Adenosine A1 Receptor Blockade Mimics Caffeine's Attenuation of Ethanol-Induced Motor Incoordination

Authors


Author for correspondence: Andrew Harkin, Department of Pharmacology and Therapeutics, School of Pharmacy, University College Cork, Ireland (fax +353 21 4904616, e-mail a.harkin@ucc.ie).

Abstract

Abstract: The effects of co-administration of caffeine and ethanol were assessed on the motor coordination of rats on the accelerating rotarod (accelerod). Ethanol (2.5 g/kg, orally) decreased motor performance on the accelerod. Co-administration of caffeine (5 and 20 mg/kg, orally) dose-dependently attenuated this ethanol-induced deficit. Caffeine (20 mg/kg, orally) alone did not affect motor performance in the test. As caffeine is a non-selective adenosine receptor antagonist the ability of adenosine A1 and A2A receptor blockade to attenuate ethanol-induced motor incoordination was determined. Pre-treatment with the adenosine A1 receptor antagonist DPCPX (5 mg/kg, intraperitoneally) attenuated ethanol (2.5 g/kg, orally)-induced motor incoordination. By contrast, prior administration of the adenosine A2A selective antagonist SCH 58261 (10 mg/kg intraperitoneally) had no effect on the ethanol-induced motor deficit. These data demonstrate that adenosine A1 receptor blockade mimics the inhibitory action of caffeine on ethanol-induced motor incorordination, and may contribute to the ability of caffeine to offset the acute intoxicating actions of ethanol.

Ethanol and caffeine are widely consumed recreational drugs. Both are frequently taken in combination through mixing alcoholic and caffeinated beverages. Reasons for combining caffeine with ethanol may stem from the popular belief that caffeine can offset the acute intoxicating actions of ethanol, enhance the stimulatory properties and ameliorate hangover effects following excessive alcohol consumption. The mechanism(s) mediating such an interaction are not presently understood.

It is widely known that ethanol mediates its intoxicating effects through the facilitation of inhibitory GABA transmission in the central nervous system (CNS) (Davies 2003) and the inhibition of excitatory glutamate ion channel receptors (Krystal et al. 2003). Numerous reports also suggest that adenosine is also involved in the acute intoxicating effect of ethanol. Studies in mice and rats have shown that adenosine modulates ethanol-induced motor incoordination primarily via central adenosine A1 receptors (Barwick & Dar 1998; Dar 1997 & 2001; El Yacoubi et al. 2003). Moreover, ethanol has been shown to increase extracellular adenosine by inhibiting adenosine re-uptake (Clark & Dar 1989a; Malliard & Diamond 2004) and facilitating release (Clark & Dar 1989b).

Caffeine produces its behavioural and physiological effects by antagonism of adenosine A1 and A2A receptors, inhibition of phosphodiesterase and mobilization of intracellular calcium (Nehlig et al. 1992; Fredholm et al. 1999). Concentrations of caffeine required to inhibit phosphodiesterase and intracellular calcium mobilization are far greater than those reached following human consumption and are generally considered irrelevant to the mechanism mediating the psychoactive properties of caffeine (Fredholm 1995). Thus it is likely that caffeine exerts it's physiological actions through blockade of adenosine receptors. Consistent with this supposition it has been previously demonstrated that the psychomotor stimulation and increased alertness that occurs following caffeine intake is associated with its ability to block adenosine receptors in the CNS (Dunwiddie & Masino 2001).

The objective of the present investigation was to determine the effects of caffeine on ethanol-induced motor incoordination in rats. As caffeine is an antagonist of adenosine receptors, the effects of adenosine receptor antagonists on ethanol-induced motor incoordination were also determined. In the present investigation motor impairment is used as a test of ethanol-induced intoxication as it is considered to be one of the best recognized central effects of ethanol in laboratory animals. The accelerating rotarod (Harvard Apparatus Coordin8®) was employed to determine the loss in motor coordination induced by ethanol. This test is considered to be highly sensitive in detecting ethanol-induced motor performance decrements in rodents (Bogo et al. 1981; Barwick & Dar 1998).

Materials and Methods

Animals. Experiments were carried out on male Sprague-Dawley rats (200–225 g). Animals were kept in a ventilated room at a temperature of 21±1 °, under a 12 hr light/12 hr dark cycle (lights on between 7 a.m. and 7 p.m.). Food and water were available ad libitum. All experiments conformed to ethical guidelines for investigation on experimental animals according to Danish law and the principles of laboratory animal care (NIH publication No. 86-23, revised 1985).

The accelerating rotarod. All animals were first trained on a fast accelerating rotarod (Harvard Apparatus Coordin8®) operating at 0.6 revolutions per minute per second on eight separate occasions, four times per day before the test. The animals were trained to remain on the rotating rod for 30 sec. Any animal that fell off the rod during training was replaced and re-tested. Any animal that failed to complete the training tasks on any day was eliminated from the study.

On the day of testing the animals were randomised into groups. Prior to drug administration they were tested on the accelerating rotarod (1 r.p.m./sec.) to determine pre-dose fall time. Animals were tested 60, 120 and 180 min. following ethanol challenge on the accelerating rotarod (1 r.p.m./sec.).

Drugs. Ethanol (20% v/v; 2.5 g/kg) was prepared in distilled H20 and was administered by the oral route throughout this investigation. The test dose of ethanol was chosen based on an in-house dose response study. Percent (%) pretreatment fall times 60 min. following ethanol administration were as follows: Vehicle 96%, 0.5 g/kg 98%; 1 g/kg 107%; 1.5 g/kg 93% and 2 g/kg 82%; N=7–8 per group. 2.5 g/kg was the lowest dose of ethanol that induced a significant reduction in % pretreatment fall time when compared to vehicle treated controls. These data are similar to those of earlier published reports which show the presence of significant motor incoordination in rodents without hypnosis and/or overt sedation (Bogo et al. 1981; Clark & Dar 1988).

Caffeine (1,3,7-trimethylxanthine) (Sigma Chemical Co.) was dissolved in the ethanol for co-administration. Injections were given in a volume of 10 ml/kg. DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (Sigma Chemical Co.) and SCH 58261 (5-amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (Schering-Plough) were suspended in 1% methylcellulose and administered via the intraperitoneal route in an injection volume of 5 ml/kg, 5 min. prior to receiving ethanol orally.

The doses of these drugs and routes of administration were chosen on the basis of several previous experiments in which the central effects of caffeine and adenosine receptor antagonists were investigated in rodents (Bogo et al. 1981; Clark & Dar 1988; Fenu et al. 1997; Ongini 1997; Marston et al. 1998; Monopoli et al. 1998; Ongini et al. 1999).

Statistical analysis. Results are expressed as group average fall times (seconds) with standard error of the mean (S.E.M.). Percent pretreatment fall times were also calculated 60 min. following ethanol administration as follows (T60/T0)*100, where T60=fall time at 60 min. after treatment and T0=pretreatment fall time. Results are also expressed as group average % pretreatment fall times with S.E.M. Fall times were analysed using a two-way repeated measures ANOVA where treatment and time were the first and second factors. If any statistically significant changes were found, the data were further analysed using a post hoc Fisher's least significant difference (LSD) test. % Pre-treatment fall times were analysed using a Student's two sample t-test or one-way ANOVA where appropriate followed by a Dunnett's post-hoc comparison test. Data were deemed significant when P<0.05.

Results

Ethanol-induced motor impairment on the accelerating rotarod.

ANOVA of fall time showed effects of ethanol (F(1,14)=5.91, P=0.03], time [F(3,42)=5.85, P=0.002] and an ethanol×time interaction [F(3,42)=3.12), P=0.036]. Post hoc comparisons revealed that ethanol (2.5 g/kg) reduced fall times 60, 120 and 180 min. after administration when compared to pretreatment fall time and corresponding controls (fig. 1A). The animals fully regained their normal motor coordination within 240 min. of ethanol treatment.

Figure 1.

The effect of ethanol (2.5 g/kg, orally) on A. fall times and B. % pretreatment fall times. Values represent mean fall time or % pretreatment fall time with standard error of 8 animals. A. *P<0.05 versus ethanol (T0); B. *P<0.05 versus Vehicle control.

Percent pretreatment fall time 60 min. following ethanol administration was reduced when compared to vehicle treated controls (fig. 1B).

Caffeine attenuates ethanol-induced motor impairment.

ANOVA of fall times showed effects of caffeine [F(1,14)=5.07, P=0.04] and time [[F(3,42)=6.02, P=0.002] in ethanol co-treated animals. Post hoc comparisons revealed that ethanol reduced fall time 60 and 180 min. following administration when compared to pretreatment fall time. Co-administration of caffeine attenuated the ethanol-induced reduction in fall times (fig. 2A).

Figure 2.

The effect of A. caffeine (20 mg/kg, orally) and B. caffeine (5 and 20 mg/kg, orally) co-treatments on ethanol (2.5 mg/kg, orally)-induced reduction in fall times and % pretreatment fall times respectively. Values represent mean fall time or % pretreatment fall time with standard error of 8 animals. A. *P<0.05 versus ethanol (T0); +P<0.05 versus corresponding vehicle + ethanol group. B. *P<0.05 versus vehicle control.

ANOVA of % pre-treatment fall time shows an effect of caffeine [F(2,21)=5.69. P=0.01]. Post hoc comparisons revealed that co-administration of caffeine (20 mg/kg) dose dependently increases % pretreatment fall time in ethanol treated rats 60 min. following co-treatment (fig. 2B).

ANOVA of fall times showed no effect of caffeine (20 mg/kg) treatment alone 60, 180 or 300 min. following its administration when compared to vehicle-treated controls. Caffeine did not affect % pretreatment fall time 60 min. after administration when compared to vehicle-treated controls [vehicle 97+16%; caffeine 84+11%, not significant].

DPCPX attenuates ethanol-induced motor impairment.

ANOVA of fall times showed effects of time [F(3,42)=6.00, P=0.002] and a DPCPX×time interaction [F(3,42)=2.98, P=0.042] in ethanol-treated animals. Post hoc comparisons revealed that ethanol reduced fall time 60 min. after administration when compared to pretreatment fall time. Pretreatment with DPCPX attenuated the ethanol-induced reduction in fall time (fig. 3A).

Figure 3.

The effect of DCPCX (5 mg/kg, intraperitoneally) pretreatment on ethanol-(2.5 g/kg, orally)-induced reduction in A. fall times and B. % pretreatment fall times. Values represent mean fall time or % pretreatment fall time with standard error of 7–8 animals. A. *P<0.05 versus ethanol (T0); +P<0.05 versus corresponding vehicle + ethanol group. B. *P<0.05 versus vehicle control; +P<0.05 versus vehicle + ethanol.

ANOVA of fall times showed no effect of DPCPX treatment alone 60, 180 or 300 min. following its administration when compared to vehicle-treated controls.

ANOVA of % pretreatment fall time 60 min. following ethanol administration shows a DPCPX×ethanol interaction [F(1,25)=4.72, P=0.039]. Post hoc comparisons revealed that ethanol reduced % pretreatment fall time when compared to corresponding controls. Pretreatment with DPCPX attenuated the ethanol-induced reduction in % pretreatment fall time. DPCPX did not affect % pretreatment fall time when compared to vehicle treated controls (fig. 3B).

SCH-58261 does not affect ethanol-induced motor impairment.

ANOVA of fall times showed effects of time [F(3,39)=6.02, P=0.002] in ethanol-treated animals. Post hoc comparisons revealed that ethanol reduced fall time 60 min. following administration when compared to pretreatment fall time. Pretreatment with SCH-58261 did not alter the ethanol-induced reduction in fall time (fig. 4A).

Figure 4.

The effect of SCH-58261 (10 mg/kg, intraperitoneally) pretreatment on ethanol (2.5 g/kg, orally)-induced reduction in A. fall times and B. % pretreatment fall times. Values represent mean fall time or % pretreatment fall time with standard error of 7–8 animals. A. *P<0.05 versus ethanol (T0). B. *P<0.05 versus vehicle control.

ANOVA of fall times showed no effect of SCH-58261 treatment alone 60, 180 or 300 min. following its administration when compared to vehicle-treated controls.

ANOVA of % pretreatment fall time 60 min. following ethanol administration shows an effect of ethanol [F(1,24)=11.3, P=0.003]. Post hoc comparisons revealed that ethanol reduced % pretreatment fall time when compared to corresponding controls. Pretreatment with SCH-58261 did not affect the ethanol-induced reduction in % pretreatment fall time. SCH-58261 did not affect % pretreatment fall time when compared to vehicle-treated controls (fig. 4B).

Discussion

In the present investigation we report that caffeine dose-dependently attenuates motor incoordination in rats assessed using the accelerod induced by an intoxicating dose of ethanol. The results are consistent with those previously reported in mice using the standard rotarod to assess motor incoordination (Dar 1988). Accelerod however is considered to be more sensitive than the standard test. The effects of ethanol on accelerod performance are significantly more disrupted at lower doses, and for longer periods of time after ingestion when compared to standard rotarod performance (Bogo et al. 1981).

Caffeine is likely to exert its actions through the antagonism of adenosine receptors, since they are known to bind caffeine at low concentrations (Fredholm 1995). As caffeine is an antagonist at adenosine A1 receptors, it is not unreasonable to suggest that caffeine might attenuate ethanol-induced motor incoordination through adenosine A1 receptor blockade. To test this hypothesis, the response to caffeine was compared to DPCPX, an antagonist with an excess of 200 times selectivity for A1 over A2A adenosine receptors which can cross the blood brain barrier (Baumgold et al. 1992; Jacobson et al. 1996 & 1997). Pretreatment with DPCPX attenuated ethanol-induced motor incoordination in rats tested on the accelerod. A number of previous studies conducted in mice also point towards the participation of the adenosine A1 receptor subtype in ethanol-induced motor impairment. For instance, central doses of adenosine A1 receptor agonists display a synergistic effect on the motor incoordinating response to ethanol (Dar 1990; Meng & Dar 1995; Dar 1997; Barwick & Dar 1998; Dar 2001), in addition, ethanol-induced motor impairment may be markedly attenuated by adenosine A1 receptor antagonists (Dar 1988; Dar 1990; Barwick & Dar 1998; Dar 2001).

As caffeine is a non-selective adenosine receptor antagonist with affinity for both A1 and A2A adenosine receptors (El Yacoubi et al. 2000a & b), the role of adenosine A2A receptors in the response to caffeine was also examined. SCH 58261 is a high affinity antagonist for the A2A adenosine receptor, lacking any affinity for A2B adenosine receptors and being 500 times more selective for A2A over A1 receptors in the rat brain. In addition SCH 58261 has been shown to successfully cross the blood-brain barrier and elicit behavioural and neuroprotective effects at doses below or equal to those used here (Ongini 1997; Fenu et al. 1997; Monopoli et al. 1998; Ongini et al. 1999). Pretreatment with SCH 58261 in the present investigation however failed to attenuate ethanol-induced motor incoordination in rats. Blockade of adenosine A1 but not A2A receptors therefore mimics the effect of caffeine on ethanol-induced motor incoordination in rats.

In addition to the ability of caffeine to attenuate ethanol-induced motor incoordination in rodents, caffeine has more recently been reported to reduce ethanol-induced hyponotic effects in mice (El Yacoubi et al. 2003). By contrast to ethanol-induced motor in-coordination however, ethanol-induced hypnosis appears to be mediated at least in part through adenosine A2A receptors. The duration of the loss of righting reflex after acute ethanol administration was reported to be shorter for A2A receptor-deficient mice than for their wild-type counterparts. In addition, SCH 58261, unlike DPCPX, shortened the duration of the loss of righting reflex induced by ethanol thus mimicking the lack of receptor in deficient mice and indicating that adenosine A2A receptors play a role in ethanol-induced hypnosis (El Yacoubi et al. 2003). Ethanol-induced intoxication consists of a number of dose-related phases of increasing intensity from motor incoordination to sedation, hypnosis, loss of consciousness and coma. Whilst caffeine has been reported to attenuate these intoxicating effects, the mechanism of caffeine may be differentiated where it acts through the A1 receptor to attenuate ethanol-induced motor incoordination and the A2A receptor to attenuate ethanol-induced loss of righting reflex.

Caffeine induces a biphasic effect on locomotion in rodents, low doses (20 mg/kg) increasing and high doses (100 mg/kg) decreasing locomotor activity (Holzman 1991; Kaplan et al. 1992; Nikodijevic & Jacobson 1993; Svenningsson et al. 1995; El Yacoubi et al. 2000a & b). A number of previous studies have concluded that the motor stimulant effects of caffeine at low doses results from adenosine A2A receptor blockade while the depressant effects seen at high doses result from adenosine A1 receptor blockade (Snyder 1981; Ledent et al. 1997; El Yacoubi et al. 2000a & b; Karcz-Kubicha et al. 2003). Attenuation of ethanol-induced motor incoordination through adenosine A1 receptor blockade thus appears unrelated to the motor stimulant actions of caffeine mediated through the blockade of adenosine A2A receptors. A basis for these differential effects may be accounted for by caffeine's albeit moderate preferential affinity for adenosine A2A receptors over the adenosine A1 receptor (Fredholm 1995; El Yacoubi et al. 2000a).

The cerebellum and the motor cortex express adenosine A1 receptors in high concentrations and these have been associated with motor coordination (Dunwiddie & Masino 2001; Ribeiro et al. 2003). By contrast adenosine A2A receptors are not highly expressed in the cerebellum or motor cortex and are confined mostly to the olfactory tubercles and striatum, an area of the brain associated with the regulation of locomotor behaviour (Moreau & Huber 1999). Linking the localisation of the adenosine A1 receptor to its effect on ethanol-induced motor incoordination is supported by studies performed in the cerebellum, striatum and motor cortex implicating adenosine A1 over A2A receptors (Dar et al. 1994; Barwick & Dar 1998; Dar 2001). In addition, intracellular biochemical pathways may contribute to the contrasting behavioural effects of adenosine A1 and A2A receptor ligands. The adenosine A1 receptor is negatively linked to adnylyl cyclase and its activation induces a consequent decrease in the levels of cAMP. Conversely, the adenosine A2A receptor subtype is positively linked to adenylyl cyclase resulting in an increase in the formation of cAMP. Evidence has been presented in support of an involvement of cerebellar cAMP signalling in the adenosinergic modulation of ethanol-induced motor incoordination in mice (Dar 1997).

In summary the present results show that caffeine antagonizes ethanol-induced motor incoordination in rats. The study indicates that adenosine, acting via the adenosine A1 receptor, plays a role in the motor incoordination that follows acute ethanol ingestion. The results lend support to the belief that caffeine is beneficial to offset the intoxicating effects of alcohol.

Acknowledgements

A.H. was supported by a postdoctoral fellowship from the Health Research Board of Ireland.

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