A role for catecholamines and 5-HT in the interaction
The present report provides evidence for a dual role of 5-HT and catecholamines in MDMA-induced hyperthermia and its exacerbation by caffeine. Initially, depletion of endogenous catecholamines attenuated MDMA-induced hyperthermia. Furthermore, a specific role for dopamine is suggested as pretreatment with the dopamine D1 receptor antagonist, SCH 23390, attenuated MDMA-induced hyperthermia and inhibited the ability of caffeine to exacerbate MDMA-induced hyperthermia. By contrast, depletion of central 5-HT failed to block MDMA-induced hyperthermia and exacerbation by caffeine. To further understand 5-HT and catecholaminergic mechanisms mediating the hyperthermic response to MDMA and its exacerbation by caffeine, we examined the effect of caffeine on the core body temperature response to two other amphetamines. d-fenfluramine induces hypothermia in rats due to its selective interactions with the 5-HT system (Cryan et al., 1999), whereas d-amphetamine increases core body temperature in rats (Jaehne et al., 2005) via catecholamine-dependent mechanisms (Glaser et al., 2005). We first determined if co-administration of caffeine with d-fenfluramine would provoke hyperthermia. Caffeine failed to alter the hypothermic response to d-fenfluramine, suggesting that 5-HT release does not play a role in mediating the ability of caffeine to promote hyperthermia. However, upon further exploration of the mechanisms, we observed that caffeine failed to increase d-amphetamine-induced hyperthermia and provoked an MDMA-like response only when d-amphetamine was co-administered with d-fenfluramine. Such a response indicates that, despite the lack of interaction following 5-HT depletion, 5-HT release is nevertheless an important contributing factor to the interaction between caffeine and MDMA.
As the 5-HT system plays a major role in mammalian thermoregulation and is one of the primary targets of the substituted amphetamines, it has often been assumed that this neurotransmitter is responsible for the hyperthermia seen following MDMA administration (Shankaran and Gudelsky, 1999). This theory is supported by studies showing that 5-HT2 receptor antagonists can prevent MDMA-induced hyperthermia (Nash et al., 1988; Schmidt et al., 1990). More recently however, Mechan et al. (2002) suggests that 5-HT may not be such a key player in MDMA-induced hyperthermia, as pretreatment with the 5-HT2 receptor antagonists methysergide, MDL 100,907, SB 242084 and ritanserin or the 5-HT re-uptake inhibitors zimeldine and fluoxetine failed to influence MDMA-induced hyperthermia in rats. The present study also suggests that 5-HT does not play a primary role in either MDMA-induced hyperthermia or its exacerbation by caffeine, as central depletion of 5-HT did not influence the response to drug challenge. Conversely, catecholamine depletion blocked the hyperthermia and provoked a switch to hypothermia in response to MDMA. A similar finding has been previously reported by Dafters and Biello (2003), where acute co-administration of αMPT or the dopamine receptor antagonist, haloperidol, reversed MDMA-induced hyperthermia to produce a hypothermic response. Interestingly, despite the profound effect of catecholamine depletion on MDMA-induced hyperthermia here, it did not fully prevent the interaction between caffeine and MDMA. As reserpine and αMPT treatment only resulted in ∼70% depletion in noradrenaline and dopamine, it is possible that the remaining catecholamine content was sufficient to mediate an interaction between caffeine and MDMA.
Dopamine is involved in thermoregulation, especially in the pre-optic area and anterior hypothalamus, which are the primary loci for maintenance of body temperature (see Hasegawa et al., 2005). In this regard, both dopamine D1 and D2 receptor subtypes are implicated in MDMA-induced changes in body temperature. MDMA-induced hypothermia in rats housed at 15°C can be blocked by pretreatment with the dopamine D2 receptor antagonist remoxipride, but not the dopamine D1 receptor antagonist SCH 23390 (Green et al., 2005). This is the converse of the hyperthermic response, which is blocked by SCH 23390 but unaltered by remoxipride (Mechan et al., 2002). It has been proposed that dopamine D2 receptor stimulation predominates in animals housed individually or at low ambient temperatures, which is why hypothermia is observed in such animals in response to MDMA. We have previously described how co-administration of caffeine switches the hypothermic response to MDMA in individually housed animals to a profound hyperthermia (McNamara et al., 2006). Under such conditions caffeine, may override dopamine D2 receptor-mediated hypothermia, and promote a switch to D1 receptor-mediated hyperthermia.
Such a mechanism is consistent with a number of the responses obtained following drug challenge in the current study. MDMA provoked a hypothermic response following catecholamine depletion or dopamine D1 receptor blockade. Dopamine itself has a greater affinity for dopamine D2-like receptors, in particular D3 and D4 (Missale et al., 1998), and it is possible that in the depletion study, although there was insufficient dopamine release to provoke an overt dopamine D1 receptor-mediated hyperthermic response to MDMA, a D2-like hypothermia occurred instead. Co-administration with caffeine may overcome the dopamine D2 receptor-mediated response by amplifying the dopamine signal. However, a switch to hyperthermia is not obtained in animals pretreated with SCH 23390, as dopamine D1 receptors are blocked under these conditions. Similar responses were obtained where the combination of d-fenfluramine and d-amphetamine provoked a hypothermic response following dopamine D1 receptor blockade (data not shown), and co-administration of caffeine failed to provoke a switch to hyperthermia. As MDMA provokes the release of dopamine in the brain (see Green et al., 2003 for review), and caffeine has also been reported to influence central dopamine release (see Cauli and Morelli, 2005; Ferré, 2008), dopamine release may represent a mechanism whereby caffeine effects a change from a D2 to a D1 receptor-mediated response. Co-administration of caffeine with MDMA may provoke dopamine release sufficient to induce a switch from a D2 receptor mediated hypothermic response to a D1 receptor-mediated hyperthermic response due to the enhanced availability of dopamine in the synapse. Further work, however, is required to clarify such a mechanism.
Despite clear effects obtained following catecholamine depletion and dopamine D1 receptor blockade, and the lack of effect following central 5-HT depletion, a role for 5-HT cannot be ruled out. This is evident where the co-administration of caffeine with a combination of d-fenfluramine and d-amphetamine provokes a hyperthermic response, akin to that described following the co-administration of caffeine with MDMA (McNamara et al., 2006). A role for 5-HT is further supported by evidence that pretreatment with the preferential 5-HT2 receptor antagonist ketanserin attenuates the hyperthermic response to MDMA and its exacerbation by caffeine, and that co-administration of the 5-HT- and dopamine-selective agonists, DOI and apomorphine, respectively, with caffeine, provokes hyperthermia, but not when either agonist is administered with caffeine alone. MDMA has direct agonist actions at 5-HT receptors, which may account for its ability to provoke toxicity in the absence of endogenous 5-HT. Such actions include the ability of pre-synaptic 5-HT receptors to influence dopamine release and thereby augment dopamine-mediated responses to MDMA (Doly et al., 2008; Gudelsky and Yamamoto, 2008). 5-HT2 receptors play an important role in the regulation of central dopaminergic function (see Di Matteo et al., 2008). It is therefore not unreasonable to suggest that ketanserin may act to reduce MDMA-induced dopamine release, resulting in the attenuation of MDMA-induced hyperthermia. Conversely, the effect of DOI may be accounted for via an enhancement of central dopamine release.
As ketanserin is also known to interact with α1 adrenoreceptors, and α1 blockade rather than 5-HT2 blockade by ketanserin has been implicated in the physiological actions of ketanserin (Orallo et al., 2000; Centurión et al., 2006), including MDMA-induced hyperthermia (Mechan et al., 2002), we examined the effects of pretreatment with the selective 5-HT2 receptor antagonist ritanserin and the α1 adrenoreceptor antagonist prazosin. Prior administration of ketanserin and prazosin, but not ritanserin, blocks MDMA-induced hyperthermia and its exacerbation by caffeine, suggesting that α1-adrenoceptor blockade plays a significant role in mediating the actions of ketanserin. In support, there is substantial evidence that noradrenaline mediates MDMA-induced hyperthermia via both peripheral and central mechanisms (Bianco et al., 1988; Sprague et al., 2004).
A role for adenosine receptors and PDE inhibition
Under normal physiological conditions, the mechanism of action of caffeine is primarily via antagonism of adenosine receptors (Fredholm et al., 1999; Fisone et al., 2004; Ferre et al., 2008). Modulation of dopamine transmission through adenosine receptors has been implicated in the psychostimulant effects of caffeine (Fuxe et al., 1998; Cauli and Morelli, 2005), and represents a putative mechanism whereby caffeine exacerbates MDMA-induced toxicity. Antagonistic A1-D1 and A2A-D2 hetereomeric receptor complexes reduce dopamine receptor recognition, coupling and signalling in the basal ganglia. Moreover, caffeine is proposed to influence dopamine release via an adenosine A1 receptor-mediated mechanism (Solinas et al., 2002; Quarta et al., 2004; Cauli and Morelli, 2005). In studies conducted to date, co-treatment with adenosine antagonists failed to provoke a caffeine-like interaction with MDMA, indicating that blockade of adenosine receptors alone does not mediate the interaction between caffeine and MDMA. While it has been reported that the inhibitory effect of caffeine on PDE is of little relevance at the concentrations of caffeine administered in vivo (Fredholm et al., 1999), the weak PDE inhibiting properties of caffeine might well be relevant against a background of increased intracellular cAMP/cGMP availability following MDMA-induced biogenic amine release in the brain. Dulloo and co-workers have extensively investigated the effects of caffeine on thermogenesis induced by ephedrine. Like MDMA, ephedrine stimulates catecholamine release, its primary effect being on noradrenaline, and caffeine exacerbates ephedrine-induced hyperthermia. Following a study of the mechanisms mediating the ability of caffeine to influence the thermogenic effects of ephedrine, PDE inhibition and not adenosine receptor antagonism resulted in a potentiation of the effects of ephedrine (Dulloo et al., 1991, 1992, 1994). In the current investigation however, similar to the adenosine receptor antagonists tested, co-treatment with PDE inhibitors failed to provoke a caffeine-like interaction with MDMA, indicating that inhibition of PDE alone is unlikely to mediate the interaction between caffeine and MDMA. The lack of interaction between MDMA and the PDE-5 inhibitor zaprinast is in line with a previous study reporting that treatment with the PDE-5 inhibitor sildenafil failed to influence MDMA-induced hyperthermia in rats (Puerta et al., 2009). In a final step, to more fully simulate the pharmacology of caffeine, we combined treatment of the adenosine receptor antagonists with the PDE inhibitor rolipram and report that co-treatment with a low dose of the PDE-4 inhibitor rolipram and the non-selective adenosine receptor antagonist CGS 15943, or the selective adenosine A2A receptor antagonist SCH 58261, exacerbate MDMA-induced hyperthermia. Thus, inhibition of PDE coupled to adenosine A2A receptor blockade provokes a caffeine-like interaction with MDMA, suggesting that these targets mediate the ability of caffeine to exacerbate MDMA-induced hyperthermia.
Although the main mechanisms of action of caffeine are adenosine receptor antagonism and PDE inhibition, caffeine has also been found to increase calcium release from the sarcoplasmic reticulum through an interaction with the ryanodine receptor. Intracellular calcium release can itself induce hyperthermia and occurs in drug-induced malignant hyperthermia (Penner and Neher, 1989; Fiege et al., 2002). Such a mechanism may be relevant in light of human studies, which have reported that MDMA intoxication and hyperthermia is associated with an elevation in myoplasmic calcium concentrations (Denborough and Hopkinson, 1997). In the present study, circulating concentrations of caffeine at their peak following drug administration were between 30 and 40 µM. Circulating caffeine concentrations following caffeine ingestion in humans rarely exceeds 100 µM. While caffeine can mobilize intracellular calcium, such a mechanism is unlikely to be applicable either to human consumption or in the current study as a minimal concentration of 250 µM is necessary to generate detectable effects on calcium shifts (see Nehlig et al., 1992). Moreover, caffeine at the dose given did not induce significant hyperthermia, although the possibility remains that co-administration of caffeine with MDMA could influence the ability of caffeine to provoke calcium release. However, as structurally related xanthines, which influence the ryanodine receptor (see Xu et al., 1998), including DPCPX and pentoxyphylline, failed to influence MDMA-induced hyperthermia, it is likely that the principal mechanism by which caffeine influences MDMA-induced hyperthermia is via the proposed mechanism involving the inhibition of adenosine A2A receptors coupled to the inhibition of PDE.
In conclusion, the results of this study show that caffeine enhances the hyperthermic response to drugs that target both 5-hydroxytryptaminergic and catecholaminergic transmission but not where either system is targeted alone. Such a mechanism may account for the ability of caffeine to more readily exacerbate the acute toxicity of MDMA when compared with other amphetamines. The ability of caffeine to exacerbate MDMA-related hyperthermia may be related to inhibitory actions on adenosine A2A receptors and PDE-4. Determination of the mechanisms mediating the toxicity associated with co-ingestion of caffeine with MDMA is an important step toward the treatment of severe hyperthermic reactions to ecstasy that can occur in some users. In accordance with our results and due to the fact that agents such as prazosin and ketanserin are available for human therapy, such agents or similar may be useful candidates for testing in the treatment of hyperthermia associated with MDMA ingestion alone or in combination with caffeine.