Abstract: The aim of this study was to investigate the effects of Ang II and captopril on altering the motivational aspects during the initiation of morphine self-administration. Male Wistar rats were first trained to receive small pellets of food by pressing the active lever in self-administration apparatus. They were anaesthetized with Ketamine and their jugular vein was cannulated. The stainless steel cannula was also inserted into the right brain ventricle and fixed with dental cement. After recovery, the animals were divided into 4 groups (saline, morphine, captopril and Ang II) and placed in self-administration apparatus and allowed to self-administer morphine (1.7 mmol per infusion all test groups) or saline (saline group) during 11 consecutive days for 2 h/sessions. Captopril (300 mmol) and Ang II (1 nmol) injected (i.c.v.) in the corresponding groups before each session. The number of active and passive levers pressed in each group was recorded. After the last session, morphine withdrawal signs were recorded following naloxone injection. In morphine group, the number of active lever pressing was significantly higher than passive one in all 11 days (p<0.01) and was also significantly higher than the saline group in the final three days (p<0.05). In captopril group, there were no significant differences between the number of active and passive lever pressings during free access to food (last 5 days). However, the number of active lever pressing was significantly lower than morphine group (p<0.05). Some of the withdrawal signs decreased and increased significantly in captopril and Ang II groups, respectively. This study implies the interaction between captopril and opioid system.
INTRODUCTION
The renin-angiotensin system (RAS) has been described initially as a circulating humoral system. Evidences have indicate that the brain is capable of synthesizing angiotensin peptides. All components of the renin-angiotensin system and enzymes which are necessary for the formation and deactivation of angiotensin peptides are present in the brain (Allen et al., 1999). Angiotensin I is enzymatically cleaved from angiotensinogen by renin and is converted to angiotensin II (Ang II) by angiotensin converting enzyme (ACE) (Voigt et al., 2005). Angiotensinogen is found locally in the brain where it is thought to be a part of the local renin- angiotensin system (Baltatu and Bader, 2003).
Based to the existence of Ang II-immunoreactive neurons in distinct brain areas, including the mesolimbic system, it has been proposed that this peptide plays the role of a neurotransmitter in the central nervous system (Winnicka, 1999). Ang II also interacts with serotonin (5-HT) and catecholamines at the synaptic level (Wright et al., 1993). There is considerable support for the notion that Ang II and its fragments facilitate cognitive processes (Braszko et al., 1991; Wright et al., 1993). These effects of Ang II on passive and active learning were abolished by dopaminergic antagonists (Winnicka and Wisniewski, 1999). It seems that Ang II exerts most of its cognition-improving effects through the activation of the central dopaminergic system (Winnicka and Wisniewski, 1999). Further investigations with 6-hydroxyl dopamine (6-OHDA) disruption of dopaminergic endings in discrete structures of the mesolimbic dopaminergic system confirmed the involvement of this system in the angiotensin facilitation of learning and memory processes (Winnicka et al., 1998). There are several reports showing that the mesolimbic dopaminergic system is involved in rewarding properties of morphine (Elmer et al., 2005; Hnasko et al., 2005).
There are also controversial reports that Ang II may interact with the opioid system. It was shown that central Ang II antagonizes the opioid-induced analgesia (Han et al., 2000). Intracerebroventricular (i.c.v) administration of Ang II exerted a dose-dependent antinociceptive effect in the acetic acid-induced abdominal writhing test in mice (Georgieva and Georgiev, 1999; Han et al., 2000). Intrathecal administration of Ang II also induced a short-lasting antinocicptive effect in rats (Toma et al., 1997). It is assumed that these effects occurred through an opioid mechanism and activation of AT1 receptor (Tchekalarova et al., 2003). These data suggest that Ang II participates in the transmission of nociceptive information and its interaction with opioid receptors (Tchekalarova et al., 2003). Administration (i.c.v.) of Ang II produced antinociceptive effects that could be blocked by pretreatment with naloxone (Tchekalarova et al., 2003).
Alteration in the brain ACE activity has been reported in several studies (Koyuncuoglu et al., 1986; Raghavendra et al., 2001). Evidence shows that ACE inhibitors such as captopril reduces endogenous opioid degradation and increases the level of these opioids in the brain (Jenkins et al., 1997). ACE inhibitors also alter dopamine level in the brain, suggesting that these drugs could be used to treat Parkinson`s disease (Reardon et al., 2000). Also, it was demonstrated that the effects of ACE inhibitors on learning and memory (Nikolova et al., 2000; Raghavendra et al., 2001) can be blocked by naloxone (Martin et al., 1990). ACE inhibitors have also been used for the treatment of cocaine-abusing populations. These drugs could reduce cocaine use by modulating the levels of dopamine in the brain (Margolin et al., 2000).
This study was designed to determine the relationship between captopril (ACE inhibitor) and Ang II (the main product of renin- angiotensin system) with the rate of morphine self-administration.
MATERIALS AND METHODS
Animals and housing conditions: Before surgery for the establishment of the infusion system, based on the ethics committee permission of Isfahan University, male Wistar rats (270-320 g) were group-housed and received food and water ad libitum. They were maintained under a day-night cycle with lights on between 07:00 and 19:00 h 3 days before starting the experiments, the day-night cycle was reversed and the animals were tested in the dark phase. Before the tests, food and water were available ad libitum in the home cages (Alaei et al., 2005) during the training phase and the first 6 days of the experimental period, they had food restriction in their cages.
Drugs: Morphine chloride was supplied by TMAD Ltd., Teheran, Iran. Angiotensin II, pentobarbital, ketamine and Naloxone hydrochloride were purchased from Sigma Co, St. Louis, USA. Captopril and penicillin G were obtained from a commercial source (Daroo-Pakhsh Pharma, Iran). The drugs were dissolved in saline solution.
Self-administration apparatus: Briefly, to aid in acquisition of drug self-administration, rats were initially trained to press a lever using food as reinforcement before being surgically implanted with a chronic intravenous (iv) jugular catheter. Training and testing were done in standard operant conditioning cages (21x21x28 cm) placed in a sound-attenuated room, ventilated with fans, based on the method used previously by others (1) with minor modifications. The apparatus was equipped with active and passive levers, 2 cm above the floor, with a red light located 4 cm above the active lever. The i.v cannula was connected to an infusion pump via a swivel, allowing the animal to move relatively freely. Pressing of the active lever, marked by a red light, resulted in a 10 s infusion of 0.1 mL fluid through an infusion pump. The fluid was saline in the saline group and morphine (1.7 mmol per infusion) in other groups. Depression of the active lever during this time (10 sec) did not affect the infusion of the drug. Pressing of the passive lever had no programmed consequences. In this study, the number of lever pressing was regarded as a measure of the reinforcing action of the drug (Alaei et al., 2002; Sahraei et al., 2004).
Experimental design: Male rats (n = 44) were randomly selected and some of them were omitted during the experiment. These rats were divided into four groups: (1) Saline group, which received 5 μL saline i.c.v before each session and also in the self-administration sessions. (2) Morphine group, which received 5 μL saline i.c.v before each session and 0.1 mL of morphine in saline solution (1.7 mmol per infusion) during the self-administration sessions. (3) Ang II group, which received 0.25 nmol Ang II i.c.v 5 mins before receiving morphine in self-administration sessions. (4) Captopril group, which received 300 mmol captopril i.c.v 30 min before receiving morphine in self-administration sessions.
Training phase: One week before the experiments, animals were transferred to a special room and 3 days before the experiment, the day-night cycle was reversed (light on at 7 pm) and the experiments was carried out during the dark phase of the cycle. Before surgery, the training program was started after 24 h food restriction. The animals were placed in the self administration apparatus where a lever filled with food pellets was available. Each lever pressing resulted in the delivery of a 100 mg pellet (Sahraei et al., 2004). Each rat allowed self-training until 40 pellets being received. Following acquisition of lever pressing behavior, rats were returned to ad libitum food and allowed to gain their weight for 3 days, before the surgery was done (Sahraei et al., 1999, 2004).
Surgical procedures: i.v cannula, animals were anaesthetized with ketamine (546 mmol kg-1) and xylazine (0.1 μmol kg-1) and a cannula was inserted into the jugular vein. The cannula was guided subcutaneously up to the skull and connected to polyethylene tubing where it was fixed to a metal tube and secured to the skull with small screws, fixed with dental acrylic cement. Then, i.c.v cannula was implanted, as described below.
Intracerebroventricular cannula, after insertion of the i.v cannula, the head of each rat was placed in a stereotaxic instrument (Stolting Instruments, USA). Stainless steel, 23-gauge guide cannulas were implanted 1 mm above the right lateral cerebral ventricle. Sterotaxy was coordinated according to the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1998) (0.9 mm posterior to the bregma, +1.6 mm lateral to the sagittal suture and 3 mm from the top of the skull). Cannulas were fixed with dental acrylic cement anchored by two screws placed in the skull. A stylet (26-gauge stainless steel) was placed into the guide cannula to allow it to maintain patency. After surgery, rats were given 300,000 units of procaine penicillin G (i.p) to prevent infection. The animals were allowed 5-7 days to recover from surgery (Kim et al., 2005).
Intracerebroventricular injection: Rats were gently restrained by hand, the style was withdrawn from the guide cannula and a 27-gauge injection needle (1 mm beyond the tip of the implanted guide cannula) was inserted. The injection needle was attached to a 10 μL Hamilton syringe by a polyethylene tube. The solutions were injected in a total volume of 5 μL during 60 sec. The injection needle was retained in the guide cannula for an additional 60 sec after injection to facilitate diffusion of the drugs.
Self-administration phase: Seven days after recovery and following 24 h of food restriction, rats were placed in the operant chambers where a lever filled with food pellets was available. Each active lever pressing resulted in the delivery of a 100 mg pellet. Following recall of lever pressing behavior, the jugular cannula of rats were connected to an infusion pump and the animals were placed in the self-administration apparatus for 2 h each day on an FR-1 schedule for 11 days (Sahraei et al., 2004). The trained animals were allowed to press active and passive levers freely. By pressing the active lever, the rats received 0.1 mL of morphine or saline and small pellets in the first 6 days and saline or morphine without pellets in the final 5 days of the experiment. Pressing the passive lever did not deliver fluid or food. In the first 6 day of self-administration period, the availability of food was restricted in order to reduce body weight by 15% which has been shown to facilitate the initiation of intravenous self-administration (Sahraei et al., 1999). On the next 5 days, the animals had free access to their ad libitum food. Catheters were flushed daily with 0.1 mL saline containing heparin sulfate (50 IU mL-1) during the recovery period as well as before and after the self-administration sessions. All operant sessions were conducted during the animals` dark cycle. Catheter potency was tested by the injection of 4 μmol of sodium pentobarbital solution into the catheter and observation of animal behavior. Animals with patent catheters exhibit prominent signs of anesthesia (loss of muscle tone) a few seconds after the administration (Sahraei et al., 2004).
Withdrawal signs: One hundred and eighty minutes after the last self administration session, the animals received naloxone (3 mg kg-1 i.p) and were placed in plastic cylinders (50x18 cm) and the signs of morphine withdrawal were recorded for 30 min by a trained observer who was blind to the treatment. Abstinence signs were precipitated by naloxone in four experimental groups and they consisted mainly of wet dog shakes, the rat shakes its body, jumping, leaping off the surface of the cage, writhing, the rat lies on the floor while the belly is firmly pressing the surface, abdominal contractions are usually present, standing, lifting the fore paws off the ground, grooming:using limbs to manipulate the head or the body, teeth chattering: teeth grinding and yawing, opening and closing the mouth (Punch et al., 1997; Wang et al., 2004).
Data analysis: Data are presented as mean±SEM. The number of active and passive lever pressings was compared in each group using paired t-test. The number of active lever pressing between different groups was compared using repeated-measures one-way analysis of variance (ANOVA) and Tukey post hoc comparisons. This method also was used to analyze the results of withdrawal signs (ANOVA and Tukey post hoc). The criterion for statistical significance was p<0.05.
Histology: Immediately after the withdrawal sign, all rats were given 2 μL of methylene blue in a lateral ventricle and were anesthetized with a high dose of ether and perfused transcardiacally with a phosphate-buffered saline solution. (pH = 7.4) followed by 100 mL of 10% formalin. The brains were removed and placed in formaldehyde (4%). After 3 days, the brains were sliced into 60 μm thin slices by a freezing microtome (Leica, Germany). The tracks left by the cannulas were identified using a light microscope and their exact positions determined by reference to a rat brain atlas (Paxinos and Watson, 1998). Data from rats with incorrect placement were excluded from the analysis.
RESULTS
Self-administration: In this study, the effects of Ang II and captopril on morphine self-administration were studied in rats. For this purpose, 0.25 nmol of Ang II and 300 mmol of captopril were injected into the right lateral ventricle as described in materials and methods. In control and morphine groups, 5 μL of normal saline were injected. The number of active and passive lever pressing were compared between and within groups.
Comparison of active and passive lever pressing in each group: In
saline group, the number of active lever pressing in the first 6 days
(with restricted food access) was significantly higher than the number
of passive lever pressing (p<0.01). But in the later 5 days (with free
access to food), the number of active lever pressing was decreased gradually,
so that, in the final three days (9, 10 and 11th days) there was no significant
difference between the number of active and passive lever pressing (Fig.
1a). In morphine group, animals received morphine for 11 days and
the number of active lever pressing was significantly higher than passive
one in all days (Fig. 1b) (p<0.001). This indicates
that the animals pressed the active lever for morphine, not something
else. In Ang II group, which received 0.25 nmol of Ang II before receiving
morphine, the number of active lever pressing was significantly higher
than passive one during all days (Fig. 1c) (p<0.01).
In captopril group, in the first 6 days the number of active lever pressing
was significantly higher than passive one (p<0.01). In those days the
animals were on restricted food in their cages. There were no significant
differences between the number of active and passive lever pressing in
the last 5 days (Fig. 1d) (with free access to food).
These findings indicate that morphine increases the number of active lever
pressing especially in the second half of the experiment when animals
had free access to food in cages. Captopril decreased this effect of morphine,
but Ang II did not have significant effect on morphine self-administration.
| Fig. 1: | Results of active and passive lever pressing in all groups (8 animals each). Rats were catheterized in to the right brain ventricle and received saline (saline group) or morphine (morphine, captopril and Ang II groups) in morphine self-administration apparatus. captopril and Ang II (test groups) and saline (morphine and saline groups) injected (i.c.v.) before each session and number of active and passive lever pressing was compared. Data are shown as mean±SEM. (a) saline group, (b) morphine group, (c) Ang II group and (d) captopril group. *p<0.05, **p<0.01, ***p<0.001 |
| Fig. 2: | Results of active lever pressing between 4 groups (saline, morphine, Ang II and captopril). Rats were catheterized in to the right brain ventricle and received saline (saline group) or morphine (morphine, captopril and Ang II groups) in morphine self-administration apparatus. captopril and Ang II (test groups) and saline (morphine and saline groups) injected (i.c.v.) before each session and number of active lever pressing was compared among 4 groups using repeated measure ANOVA Data are presented as mean±SEM. F = 15.79, n = 8. #p<0.05 and ###p<0.001: The difference of morphine group to saline group.*p<0.05, **p<0. 01 and ***p<0.001: The difference of captopril group to morphine group.+p<0.05, ++p<0. 01: The difference of captopril group to Ang II group |
Comparison of active and passive lever pressing between groups: The number of active lever pressing in morphine group in the last three days of experiments in which animals had free access to food was higher than in the saline group (9, 10 and 11th days) (Fig. 2, p<0.01). Figure 2 also shows that Ang II had insignificant effect on the number of active lever pressing in comparison with morphine group. The number of active lever pressing in captopril group was lower than in the morphine group in the last 5 days (Fig. 2, p<0.01). These findings indicate that captopril could decrease the tendency to intake morphine. As Fig. 3 shows, there were no significant differences in the number of passive lever pressing between all groups (p>0.05).
Figure 4 shows that the mean number of active lever
pressing in morphine, Ang II and captopril groups was significantly greater
than in the saline group in the first 6 days in which the animals had
restricted food access (p<0.001). In the last 5 days in which animals
had free access to food, the number of active lever pressing in morphine
(p<0.05) and Ang II (p<0.001) groups were higher than in the saline
group, but in captopril group it was lower than morphine (p<0.001)
and saline (p<0.05) groups.
| Fig. 3: | Results of passive lever pressing between all groups of animals (Saline, morphine, Ang II and captopril). Data are presented as mean±SEM. No significant difference in the number of passive lever pressing was observed between groups |
Results of active lever pressing in restricted food period and free food access period is shown in Fig. 4. In saline and captopril groups active lever bar is significantly reduced after receiving food (p<0.001), but in morphine and Ang II groups it increased (p<0.05).
Withdrawal signs: Table 1 shows that the numbers of all withdrawal signs including standing, teeth chattering and the percentage of weight loss in morphine group were significantly greater than those of the control group (p<0.05). In captopril group, the number of standing, grooming and genital grooming, writhing, defecation, jumping and the percentage of weight loss was lower than in the morphine group and the differences of standing; grooming and weight loss were significant (p<0.05).
DISCUSSION
In present study, the effects of Ang II and captopril on morphine self-administration
were investigated. Based on our results, the number of active lever pressing
in the last 3 days in morphine group was higher than in the saline group
but decreased when captopril was used (Fig. 2). Administration
of captopril in the last 5 days, before each session, could reduce the
tendency of animals to uptake morphine because the number of active ever
pressing was lower than passive ones during those days in contrast to
the morphine group. Again, it should be reminded that animals had free
access to food during those days and pressed the active lever to get morphine.
| Fig. 4: | Results of active lever pressing between 4 different groups (saline, morphine, Ang II and captopril) in different periods. Data are presented as mean±SEM. The fig shows that the mean of active lever pressing number in morphine, Ang II and captopril groups is significantly greater than saline in the first 6 days (p<0.001). In those days animals had restricted access to food. In the last 5 days with free access to food, the number of active lever pressing in morphine and Ang II groups were higher than saline group, but in captopril group it was lower than morphine and saline groups. Comparing the number of active lever pressing between before and after receiving food in each group, shows that in saline and captopril groups the number of active bar pressing in the final 5 days (after food) was significantly lower than the first 6 days (before food, p<0.001) but in morphine group it was greater than that in final 6 days (p<0.05). *: comparing different groups to saline group. +: comparing different groups to Morphine group. #: comparing before and after receiving food in each group |
Morphine Withdrawal Signs (MWS) were also decreased in the captopril group in comparison with the morphine treated rats (Table 1). These findings are in agreement with previous studies which have shown that intracerebroventricular injection of captopril in doses of 100, 300, 500 and 1000 μg induced a dose dependent antinociceptive effect in rats (Gupta et al., 1991; Punch et al., 1997) and naloxone pretreatment (10 mg kg-1, i.p) completely antagonized this effect (Gupta et al., 1991). Others have also shown that the antinociceptive effect of repeated doses of captopril was reversed by naloxone (Takai et al., 1996). It has also been indicated that administration of 300 μg i.c.v of captopril potentiates the antinociceptive effect of morphine in intact animals. This potentiation is most likely due to increase in brain enkephalin levels (Gupta et al., 1991). In our results, the tendency for receiving morphine was attenuated by injection of captopril (Fig. 1d). It is possible that the decrease in this tendency after using captopril is due to a change in the concentration of enkephalin or other endogenous opioids in the brain. In fact, it has been demonstrated that morphine increases the degradation of leu-enkephalin in bovine aortic endothelial cells in a concentration dependent manner which may support the above hypothesis. This enhanced leu-enkephalin degradation was due to an increase in the activity of ACE (Melzig et al., 1998). In previous experiments also captopril, 0.3 mg kg-1 s.c., has enhanced some of the naloxone-precipitated withdrawal signs such as rhinorrhea, lacrimation and salivation; but other withdrawal signs have not been affected (Sharpe and Jaffe, 1989). In contrast, in this study captopril decreased some of the withdrawal signs such as standing, grooming and weight loss (Table 1). These controversial results may be due to the dose and the method of captopril administration. On the other hand in the present study the animals which received captopril (i.c.v) received less morphine and showing lower withdrawal signs compare d to morphine group.
The activity of brain ACE has been decreased in rats implanted with morphine containing pellets. This effect has been abolished after injection of 10 mg kg-1 of naloxone (Koyuncuoglu et al., 1986). These findings are probably due to permissive interaction between morphine and renin-angiotensin system. Captopril increased morphine-induced water intake. It is possible that this response is due to an interaction between morphine and circulating angiotensin I and captopril. The competitive antagonist of Ang II, saralasin, has no effect on morphine-induced drinking. This result has pointed once again to a permissive interaction between morphine and circulating angiotensin I or renin (Lal and Atkinson, 1985).
The involvement of the angiotensin-adrenal system as a possible mechanism
in the potentiation of morphine analgesia by captopril was studied in
rats. Captopril pretreatment has sensitized the animals to the analgesic
effects of morphine, while Ang II has exerted an attenuating effect (Das
et al., 1982). Present results demonstrated that the injection
of 0.25 nmol i.c.v of Ang II had no significant effect on morphine uptake
(Fig. 2). However, some withdrawal signs were changed
after using AngII (Table 1). Others investigators have
suggested that the brain endogenous Ang II is likely to be involved in
the central nociceptive mechanisms by its antagonistic action on the endogenous
opioid system (Takai et al., 1996).
| Table 1: | Results of morphine withdrawal signs between saline, morphine, Ang II and captopril groups in 11th day of self-administration 30 min after injection of 3 mg kg-1 naloxone in rats (n = 8) |
| The number of withdrawal signs in morphine group was more than saline group and the differences of standing, teeth chattering and percent of weight loss were significant (*p<0.05, *** p<0.001). In the captopril group the number of standing, grooming and percent of weight loss were lower than morphine group (+p<0.05, +++ p<0.001). Data presented as mean±SEM | |
Intrathecal and i.c.v injection of Ang II exerted antinociceptive effects that could be blocked by naloxone. The analgesic action of morphine could also be reversed by Ang II (Han et al., 2000). It seems that these effects have been exerted through an endogenous opioid mechanism, although they have been sometimes short lasting (Prado et al., 2003; Tchekalarova et al., 2003). Present results may be influenced by this short lasting effect. Different mechanisms are suggested for anti-opioid activity of Ang II and captopril. Some researchers have indicated that the release of GABA could be changed after using these drugs (Hadjiivanova and Georgiev, 1998; Prado et al., 2003).
In conclusion, we showed, for the first time, that captopril pretreatment is capable of reducing morphine tendency and some signs of withdrawal syndrome in rats. Conversely, AngII increased some of these signs, such as, the number of defecation and standing, suggesting that captopril and Ang II may have a role in the modulation of rewarding system.
ACKNOWLEDGMENTS
Thanks are due to Dr. Ali nasimi for help in preparation of manuscript and the Vice Presidency of Research of Isfahan University of Medical Sciences, for financial assistance.