Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans.

Caffeine induces locomotor activation by its ability to block adenosine receptors. Caffeine is metabolized to several methylxanthines, with paraxanthine being the main metabolite in humans. In this study we show that in rats paraxanthine has a stronger locomotor activating effect than caffeine or the two other main metabolites of caffeine, theophylline and theobromine. As previously described for caffeine, the locomotor activating doses of paraxanthine more efficiently counteract the locomotor depressant effects of an adenosine A(1) than an adenosine A(2A) receptor agonist. In drug discrimination experiments in rats trained to discriminate a maximal locomotor activating dose of caffeine, paraxanthine, unlike theophylline, generalized poorly to caffeine suggesting the existence of additional mechanisms other than adenosine antagonism in the behavioral effects of paraxanthine. Pretreatment with the nitric oxide inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) reduced the locomotor activating effects of paraxanthine, but not caffeine. On the other hand, pretreatment with the selective cGMP-preferring phosphodiesterase PDE9 inhibitor BAY 73-6691, increased locomotor activity induced by caffeine, but not paraxanthine. Ex vivo experiments demonstrated that paraxanthine, but not caffeine, can induce cGMP accumulation in the rat striatum. Finally, in vivo microdialysis experiments showed that paraxanthine, but not caffeine, significantly increases extracellular levels of dopamine in the dorsolateral striatum, which was blocked by l-NAME. These findings indicate that inhibition of cGMP-preferring PDE is involved in the locomotor activating effects of the acute administration of paraxanthine. The present results demonstrate a unique psychostimulant profile of paraxanthine, which might contribute to the reinforcing effects of caffeine in humans.

Developmental lead exposure impairs extinction of conditioned fear in young adult rats.

Pavlovian fear conditioning is a model of emotional learning in which a neutral stimulus such as a tone is paired with an aversive stimulus such as a foot shock. Presentation of a tone with a foot shock in a context (test box) elicits a freezing response representative of stereotypic fear behavior. After conditioning has occurred, presentation of the context (test box) or tone in the absence of the unconditioned stimulus (shock) causes extinction of the fear response. Rats chronically exposed to environmentally relevant levels of lead (Pb(2+)) and controls were tested in a fear-conditioning (FC) paradigm at 50 days of age (PN50). Littermates to FC rats received an immediate shock (IS) when placed in the test box with no tone. Blood Pb(2+) levels in control and Pb(2+)-exposed animals were (mean+/-S.E.M.): 0.76+/-0.11 (n=15) and 25.8+/-1.28microg/dL (n=14). Freezing behavior was recorded during acquisition (day of training) or during 4 consecutive extinction days. Control and Pb(2+)-exposed FC rats exhibited the same level of freezing time on the acquisition day. No freezing behavior occurred in IS rats regardless of treatment. Presentation of context 24h later produced a freezing response on both control and Pb(2+)-exposed FC rats but not in IS rats. When tested in the extinction phase, Pb(2+)-exposed FC rats exhibited deficits in extinction compared to control FC rats. That is, when presented with context on 4 consecutive days after acquisition of the fear response, Pb(2+)-exposed FC rats exhibited a greater freezing response than control FC rats. These findings indicate that chronic Pb(2+) exposure produces a deficit in extinction learning and the animals remain more fearful than controls.

Stimulation of adenosine receptors selectively activates gene expression in striatal enkephalinergic neurons.

In the striatum, adenosine A2A and dopamine D2 receptors exert reciprocal antagonistic interactions that modulate the function of GABAergic enkephalinergic neurons. We have previously shown that stimulation of adenosine A1 receptors allows the stimulation of A2A receptors to overcome a tonic inhibitory effect of D2 receptors and induce striatal expression of c-fos. In the present work, by studying co-localization of c-Fos immunoreactivity and preproenkephalin and preprodynorphin transcripts, we show that co-administration of the A1 receptor agonist CPA and the A2A receptor agonist CGS 21680 increases the striatal expression of c-fos in GABAergic enkephalinergic but not in GABAergic dynorphinergic neurons. Co-administration of CPA and CGS 21680 also induced a significant increase in the striatal expression of preproenkephalin. The results underscore the role of adenosine in the activation of gene expression in the GABAergic enkephalinergic neuron.

Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery.

Lack of adenosine A1 and dopamine D2 receptor-mediated modulation of the cardiovascular effects of the adenosine A2A receptor agonist CGS 21680.

Some behavioral and biochemical effects of the systemically administered adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) in rats are potentiated by adenosine A(1) receptor agonists and counteracted by dopamine D2 receptor agonists. In the present study we compared potentiating and antagonistic interactions between CGS 21680 and adenosine A(1) and dopamine D2 receptor agonists on motor activity and on cardiovascular responses (arterial blood pressure and heart rate). The motor-depressant effects produced by CGS 21680 (0.5 mg/kg, i.p.) were potentiated by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 0.3 mg/kg, i.p.) and counteracted by the dopamine D2 receptor agonist quinpirole (0.5 mg/kg, i.p.). In contrast, neither CPA nor quinpirole significantly modified the decrease in arterial pressure or the increase in heart rate induced by CGS 21680. However, the adenosine A(2A) receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3, 3 mg/kg, i.p.) counteracted both the motor-depressant and cardiovascular effects of CGS 21680. Therefore, the effects of the systemically administered adenosine A(2A) receptor agonist CGS 21680 on cardiovascular function, in contrast to its effects on motor behavior, appear to be independent of the effects of adenosine A(1) and dopamine D2 receptor activity.

Enabling role of adenosine A1 receptors in adenosine A2A receptor-mediated striatal expression of c-fos.

When striatal neurons are strongly activated they produce adenosine, which activates nearby adenosine A1 receptors (A1Rs) and adenosine A2A receptors (A2ARs). Although the effects of A1R or A2AR activation on neural activity in the striatum have been examined separately, the effects of coactivating both receptors has not been investigated. Using c-Fos immunohistochemistry as an indicator of neural activity, we examined the effects of coactivation of A1Rs and A2ARs on neural activity and their mechanism of interaction in the caudate-putamen, nucleus accumbens (NAc) and prefrontal cortex in rats. Administration of a motor-depressant dose of the A2AR agonist CGS 21680 (0.5 mg/kg i.p.) did not significantly induce c-fos expression in any of these brain regions. Administration of a motor-depressant dose of the A1R agonist CPA (0.3 mg/kg, i.p.) produced a small but significant induction of c-fos expression only in the shell of the NAc. Coadministration of CGS 21680 and CPA produced a synergistic induction of c-fos expression in the caudate-putamen, cingulate cortex, and especially the NAc. In the shell of the NAc administration of CPA significantly decreased extracellular dopamine levels measured by in vivo microdialysis and blocked CGS 21680-induced increases in dopamine levels. Because it has been previously shown that activation of dopamine D2 receptors (D2Rs) by endogenous dopamine blocks A2AR-mediated c-fos expression, it is hypothesized that the enabling role of A1Rs in A2AR-mediated striatal c-fos expression is related to the A1R-mediated inhibition of dopamine release.

Involvement of adenosine A1 and A2A receptors in the motor effects of caffeine after its acute and chronic administration.

The involvement of adenosine A(1) and A(2A) receptors in the motor effects of caffeine is still a matter of debate. In the present study, counteraction of the motor-depressant effects of the selective A(1) receptor agonist CPA and the A(2A) receptor agonist CGS 21680 by caffeine, the selective A(1) receptor antagonist CPT, and the A(2A) receptor antagonist MSX-3 was compared. CPT and MSX-3 produced motor activation at the same doses that selectively counteracted motor depression induced by CPA and CGS 21680, respectively. Caffeine also counteracted motor depression induced by CPA and CGS 21680 at doses that produced motor activation. However, caffeine was less effective than CPT at counteracting CPA and even less effective than MSX-3 at counteracting CGS 21680. On the other hand, when administered alone in habituated animals, caffeine produced stronger motor activation than CPT or MSX-3. An additive effect on motor activation was obtained when CPT and MSX-3 were coadministered. Altogether, these results suggest that the motor-activating effects of acutely administered caffeine in rats involve the central blockade of both A(1) and A(2A) receptors. Chronic exposure to caffeine in the drinking water (1.0 mg/ml) resulted in tolerance to the motor effects of an acute administration of caffeine, lack of tolerance to amphetamine, apparent tolerance to MSX-3 (shift to the left of its 'bell-shaped' dose-response curve), and true cross-tolerance to CPT. The present results suggest that development of tolerance to the effects of A(1) receptor blockade might be mostly responsible for the tolerance to the motor-activating effects of caffeine and that the residual motor-activating effects of caffeine in tolerant individuals might be mostly because of A(2A) receptor blockade.

Synergistic interaction between adenosine A2A and glutamate mGlu5 receptors: implications for striatal neuronal function.

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in gamma- aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor/receptor interactions. Colocalization of A2AR and mGluR5 at the membrane level was demonstrated in nonpermeabilized human embryonic kidney (HEK)-293 cells transiently cotransfected with both receptors by confocal laser microscopy. Complexes containing A2AR and mGluR5 were demonstrated by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparations from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparations. In cotransfected HEK-293 cells a synergistic effect on extracellular signal-regulated kinase 1/2 phosphorylation and c-fos expression was demonstrated upon A2AR/mGluR5 costimulation. No synergistic effect was observed at the second messenger level (cAMP accumulation and intracellular calcium mobilization). Accordingly, a synergistic effect on c-fos expression in striatal sections and on counteracting phencyclidine-induced motor activation was also demonstrated after the central coadministration of A2AR and mGluR5 agonists to rats with intact dopaminergic innervation. The results suggest that a functional mGluR5/A2AR interaction is required to overcome the well-known strong tonic inhibitory effect of dopamine on striatal adenosine A2AR function.

Caffeine induces dopamine and glutamate release in the shell of the nucleus accumbens.

An increase in the extracellular concentration of dopamine in the nucleus accumbens (NAc) is believed to be one of the main mechanisms involved in the rewarding and motor-activating properties of psychostimulants such as amphetamines and cocaine. Using in vivo microdialysis in freely moving rats, we demonstrate that systemic administration of behaviorally relevant doses of caffeine can preferentially increase extracellular levels of dopamine and glutamate in the shell of the NAc. These effects could be reproduced by the administration of a selective adenosine A1 receptor antagonist but not by a selective adenosine A2A receptor antagonist. This suggests that caffeine, because of its ability to block adenosine A1 receptors, shares neurochemical properties with other psychostimulants, which could contribute to the widespread consumption of caffeine-containing beverages.