International Journal of Neuroscience, 122, 17–21, 2012Copyright © 2012 Informa Healthcare USA, Inc.ISSN: 0020-7454 print / 1543-5245 onlineDOI: 10.3109/00207454.2011.613550

Dopamine Release is Involved in Antinociceptive Effectof Theophylline

Jatinder Katyal∗ and Yogendra Kumar Gupta

Neuropharmacology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India

ABSTRACT

The methylxanthines, e.g., theophylline, are widely used for the treatment of bronchial asthma. Additionally, a painrelieving effect of theophylline has been reported in patients as well as in experimental animals. The mechanism ofthis antinociceptive action is not clear. In this study, involvement of dopaminergic system in theophylline-inducedantinociception was evaluated using tail flick test model. Swiss albino mice, (either sex, weighing 25–30 g) withbase line tail flick latencies (TFL) between 2.0 and 3.5 s, were used. TFL was recorded before and at intervals of15, 30, 45, and 60 min. after drug treatment. The experimental protocol was duly approved by the InstitutionalAnimal Ethics Committee, All India Institute of Medical Sciences, New Delhi, India. To determine the role ofdopaminergic system, the mice were pretreated with either D1 or D2 dopaminergic receptor antagonists SCH23390 and haloperidol, respectively, prior to treatment with theophylline. Another group of animals receivedapomorphine along with theophylline. The dose of theophylline used, i.e., 10 mg/kg, intraperitoneally (i.p.), hadshown a significant increase in TFLs. The theophylline-induced antinociception, 10 mg/kg, i.p., was reversed bypretreatment with both D1 and D2 dopaminergic receptor antagonists SCH 23390 and haloperidol as well aswith apomorphine (1 mg/kg) pretreatment. The results suggest that theophylline-induced antinociception is dueto release of dopamine.

KEYWORDS: mice, pain, tail flick latency

INTRODUCTION

We have previously shown an antinociceptive effectof adenosinergic system mediated via adenosine A1receptor, in tail flick latency (TFL) test, in mice.Further, this effect could be antagonized by theclassical adenosine receptor antagonist theophylline(Malhotra, Chaudhary, & Gupta, 2000). Interestingly,in the same study, theophylline per se in higher dosesshowed an antinociceptive effect, at doses greater thanthose required for adenosine receptor antagonism.That, methylxanthines have an antinociceptive effectin experimental animals has been demonstrated inother studies also (Sawynok & Reid, 1996; Zarrindast,Matinrokh, & Mojtahedzadeh-Ardebili, 2003). Apartfrom this, in patients, theophylline has been shown toimprove chest pain symptoms in subjects with hyper-sensitive esophagus (Rao, Mudipalli, Remes-Troche,

Received 26 May 2011

Address correspondence to Dr. Jatinder Katyal, NeuropharmacologyLaboratory, Department of Pharmacology, All India Institute of MedicalSciences, New Delhi, 110029, India. E-mail: jatinderkatyal.aiims@gmail.com

Utech, & Zimmerman, 2007) and offered benefit inpostdural puncture headache (Ergun et al., 2008).Recently, theophylline has been suggested as an optionin patients with esophageal pain who do not respond toproton pump inhibitors and patients with esophagealhypersensitivity (Hong, 2010). In another double blindplacebo-controlled, pilot study, as well, theophylline re-lieved headache following lumbar puncture (Feuerstein& Zeides, 1986).

The mechanism(s) underlying these antinociceptiveeffects of theophylline is however not clear. Several pos-sibilities have been suggested for the nociceptive effectsof theophylline viz. adenosinergic modulation (Raoet al., 2002), phosphodiesterase inhibition (Kumar,Jain, & Kulkarni, 2000), interaction with N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid(GABA), and noradrenergic or dopaminergic system(Nagaoka et al., 1993; Paalzow, 1994; Sabetkasai& Zarrindast, 1993; Steardo & Sawynok, 1985).Dopaminergic system is known to be a key player in no-ciceptive processing (Lapirot et al., 2011). However, tilldate no study has been carried out to evaluate the role ofdopaminergic system in theophylline antinociception.

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18 J. Katyal and Y. K. Gupta

In the present study, we examined the influence ofdopaminergic agents on theophylline antinociception.

METHODS

Experimental Animals

The study was conducted using Swiss albino miceof either sex, weighing 25–35 g. Mice were procuredfrom the Central Animal Facility, All India Instituteof Medical Sciences, and group housed in polypropy-lene cages under standard laboratory conditions witha natural light–dark cycle and controlled temperature(20◦C–25◦C) and humidity. They were allowed free ac-cess to standard dry rodent diet (Aashirwad Industries,Chandigarh, India) and tap water ad libitum. The micewere acclimatized to the environment for at least a weekprior to experimentation. Each treatment group com-posed of eight animals. Mice were used only once in thestudy. The study protocol and all study-related proce-dures were approved by the Institutional Animal EthicsCommittee at the All India Institute of Medical Sci-ences, New Delhi, India. All experimental procedureswere in accordance with the Indian National ScienceAcademy guidelines for the use and care of experimentalanimals in research.

Determination of Tail Flick Latencies (TFL)

A simple modification of the technique of D’Amourand Smith (1941) as described by Davies, Raventos,and Wealpole (1946) was used to induce pain by heatstimulus using an analgesiometer (Techno, Lucknow,India). A constant intensity heat stimulus was appliedand TFLs were recorded. The level of heat intensitywas such that the baseline TFL ranged between 2.0and 3.5 s. Animals showing higher or lower controlvalues were rejected. After drug treatment, TFLs weredetermined at 15 min intervals, i.e., 15, 30, 45 and 60min. A cut-off time of double the baseline value wasused to prevent blistering. The latencies were convertedto percent (%) change from baseline as described byYang, Zhang, Zhang, Qiao, and Dafny (1996), usingthe following formula:

% Change = TFL after drug administration − Baseline TFLBaseline TFL

× 100

Drugs and Treatment Schedules

All drugs were prepared fresh and administered,intraperitoneally (i.p.), in a volume not exceeding1 ml/100 g. Control experiments were performed with

the respective vehicles for different drugs that includedsaline and 3% citric acid.

Experiments With Theophylline

Theophylline (courtesy of Sun Pharma, Baroda, India),the classical, nonspecific adenosine receptor antagonist,was dissolved in saline and was given in a dose of10 mg/kg, i.p.

Experiments With Dopaminergic Agents

Apomorphine (Sigma, St. Louis, MO, USA), a mixeddopamine D1/D2 receptor agonist, and SCH 23390(Sigma), a dopamine D1 receptor antagonist, were dis-solved in normal saline, while haloperidol (Sigma),a D2 antagonist, was dissolved in 3% citric acid.They were administered at doses of 1 mg/kg for apo-morphine, 0.05 mg/kg for SCH 23390, and 0.25/0.5mg/kg for haloperidol. The effect of these doses given10 min prior to theophylline (10 mg/kg) administrationwas determined.

The doses and treatment schedules of different drugswere selected from the result of our previous studies(Gupta, Chugh, & Seth, 1989; Malhotra et al., 2000).

Statistical Analysis

The results are expressed as mean ± SEM (standard er-ror of mean) and were analyzed using analysis of vari-ance (ANOVA), followed by Bonferoni post hoc test us-ing SPSS 11.5 software.

RESULTS

No significant effect on TFLs was observed in micetreated with the vehicles.

Table 1 shows the effect of various drug treatmentson TFLs. Theophylline, 10 mg/kg, i.p., consistentlydemonstrated an increase in TFLs at all time points con-sidered, i.e., 15, 30, 45, and 60 min.

Administration of apomorphine (1 mg/kg), on theother hand, was associated with a short-lived hyper-algesic effect. The TFL values were less than thebaseline values at 15 and 30 min but returned to nearbaseline at 45 min. Both dopamine D1 and D2 receptorantagonists, SCH 23390 and haloperidol, respectively,in doses used, failed to show any significant effect onTFLs. However, the dopaminergic agonist as well asboth the antagonists reversed the antinociceptive effectof theophylline.

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Dopamine in Theophylline Antinociception 19

TABLE 1. The effect of different dopaminergic agents on theophylline-induced increases, 10 mg/kg, in TFLs in mice at different timepoints posttreatment. Values are mean ± SEM, n = 8

% Change of TFL

Drug Dose (mg/kg) 15 min 30 min 45 min 60 min

Normal saline 00.30 ml 04.31 ± 01.80 −05.95 ± 06.57 02.24 ± 06.87 05.13 ± 03.773% Citric acid 00.30 ml −04.44 ± 03.56 −05.97 ± 04.61 −07.90 ± 01.43 −02.21 ± 04.59Theophylline 10 62.69 ± 15.18# 77.15 ± 10.83## 58.07 ± 13.98# 68.77 ± 13.99#

Apomorphine 1 −22.39 ± 09.98 −36.63 ± 08.11 −04.09 ± 06.44 −02.06 ± 07.29Haloperidol 00.50 16.02 ± 03.47 10.09 ± 05.00 28.91 ± 04.86 24.51 ± 09.46Haloperidol 00.25 03.18 ± 08.20 06.38 ± 16.90 14.62 ± 17.79 07.84 ± 12.66SCH 23390 00.05 09.66 ± 05.87 18.77 ± 04.27 10.68 ± 08.81 14.82 ± 12.89Theophylline +

Apomorphine10 + 1 −07.98 ± 16.70∗∗∗ −18.56 ± 14.22∗∗∗ −28.06 ± 11.43∗∗ −26.17 ± 16.10∗∗∗

Theophylline +Haloperidol

10 + 00.25 −01.92 ± 08.87∗∗ 14.39 ± 12.77∗∗ 06.32 ± 12.75∗∗ −07.59 ± 09.79∗∗∗

Theophylline + SCH23390

10 + 00.05 −04.73 ± 08.49∗∗∗ 08.74 ± 02.36∗∗∗ 19.70 ± 05.57∗∗ 31.95 ± 13.80∗

∗p < .05, ∗∗p < .005, ∗∗∗p < .001 (group vs. theophylline).#p < .01, ##p < .001 (theophylline versus control).

DISCUSSION

The dopaminergic system plays a major role in process-ing nociceptive information (Lapirot et al., 2011). It hasalso been suggested that an altered dopaminergic con-trol may not only be responsible for certain pathologicalconditions such as parkinsons disease, chronic cephalicpain, and fibromyalgia, but also contribute to the asso-ciated pain (Mascia, Afra, & Schoenen, 1998; Potvin,Grignon, & Marchand, 2009; Wood, 2008).

A theophylline–dopamine interaction has beendemonstrated in many different experimental paradigmsinvolving the central nervous system. Thus, theophyllinereverses the neuroleptic-induced catalepsy and potenti-ates anticataleptic effects of dopamine agonists (Hauber& Munkle, 1996). In fact theophylline has been shownto improve Parkinsonian symptoms in experimentalanimals (Mathur & Gupta, 1999). Theophylline isbelieved to elicit grooming in rats through an indirectdopaminergic mechanism (Zarrindast, Sharifzadeh,& Sadeghi, 1996). A dopaminergic component isimplicated in the effect of theophylline on food andwater intake in bilateral 6-OHDA-lesioned rats (Casaset al., 2000).

Theophylline has an adenosine receptor antagonistactivity, and many of these actions have been postulatedto occur via this mechanism. Adenosine has an antag-onistic interaction with dopamine by virtue of multiplemechanisms, e.g., inhibition of dopamine release pos-sibly via adenosine A1 receptors (Ballarin, Reiriz, Am-brosio, & Mahy, 1995), receptor–receptor interactionbetween adenosine A2 and dopamine D2 receptors inbrain, wherein stimulation of adenosine A2 receptorsinhibits and their blockade potentiates the effects of D2

receptor stimulation (Ferre, O’Connor, Fuxe, & Unger-stedt, 1993).

In the present study, however, adenosine receptor in-volvement appears unlikely in the antinociceptive effectof theophylline. This contention is based on our earlierstudy, wherein we have shown an increase in TFLs withboth adenosine and the specific adenosine A1 recep-tor agonist, N6-cyclopentyl adenosine (CPA), whichcould be reversed by theophylline 5 mg/kg but higherdoses of theophylline, i.e., 10 and 50 mg/kg, per se,had an antinociceptive effect (Malhotra et al., 2000).The dose of theophylline used in the present study hasbeen derived from this earlier study, and it was previ-ously hypothesized by us that since theophylline hasdose-dependent multiple actions and effector systems,namely, phosphodiesterase inhibition, calcium mobi-lization, and modulation of dopamine release (Morgan& Vestal, 1989), it is likely that different mechanismsmay be involved in nociceptive and antinociceptiveeffects of theophylline. Thus, for the nociceptive effect,adenosinergic system involvement is likely, while for theantinociceptive effect, dopaminergic involvement maybe a possibility (Malhotra et al., 2000). Other studieshave also reported both antinociceptive (Ergun et al.,2008; Feuerstein & Zeides, 1986; Hong, 2010; Raoet al., 2007) as well as nociceptive effects for theo-phylline (Nagaoka et al., 1993). Interestingly, Nagaokaet al. (1993) have also related the nociceptive action oftheophylline with adenosine receptor antagonism pri-marily; though, NMDA involvement is also a possibility.

To the best of our knowledge, till date, none hasdemonstrated a dopaminergic component in antinoci-ceptive effect of theophylline. In the present study,apomorphine 1 mg/kg per se pretreatment decreased

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20 J. Katyal and Y. K. Gupta

the TFLs. This is consistent with the results of workcarried out in our laboratory earlier (Gupta et al.,1989) and is most likely due to the fact that apomor-phine has dose-dependent opposing effects, such thatlower doses cause preferential activation of presynapticdopamine receptors and consequently decrease the re-lease of dopamine, while only at higher doses postsynap-tic response becomes apparent. Such opposing effectshave been demonstrated in other studies as well as indifferent experimental paradigms (Paalzow & Paalzow,1983; Yamada & Furukawa, 1986). Both D1 and D2antagonists, SCH 23390 and haloperidol, respectively,per se, were devoid of any effect on TFLs. It is how-ever noteworthy that theophylline-induced antinocicep-tion was completely reversed by all the dopaminergicagents used, i.e., the mixed D1/D2 agonist and bothdopamine D1 and D2 antagonists. A role for both D1and D2 receptors in antinociception has been demon-strated in other studies also albeit in different models(Dang et al., 2011).

Here it is worthwhile to mention that the mouse tailflick test used in this study involves a spinal nocicep-tive reflex (Malhotra et al., 2000), and all subtypes ofdopamine receptors are present in the spinal cord, andit has been suggested that of these, D2 receptor may bemore relevant for pain (Lapirot et al., 2011).

The finding that apomorphine as well as dopamineD1 and D2 receptor could reverse theophyllineantinociception would imply that theophylline inducesantinociception by augmenting dopamine release.Since apomorphine at the doses used can block presy-naptic dopamine receptors, it decreases the release ofdopamine and hence reverses theophylline antinoci-ception. On the other hand, the reversal by dopaminereceptor antagonists is due to blockade of postsynap-tic receptors, which does not permit the dopaminereleased by theophylline to bind with receptors andinduce antinociception.

CONCLUSIONS

Thus, it can be concluded that theophylline-inducedantinociception is via the activation of dopaminergicsystem.

Declaration of interest: The authors declare thatthere is no conflict of interest.

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Dopamine in Theophylline Antinociception 21

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