Adenosine Antagonist Caffeine Research Articles

Citrulline activates adenosine receptors: New insight into metabolic pathways interaction

Citrulline is a non-proteinogenic amino acid that forms as by-product in nitric oxide (NO) synthesis from arginine and may act in concert with NO as an independent signaling molecule that involves in the mechanism of vascular smooth muscle vasodilation.In this study we examined the effects of citrulline on pulmonary artery smooth muscles. Experimental design comprised outward potassium currents measurements in enzymatically isolated rat pulmonary artery smooth muscle (PASMc) cells using whole-cell patch clamp technique, isometric contractile force recordings on rat pulmonary artery rings and method of molecular docking simulation.Citrulline in a concentration 10−9-10−5 M relaxed phenylephrine (PHE)-preactivated SM of rat pulmonary artery in a dose-dependent manner (EC50 0,67 μM). This citrulline-induced relaxation was dependent on an intact endothelium. Bath application of citrulline (10-8-10-5 M) on isolated PASMc induced a significant increase in the amplitude of outward potassium current (Ik). The adenosine antagonist caffeine (10-6 M) effectively blocked both the citrulline-induced relaxation response and Ik increment. Molecular docking modeling suggests that caffeine blocking the potent activity of citrulline results from competitive interactions at the A2 adenosine receptor binding site.In summary, our data suggest that citrulline, released with NO at low concentrations, can effectively interact with adenosine receptors in smooth muscle cells, causing their relaxation, indicating surprising interaction between NO and adenosine pathways.

Abstract 14180: RyR2 Hyperactivity Contributes to Arrhythmogenic Triggers in the ex vivo Human Right Ventricle

Introduction: Sudden cardiac death (SCD) caused by VT/VF is a major cause of mortality and morbidity, accounting for up to 20% of all deaths in the United States. Novel antiarrhythmic targets and relevant translational models of SCD are urgently needed for basic and translational arrhythmia research, and ex vivo donor human hearts are invaluable resources for such pharmacological studies. It was the goal of this study to investigate the occurrence of VT/VF in excised human hearts and to test a novel pharmacological agent to suppress arrhythmias. Hypothesis: We assessed the hypothesis that RyR2 hyperactivity contributes to arrhythmogenic triggers and substrates in the human RV, and we analyzed the antiarrhythmic effects of pharmacological RyR2 suppression. Methods: Adult human hearts (n = 10; 3F, 7M) were procured from the Washington Regional Transplant Community. The entire RV free wall was isolated, coronary perfused, and placed in an oxygenated, 37°C bath of Tyrode’s solution for optical mapping with the addition of Di-4-ANBDQBS and blebbistatin. Pseduo ECGs were recorded, and action potentials (APs) were spatiotemporally mapped using high-speed CMOS cameras. Optical recordings were taken at baseline and after exposure to the adrenergic agonist isoproterenol (ISO, 250 nM) and the adenosine antagonist caffeine (CAF, 200 μM) to elicit RyR2 hyperactivity. We also evaluated pharmacological RyR2 suppression (2 μM dantrolene) on arrhythmia inhibition. Results: ISO and ISO+CAF significantly shortened tissue APDs. ISO+CAF increased the incidence of PVCs at low pacing frequencies as well as increased the incidence of VT/VF at high pacing frequencies. We observed non-sustained arrhythmias in roughly a third of the hearts examined. No significant differences in conduction velocities were observed. While dantrolene did not return APDs to baseline conditions, it did significantly reverse the effect of caffeine on inducing PVCs. Conclusions: In conclusion, caffeine contributes to arrhythmogenic triggers in the ex vivo human right ventricle, and dantrolene may be used as a pharmacological tool to suppress RyR2 hyperactivity and thus prevent SCD.

Purinergic Signaling and Related Biomarkers in Depression.

It is established that purinergic signaling can shape a wide range of physiological functions, including neurotransmission and neuromodulation. The purinergic system may play a role in the pathophysiology of mood disorders, influencing neurotransmitter systems and hormonal pathways of the hypothalamic-pituitary-adrenal axis. Treatment with mood stabilizers and antidepressants can lead to changes in purinergic signaling. In this overview, we describe the biological background on the possible link between the purinergic system and depression, possibly involving changes in adenosine- and ATP-mediated signaling at P1 and P2 receptors, respectively. Furthermore, evidence on the possible antidepressive effects of non-selective adenosine antagonist caffeine and other purinergic modulators is reviewed. In particular, A2A and P2X7 receptors have been identified as potential targets for depression treatment. Preclinical studies highlight that both selective A2A and P2X7 antagonists may have antidepressant effects and potentiate responses to antidepressant treatments. Consistently, recent studies feature the possible role of the purinergic system peripheral metabolites as possible biomarkers of depression. In particular, variations of serum uric acid, as the end product of purinergic metabolism, have been found in depression. Although several open questions remain, the purinergic system represents a promising research area for insights into the molecular basis of depression.

Caffeine Is Ergogenic for Adenosine A2A Receptor Gene (ADORA2A) T Allele Homozygotes: A Pilot Study

Background: Caffeine improves exercise performance for some people, but not all. The reason for this variability is unknown. Differences in caffeine sensitivity have been explained by variation in a single nucleotide polymorphism in ADORA2A (rs5751876), a gene that codes for the adenosine A2A receptor. Carriers of the C allele (CT/CC) consume more caffeine and are less sensitive to caffeine than T allele homozygotes (TT). The study aim was to determine if the polymorphism in ADORA2A influences the effect of the adenosine antagonist caffeine on total work performed during a cycling time trial. Methods: Women with high self-reported caffeine sensitivity and low daily caffeine consumption participated in a randomized, double-blinded, crossover experiment. On visit 1, participants performed a cycling \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\use...

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Energy drinks are highly consumed beverages rich in caffeine. In humans, energy drinks are very frequently consumed with alcohol in order to reduce the sedation and incoordination induced by high doses of this drug of abuse. Both drugs have shown to exert AG OR A DE SALU T I ISSN: 2443-9827. DOI: http://dx.doi.org/10.6035/AgoraSalut.2015.1.30 pp. 379-385 380 AGORA DE SALUT. VOL. I. ISSN: 2443-9827. DOI:http://dx.doi.org/10.6035/AgoraSalut.2015.1.30 pp. 379-385 effects on the adenosine system. Ethanol is a central nervous system (CNS) depressant that increases the adenosinergic tone, and this effect is thought to mediate the sedative and incoordinating effects observed after alcohol consumption. On the other hand, caffeine is a methylxanthine that acts as a non-selective adenosine antagonist, binding to the two main types of adenosine receptors present in the cns: the A1 and A2A subtypes. The aim of the present study was to fold: to explore the effect of an acute caffeine administration (15 or 30 mg/kg, administered intraperitoneally, IP) on the incoordination effects of ethanol, and to study the acute effect of this methylxanthine in animals that had been repeatedly pre-exposed to ethanol before being tested. The acute administration of ethanol (1.5 g/kg, IP) produced motor impairment in the rotarod when is compared to the control vehicle group. However, caffeine was unable to reverse the impairing effects of an acute dose of ethanol, and did not improved the ataxia produced by a repeated administration of ethanol (1.5 g/kg). Thus, these results show that the non-selective adenosine antagonist caffeine at moderate to high doses (15 and 30 mg/kg) is not efficacious at counteracting the incoordination produced by alcohol in mice, neither after acute, nor after repeated pre-exposure to alcohol.

Regulation of Cardiovascular Development by Adenosine and Adenosine-Mediated Embryo Protection

Few signaling molecules have as much potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methylxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (A1AR, A2aAR, A2bAR, and A3AR). We examined how adenosine acts via A1ARs to influence embryo development. Transgenic mice were studied along with embryo cultures. Embryos lacking A1ARs were markedly growth retarded following intrauterine hypoxia exposure. Studies of mice selectively lacking A1AR in the heart identify the heart as a key site of adenosine's embryo-protective effects. Studies of isolated embryos showed that adenosine plays a key role in modulating embryo cardiac function, especially in the setting of hypoxia. When pregnant mice were treated during embryogenesis with the adenosine antagonist caffeine, adult mice had abnormal heart function. Adenosine acts via A1ARs to play an essential role in protecting the embryo against intrauterine stress, and adenosine antagonists, including caffeine, may be an unwelcome exposure for the embryo.

The role of adenosine in cerebral vascular regulation during reductions in perfusion pressure.

The adenosine antagonist caffeine was used to test the hypothesis that endogenous adenosine contributes to the regulation of cerebral blood flow during acute reductions in mean arterial blood pressure. Caffeine, administered intraperitoneally in 20 and 40 mg kg-1 doses, failed to alter the autoregulatory flow responses to marked reductions in arterial blood pressure, showing that adenosine is not essential for cerebrovascular regulation during severe hypotension.

Prior Treatment (Priming) with Caffeine Sensitizes D2-Dopamine-Mediated Contralateral Rotational Behavior in 6-Hydroxydopamine-Lesioned Rats

Background/Aims: In unilaterally dopamine-depleted rats, repeated treatment with dopamine agonists sensitizes contralateral rotational behavior. Since A2a adenosine receptors are co-localized with D2 dopamine receptors in the brain, it was hypothesized that repeated treatment with the adenosine antagonist caffeine could sensitize D2 dopamine-mediated rotational behavior. Methods: Rats were unilaterally lesioned with 6-hydroxydopamine (6-OHDA), and pretreated (primed) with 3 injections of caffeine or water. One week later, rats were challenged with the D2 agonist quinpirole (0.25 mg/kg). Results: 6-OHDA rats primed with caffeine (50 mg/kg) displayed contralateral rotational behavior following challenge with quinpirole – an effect not observed with caffeine (10 or 75 mg/kg) or water. Conclusions: These results suggest that prior administration of caffeine can sensitize D2 dopamine-mediated rotational behavior in dopamine-depleted rats.

Interactions between adenosine and dopamine receptor antagonists with different selectivity profiles: Effects on locomotor activity

Forebrain dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation. Adenosine A 2A antagonists reverse many of the behavioral effects of DA antagonists, and A 2A receptors are co-localized with D 2 receptors on striatal medium spiny neurons. The present work was undertaken to determine if the ability of an A 2A antagonist, a non-selective adenosine antagonist, or an A 1 antagonist to reverse the locomotor effects of DA blockade in rats differed depending upon whether D 1 or D 2 family receptors were being antagonized. The adenosine antagonists MSX-3, caffeine, DPCPX and CPT were studied for their ability to reverse the locomotor suppression induced by the D 1 antagonist SCH 39166 (ecopipam) and the D 2 antagonist eticlopride. The D 1 and D 2 antagonists suppressed locomotion in all experiments. The adenosine A 2A receptor antagonist MSX-3 (0.5–2.0 mg/kg IP) significantly reversed the suppression of locomotion induced by eticlopride. The non-selective adenosine antagonist caffeine (5.0–20.0 mg/kg IP) also reversed the effect of eticlopride, though the effect was not as robust as that seen with MSX-3. The adenosine A 1 antagonists DPCPX (0.375–1.5 mg/kg) and CPT (3.0–12.0 mg/kg IP) were unable to reverse the locomotor impairment elicited by eticlopride. Furthermore, the attenuation of locomotion induced by the D 1 antagonist could only be reversed by the highest dose of MSX-3, but not by caffeine, DPCPX or CPT. DA and adenosine receptor antagonists interact in the regulation of locomotor activation, but the nature of this interaction appears to depend upon the receptor selectivity profiles of the specific drugs being tested.

Caffeine protects against combined paraquat and maneb-induced dopaminergic neuron degeneration

Environmental exposures suspected of contributing to the pathophysiology of Parkinson's disease (PD) include potentially neurotoxic pesticides, which have been linked to an increased risk of PD. Conversely, possible protective factors such as the adenosine antagonist caffeine have been linked to a reduced risk of the disease. Here we assessed whether caffeine alters dopaminergic neuron loss induced by exposure to environmentally relevant pesticides (paraquat and maneb) over 8 weeks. The number of nigral neurons positive for tyrosine hydroxylase immunoreactivity (TH+) was assessed using stereological methods and found to be significantly reduced (to 60% of control) by combined pesticide treatment. Caffeine at 20 mg/kg significantly reduced TH+ neuron loss (to 85% of the respective control). The results demonstrate the neuroprotective potential of caffeine in a chronic pesticide exposure model of model of PD.

Rosuvastatin Increases Extracellular Adenosine Formation in Humans In Vivo

Statins may increase extracellular adenosine formation from adenosine monophosphate by enhancing ecto-5'-nucleotidase activity. This theory was tested in humans using dipyridamole-induced vasodilation as a read-out for local adenosine formation. Dipyridamole inhibits the transport of extracellular adenosine into the cytosol resulting in increased extracellular adenosine and subsequent vasodilation. In addition, we studied the effect of statin therapy in a forearm model of ischemia-reperfusion injury. Volunteers randomly received rosuvastatin or placebo in a double-blind parallel design (n=21). The forearm vasodilator response to intraarterial dipyridamole was determined in the absence and presence of the adenosine antagonist caffeine. During a separate visit the vasodilator response to nitroprusside and adenosine was established. In addition, healthy men were randomly divided in 3 groups to receive either placebo (n=10), rosuvastatin (n=22), or rosuvastatin combined with intravenous caffeine (n=12). Subsequently, volunteers performed forearm ischemic exercise. At reperfusion, Tc-99m-labeled annexin A5 was infused intravenously and scintigraphic images were acquired, providing an early marker of cell injury. Rosuvastatin treatment significantly increased the vasodilator response to dipyridamole, which was prevented by caffeine. Rosuvastatin did not influence the response to either sodium nitroprusside or adenosine indicating a specific interaction between rosuvastatin and dipyridamole, which does not result from an effect of rosuvastatin on adenosine clearance nor adenosine-receptor affinity or efficacy. Rosuvastatin increased tolerance to ischemia-reperfusion injury, which was attenuated by caffeine. Rosuvastatin increases extracellular adenosine formation, which provides protection against ischemia-reperfusion injury in humans in vivo. Therefore, statins and dipyridamole may interact synergistically.

Differential actions of adenosine A 1 and A 2A antagonists on the effort-related effects of dopamine D 2 antagonism

Adenosine and dopamine receptors in striatal areas interact to regulate a number of different functions, including aspects of motor control and motivation. Recent studies indicate that adenosine A 2A receptor antagonists can reverse the effects of dopamine (DA) D 2 antagonists on instrumental tasks that provide measures of effort-related choice behavior. The present experiments compared the ability of the adenosine A 2A antagonist KW6002, the nonselective adenosine antagonist caffeine, and the adenosine A 1 receptor selective antagonist DPCPX, to reverse the behavioral effects of the DA D 2 antagonist haloperidol. For these studies, a concurrent choice procedure was used in which rats could select between lever pressing on a fixed ratio 5 schedule for a preferred food or approaching and consuming a less preferred lab chow that was concurrently available in the chamber. Under baseline or control conditions, rats show a strong preference for lever pressing, and eat little of the chow; IP injections of 0.1 mg/kg haloperidol significantly reduced lever pressing and substantially increased chow intake. The adenosine A 2A antagonist KW6002 (0.125–0.5 mg/kg IP) and the nonselective adenosine antagonist caffeine (5.0–20.0 mg/kg) significantly reversed the effects of haloperidol. However, the adenosine A 1 antagonist DPCPX (0.1875–0.75 mg/kg IP) failed to reverse the effects of the D 2 antagonist. The rank order of effect sizes in the reversal experiments was KW6002 > caffeine > DPCPX. None of these drugs had any effect on behavior when they were injected in the absence of haloperidol. These results indicate that the ability of an adenosine antagonist to reverse the effort-related effects of a D 2 antagonist depends upon the subtype of adenosine receptor being blocked. Together with other recent results, these experiments indicate that there is a specific interaction between DA D 2 and adenosine A 2A receptors, which could be related to the co-localization of these receptors on the same population of striatal neurons.

Adenosine antagonists reverse the cataleptic effects of haloperidol: Implications for the treatment of Parkinson's disease

The effects of adenosine antagonists were compared in two rodent models of Parkinsonian symptoms. In the first experiment the dopamine D 2 antagonist, haloperidol, was used to induce catalepsy. It was found that treatment with the non-selective adenosine antagonist caffeine significantly reduced catalepsy at each dose. Treatment with the selective A 1 antagonist CPT also produced a significant reduction in catalepsy, as did treatment with the selective A 2A antagonist SCH58261. In the second experiment haloperidol was used to suppress locomotor activity in an open field test. Treatment with caffeine significantly increased locomotion reduced by haloperidol, but not at all doses tested. Treatment with CPT also increased haloperidol-suppressed locomotor activity in dose-dependent manner. Surprisingly, treatment with SCH58261 did not significantly increase locomotor activity in animals treated with haloperidol at any dose tested. While some of these results were unexpected, the overall pattern suggests that adenosine antagonists would be useful as therapies for Parkinsonian patients as they appear to increase movement. The results also suggest that in acute timelines A 1 antagonists may be more beneficial than previously supposed.

Rosuvastatin increases extracellular adenosine in humans in vivo: A New perspective on cardiovascular protection

Objective—Statins may increase extracellular adenosine formation from adenosine monophosphate by enhancing ecto-5 -nucleotidase activity. This theory was tested in humans using dipyridamole-induced vasodilation as a read-out for local adenosine formation. Dipyridamole inhibits the transport of extracellular adenosine into the cytosol resulting in increased extracellular adenosine and subsequent vasodilation. In addition, we studied the effect of statin therapy in a forearm model of ischemia-reperfusion injury. Methods and Results—Volunteers randomly received rosuvastatin or placebo in a double-blind parallel design (n 21). The forearm vasodilator response to intraarterial dipyridamole was determined in the absence and presence of the adenosine antagonist caffeine. During a separate visit the vasodilator response to nitroprusside and adenosine was established. In addition, healthy men were randomly divided in 3 groups to receive either placebo (n 10), rosuvastatin (n 22), or rosuvastatin combined with intravenous caffeine (n 12). Subsequently, volunteers performed forearm ischemic exercise. At reperfusion, Tc-99m–labeled annexin A5 was infused intravenously and scintigraphic images were acquired, providing an early marker of cell injury. Rosuvastatin treatment significantly increased the vasodilator response to dipyridamole, which was prevented by caffeine. Rosuvastatin did not influence the response to either sodium nitroprusside or adenosine indicating a specific interaction between rosuvastatin and dipyridamole, which does not result from an effect of rosuvastatin on adenosine clearance nor adenosine-receptor affinity or efficacy. Rosuvastatin increased tolerance to ischemia-reperfusion injury, which was attenuated by caffeine. Conclusions—Rosuvastatin increases extracellular adenosine formation, which provides protection against ischemiareperfusion injury in humans in vivo. Therefore, statins and dipyridamole may interact synergistically. (Arterioscler Thromb Vasc Biol. 2009;29:963-968.)

Differential glutamate‐dependent and glutamate‐independent adenosine A1 receptor‐mediated modulation of dopamine release in different striatal compartments

Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, we provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, we demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission.

Cross-sensitization between caffeine- and l-dopa-induced behaviors in hemiparkinsonian mice

Adenosine A 2A receptor (A 2AR) antagonists, including the non-specific adenosine antagonist caffeine, have been proposed as a novel, non-dopaminergic treatment strategy for Parkinson's disease (PD). However, the long-term interaction between caffeine and l-dopa treatment in PD models has not been characterized. We examined the interaction between caffeine and l-dopa following a repeated treatment paradigm in hemiparkinsonian mice. In contrast to the progressively sensitized rotational behavior induced by daily l-dopa (2.0 mg/kg) treatment, tolerance for the rotational response to daily caffeine (2.5 or 10 mg/kg) treatment tended to develop over several weeks. However, after a subsequent two-week washout, challenge with same drug demonstrated an extinction of the sensitized l-dopa-induced rotation, but a sensitization of the caffeine-induced rotation. In a cross-challenge paradigm, daily treatment of mice with l-dopa (compared to daily saline) produced a three-fold enhancement in the rotational response to a subsequent re-challenge with caffeine. Similarly, daily treatment of mice with caffeine produced a six-fold enhancement in the rotational response to a subsequent re-challenge with l-dopa. Furthermore, daily co-administration of caffeine plus l-dopa produced enhanced rotational behavior, compared to caffeine or l-dopa alone, indicating an additive or synergistic interaction between caffeine and l-dopa during repeated treatment. Cross-sensitization between caffeine and l-dopa following repeated treatment and their positive interaction during chronic co-adminstration in hemiparkinsonian mice suggest that repeated exposure to caffeine may alter l-dopa responses in PD.

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.

Contraction-related factors affect the concentration of a kallidin-like peptide in rat muscle tissue.

In order to study the effects of the manipulation of various factors related to muscular activity on the concentration of kinins in muscular tissue, a microdialysis probe was implanted in the adductor muscle of the hindlimb in anaesthetized rats. After collection of baseline samples, the perfusion fluid was changed to a Ringer solution containing sodium lactate (10 or 20 mM), adenosine (50 or 100 microM) or a lower pH (7.0 or 6.6). Whereas perfusion with lactate did not have any significant effect on the concentration of kinins in the dialysate, the perfusion with a lower pH or with adenosine dose-dependently increased the kinin content in the samples. In a second microdialysis experiment, by using specific radioimmunoassays (RIA) for bradykinin and kallidin, we observed that about 70 % of the total kinins dialysed from rat muscle are a kallidin-like peptide. Also, the simultaneous perfusion with 100 microM caffeine totally abolished the increase in kinin levels induced by the perfusion at pH 6.6. In a third experiment, soleus muscles from rat were stimulated in vitro during 30 min in the presence or absence of 77 microM caffeine. Electrically stimulated contraction, but not the addition of 10 mU ml(-1) insulin, induced an increase in the concentration of the kallidin-like peptide in the buffer. This effect was totally prevented by the addition of the adenosine antagonist caffeine. These results show that a kallidin-like peptide is released from rat muscle, and that its production is enhanced by muscle activity. Furthermore, the increase in kinin peptides during muscle contraction may be mediated by an increase in adenosine levels.

8-(3-Chlorostyryl)caffeine May Attenuate MPTP Neurotoxicity through Dual Actions of Monoamine Oxidase Inhibition and A2A Receptor Antagonism

Caffeine and more specific antagonists of the adenosine A(2A) receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A(2A) antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP(+), we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP(+) and of MPP(+), suggesting that CSC blocks the conversion of MPTP to MPDP(+) in vivo. In assessing the direct effects of CSC and A(2A) receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with a K(i) value of 100 nm, whereas caffeine and another relatively specific A(2A) antagonist produced little or no inhibition. The A(2A) receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A(2A) receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A(2A) receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A(2A) receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.

Adenosinergic modulation of the discriminative-stimulus effects of methamphetamine in rats.

A(1) and A(2A) adenosine receptors are co-localized with dopamine D(1) and D(2) receptors, respectively, and their stimulation attenuates dopaminergic functioning. To test whether adenosine antagonists with different selectivities for A(1) and A(2A) receptors mimic the discriminative-stimulus effects of dopamine releaser methamphetamine. Effects of the A(1) antagonist DPCPX, the preferential A(2A) antagonist DMPX and the non-selective adenosine antagonist caffeine were evaluated in Sprague-Dawley rats trained to discriminate 1.0 mg/kg, IP, methamphetamine from saline under a fixed-ratio 10 schedule of food presentation. The A(1) antagonist DPCPX (1.0-10.0 mg/kg) failed to substitute for methamphetamine. However, 5.6 mg/kg DPCPX shifted the methamphetamine dose-response curve to the left. The A(2A) antagonist DMPX (1.8-18.0 mg/kg) produced about 70% methamphetamine-appropriate responding and the non-selective antagonist caffeine (3.0-56.0 mg/kg) about 50% methamphetamine-appropriate responding at the highest tested doses. Both DMPX (5.6 mg/kg) and caffeine (30.0 mg/kg) shifted the methamphetamine dose-response curve to the left. Methamphetamine-like effects of DMPX were blocked fully by the D(2) antagonist spiperone (0.18 mg/kg) and partially by the D(1) antagonist SCH-23390 (0.018 mg/kg). Antagonism at A(2A) adenosine receptors directly mimics the discriminative-stimulus effects of methamphetamine through the interaction with dopamine receptors. Antagonism at A(1) adenosine receptors potentiates effects of lower methamphetamine doses and thus plays a rather indirect, modulatory role.