Role of D 1 / D 2 dopamin receptors antagonist perphenazine in morphine analgesia and tolerance in rats

While opioid receptors have been implicated in the development of tolerance, the subsequent mechanisms involved in these phenomena have not been completely understood. Th e purpose of this study was to investigate eff ects of D/D dopamine receptors antagonist perphenazine on morphine analgesia and tolerance in rats. Male Wistar albino rats weighing – g were used in these experiments. To constitute of morphine tolerance, animals received morphine ( mg/kg) once daily for  days. After last dose of morphine was injected on day , morphine tolerance was evaluated by the analgesia tests. Th e analgesic eff ects of perphenazine (, , and  mg/kg ), D-dopamine receptor antagonist SCH  ( mg/kg), D-dopamine receptor antagonist eticlopride ( mg/kg), and morphine were considered at -min intervals (, , , , and  min) by tail-fl ick and hot-plate analgesia tests. Obtained data suggested that D/D dopamine receptors antagonist perphenazine was capable of suppressing opioid tolerance, possibly by the mechanism of inhibiting D-dopamine receptor. Because the data indicated that D-dopamine receptor antagonist eticloride, but not D-dopamine receptor antagonist SCH , signifi cantly decreased morphine tolerance in analgesia tests. In addition, administration of perphenazine with morphine increased morphine analgesia. Results from the present study suggested that dopamine receptors play a signifi cant role in the morphine analgesic tolerance. In particular, Ddopamine receptor has an important role rather than D-dopamine receptor in development tolerance to morphine. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Opioids are highly efficacious analgesic drugs.However, repeated use of these drugs leads to the development of tolerance, thereby limiting their effectiveness and usage.The mechanisms underlying opioid tolerance are not entirely understood.Up to the present, several studies carried out on the mechanism of morphine tolerance.It has been shown that NMDA receptors [] and cannaboid receptors [] are involved in tolerance to opiate-induced antinociception.Tang et al. [] reported that Ca + /calmodulin-dependent protein kinase II (CaMKII) can modulate opioid tolerance and dependence via its action on learning and memory.Furthermore, it has been suggested that transient receptor potential vanilloid- (TRPV) plays a critical role in morphine tolerance [].Recently, we demonstrated that the serotonergic [, ] and noradrenergic system [] play a signifi cant role in the morphine tolerance.Th e opioid system has close functional links to the dopaminergic system in several brain areas, including the substantia nigra [], striatum, and mesolimbic projections [].Opioids are known to modulate dopamine release in a variety of brain areas [].Moreover, some properties of opioids, including hyperlocomotion and reward, are at least partly mediated through dopamine receptors [], and morphine is reported to increase the metabolism of dopamine in the septum and nucleus accumbens [].Dopamine has been implicated in the development of tolerance and dependence to opioids [].It has been demonstrated that activation of opioid receptors located on dopaminergic neurones in the striatum and nucleus accumbens may play an important role in mediating tolerance and sensitization to opiates [].Dopamine exerts its action by binding to its specifi c membrane receptors (DA-Rs) which are subdivided into two subfamilies, D-and D-like, on the basis of their biochemical and pharmacological properties [,].Th e D-like subfamily comprises D-and D-R, while the D-like subfamily includes D-, D-and D-R [].Th e two DA-R subfamilies diff erentially couple to adenylate cyclase [] and exert different eff ects in many conditions [].Dopamine regulates the two major striatal neuronal pathways through diff erential dopamine receptors.It regulates the striatonigral neurons via D-dopamine receptors but regulates the striatopallidal neurons via D-dopamine receptors [].In the striatum, dopaminergic and opioidergic neurons display interactions in regulating the function of eff erent striatal neurons.For example, morphine acutely increases dopamine release in caudate putamen and nucleus accumbens [].Th erefore, the D-like dopamine receptor antagonist SCH  can block a short-term morphine-induced increase in c-fos expression in the nucleus accumbens and caudate putamen [].Accordingly, it has been shown that D-and D-like dopamine receptors have opposing infl uences on morphine-induced antinociception [].D-like dopamine receptors, but not the D-dopamine receptor, exert antinociceptive effects and have a potentiating eff ect on morphine antinociception [].However, the eff ect of D-dopamine receptors on the expression of morphine tolerance has not been elucidated.Perphenazine has been used in clinical practice as antipsychotic drugs for more than  years, and appears to induce their effects by acting as dopamine receptor antagonist [].In this study, we examined eff ects of D/D dopamin receptors antagonist perphenazine on morphine analgesia and tolerance using the tail-flick and hot-plate tests in rats.In addition, we compared separately the roles of D-and D-dopamin receptor in these effects of perphenazine using D-dopamine receptor antagonist SCH  and D-dopamine receptor antagonist eticlopride.

Animals
Wistar Albino rats (weighing - g) were obtained from the Laboratory Animal Center, Cumhuriyet University School of Medicine (Sivas, Turkey).Rats were maintained under standard conditions: -h light-dark schedule (lights on at : A.M.) with ad libitum food and water and constant temperature (±°C).All experiments were carried out blindly between : and : h.Procedures and animal handling met the guidelines of the National Institutes of Health detailed in the "Principles of animal laboratory care".Th e experimental protocols were approved by the Cumhuriyet University Animal Ethics Committee (Licence number: /Ethic).

Induction of morphine tolerance
Th e animals were rendered tolerant to morphine using the method by a previous study on the induction of morphine tolerance [].For tolerance induction, groups of - rats were randomly chosen and treated subcutaneously (s.c.) with morphine  mg/kg, once a day for  days.To evaluate the degree of tolerance, the analgesic eff ect of the test doses of morphine ( mg/kg, s.c.) were measured by the hot-plate and tail-flick tests  h after the last morphine injection (day ).In addition, to determine effects of perphenazine, SCH  and eticlopride on the morphine tolerance, morphine applied with perphenazine, SCH  and eticlopride to the morphine tolerant animals on day .

Antinociception tests
Tail-fl ick test We used a standardised tail fl ick apparatus (May TF  Tail-flick Unit, Commat, Turkey) to evaluate thermal nociception.Th e radiant heat source was focused on the distal portion of the tail at  cm after administration of the vehicle and study drugs.Following vehicle or compound administration, tail-flick latencies (TFL) were obtained.The infrared intensity was adjusted so that basal TFL occurred at .±.s.Animals with a baseline TFL below . or above .s were excluded from further testing.The cutoff latency was set at  s to avoid tissue damage.Any animal not responding after  s was excluded from the study.The hyperalgesic response in the tail-withdrawal test is generally attributed to central mechanisms [, ].

Hot-plate test
Th e antinociceptive response on the hot-plate is considered to result from a combination of central and peripheral mechanisms ().In this test, animals were individually placed on a hot-plate (May AHP  Analgesic Hot-plate Commat, Turkey) with the temperature adjusted to ±°C.Th e latency to the fi rst sign of paw licking or jump response to avoid the heat was taken as an index of the pain threshold; the cut-off time was  s in order to avoid damage to the paw.

Experimental protocols
The analgesic effects of perphenazine, SCH , eticlo-pride, and morphine were considered at -min intervals (, , , , and  min) by tail-fl ick and hot-plate test in rats.To evaluate the effects of perphenazine, SCH , and eticlopride on development of morphine tolerance, morphine tolerant animals received perphenazine (,  and  mg/kg), SCH  ( mg/kg), and eticlopride ( mg/ kg).In the saline-treated group, animals received saline ( ml/kg) instead of morphine during the induction session.

Data analysis
In order to calculate  maximal antinociceptive effects ( MPE), tail-flick and hot-plate latencies (in seconds) were converted to percent antinociceptive effect using the following equation:  MPE = [(Postdrug latency -Baseline latency) / (Cutoff value -Baseline latency)]×.

Statistical analysis
All experimental results were expressed as mean±SEM (standard error of mean).The effect of antinociception was measured and the mean of  MPEs in all groups was calculated.The data were analysed by analysis of variance followed by Tukey test.A significant difference was defined as a p value <..

Th e analgesic eff ects of diff erent doses of perphenazine
To determine the effective dose of perphenazine, the analgesic response were measured for the three different doses of perphenazine (, , and  mg/kg) at min intervals (, , , , and  min) by the analgesia tests.The maximum analgesic effect was observed at  min after administration  mg/kg dose of perphenazine (.±. for tail-fl ick and .±. for hot-plate test).Th e  MPE produced by perphenazine ( mg/kg) was signifi cantly higher than in the other groups ( mg/kg,  mg/ kg perphenazine, and saline group) in both tail-flick test (p<.;

Eff ect of eticlopride on morphine analgesia
The findings demonstrated that D-dopamin receptor antagonist eticlopride significantly increased morphine analgesic effect in tail-flick (p<.; Figure A) and hotplate test (p<.; Figure B) compared to morphine administration group.Th e peak value of this group was also observed at  min after administration of morphine in analgesia tests (tail-flick: .±. and hot-plate: .±.).Furthermore, these data suggested that eticlopride alone has a signifi cant analgesic eff ect compared to the saline group.hot-plate (Figure B) assays as compared to morphine group rats.Also, SCH  alone had no a signifi cant analgesic eff ect compared to the saline treatment group rats.

Eff ects of perphenazine, eticlopride, and SCH  on tolerance to the morphine antinociception
Perphenazine and eticlopride in combination with morphine produced a signifi cant decrease (increase in  MPE value) morphine tolerance in both the tail-fl ick (p<., for perphenazine and p<., for eticlopride; Figure A) and hot-plate assays (p<., for perphenazine and p<., for eticlopride;

DISCUSSION
In the present study, blockade of D-and D-dopamine receptors by perphenazine led to a decreased degree of tolerance to morphine-induced antinociception.Likewise, selective D-dopamine receptor antagonist eticlopride produced a significant decrease morphine analgesic tolerance.In contrast, D-dopamine receptor antagonist SCH  in combination with morphine did not signifi cantly decrease morphine tolerance.Th ese fi ndings demonstrate that D-dopamine receptor has an important role rather than D-dopamine receptor in morphine analgesic tolerance.The last two decades, many mechanisms have been proposed about the development of morphine tolerance.Recently, one of the discussed topics is the dopaminergic system on the analgesia.Initially, the analgesic eff ect is caused by inhibition of dopamine receptors [].Accordingly, our fi ndings indicate that D/D dopamine receptor antagonist perphenazine and D-dopamin receptor antagonist eticlopride showed a signifi cant analgesic eff ect in analgesia tests.Th e interaction between the dopamine and opiate systems is well recognized [].Considerable evidence suggests that dopamine activity aff ects the opioid system by modulating opiate peptide transcripts [], synthesis [], release, and biotransformation [].In contrast, opioids modulate the dopamine system by several mechanisms, such as dopamine synthesis [], release [], biotransformation [], and activity of dopaminergic neurons [].It has been reported that there is functional interrelationship between morphine and the dopaminergic system [, ].Locomotion induced by morphine [] and changes in temperature [] may be mediated via the dopaminergic system.Furthermore, the dopaminergic system also has been implicated in the antinociceptive eff ect and in the expression of signs of morphine withdrawal [].It seems that mesolimbic dopaminergic pathway is essential in the sensitization process.For example, it has been shown that dopamine alters the acquisition of sensitization to the motor eff ects of morphine in mice [].It has been suggested that D-dopamine receptor antagonist sulpiride decreased the response to morphine ( and  mg/kg) in the formalin test, whereas SCH  did not infl uence the antinociception induced by morphine [].In contrast, our data demonstrated that D-dopamine receptor antagonist eticlopride significantly increased morphine analgesic eff ect in the analgesia tests.In addition, several studies have demonstrated that CaMKII has a critical role in opioid tolerance and dependence [, ].Supraspinal and spinal inhibition of CaMKII not only prevented but also reversed opioid-antinociceptive tolerance and physical dependence in several rodent models [, ].Furthermore, it has been reported that dopamin receptor antagonist haloperidol attenuates opioid tolerance and dependence by suppressing CaM-KII activity [].Th ese data support a critical role of CaMKII in the development and maintenance of opioid tolerance.It has been suggested that cAMP-dependent protein kinase (PKA) signal pathway was involved in dopamine-mediated change of Na + , K + -ATPase activity after short-term or longterm morphine treatment [].Opioid and D-dopamine receptors both couple to inhibitory G protein (Gi/o).Activation of both receptors by their agonists inhibits adenylyl cyclase and decreases PKA activation, leading to an enhancement of Na + , K + -ATPase activity.Eticlopride only reverses D-receptor-mediated increase in Na + , K + -ATPase activity.Opioid receptor antagonist naltrexone reversed both short-term and long-term morphine-induced changes of Na + , K + -ATPase activity.Th ese results suggest that D-dopamine receptors are implicated in regulating striatal Na + , K + -ATPase activity after shorterm morphine treatment, whereas D-dopamine receptors are involved in regulating striatal Na + , K + -ATPase activity upon long-term morphine treatment.Involvement of dopamine receptors in regulating Na + , K + -ATPase activity in vivo by morphine is further supported by the observation that eticlopride failed to suppress the enhancement of Na + , K + -ATPase activity induced by in vitro direct administration of morphine to isolated striatal synaptosomes [].Dopamine is a general dopamine receptor agonist and activates both D and D dopamine receptors.Activation of D dopamine receptors stimulates Na + , K + -ATPase activity [], whereas activation of D dopamine receptors inhibits Na + , K + -ATPase activity [].Th is dopamine-PKA signal pathway may explain the mechanism of tolerance to morphine.In the same way, dopamin receptor antagonist perphenazine may decrease tolerance to morphine by means of D-receptor.

CONCLUSIONS
Results from the present study suggested that dopamine receptors play a significant role in the morphine analgesic tolerance.In particular, D-dopamine receptor has an important role rather than D-dopamine receptor in development tolerance to morphine.The most important limitation of this study is no adequate data on human study.In the future, dopamin D-receptor antagonist perphenazine may be used to decrease the development of tolerance to morphine.However, further investigation is needed to explain the mechanism of morphine tolerance.
Bosn J Basic Med Sci 2013; 13 (2): 120-125 ERCAN OZDEMIR ET AL.: ROLE OF D 1 /D 2 DOPAMIN RECEPTORS ANTAGONIST PERPHENAZINE IN MORPHINE ANALGESIA AND TOLERANCE IN RATS Figure A) and hot-plate test (p<.; Figure B).Effect of perphenazine on morphine analgesia Statistical analysis indicated that pretreatment of animals with perphenazine significantly increased morphine analgesic eff ect in both tail-fl ick (p<.; Figure A) and hot-plate test (p<.; Figure B) compared to morphine administration group.Th e peak value of this group was observed at  min after administration of morphine and perphenazine in analgesia tests (tail-flick: .±. and hot-plate: .±.).

FIGURE 1 .
FIGURE 1.The analgesic eff ects of diff erent doses of perphenazine.(A) shows eff ect of three diff erent doses of perphenazine (1, 5, and 10 mg/kg) in the tail-fl ick test, and (B) shows eff ect of perphenazine in the hot-plate test.Each point represents the mean±SEM of percent of maximal possible eff ect (% MPE) for 8 rats.*, p<0.05 and **, p<0.01 compared to saline-treated group.

FIGURE 2 .
FIGURE 2. Eff ect of perphenazine on the morphine analgesia.(A) shows eff ect of perphenazine (10 mg/kg) in the tail-fl ick test, and (B) shows eff ect of perphenazine in the hot-plate test.Each point represents the mean±SEM of percent of maximal possible eff ect for 7 rats.*, p<0.05 compared to morphine treated group and **, p<0.01 compared to saline-treated group.PERP, perphenazine.
Systemic administration of SCH  (D-dopamin receptor antagonist) with morphine did not significantly increase in  MPE in both the tail-flick (Figure A) and

FIGURE 3 .
FIGURE 3. Eff ect of eticlopride on the morphine analgesia.(A) shows eff ect of eticlopride (1 mg/kg) in the tail-fl ick test, and (B) shows eff ect of eticlopride in the hot-plate test.Each point represents the mean±SEM of percent of maximal possible eff ect for 8 rats.*, p<0.05 compared to morphine treated group and **, p<0.01 compared to saline-treated group.ETI, eticlopride.

FIGURE 4 .
FIGURE 4. Eff ect of SCH 23390 on the morphine analgesia.(A) shows eff ect of SCH 23390 (1 mg/kg) in the tail-fl ick, and (B) shows eff ect of SCH 23390 in the hot-plate test.Each point represents the mean±SEM of percent of maximal possible eff ect for 7 rats.*, p <0.01 compared to saline-treated group.SCH, SCH 23390.

FIGURE 5 .
FIGURE 5. Eff ects of perphenazine, eticlopride, and SCH 23390 on the morphine tolerance.(A) shows eff ects of perphenazine, eticlopride, and SCH 23390 in the tail-fl ick, and (B) shows eff ects of perphenazine, eticlopride, and SCH 23390 in the hot-plate test.Each point represents the mean±SEM of percent of maximal possible eff ect for 8 rats.*, p<0.05 and **, p<0.01 compared to the morphine tolerant group.PERP, perhanazine; ETI, eticlopride; SCH, SCH 23390.
Figure B) as compared to the morphine tolerant rats.On the contrary, D-receptor antagonist SCH  in combination with morphine produced no signifi cant eff ect on morphine tolerance in the tail-fl ick (Figure A) and hot-plate assays (Figure B) as compared to the morphine tolerant rats.Th e maximum  MPE was observed at  min after administration of morphine by analgesia tests in all groups rats.