Adenosine Receptor Blockade Research Articles

CD73 on Tumor Cells Impairs Antitumor T-Cell Responses: A Novel Mechanism of Tumor-Induced Immune Suppression

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a cosignaling molecule on T lymphocytes and an adhesion molecule that is required for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and is upregulated in cancerous tissues. Because the ecto-5'-nucleotidase activity of CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we hypothesized that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that combining tumor CD73 knockdown and tumor-specific T-cell transfer cured all tumor-bearing mice. In striking contrast, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. Moreover, blockade of the A2A adenosine receptor with a selective antagonist also augmented the efficacy of adoptive T-cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting the enzymatic activity of tumor CD73.

A novel mouse model for sudden unexpected death in epilepsy (SUDEP): Role of impaired adenosine clearance

Sudden unexpected death in epilepsy (SUDEP) is a significant cause of mortality in people with epilepsy. Two postulated causes for SUDEP, cardiac and respiratory depression, can both be explained by overstimulation of adenosine receptors. We hypothesized that SUDEP is a consequence of a surge in adenosine as a result of prolonged seizures combined with deficient adenosine clearance; consequently, blockade of adenosine receptors should prevent SUDEP. Here we induced impaired adenosine clearance in adult mice by pharmacologic inhibition of the adenosine-removing enzymes, adenosine kinase and deaminase. Combination of impaired adenosine clearance with kainic acid-induced seizures triggered sudden death in all animals. Most importantly, the adenosine receptor antagonist caffeine, when given after seizure onset, increased survival from 23.75 +/- 1.35 min to 54.86 +/- 6.59 min (p < 0.01). Our data indicate that SUDEP is due to overactivation of adenosine receptors and that caffeine treatment after seizure onset might be beneficial.

Improvement of postural adjustment steps in hemiparkinsonian rats chronically treated with caffeine is mediated by concurrent blockade of A 1 and A 2A adenosine receptors

Chronic treatment with the non-selective adenosine receptor antagonist caffeine produces full recovery of the contralateral adjusting steps in hemiparkinsonian rats. In order to disclose which adenosine receptor subtype mediates this effect, a group of hemiparkinsonian rats ( n=9) was treated with caffeine (5.15 μmol/kg/day), or equimolar doses of selective A1 (DPCPX) or A2A (ZM 241385) adenosine receptor antagonists, administered in a counterbalanced order over periods of 3 weeks, interspersed with equivalent washout intervals. Treatment with ZM 241385 caused full recovery (102±6%) of the contralateral forepaw stepping, while the maximal effect of DPCPX was only 73±7% of that produced by caffeine. The maximal effect of caffeine and ZM 241385 remained stable throughout the treatment period. The response to DPCPX showed more fluctuations, but tolerance did not develop. Stepping improvement was significantly faster with DPCPX than with ZM 241385, while caffeine had intermediate values. Stepping decrease after treatment interruption was faster with ZM 241385 than with caffeine, while DPCPX had intermediate values. In other experiments with the same rats, addition of the A2AR agonist CGS 21680 (5.15 μmol/kg) or the A1R agonist CCPA (2.71 μmol/kg) during the second week of caffeine treatment reversed the improvement of contralateral stepping by 59±4% and 30±3%, respectively. The combined treatment with CGS 21680 and CCPA caused complete reversal of the contralateral stepping recovery afforded by caffeine, which was more than additive (114±5%) compared with the sum of the maximal inhibition produced by either agonist administered alone (89±4%). In all cases, after interrupting the adenosine agonists, the effect of caffeine was fully restored. None of the aforementioned treatments induced significant changes in the stepping of the ipsilateral forepaw. Collectively, these results suggest that the improvement of postural adjustments induced by chronic treatment with low doses of caffeine in hemiparkinsonian rats is mediated by concurrent blockade of A1 and A2A adenosine receptors, with a larger involvement of the latter.

Mitogen-activated protein kinase p38α and retinal ischemic preconditioning

In previous studies, inhibition of mitogen-activated protein kinase (MAP) p38 significantly improved recovery and attenuated apoptosis after retinal ischemia in rats. Yet, ischemic preconditioning (IPC) attenuated the ischemia-induced increase in p38 expression. We hypothesized that p38 was required for induction of ischemic tolerance by IPC. We examined the mechanisms of involvement of p38 in IPC neuroprotection. IPC or ischemia was induced in rat retina in vivo. Recovery after ischemia performed 24h after IPC was assessed functionally (electroretinography) and histologically at 7d after ischemia in the presence or absence of inhibition of p38. We examined the role of p38α in the mimicking of IPC produced by opening mitochondrial KATP channels using diazoxide, or stimulation of p38 activation by anisomycin. The importance of adenosine receptors in p38 activation after IPC was assessed using specific blockers of adenosine A1 and A2a receptors. Interfering RNA (siRNA) or SB203580 was used to block p38α. Phosphorylated p38 levels were measured. Phosphorylated p38 protein increased with IPC. Interfering RNA (siRNA) to p38α prior to IPC, or inhibiting p38 activation with SB203580, with ischemia following 24h later, significantly attenuated the neuroprotective effect of IPC. Anisomycin administered to increase p38 mimicked IPC, an effect blocked by SB203580. IPC-mimicking with diazoxide, an opener of mitochondrial KATP channels, was diminished with p38α siRNA. Adenosine receptor blockade did not decrease the elevated levels of phosphorylated p38 after IPC. Specific inhibition of p38α suggests that this MAPK is involved in the protective effects of IPC, and that p38 is downstream of mitochondrial KATP channels, but not adenosine receptors, in this neuroprotection.

Adenosine Contributes to Blood Flow Regulation in the Exercising Human Leg by Increasing Prostaglandin and Nitric Oxide Formation

Adenosine can induce vasodilation in skeletal muscle, but to what extent adenosine exerts its effect via formation of other vasodilators and whether there is redundancy between adenosine and other vasodilators remain unclear. We tested the hypothesis that adenosine, prostaglandins, and NO act in synergy to regulate skeletal muscle hyperemia by determining the following: (1) the effect of adenosine receptor blockade on skeletal muscle exercise hyperemia with and without simultaneous inhibition of prostaglandins (indomethacin; 0.8 to 1.8 mg/min) and NO (N(G)-mono-methyl-l-arginine; 29 to 52 mg/min); (2) whether adenosine-induced vasodilation is mediated via formation of prostaglandins and/or NO; and (3) the femoral arterial and venous plasma adenosine concentrations during leg exercise with the microdialysis technique in a total of 24 healthy, male subjects. Inhibition of adenosine receptors (theophylline; 399+/-9 mg, mean +/- SEM) or combined inhibition of prostaglandins and NO formation inhibited the exercise-induced increase in leg blood flow by 14+/-1% and 29+/-2% (P<0.05), respectively, but combined inhibition of prostaglandins, NO, and adenosine receptors did not result in an additive reduction of leg blood flow (31+/-5%). Femoral arterial infusion of adenosine increased leg blood flow from approximately 0.3 to approximately 2.5 L/min. Inhibition of prostaglandins or NO, or prostaglandins and NO combined, inhibited the adenosine-induced increase in leg blood flow by 51+/-3%, 39+/-8%, and 66+/-8%, respectively (P<0.05). Arterial and venous plasma adenosine concentrations were similar at rest and during exercise. These results suggest that adenosine contributes to the regulation of skeletal muscle blood flow by stimulating prostaglandin and NO synthesis.

Adenosine receptor blockade accentuates the responses of muscle sympathetic nerve activity to fatiguing exercise

The role adenosine plays in evoking the exercise pressor reflex in humans remain controversial. Interestingly, animal studies suggest that adenosine can inhibit type 1 vanilloid receptors activity. Thus, we hypothesized that adenosine receptor blockade would accentuate muscle sympathetic nerves activity (MSNA) response to fatiguing exercise in humans. Blood pressure (Finometer), heart rate, and MSNA from peroneal nerve were assessed in 11 healthy young volunteers during fatiguing isometric handgrip, post exercise muscle ischemia (PEMI), and passive muscle stretch during PEMI. The protocol was performed before and after adenosine receptor blockade by local infusion of 40 mg aminophylline in saline via forearm Bier block (regional intravenous anesthesia). In the second experiment, the same amount of saline was infused via the Bier block. After aminophylline, the MSNA and blood pressure responses to fatiguing handgrip, PEMI and passive stretch (all P&lt;0.05) were significantly greater than those in control condition. Saline Bier block had no similar effects on the MSNA and blood pressure responses. These data verified our hypothesis, and suggest that adenosine may act to inhibit MSNA response in exercise pressor reflex. Supported by NIH P01 HL077670, M01 RR010732 and AHA 0635245 N.

Modulation of migration of immune cells and molecules into the central nervous system by adenosine receptor signaling (50.5)

Abstract We have shown that mice lacking CD73, an extracellular enzyme of the purine catabolic pathway that catalyzes the breakdown of adenosine monophosphate (AMP) to adenosine, were resistant to EAE, the animal model of the central nervous system (CNS) inflammatory disease multiple sclerosis (MS). Furthermore, blockade of adenosine receptor signaling with an antagonist specific for the A2a adenosine receptor, which is expressed in the CNS, inhibited lymphocyte migration into the CNS and protected wild type mice from EAE. We now show that blockade of adenosine receptors with broad spectrum or a specific A2a receptor antagonist prohibits the entry of immune cells into the CNS of (SJL/J x PL/J)F1 mice (that develop a relapsing-remitting form of EAE), inhibits the development of EAE, and halts EAE relapse. Importantly, we have strong data showing that adenosine receptor blockade can inhibit the entry of molecules such as FITC-Dextran and Evans blue dye across CNS barriers. These data provide convincing evidence that modulation of adenosine receptors can have a profound effect on CNS mediated diseases such as MS and drug delivery across CNS barriers. This work was supported by NIH grants AI 57854 and NS 063011 (to MSB) and AI 18220 (to LFT).

CD73 on tumor cells impairs anti-tumor T cell immunity: a novel mechanism of tumor-induced immune suppression (40.17)

Abstract CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a costimulatory molecule on T lymphocytes and an adhesion molecule that is important for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and upregulated in cancerous tissues. Because CD73 ecto-5'-nucleotidase catalyzes AMP breakdown to immunosuppressive adenosine, we propose that CD73-generated adenosine may prevent tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that genetic ablation of CD73 on ID8 ovarian tumor cells by siRNA reduced its enzymatic activity and increased survival and decreased tumor burden in immunocompetent but not immunodificient Rag1-/- mice. CD73 knockdown improved tumor antigen-specific T cell proliferation and CTL in vivo killing function and decreased apoptosis of tumor activated microenvironmental T cells. Notably, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. By contrast, combining tumor CD73 knockdown and T cell transfer cured all mice. Moreover, blockade of A2A adenosine receptor with its antagonist SCH58261 also augmented the efficacy of T cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting tumor CD73.

Adenosine receptor antagonists improve short-term object-recognition ability of spontaneously hypertensive rats: a rodent model of attention-deficit hyperactivity disorder

The strain of spontaneously hypertensive rats (SHR) is considered a genetic model for the study of attention-deficit hyperactivity disorder (ADHD), as it displays hyperactivity, impulsivity and poorly sustained attention. Recently, we have shown the involvement of adenosinergic neuromodulation in the SHR's short-term and long-term memory impairments. In this study, we investigated the performance of male and female SHR in a modified version of the object-recognition task (using objects with different structural complexity) and compared them with Wistar rats, a widely used outbred rat strain for the investigation of learning processes. The suitability of the SHR strain to represent an animal model of ADHD, as far as mnemonic deficits are concerned, was pharmacologically validated by the administration of methylphenidate, the first-choice drug for the treatment of ADHD patients. The role of adenosine A1 and A2A receptors in object discrimination was investigated by the administration of caffeine (nonselective antagonist) or selective adenosine receptor antagonists. Wistar rats discriminated all the objects used (cube vs. pyramid; cube vs. T-shaped object), whereas SHR only discriminated the most structurally distinct pairs of objects (cube vs. pyramid). Pretraining administration of methylphenidate [2 mg/kg, intraperitoneal (i.p.)], caffeine (1-10 mg/kg, i.p.), the selective adenosine receptor antagonists DPCPX (8-cyclopenthyl-1,3-dipropylxanthine; A1 antagonist, 5 mg/kg, i.p.) and ZM241385 (A2A antagonist, 1.0 mg/kg, i.p.), or the association of ineffective doses of DPCPX (3 mg/kg) and ZM241385 (0.5 mg/kg), improved the performance of SHR in the object-recognition task. These findings show that the discriminative learning impairments of SHR can be attenuated by the blockade of either A1 or A2A adenosine receptors, suggesting that adenosinergic antagonists might represent potentially interesting drugs for the treatment of ADHD.

Caffeine Modulates TNF-α Production by Cord Blood Monocytes: The Role of Adenosine Receptors

Caffeine, a nonspecific adenosine receptor (AR) antagonist is widely used to treat apnea of prematurity. Because adenosine modulates multiple biologic processes including inflammation, we hypothesized that AR blockade by caffeine would increase cytokine release from neonatal monocytes. Using cord blood monocytes (CBM), we investigated 1) the changes in AR mRNA profile by real time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and protein expression (western blot) after in vitro culture, caffeine or lipopolysaccharide (LPS) exposure, and 2) the modulation of cytokine release and cyclic adenosine monophosphate (cAMP) production by enzyme-linked immunosorbent assay (ELISA) induced by caffeine and specific AR antagonists: DPCPX(A1R), ZM241385(A2aR), MRS1754(A2bR), and MRS1220(A3R). After 48 h in culture, A2aR and A2bR gene expression increased 1.9 (p = 0.04) and 2.5-fold (p = 0.003), respectively. A1R protein expression directly correlated with increasing LPS concentrations (p = 0.01), with minimal expression preexposure. Only caffeine (50 microM) and DPCPX (10 nM) decreased tumor necrosis factor-alpha (TNF-alpha) release from LPS activated-CBM by 20 and 25% (p = 0.01) and TNF-alpha gene expression by 30 and 50%, respectively, in conjunction with a > or =2-fold increase in cAMP (p < 0.05). AR blockade did not modulate other measured cytokines. The induction of A1R after LPS exposure suggests an important role of this receptor in the control of inflammation in neonates. Our findings also suggest that caffeine, via A1R blockade, increases cAMP production and inhibits pretranscriptional TNF-alpha production by CBM.

Adenosine A1and A2AReceptors in Mouse Prefrontal Cortex Modulate Acetylcholine Release and Behavioral Arousal

During prolonged intervals of wakefulness, brain adenosine levels rise within the basal forebrain and cortex. The view that adenosine promotes sleep is supported by the corollary that N-methylated xanthines such as caffeine increase brain and behavioral arousal by blocking adenosine receptors. The four subtypes of adenosine receptors are distributed heterogeneously throughout the brain, yet the neurotransmitter systems and brain regions through which adenosine receptor blockade causes arousal are incompletely understood. This study tested the hypothesis that adenosine A(1) and A(2A) receptors in the prefrontal cortex contribute to the regulation of behavioral and cortical arousal. Dependent measures included acetylcholine (ACh) release in the prefrontal cortex, cortical electroencephalographic (EEG) power, and time to waking after anesthesia. Sleep and wakefulness were also quantified after microinjecting an adenosine A(1) receptor antagonist into the prefrontal cortex. The results showed that adenosine A(1) and A(2A) receptors in the prefrontal cortex modulate cortical ACh release, behavioral arousal, EEG delta power, and sleep. Additional dual microdialysis studies revealed that ACh release in the pontine reticular formation is significantly altered by dialysis delivery of adenosine receptor agonists and antagonists to the prefrontal cortex. These data, and early brain transection studies demonstrating that the forebrain is not needed for sleep cycle generation, suggest that the prefrontal cortex modulates EEG and behavioral arousal via descending input to the pontine brainstem. The results provide novel evidence that adenosine A(1) receptors within the prefrontal cortex comprise part of a descending system that inhibits wakefulness.

Blockade of Adenosine Receptors by Caffeine Provides Neuroprotection in a Lentiviral Mouse Model of Machado-Joseph Disease

Event Abstract Back to Event Blockade of Adenosine Receptors by Caffeine Provides Neuroprotection in a Lentiviral Mouse Model of Machado-Joseph Disease Nelio Goncalves1, 2, 3*, Ana T. Simoes2, 3, Rodrigo A. Cunha2, 4 and Luis Pereira-de-Almeida2, 3 1 University of Coimbra, PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Portugal 2 University of Coimbra, Center for Neuroscience and Cell Biology, Portugal 3 University of Coimbra, Faculty of Pharmacy, Portugal 4 University of Coimbra, Institute of Biochemistry, Faculty of Medicine, Portugal Machado Joseph Disease (MJD) or spinocerebellar ataxia type 3, a devastating brain disease, is one of the nine dominantly-inherited polyglutamine neurodegenerative disorders presently known, and it is considered to result from a toxic gain of function of mutant ataxin-3, a polyubiquitin-binding protein. The mechanisms accounting for neuronal degeneration are still largely unknown and there is currently no treatment available. One candidate strategy to manage MJD neurodegeneration might be the antagonism of adenosine A2A receptors (A2ARs). In fact, A2AR blockade has been shown to afford robust neuroprotection in different neurodegenerative disorders based on the key role of A2ARs to control glutamatergic transmission, apoptotic neuronal death, astrocytic function and neuroinflammation. This prompts the hypothesis that the manipulation of A2AR function might also be beneficial in MJD.A non-selective antagonism of the A2AR function promoted by the chronic consumption of caffeine (applied through the drinking water – 1g/L) was carried out in a lentiviral mouse model of MJD (Alves et al, 2008). Preliminary data suggest that caffeine led to an increase in the number of mutant ataxin-3 intranuclear inclusions in the striatum, a reduction in the loss of immunoreactivity for the dopaminergic marker DARPP-32, a decrease in the number of pycnotic nuclei and a reduction of the MJD-associated astrogliosis over time, when compared to the control group that only drank water. Further studies are necessary to confirm if blockade of A2ARs is indeed able to control striatal pathology associated with MJD. Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neurodegenerative Disorders Citation: Goncalves N, Simoes AT, Cunha RA and Pereira-de-Almeida L (2009). Blockade of Adenosine Receptors by Caffeine Provides Neuroprotection in a Lentiviral Mouse Model of Machado-Joseph Disease. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.105 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Aug 2009; Published Online: 10 Aug 2009. * Correspondence: Nelio Goncalves, University of Coimbra, PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Coimbra, Portugal, neliomotag@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nelio Goncalves Ana T Simoes Rodrigo A Cunha Luis Pereira-de-Almeida Google Nelio Goncalves Ana T Simoes Rodrigo A Cunha Luis Pereira-de-Almeida Google Scholar Nelio Goncalves Ana T Simoes Rodrigo A Cunha Luis Pereira-de-Almeida PubMed Nelio Goncalves Ana T Simoes Rodrigo A Cunha Luis Pereira-de-Almeida Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Paraxanthine, the Primary Metabolite of Caffeine, Provides Protection against Dopaminergic Cell Death via Stimulation of Ryanodine Receptor Channels

Epidemiological evidence suggests that caffeine or its metabolites reduce the risk of developing Parkinson's disease, possibly by protecting dopaminergic neurons, but the underlying mechanism is not clearly understood. Here, we show that the primary metabolite of caffeine, paraxanthine (PX; 1, 7-dimethylxanthine), was strongly protective against neurodegeneration and loss of synaptic function in a culture system of selective dopaminergic cell death. In contrast, caffeine itself afforded only marginal protection. The survival effect of PX was highly specific to dopaminergic neurons and independent of glial cell line-derived neurotrophic factor (GDNF). Nevertheless, PX had the potential to rescue dopaminergic neurons that had matured initially with and were then deprived of GDNF. The protective effect of PX was not mediated by blockade of adenosine receptors or by elevation of intracellular cAMP levels, two pharmacological effects typical of methylxanthine derivatives. Instead, it was attributable to a moderate increase in free cytosolic calcium via the activation of reticulum endoplasmic ryanodine receptor (RyR) channels. Consistent with these observations, PX and also ryanodine, the preferential agonist of RyRs, were protective in an unrelated paradigm of mitochondrial toxin-induced dopaminergic cell death. In conclusion, our data suggest that PX has a neuroprotective potential for diseased dopaminergic neurons.

Blockade of A1 adenosine receptors with BG9928 prevents acute renal injury in rats

Blockade of A1 adenosine receptors with BG9928 prevents acute renal injury in rats

Chronic adenosine receptor blockade with BG9928 attenuates cardiac and renal histopathology and improves diabetes in animals with the metabolic syndrome

Chronic adenosine receptor blockade with BG9928 attenuates cardiac and renal histopathology and improves diabetes in animals with the metabolic syndrome

Partial hindlimb occlusion during coronary artery occlusion reduces the susceptibility to ventricular arrhythmias via the intrinsic adenosine receptor system in conscious rats

Temporary occlusion of coronary arteries can lead to potentially lethal arrhythmias. The arrhythmias are triggered directly by ischemia or during the reperfusion phase. Remote conditioning induced by ischemia in distant organs protects the heart from ischemic/reperfusion injury. However, the effect of remote conditioning on ischemia‐induced ventricular arrhythmias is unknown. Therefore, we tested the hypothesis that partial hindlimb occlusion during coronary artery occlusion reduces the susceptibility to ventricular arrhythmias. To test this hypothesis, 7 rats were instrumented for recording arterial pressure and ECG. A Doppler ultrasonic flow probe and vascular occluder were placed around the terminal aorta. Finally, a snare was placed around the left main coronary artery. The response to coronary artery occlusion during partial hindlimb occlusion was determined in conscious rats. Occlusion of the left main coronary artery resulted in tachy‐arrhythmias that culminated in ventricular fibrillation within 4.56 ± 0.15 min. Partial hindlimb occlusion during coronary artery occlusion culminated in ventricular fibrillation within 6.29 ± 0.49 min. Adenosine receptor blockade (theophylline, 10 mg/kg) attenuated the cardioprotective effects of remote conditioning (4.62 ± 0.79 min). Supported by HL67713

Caffeine‐induced hyperactivity in the horse: comparisons of drug and metabolite concentrations in blood and cerebrospinal fluid

The goals of this study were to elucidate the temporal and quantitative relationships between caffeine and its major bioactive metabolites in blood and cerebrospinal fluid (CSF) and to characterize the pharmacokinetic-pharmacodynamic relationship for caffeine-induced changes in spontaneous locomotor activity in the horse. We hypothesized that caffeine and its metabolites distribute efficiently into the CSF to antagonize adenosine A1 and A2a receptors and that spontaneous locomotor activity correlates well with caffeine and/or metabolite concentrations in CSF and blood. A microdialysis system was developed to allow simultaneous monitoring of locomotor activity and collection of CSF and blood samples for pharmacokinetic analysis. CSF concentrations of caffeine and its metabolites were evaluated to determine the percentage of central adenosine receptor blockade by the established standard inhibition curves. Caffeine increased the spontaneous locomotor activity for up to 4 h in a dose-dependent manner. After 3 mg/kg caffeine administration, blood caffeine concentration as well as locomotor activity increased sharply to near peak level while CSF caffeine concentrations exhibited a slow rise to a steady-state 75 min later. High correlation coefficient was found between locomotor activity and caffeine concentrations in blood (R(2 )=0.95) and in CSF (R(2) = 0.93). At 3 mg/kg dosage, theophylline was the only detectable caffeine metabolite in the CSF. The concentrations reached in the CSF were sufficient to partially block central adenosine A1 (14% blockade) and A2a (11% blockade) receptors. There were no statistically significant differences between the pharmacokinetics of caffeine in the blood and CSF. This study provides novel evidence that locomotor stimulation in horses is closely correlated with caffeine concentrations in the blood and CSF and, furthermore, is consistent with blockade of central adenosine receptors.

Ecto‐5′‐nucleotidase (CD73) ‐mediated extracellular adenosine production plays a critical role in hepatic fibrosis

Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A(2A) receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl(4), ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl(4) or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl(4), ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl(4) or TAA. There were far fewer alpha-smooth muscle actin positive hepatic stellate cells in CCl(4)-treated KO mice than that in WT mice. After CCl(4) treatment, the mRNA level of A(1), A(2A), A(2B), and A(3) adenosine receptors, tumor necrosis factor-alpha, interleukin (IL) -1beta, IL-13r alpha1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1 alpha1, Col3 alpha1, and transforming growth factor-beta1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13r alpha2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.

Drugs and ergogenic aids to improve sport performance

The use of biochemical aids to enhance athletic performance has a long history. In our current sporting culture we attempt to divide these between the accepted legal ‘ergogenic aids’ and the unacceptable performance-enhancing ‘drugs’. It is unclear whether this distinction would have been made 2000 years ago when Pliny the Elder reported the effects of Horsetail juice on performance. Interestingly, the sporting ergogenic effects of horsetail haven’t passed the test of time. In the middle ages its astringency, due to its high silica content, made it ideal for scouring pewter and wooden kitchen utensils. The juice's current ergogenic properties are more refined, its main use being in bath and shower products where a ‘natural conditioning effect’ is required. Perhaps more controversially, hidden amongst his 600 books, Claudius Galen, the 2nd century Greek physician to the gladiators, mentioned the positive effects of eating herbs, mushrooms and testicles. Galen believed that the right testicle was hotter and purer than the left, though whether this led to differential performance-enhancing effects was not rigorously tested. We shouldn't think that modern people are unusual in being obsessed with winning at all costs. Philostratos's (200 AD) view of the Ancient Greeks was that “They made war training for sport and sport training for war.” He was less impressed with his generation of sportsmen who “spent too much time eating, drinking and fornicating instead of actually training”. This was reflected in their poor choice of ergogenic aids. Although the ancient Spartan athletes trained on a meat-full diet of bulls, oxen, goats and deer, athletes of his generation ate white bread, poppy seeds, fish and pork [1]. They treated sports as “more of a hobby than a way of life”. Grumpy old men are clearly not a modern phenomenon! Clearly, if not actually preparing for war, … The use of biochemical aids to enhance athletic performance has a long history. In our current sporting culture we attempt to divide these between the accepted legal ‘ergogenic aids’ and the unacceptable performance-enhancing ‘drugs’. It is unclear whether this distinction would have been made 2000 years ago when Pliny the Elder reported the effects of Horsetail juice on performance. Interestingly, the sporting ergogenic effects of horsetail haven’t passed the test of time. In the middle ages its astringency, due to its high silica content, made it ideal for scouring pewter and wooden kitchen utensils. The juice's current ergogenic properties are more refined, its main use being in bath and shower products where a ‘natural conditioning effect’ is required. Perhaps more controversially, hidden amongst his 600 books, Claudius Galen, the 2nd century Greek physician to the gladiators, mentioned the positive effects of eating herbs, mushrooms and testicles. Galen believed that the right testicle was hotter and purer than the left, though whether this led to differential performance-enhancing effects was not rigorously tested. We shouldn't think that modern people are unusual in being obsessed with winning at all costs. Philostratos's (200 AD) view of the Ancient Greeks was that “They made war training for sport and sport training for war.” He was less impressed with his generation of sportsmen who “spent too much time eating, drinking and fornicating instead of actually training”. This was reflected in their poor choice of ergogenic aids. Although the ancient Spartan athletes trained on a meat-full diet of bulls, oxen, goats and deer, athletes of his generation ate white bread, poppy seeds, fish and pork [1]. They treated sports as “more of a hobby than a way of life”. Grumpy old men are clearly not a modern phenomenon! Clearly, if not actually preparing for war, …

Caffeine and other sympathomimetic stimulants: modes of action and effects on sports performance

Stimulants, illegal and legal, continue to be used in competitive sport. The evidence for the ergogenic properties of the most potent stimulants, amphetamines, cocaine and ephedrine, is mostly insubstantial. Low doses of amphetamines may aid performance where effects of fatigue adversely affect higher psychomotor activity. Pseudoephedrine, at high doses, has been suggested to improve high intensity and endurance exercise but phenylpropanolamine has not been proven to be ergogenic. Only caffeine has substantial experimental backing for being ergogenic in exercise. The mode of action of these stimulants centres on their ability to cause persistence of catecholamine neurotransmitters, with the exception of caffeine which is an adenosine receptor antagonist. By these actions, the stimulants are able to influence the activity of neuronal control pathways in the central (and peripheral) nervous system. Rodent models suggest that amphetamines and cocaine interact with different pathways to that affected by caffeine. Caffeine has a variety of pharmacological effects but its affinity for adenosine receptors is comparable with the levels expected to exist in the body after moderate caffeine intake, thus making adenosine receptor blockade the favoured mode of ergogenic action. However, alternative modes of action to account for the ergogenic properties of caffeine have been supported in the literature. Biochemical mechanisms that are consistent with more recent research findings, involving proteins such as DARPP-32 (dopamine and cAMP-regulated phosphoprotein), are helping to rationalize the molecular details of stimulant action in the central nervous system.