Anti-inflammatory effects of inosine in allergic lung inflammation in mice: evidence for the participation of adenosine A2A and A3 receptors

Inosine, a naturally occurring purine formed from the breakdown of adenosine, is associated with immunoregulatory effects. Evidence shows that inosine modulates lung inflammation and regulates cytokine generation. However, its role in controlling allergen-induced lung inflammation has yet to be identified. In this study, we aimed to investigate the role of inosine and adenosine receptors in a murine model of lung allergy induced by ovalbumin (OVA). Intraperitoneal administration of inosine (0.001–10 mg/kg, 30 min before OVA challenge) significantly reduced the number of leukocytes, macrophages, lymphocytes and eosinophils recovered in the bronchoalveolar lavage fluid of sensitized mice compared with controls. Interestingly, our results showed that pre-treatment with the selective A_2A receptor antagonist (ZM241385), but not with the selective A_2B receptor antagonist (alloxazine), reduced the inhibitory effects of inosine against macrophage count, suggesting that A_2A receptors mediate monocyte recruitment into the lungs. In addition, the pre-treatment of mice with selective A₃ antagonist (MRS3777) also prevented inosine effects against macrophages, lymphocytes and eosinophils. Histological analysis confirmed the effects of inosine and A_2A adenosine receptors on cell recruitment and demonstrated that the treatment with ZM241385 and alloxazine reverted inosine effects against mast cell migration into the lungs. Accordingly, the treatment with inosine reduced lung elastance, an effect related to A₂ receptors. Moreover, inosine reduced the levels of Th₂-cytokines, interleukin-4 and interleukin-5, an effect that was not reversed by A_2A or A_2B selective antagonists. Our data show that inosine acting on A_2A or A₃ adenosine receptors can regulate OVA-induced allergic lung inflammation and also implicate inosine as an endogenous modulator of inflammatory processes observed in the lungs of asthmatic patients.     

The role of peripheral adenosine receptors in glutamate-induced pain nociceptive behavior

The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 μmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 μg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8−10). In contrast, i.pl. injection of hydrochloride hydrate ({"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053"}}CGS21680, an adenosine A2A receptor agonist) (1 μg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 μg/paw) caused a significant reduction (p<0.05, n=7−8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs—8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1–10 μg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1–100 μg/paw), alloxazine (an A2B antagonist, 0.1–3 μg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1–100 μg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 μg/paw) significantly blocked the anti-nociceptive effect of CHA (1 μg/paw) (p<0.05, n=7–9). Similarly, ZM241385 (20 μg/paw) administered prior to {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053"}}CGS21680 (1 μg/paw) significantly blocked {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053"}}CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 μg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7–8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 μg/paw) (p<0.05, n=7−9) but not by the A2A antagonist ZM241385 (10 μg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.     

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A_2A and A₃ receptor expression was observed in the arterial wall and A_2A-immunoreactivity was identified in the adventitia–media junction and endothelium. A₁ and A_2B receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA) = {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A_2A antagonist, reduced NECA relaxations that were not modified by A₁, A_2B, and A₃ receptor antagonists. Neuronal voltage-gated Ca²⁺ channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK_Ca)- and small (SK_Ca)-conductance Ca²⁺-activated K⁺ channels. Inhibition of cyclooxygenase (COX), large-conductance Ca²⁺-activated-, ATP-dependent-, and voltage-gated-K⁺ channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A_2A purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK_Ca and SK_Ca channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.     

Static magnetic field exposure reproduces cellular effects of the Parkinson's disease drug candidate ZM241385

Background

This study was inspired by coalescing evidence that magnetic therapy may be a viable treatment option for certain diseases. This premise is based on the ability of moderate strength fields (i.e., 0.1 to 1 Tesla) to alter the biophysical properties of lipid bilayers and in turn modulate cellular signaling pathways. In particular, previous results from our laboratory (Wang et al., BMC Genomics, 10, 356 (2009)) established that moderate strength static magnetic field (SMF) exposure altered cellular endpoints associated with neuronal function and differentiation. Building on this background, the current paper investigated SMF by focusing on the adenosine A_2A receptor (A_2AR) in the PC12 rat adrenal pheochromocytoma cell line that displays metabolic features of Parkinson''s disease (PD).

Methodology and Principal Findings

SMF reproduced several responses elicited by ZM241385, a selective A_2AR antagonist, in PC12 cells including altered calcium flux, increased ATP levels, reduced cAMP levels, reduced nitric oxide production, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake. SMF also counteracted several PD-relevant endpoints exacerbated by A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 in a manner similar to ZM241385; these include reduction of increased expression of A_2AR, reversal of altered calcium efflux, dampening of increased adenosine production, reduction of enhanced proliferation and associated p44/42 MAPK phosphorylation, and inhibition of neurite outgrowth.

Conclusions and Significance

When measured against multiple endpoints, SMF elicited qualitatively similar responses as ZM241385, a PD drug candidate. Provided that the in vitro results presented in this paper apply in vivo, SMF holds promise as an intriguing non-invasive approach to treat PD and potentially other neurological disorders.     

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A2A receptors and endogenous adenosine

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N⁶‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A_2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A_2A receptors. The A_2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A_2A receptors modulators.     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.     

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A_2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A_2B and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A_2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A_2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A_2A and A₃) receptors by endogenous adenosine. The selective A_2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1–10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A_2A receptors. In contrast to 2-Cl-IB MECA, activation of A_2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A_2A receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A_2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.     

Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A₁ and A₂ as well as forkolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A₁ receptors were specifically labeled with [³H]chloro-N⁶-cyclopentyladenosine (CCPA), whereas [³H]CGS 21680 was used for the analysis of A₂ receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax=160 fmol/mg protein; Kd=2.9 nM), and for CGS 21680 (Bmax=14 fmol/mg protein; Kd=1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [³H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.     

Functional Coupling of the Gαolf Variant XLGαolf with the Human Adenosine A2A Receptor

A recently identified novel Gα_olf variant, XLGα_olf, is shown to functionally couple to the human adenosine A_2A receptor (A_2AR). In Sf9 cells expressing A_2AR, β1, and γ2, co-expression of XLGα_olf increased NECA-induced [³⁵S]GTPγS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A_2AR ligands on these cells were evaluated by using [³H]ZM241385- and [³⁵S]GTPγS- binding assays. The rank order of the equilibrium binding constants (K_d or K_i) of adenosine receptor ligands were [³H]ZM241385 ≈ CGS15943 < MRS1220 < < CV1808 ≈ NECA < CGS21680 ≈ adenosine < IBMECA < HEMADO ≈ CPA ≈ CCPA. The rank order of EC₅₀ values for agonists were CV1808 ≈ NECA < adenosine ≈ CGS26180 < IBMECA < HEMADO ≈ CPA ≈ CCPA. This pharmacology is consistent with the literature for A_2AR and suggests that Sf9 cells co-expressing A_2AR, β1, γ2, and XLGα_olf could serve as a heterologous expression system for A_2AR drug screening.     

Is the functional interaction between adenosine A2A receptors and metabotropic glutamate 5 receptors a general mechanism in the brain? Differences and similarities between the striatum and the hippocampus

The aim of the present paper was to examine, in a comparative way, the occurrence and the mechanisms of the interactions between adenosine A_2A receptors (A_2ARs) and metabotropic glutamate 5 receptors (mGlu5Rs) in the hippocampus and the striatum. In rat hippocampal and corticostriatal slices, combined ineffective doses of the mGlu5R agonist 2-chloro-5-hydroxyphenylglycine (CHPG) and the A_2AR agonist CGS 21680 synergistically reduced the slope of excitatory postsynaptic field potentials (fEPSPs) recorded in CA1 and the amplitude of field potentials (FPs) recorded in the dorsomedial striatum. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway appeared to be involved in the effects of CGS 21680 in corticostriatal but not in hippocampal slices. In both areas, a postsynaptic locus of interaction appeared more likely. N-methyl-D-aspartate (NMDA) reduced the fEPSP slope and FP amplitude in hippocampal and corticostriatal slices, respectively. Such an effect was significantly potentiated by CHPG in both areas. Interestingly, the A_2AR antagonist ZM 241385 significantly reduced the NMDA-potentiating effect of CHPG. In primary cultures of rat hippocampal and striatal neurons (ED 17, DIV 14), CHPG significantly potentiated NMDA-induced lactate dehydrogenase (LDH) release. Again, such an effect was prevented by ZM 241385. Our results show that A_2A and mGlu5 receptors functionally interact both in the hippocampus and in the striatum, even though different mechanisms seem to be involved in the two areas. The ability of A_2ARs to control mGlu5R-dependent effects may thus be a general feature of A_2ARs in different brain regions (irrespective of their density) and may represent an additional target for the development of therapeutic strategies against neurological disorders.     

Increase in the Number, G Protein Coupling, and Efficiency of Facilitatory Adenosine A2A Receptors in the Limbic Cortex, but not Striatum, of Aged Rats

Adenosine's effects result from a balanced activation of inhibitory A1 and facilitatory A2A receptors. Because in aged animals there is an increased number of A2A receptors, we now compared the efficiency of A2A receptors in cortical and striatal preparations of young adult (6-week-old) and aged (2-year-old) rats. In cortical, in contrast to striatal, membranes from aged rats, A2A receptors were more tightly coupled to G proteins, because 5'-guanylylimidodiphosphate (100 microM) increased by 321% the Ki of the A2A agonist CGS21680 as a displacer of binding of the A2A antagonist [3H]ZM241385 (1 nM), compared with a 112% increase in young rats. In cortical slices, CGS21680 (30-1,000 nM) was virtually devoid of effect on cyclic AMP accumulation in young rats but increased cyclic AMP accumulation with an EC50 of 153 nM in aged rats, whereas the efficiency of CGS21680 was similar in striatal slices of young and aged rats. CGS21680 (30 nM) was virtually devoid of effect on acetylcholine release from hippocampal CA1 slices of young rats but caused a 55% facilitation in aged rats. These results show that the number of A2A receptors, their coupling to G proteins, and their efficiency are enhanced in the limbic cortex of aged rats, suggesting a greater involvement of facilitation in adenosine responses.     

Apremilast,a novel phosphodiesterase 4 (PDE4) inhibitor,regulates inflammation through multiple cAMP downstream effectors

IntroductionThis work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A_2A receptors (A_2AR).MethodsAfter apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A_2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student’s t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant.ResultsIn vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC₅₀=104nM) and the specific A_2AR-agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (1μM) increased apremilast potency (IC₅₀=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone.ConclusionsThe immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0771-6) contains supplementary material, which is available to authorized users.     

Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries

This study aimed to evaluate the role for adenosine A2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 micromol/l, A2A antagonist) and alloxazine (1 micromol/l, A2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 micromol/l, A1 antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5'-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 micromol/l of ZM 241385 (53.0+/-3.9 mm Hg to 69.2+/-2.9 mm Hg, P<0.01) and 10 micromol/l of glibenclamide (54.7+/-6.5 mm Hg to 77.9+/-4.2 mm Hg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A2A and A2B receptors, but not by A1 subtype, and activation of adenosine A2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K+ channels in response to hypotension and maintenance of CBF autoregulation.     

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X_1–5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X₁, P2X₃, P2X_2/3, and P2X₇ receptors, respectively. Moreover, adenosine receptors blockade with A₁ receptor antagonist DPCPX (100 nM) or with A_2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A_2A receptor antagonist ZM241385 (50 nM), or A₃ receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A₁, and A_2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Activation of adenosine A2 receptors enhances high K+-evoked taurine release from rat hippocampus: A microdialysis study

Summary The present study was designed to examine which type of adenosine receptors was involved in enhancement of high K⁺-evoked taurine release fromin vivo rat hippocampus using microdialysis. Perfusion with 0.5 or 5.0 mM adenosine enhanced high K⁺-evoked taurine release. Perfusion with 2M R(–)-N⁶-2-phenylisopropyladenosine (PIA), a selective adenosine A₁ receptor agonist, did not modulate taurine release. Perfusion with 1M 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A₁ receptor antagonist, increased taurine release. On the other hand, perfusion with 20M 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethyl-carboxamideadenosine (CGS21680), a selective adenosine A_2A receptor agonist, enhanced taurine release, while perfusion with 1 mM 3,7-dimethyl-propagylxanthine (DMPX), an adenosine A₂ receptor antagonist, did not affect taurine release. These results demonstrate that adenosine enhances high K⁺-evoked taurine release via activation of adenosine A_2A receptors from both neurons and glial cells ofin vivo rat hippocampus.     

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A_2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A₁ receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4–6 weeks old) and symptomatic (12–14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg²⁺ paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N⁶-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.     

Molecular modeling of a PAMAM-CGS21680 dendrimer bound to an A2A adenosine receptor homodimer

The theoretical possibility of bivalent binding of a dendrimer, covalently appended with multiple copies of a small ligand, to a homodimer of a G protein-coupled receptor was investigated with a molecular modeling approach. A molecular model was constructed of a third generation (G3) poly(amidoamine) (PAMAM) dendrimer condensed with multiple copies of the potent A_2A adenosine receptor agonist CGS21680. The dendrimer was bound to an A_2A adenosine receptor homodimer. Two units of the nucleoside CGS21680 could occupy the A_2A receptor homodimer simultaneously. The binding mode of CGS21680 moieties linked to the PAMAM dendrimer and docked to the A_2A receptor was found to be similar to the binding mode of a monomeric CGS21680 ligand.     

Functional coupling of the Galpha(olf) variant XLGalpha(olf) with the human adenosine A2A receptor

A recently identified novel Galphaolf variant, XLGalphaolf, is shown to functionally couple to the human adenosine A2A receptor (A2AR). In Sf9 cells expressing A2AR, beta1, and gamma2, co-expression of XLGalphaolf increased NECA-induced [35S]GTPgammaS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A2AR ligands on these cells were evaluated by using [3H]ZM241385- and [35S]GTPgammaS- binding assays. The rank order of the equilibrium binding constants (Kd or Ki) of adenosine receptor ligands were [3H]ZM241385 approximately CGS15943 < MRS1220 < < CV1808 approximately NECA < CGS21680 approximately adenosine < IBMECA < HEMADO approximately CPA approximately CCPA. The rank order of EC50 values for agonists were CV1808 approximately NECA < adenosine approximately CGS26180 < IBMECA < HEMADO approximately CPA approximately CCPA. This pharmacology is consistent with the literature for A2AR and suggests that Sf9 cells co-expressing A2AR, beta1, gamma2, and XLGalphaolf could serve as a heterologous expression system for A2AR drug screening.     

The antidepressant-like effect of inosine in the FST is associated with both adenosine A1 and A2A receptors

Inosine is an endogenous purine nucleoside, which is formed during the breakdown of adenosine. The adenosinergic system was already described as capable of modulating mood in preclinical models; we now explored the effects of inosine in two predictive models of depression: the forced swim test (FST) and tail suspension test (TST). Mice treated with inosine displayed higher anti-immobility in the FST (5 and 50 mg/kg, intraperitoneal route (i.p.)) and in the TST (1 and 10 mg/kg, i.p.) when compared to vehicle-treated groups. These antidepressant-like effects started 30 min and lasted for 2 h after intraperitoneal administration of inosine and were not accompanied by any changes in the ambulatory activity in the open-field test. Both adenosine A₁ and A_2A receptor antagonists prevented the antidepressant-like effect of inosine in the FST. In addition, the administration of an adenosine deaminase inhibitor (1 and 10 mg/kg, i.p.) also caused an antidepressant-like effect in the FST. These results indicate that inosine possesses an antidepressant-like effect in the FST and TST probably through the activation of adenosine A₁ and A_2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.