus. It is believed that LHPA axis and several other neuronal pathways participate in the precipitation of stress response. But intensities of stress response decrease if body is repeatedly subjected to a similar stimulus. This phenomenon suggests the adaptive mechanism and effect of adaptive mechanism broadly depends on the nature, period of application and the intensity of the stressor (Armario et al., 1984). It is evidenced that there is substantial attenuation of CRF expression due to chronic or repeated exposure to a stressor (Bonaz and Rivest, 1998). This observation suggests that the adaptive mechanism may be responsible for decreased adrenocorticotropic hormone level and its response.
Anatomical and molecular studies have suggested the interactive relationship of LHPA axis with wide variety of neurons. The commonly found neurons are serotonergic, dopaminergic and GABAergic. Besides the neurotransmitters several neurohormones, neurosteroids, neuropeptides and biochemical end products are known to monitor the activity of LHPA axis. Some of these agents are positive modulators and some are likely to be negative. The positive modulators are responsible for expression of stress response whereas the negative modulators either may have a limiting role or may participate in the adaptive mechanism.
The exhaustive literature survey reveals that the mechanism of stress response has been duly emphasized whereas not much attention has been paid to the mechanism of adaptation. Nitric oxide is a diffusible gas, which is produced by any cell from arginine by expressing an enzyme, nitric oxide synthase. The evidences indicate that it can influence the LHPA axis and may down regulate the release of various hormones, which characterizes LHPA axis, such as CRF, Adrenocorticotropic hormone and corticosteroids (Kishimoto et al., 1996; Lopez-Figueroa et al., 1998; Weidenfeld et al., 1999; McLeod et al., 2001). Incidentally, it has been shown that nitric oxide synthase containing neurons are histochemically located in magnocellular and parvocellular subdivision of hypothatlamic nuclei (Riedel, 2000). These neclei control the neurosecretory processes, especially CRF. Probably, therefore, nitric oxide is known to inhibit the CRF release in median eminence (Weidenfeld et al., 1999).
In order to develop to the environment of elevated plus maze, the control group of animals was daily subjected to elevated plus maze at a specified time each day until a consistent stay of the animals in the open arm was observed. Interestingly it was observed that there is a gradual increase in the time spent in the open arm from day 10 onward indicated the lowering of anxiety in the mice. Since, this behavior was consistently shown by all the animals of control group, the working protocol, was found to be dependable to establish adaptation in animals.
Since the objective was to study the mechanism of adaptation to a stressful situation, an additional stressor was introduced to confirm whether the subjection of mice to elevated plus maze is stressful or is the natural behavior of the animals to stay in the open arm of the maze.
In the audiogenic stressed group of mice, it was observed that the additional stressor as compared to the control slowed the development of adaptation down. Further it was seen that animal could not attain same level of adaptation as that of control even upto 14 days. Though the level of adaptation in the stressed group is not same as that of the control but the gradual increase in stay of the animals in open arm indicated the partial development of adaptation in mice. These observations in the control and stressed group suggests that repeated subjection of animal to elevated plus maze can be a dependable model to study the adaptation mechanism. The present investigation has substantiated the fact that exposure to elevated plus maze develops anxiety. Further, the investigation documented that repeated exposure to the same stressor declined the level of anxiety indicating the development of adaptation in mice.
Using the above two protocols, the basic objective of finding the involvement of nitric oxide in adaptation was investigated. For the purpose to eliminate nitric oxide, the nitric oxide synthase was inhibited by L-NAME. The mice were treated with L-Arginine, nitric oxide donor, prior to the test. The studies revealed that L-NAME significantly impaired adaptation in the control and stressed mice. It was further observed that L-arginine treatment though did not produce much alteration in the control mice, but ameliorated adaptation in the stressed mice. It has been reflected that the level of adaptation in the vehicle treated stressed mice were much lesser than the vehicle treated control group. These findings indicate that the nitric oxide is probably involved in the process of adaptation. The pretreatment with L-Arginine did not eliminate the anxiety levels in both the groups in the initial days of the test; suggest that L-Arginine does not posses any anti-stress effect. However, it has been reported that nitric oxide had an anxiolytic effect(Li and Quock, 2001; Spiacci et al., 2008) and nitric oxide synthase inhibitor shows anxiogenic property (Vale et al., 1998; Monzon et al., 2001; Workman et al., 2008). The anxiogenic effect is dose dependant. Even though nitric oxide is anxiolytic in nature but the nitric oxide donor L-Arginine, did not show any anxiolysis in the initial phase of adaptation. This is probably because of maximum release of CRF, which in itself is known for anxiogenesis. This also supports the possibility of reduced release of CRF in the event of adaptation. The effect of L-NAME and L-Arginine on the adaptation process in control and stressed mice suggested the involvement of nitric oxide in the process of adaptation. Since, nitric oxide has been found to play a major role in adaptation, studies to find whether inducible and constitutional nitric oxide synthase inhibitor is responsible for adaptation.
A continued/chronic/repeated stress involves over activation of various neuronal systems, irrespective of its metabolic status and the available blood supply. In the event of over activation, the metabolic substrates in the neurons go up and the increased circulation can only meet this increased demand of the substrate. Incidentally, nitric oxide is known for its vasodilatory effects (Rang et al., 2007). At the same time, the process those are responsible for over activation if down regulated by nitric oxide, the need for increased demand of metabolic substrate can also be avoided. Literature documents that nitric oxide reduces the responsiveness of various components of LHPA axis to their proximal sites (Riedel, 2000).
Since, L-Arginine is an amino acid, the possibility of gluconeogenesis in the over activation of any cell cannot be ruled out. In view of this, it can be hypothesized that in the event of repeated stress the over utilization of metabolic substrate is probably derived by breaking the protein, which leads to the exhaustion of its store and energy. This may result in the release of arginine. The eventually formed nitric oxide, may improve the circulation and/or prevent the proximally situated excitatory or down regulate the proximal stimulus of the response of the affected neuron. It is also possible that the nitric oxide, thus produced may also activate the release of LHRH (McCann et al., 2003), and on the other side down regulate the release of CRF. The present observation, that prior treatment of mice with LHRH antagonist significantly impaired the adaptation in control as well as in the stressed group, further substantiates the possible involvement of LHRH in the process of adaptation. Incidentally some reports have already indicated the role of nitric oxide in the release of LHRH (Moretto et al., 1993). Moreover it has been seen the behavioral activities of LHRH is opposite to that of CRF. LHRH is involved with the anxiolytic effect (Umathe et al., 2008). However to confirm the involvement of nitric oxide mediated release of LHRH, in adaptation, further studies are required.
Concluding it can be said that nitric oxide and LHRH are probably involved in down regulating expressions of stress response when mice are repeatedly exposed to a stressor. It is the primary attempt to find the role of nitric oxide and LHRH in the process of adaptation. In order to confirm the role of LHRH and its relationship with nitric oxide, interactive studies with nitric oxide and LHRH modulators should be carried out.
Acknowledgements
The authors are grateful to the staff and students of the Department of Pharmacology in Nagpur University for the facilities provided to carry out the entire work on adaptation.
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