Effects Of Adenosine Receptor Agonists Research Articles

Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists.

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.

Adenosine receptor agonists deepen the inhibition of platelet aggregation by P2Y12 antagonists

Several adenosine receptor (AR) agonists have been shown in the past to possess anti-platelet potential; however, the adjunctive role of AR agonists in anti-platelet therapy with the use of P2Y12 receptor inhibitors has not been elucidated so far.This in vitro aggregation-based study investigates whether the inhibition of platelet function mediated by cangrelor or prasugrel metabolite can be potentiated by AR agonists. It evaluates the effect of non-selective (2-chloroadenosine), A2A-selective (UK 432097, MRE 0094, PSB 0777) and A2B-selective AR agonists (BAY 60-6583) on platelet function in relation to their toxicity, specificity towards adenosine receptor subtypes, structure and solubility.UK 432097, 2-chloroadenosine, MRE 0094 and PSB 0777 were found to be more or less potent inhibitors of ADP-induced platelet aggregation when acting alone, and that they remained non-cytotoxic to the cells. These AR agonists were also effective in the potentiation of the effects exerted by P2Y12 antagonists. Considering the estimated IC50 value, UK 432097, showing a relatively high binding affinity to the A2A adenosine receptor, has been identified as the most potent anti-aggregatory agent. This compound diminished platelet aggregation at nanomolar concentrations and further augmented platelet inhibition by P2Y12 antagonists by approx. 60% (P < .01).Our results indicate the importance of adenosine receptors as therapeutic targets and point out challenges and potential benefits of therapeutic use of a combined therapy of P2Y12 antagonist and AR agonist in cardioprotection. Our comparative analysis of the effects of AR agonists on platelet response in plasma and whole blood may indirectly suggest that other blood morphology elements contribute little to the inhibition of platelet function by AR agonists.

Effects of adenosine receptor agonist on the rocuroniuminduced neuromuscular block and sugammadex-induced recovery.

BackgroundSeveral types of receptors are found at neuromuscular presynaptic membranes. Presynaptic inhibitory A1 and facilitatory A2A receptors mediate different modulatory functions on acetylcholine release. This study investigated whether adenosine A1 receptor agonist contributes to the first twitch tension (T1) of train-of-four (TOF) stimulation depression and TOF fade during rocuronium-induced neuromuscular blockade, and sugammadex-induced recovery.MethodsPhrenic nerve-diaphragm tissues were obtained from 30 adult Sprague-Dawley rats. Each tissue specimen was randomly allocated to either control group or 2-chloroadenosine (CADO, 10 μM) group. One hour of reaction time was allowed before initiating main experimental data collection. Loading and boost doses of rocuronium were sequentially administered until > 95% depression of the T1 was achieved. After confirming that there was no T1 twitch tension response, 15 min of resting time was allowed, after which sugammadex was administered. Recovery profiles (T1, TOF ratio [TOFR], and recovery index) were collected for 1 h and compared between groups.ResultsThere were statistically significant differences on amount of rocuronium (actually used during experiment), TOFR changes during concentration-response of rocuronium (P = 0.04), and recovery profiles (P < 0.01) of CADO group comparing with the control group. However, at the initial phase of this experiment, dose-response of rocuronium in each group demonstrated no statistically significant differences (P = 0.12).ConclusionsThe adenosine A1 receptor agonist (CADO) influenced the TOFR and the recovery profile. After activating adenosine receptor, sugammadex-induced recovery from rocuronium-induced neuromuscular block was delayed.

Stimulation of Adenosine A2B Receptor Inhibits Endothelin-1-Induced Cardiac Fibroblast Proliferation and α-Smooth Muscle Actin Synthesis Through the cAMP/Epac/PI3K/Akt-Signaling Pathway.

Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified.Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA.Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation.Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis.

Human adenosine deaminases control the immune cell responses to activation signals by reducing extracellular adenosine concentration

Abstract Adenosine rapidly accumulates in the sites of inflammation and tumor growth. It binds to adenosine receptors expressed on the cell surface of immune cells and induces either suppression or activation of inflammatory responses to pathogens. In humans the level of extracellular adenosine is regulated by two adenosine deaminases ADA1 and ADA2. Decrease in ADAs concentration due to genetic defects in the ADA genes leads to serious perturbation in the immune system function while increase in ADA activity associates with numerous immune diseases and cancers. The immune responses to extracellular adenosine have largely been studied using pharmacological approach where non-hydrolysable adenosine receptors agonists substitute adenosine to form the activated state of adenosine receptors. On contrary, adenosine receptors bound to adenosine receptor antagonists mimic inactivated state of adenosine receptors. Here, the effect of adenosine receptor agonists and antagonists on the monocytes function as well as and T helper cell proliferation and differentiation was compared with the effect of adenosine and adenosine deaminases. It was demonstrated that adenosine deaminases control the immune cells responses to activation signals by reducing the concentration of extracellular adenosine and that the cells sensitivity to adenosine greatly depends on the type of the cell activation. Therefore, our data suggests that ADAs could be considered as new drug candidates for the treatment of immune disorders and cancers.

A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c(+)Gr-1(+) dendritic cell subset that promotes the Th17 response.

Adenosine is one of the major molecules associated with inflammation. We have previously reported that an adenosine receptor (AR) agonist has an enhancing effect on Th17 autoimmune responses, even though it suppressed Th1 responses. To determine the mechanism involved, we have examined the effect of AR agonists on mouse bone marrow dendritic cell (BMDC) differentiation and function. We show that mouse bone marrow cells (BMCs) differentiated into CD11c(+)Gr-1(+) dentritic cells (DCs) when cultured in granulocyte macrophage colony-stimulating factor (GM-CSF)-containing medium containing an AR agonist. The non-selective AR agonist NECA and an A2BR-specific agonist had a similar effect, and the effect of NECA could be blocked by an A2BR-specific antagonist. Unlike CD11c(+)Gr-1(-) BMDCs, which have a greater stimulatory effect on Th1 T cells than Th17 cells, CD11c(+)Gr-1(+) BMDCs had a greater stimulatory effect on Th17 autoreactive T cells than on Th1 autoreactive T cells and this effect depended on γδ T cell activation.

Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer's disease, an effect mimicked by adenosine A2 A receptor, but not A1 receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice. We determined whether A2 A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2 A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. Scopolamine (1.0 mg·kg(-1) , i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A2 A receptor antagonist (SCH 58261, 0.1-1.0 mg·kg(-1) , i.p.) and by the A1 receptor antagonist (DPCPX, 0.2-5.0 mg·kg(-1) , i.p.), except in the modified Y-maze where only SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A2 A receptors with CGS 21680 (0.1-0.5 mg·kg(-1) , i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg(-1) , i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2 A receptors are necessary and sufficient to trigger memory impairment and further suggest that A1 receptors might also be selectively engaged to control the cholinergic-driven memory impairment.

Prevention of RhoA activation and cofilin-mediated actin polymerization mediates the antihypertrophic effect of adenosine receptor agonists in angiotensin II- and endothelin-1-treated cardiomyocytes

Adenosine receptor activation has been shown to be associated with diminution of cardiac hypertrophy and it has been suggested that endogenously produced adenosine may serve to blunt pro-hypertrophic processes. In the present study, we determined the effects of two pro-hypertrophic stimuli, angiotensin II (Ang II, 100 nM) and endothelin-1 (ET-1, 10 nM) on Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK) activation in cultured neonatal rat ventricular myocytes and whether the latter serves as a target for the anti-hypertrophic effect of adenosine receptor activation. Both hypertrophic stimuli potently increased RhoA activity with peak activation occurring 15-30 min following agonist addition. These effects were associated with significantly increased phosphorylation (inactivation) of cofilin, a downstream mediator of RhoA, an increase in actin polymerization, and increased activation and nuclear import of p38 mitogen activated protein kinase. The ability of both Ang II and ET-1 to activate the RhoA pathway was completely prevented by the adenosine A1 receptor agonist N (6)-cyclopentyladenosine, the A2a receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine, the A3 receptor agonist N (6)-(3-iodobenzyl)adenosine-5'-methyluronamide as well as the nonspecific adenosine analog 2-chloro adenosine. All effects of specific receptor agonists were prevented by their respective receptor antagonists. Moreover, all adenosine agonists prevented either Ang II- or ET-1-induced hypertrophy, a property shared by the RhoA inhibitor Clostridium botulinum C3 exoenzyme, the ROCK inhibitor Y-27632 or the actin depolymerizing agent latrunculin B. Our study therefore demonstrates that both Ang II and ET-1 can activate the RhoA pathway and that prevention of the hypertrophic response to both agonists by adenosine receptor activation is mediated by prevention of RhoA stimulation and actin polymerization.

Attenuating effect of adenosine receptor agonists on the development of behavioral sensitization induced by sporadic treatment with morphine

The aim of the study is to investigate the effect of adenosine receptor agonists on the development of morphine-induced sensitization to the locomotor activity of mice. Selective A1 (N6-cyclopentyladenosine – CPA) and A2A (2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride – CGS 21680) adenosine receptor agonists or non-selective A1/A2A (5'-N-ethylcarboxamidoadenosine – NECA) adenosine agonists as representatives of adenosinergic drugs have been used in the experiment. Behavioral sensitization has been obtained by sporadic treatment with morphine (10.0mg/kg, i.p.). We have shown that adenosine receptor agonists co-administered with morphine significantly attenuates increase in the locomotor activity of mice evoked by challenge dose of morphine. These effects have been observed after stimulation of the selective A1 or A2A and non-selective A1/A2A adenosine receptors, namely both receptors were involved in morphine-induced sensitization. Thus, we have demonstrated that adenosine agonists are able to inhibit behavioral sensitization induced by sporadic applications of morphine.

Adenosine A1 Receptor Activation as a Brake on the Microglial Response after Experimental Traumatic Brain Injury in Mice

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.

Differential AMPK phosphorylation sites associated with phenylephrine vs. antihypertrophic effects of adenosine agonists in neonatal rat ventricular myocytes

Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic alpha-subunit of AMPK at two different sites, Thr(172) and Ser(485/491), in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser(485/491), in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr(172). Furthermore, the phosphorylation at Ser(485/491) by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr(172) by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser(485/491) and at Thr(172), associated with prohypertrophic and antihypertrophic responses, respectively.

Effects of adenosine receptor agonists on guinea-pig isolated working hearts and the role of endothelium and NO.

The hypothesis that the coronary vasodilator effects of adenosine receptor agonists are independent of the vascular endothelium or mediators derived therefrom was examined in guinea-pig isolated working hearts. Adenosine receptor agonists, 5'-(N-ethylcarboxamido)-adenosine (NECA; two-fold selective for A2 over A1 receptors), 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; A2A selective), N6-cyclopentyl-adenosine (CPA; A1 selective) and N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; A3 selective), were infused (3 x 10(-7) M) after endothelium removal by passing oxygen through the coronary circulation. In spontaneously beating hearts, CGS21680 and NECA increased, while CPA decreased, coronary flow. NECA and CPA reduced heart rate, left ventricular pressure and aortic output. The nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NOARG; 3 x 10(-5) M) abolished the vasodilatation by NECA but not CGS21680, indicating that nitric oxide (NO) of a non-endothelial source mediated the NECA response. Coronary vasodilatation by CGS21680 was inhibited bythe A2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). Indometacin (10(-6) M) attenuated the coronary vasodilatation to CGS21680, suggesting a partial role for cyclooxygenase products. IB-MECA had no effect, indicating no A3 receptor involvement. In paced working hearts, the responses were similar except CPA had no effect on coronary flow or aortic output and CGS21680 increased left ventricular pressure and the maximum rate of ventricular pressure rise. This study has demonstrated functionally effective removal of the endothelium by a novel method of passing oxygen through the coronary vasculature. A coronary vasodilator action of adenosine receptor agonists mediated via A2A receptors is endothelium- and NO-independent, but partially involves cyclooxygenase products.

Adenosine receptor agonists affect taurine release from mouse brain stem slices in ischemia

The release of the inhibitory amino acid taurine is markedly enhanced under ischemic conditions in both adult and developing brain stem, together with a pronounced increase in the release of the neuromodulator adenosine. We now studied the effects of adenosine receptor agonists and antagonists on [(3)H]taurine release in the brain stem in normoxia and ischemia, using a superfusion system. Under standard conditions, the adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) potentiated basal taurine release in adult mice, which response was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CHA and the A(2a) receptor agonist 2-p-(2-carboxyethyl)phenylamino-5'-N-ethylcarboxaminoadenosinehydrochloride (CGS 21680) had no effect on the release in developing mice. In ischemia, CHA depressed both basal and K(+)-stimulated taurine release in developing mice in a receptor-mediated manner, blocked by DPCPX. The A(2a) receptor agonist CGS 21680 was also inhibitory. Taurine and adenosine may thus not cooperate in developing mice to prevent ischemic neuronal damage. On the other hand, CGS 21680 enhanced taurine release in the adult brain stem in ischemia, both basal and K(+)-stimulated release being affected. These effects were abolished by the antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicating a receptor-mediated process. In this case elevated levels of taurine could be beneficial, protecting against hyperexcitation and excitotoxicity.

Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA

We have shown that the cellular process of macroautophagy plays a protective role in HL-1 cardiomyocytes subjected to simulated ischemia/reperfusion (sI/R) (Hamacher-Brady et al. in J Biol Chem 281(40):29776-29787). Since the nucleoside adenosine has been shown to mimic both early and late phase ischemic preconditioning, a potent cardioprotective phenomenon, the purpose of this study was to determine the effect of adenosine on autophagosome formation. Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles, and can be monitored by imaging the incorporation of microtubule-associated light chain 3 (LC3) fused to a fluorescent protein (GFP or mCherry) into nascent autophagosomes. We investigated the effect of adenosine receptor agonists on autophagy and cell survival following sI/R in GFP-LC3 infected HL-1 cells and neonatal rat cardiomyocytes. The A(1) adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) (100 nM) caused an increase in the number of autophagosomes within 10 min of treatment; the effect persisted for at least 300 min. A significant inhibition of autophagy and loss of protection against sI/R measured by release of lactate dehydrogenase (LDH), was demonstrated in CCPA-pretreated cells treated with an A(1) receptor antagonist, a phospholipase C inhibitor, or an intracellular Ca(+2) chelator. To determine whether autophagy was required for the protective effect of CCPA, autophagy was blocked with a dominant negative inhibitor (Atg5(K130R)) delivered by transient transfection (in HL-1 cells) or protein transduction (in adult rat cardiomyocytes). CCPA attenuated LDH release after sI/R, but protection was lost when autophagy was blocked. To assess autophagy in vivo, transgenic mice expressing the red fluorescent autophagy marker mCherry-LC3 under the control of the alpha myosin heavy chain promoter were treated with CCPA 1 mg/kg i.p. Fluorescence microscopy of cryosections taken from the left ventricle 30 min after CCPA injection revealed a large increase in the number of mCherry-LC3-labeled structures, indicating the induction of autophagy by CCPA in vivo. Taken together, these results indicate that autophagy plays an important role in mediating the cardioprotective effects conferred by adenosine pretreatment.

Adenosine Receptor Agonists Modulate Visceral Hyperalgesia in the Rat

Adenosine is an endogenous modulator of nociception. Its role in visceral nociception, particularly in visceral hyperalgesia, has not been studied. The aim of this study was to determine the effects of adenosine receptor agonists in a model of visceral hyperalgesia. The visceromotor response (VMR) in rats to colorectal distension (CRD; 80 mmHg, 20 seconds) was quantified by electromyographic recordings from the abdominal musculature. Three hours after the intracolonic administration of zymosan (25 mg/mL, 1 mL), VMRs to CRD were measured before and after either subcutaneous or intrathecal administration of an adenosine receptor agonist. Subcutaneous injection of 5'-N-ethylcarboxyamidoadenosine (NECA; an A1 and A2 receptor agonist), R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA; a selective A1 receptor agonist), or CGS-21680 hydrochloride (a selective A2a receptor agonist) dose-dependently (10-100 mg/kg) attenuated the VMR to CRD, although hindlimb weakness occurred at the higher doses tested. Intrathecal administration of NECA or R-PIA dose-dependently (0.1-1.0 microg/kg) decreased the VMR, whereas CGS-21680 hydrochloride was ineffective over the same concentration range. Higher intrathecal doses of the A1/A2 receptor agonist NECA produced motor weakness. Adenosine receptor agonists are antihyperalgesic, but also produce motor weakness at high doses. However, activation of the spinal A1 receptor significantly attenuates the VMR to CRD without producing motor weakness.

Effect of adenosine receptor agonists on anticonvulsive action of antiepileptic drugs in a test of maximal electroshock in mice

Effect of adenosine receptor agonists on anticonvulsive action of antiepileptic drugs in a test of maximal electroshock in mice

Adenosine A 3 receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis

The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A 3 receptor agonist, N 6-(3-iodobenzyl)adenosine-5′- N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200 nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions.

The effect of adenosine receptor agonists on cytokine release by human mononuclear cells depends on the specific Toll-like receptor subtype used for stimulation

In the present study, we determined whether the immunomodulatory effect of adenosine receptor stimulation depends on the Toll-like Receptor (TLR) used for stimulation of cytokine release. Therefore, human mononuclear cells were stimulated by different TLR agonists in the absence and presence of A 1 (CPA), A 2a (CGS21680), and A 3 (Cl-IB-MECA) adenosine receptor agonists. Effects of these agonists on Il-6, Il-10, IFN-γ, TNF-α, and Il-1β production were expressed as percentage inhibition/stimulation after TLR stimulation. CGS21680 inhibited TLR4-mediated TNF-α release and potentiated TLR3- and TLR5-mediated IL-6 release. Cl-IB-MECA inhibited TLR4-agonist-induced IFN-γ release. Interestingly, CPA en Cl-IB-MECA tended to inhibit cytokine release only after TLR4 stimulation. In more detail, CPA potentiated TLR5-mediated IL-6 production, TLR3-mediated IFN-γ production and TLR3-mediated Il-1β-production compared to TLR4-mediated stimulation. Cl-IB-MECA potentiated TLR5-mediated IL-6 and Il-1β formation as compared to TLR4-mediated stimulation. Finally, CGS21680 potentiated TLR5-mediated IL-6 production compared to TLR1-2 stimulation, and potentiated TLR3- and TLR5-mediated IL-10 production compared to TLR1-2-mediated stimulation. In conclusion, the effect of adenosine agonists on cytokine production depends on the specific TLR agonist used for stimulation. These findings suggest that well-known anti-inflammatory effects of adenosine agonists on LPS-induced inflammation cannot be extrapolated to situations in which stimulation of other TLR subtypes is involved.

Human Osteoblast Precursors Produce Extracellular Adenosine, Which Modulates Their Secretion of IL-6 and Osteoprotegerin

We showed that human osteoprogenitor cells produced adenosine and expressed ecto-5'-nucleotidase and all four adenosine receptor subtypes. Adenosine stimulated IL-6 but inhibited osteoprotegerin secretion, suggesting that adenosine is a newly described regulator of progenitor cell function. Maintaining skeletal homeostasis relies on there being a balance between bone formation and resorption; an imbalance between these processes can lead to diseases such as osteoporosis and rheumatoid arthritis. Recent reports showed that locally produced ATP, acting through P2 receptors, has pronounced effects on bone formation. However, ATP can be enzymatically cleaved to adenosine that has little or no activity at P2 receptors but mediates its action through the P1 family of receptors. We studied whether adenosine may also have an important role in controlling bone cell differentiation and function. Extracellular adenosine levels were analyzed by high-performance liquid chromatography in HCC1 and bone marrow stromal (BMS) cells. Ecto-5'-nucleotidase (CD73) expression and activity was determined by RT-PCR, immunocytochemistry, and the cleavage of etheno-AMP to ethenoadenosine. Adenosine receptor expression and activity were determined by RT-PCR and cAMP measurements. The effects of adenosine receptor agonists on IL-6, osteoprotegerin (OPG), and RANKL expression were determined by ELISA and QRT-PCR. HCC1 and BMS cells produce adenosine and express CD73 and all four adenosine receptor subtypes. The A2b receptor was shown to be functionally dominant in HCC1 cells, as determined by cAMP production and in its stimulation of IL-6 secretion. Adenosine receptor agonism also inhibited OPG secretion and OPG but not RANKL mRNA expression. Our findings show that HCC1 and primary BMS cells produce adenosine, express CD73 and all four adenosine receptor subtypes. In HCC1 cells, adenosine has a potent stimulatory action on IL-6 secretion but an inhibitory action on OPG expression. These data show for the first time that adenosine may be an important regulator of progenitor cell differentiation and hence an important local contributor to the regulation of bone formation and resorption.

Characterization of the Effects of Adenosine Receptor Agonists on Cerebral Blood Flow in Uninjured and Traumatically Injured Rat Brain using Continuous Arterial Spin-Labeled Magnetic Resonance Imaging

Hypoperfusion after traumatic brain injury may exacerbate damage. Adenosine, a vasodilator, regulates cerebral blood flow (CBF). Treatment with adenosine receptor agonists has shown benefit in experimental CNS trauma; however, their effects on CBF after injury remain undefined. We used magnetic resonance imaging to assess CBF in uninjured rats both early and at 24 h after intrahippocampal administration of either the nonselective adenosine receptor agonist 2-chloroadenosine (2-CA, 12 nmol) or the A(2A)-receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarbox-amidoadenosine (CGS 21680, 6 nmol). We also assessed the effects of these agents on cerebral metabolic rate for glucose (CMRglu). We then assessed the effect of 2-CA on CBF at 3.5 to 5 h after controlled cortical impact (CCI). Injection of 2-CA into uninjured rat brain produced marked increases in CBF in ipsilateral hippocampus and cortex versus vehicle (P<0.05); CBF increases persisted even at 24 h. Measurement of hippocampal levels of 2-CA showed persistent increases to 24 h. CGS 21680 produced even more marked global increases in CBF than seen with 2-CA (2-6-fold versus vehicle, P<0.05 in 10/12 regions of interest (ROIs)). Neither agonist altered CMRglu versus vehicle. After CCI, 2-CA increased CBF in ipsilateral hippocampal and hemispheric ROIs (P<0.05 versus vehicle), but the response was attenuated at severe injury levels. We report marked increases in CBF after injection of adenosine receptor agonists into uninjured rat brain despite unaltered CMRglu. 2-Chloroadenosine produced enduring increases in CBF in uninjured brain and attenuated posttraumatic hypoperfusion. Future studies of adenosine-related therapies in CNS injury should address the role of CBF.