Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice.

Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03-1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.

Effect of trans-resveratrol on dexamethasone-induced changes in the expression of MMPs by human trabecular meshwork cells: Involvement of adenosine A1 receptors and NFkB.

Intraocular pressure (IOP) lowering in glaucomatous eyes is currently achieved mainly by improved aqueous outflow via alternate drainage pathways. However, the focus is now shifting to trabecular meshwork (TM), the site or major pathological changes including increased extracellular matrix (ECM) deposition and reduced matrix metalloproteinases (MMPs) secretion by TM cells. Trans-resveratrol was previously shown to lower IOP and reduce ECM deposition; however, the mechanisms of action remain unclear. Therefore, we determined the effect of trans-resveratrol on MMP-2 and -9 expression by human TM cells (HTMCs) in the presence of dexamethasone and whether it also affects adenosine A1 receptors (A1AR) expression and nuclear factor kappa B (NFkB) activation. We observed that trans-resveratrol, 12.5 µM, increased MMP-2 and -9 protein expression by HTMCs despite exposure to dexamethasone (1.89- and 1.53-fold, respectively; P < 0.001). Further it was observed that trans-resveratrol increases A1AR expression in HTMC in the presence of dexamethasone (1.55-fold; P < 0.01). Trans-resveratrol also increased NFkB activation in the presence of dexamethasone and A1AR antagonist (P < 0.01 versus dexamethasone group). These effects of trans-resveratrol were associated with increased MMP -2 and -9 expression. It could be concluded that trans-resveratrol prevents dexamethasone-induced reduction in MMP-2 and -9 secretion by NFkB activation in HTMCs. This effect of trans-resveratrol is likely to involve increased A1AR expression.

Adenosine A1 Receptor mRNA Expression by Neurons and Glia in the Auditory Forebrain.

In the brain, purines such as ATP and adenosine can function as neurotransmitters and co-transmitters, or serve as signals in neuron-glial interactions. In thalamocortical (TC) projections to sensory cortex, adenosine functions as a negative regulator of glutamate release via activation of the presynaptic adenosine A1 receptor (A1 R). In the auditory forebrain, restriction of A1 R-adenosine signaling in medial geniculate (MG) neurons is sufficient to extend LTP, LTD, and tonotopic map plasticity in adult mice for months beyond the critical period. Interfering with adenosine signaling in primary auditory cortex (A1) does not contribute to these forms of plasticity, suggesting regional differences in the roles of A1 R-mediated adenosine signaling in the forebrain. To advance understanding of the circuitry, in situ hybridization was used to localize neuronal and glial cell types in the auditory forebrain that express A1 R transcripts (Adora1), based on co-expression with cell-specific markers for neuronal and glial subtypes. In A1, Adora1 transcripts were concentrated in L3/4 and L6 of glutamatergic neurons. Subpopulations of GABAergic neurons, astrocytes, oligodendrocytes, and microglia expressed lower levels of Adora1. In MG, Adora1 was expressed by glutamatergic neurons in all divisions, and subpopulations of all glial classes. The collective findings imply that A1 R-mediated signaling broadly extends to all subdivisions of auditory cortex and MG. Selective expression by neuronal and glial subpopulations suggests that experimental manipulations of A1 R-adenosine signaling could impact several cell types, depending on their location. Strategies to target Adora1 in specific cell types can be developed from the data generated here. Anat Rec, 301:1882-1905, 2018. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

BACKGROUND: Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). METHODS: We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). RESULTS: Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. CONCLUSIONS: This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine.

Differential Expression of Adenosine P1 Receptor ADORA1 and ADORA2A Associated with Glioma Development and Tumor-Associated Epilepsy.

Level of adenosine, an endogenous astrocyte-based neuromodulator, is primarily regulated by adenosine P1 receptors. This study assessed expression of adenosine P1 receptors, ADORA1 (adenosine A1 receptor) and ADORA2A (adenosine A2a receptor) and their association with glioma development and epilepsy in glioma patients. Expression of ADORA1/ADORA2A was assessed immunohistochemically in 65 surgically removed glioma tissue and 21 peri-tumor tissues and 8 cases of normal brain tissues obtained from hematoma patients with cerebral trauma. Immunofluorescence, Western blot, and qRT-PCR were also used to verify immunohistochemical data. Adenosine P1 receptor ADORA1 and ADORA2A proteins were localized in the cell membrane and cytoplasm and ADORA1/ADORA2A immunoreactivity was significantly stronger in glioma and peri-tumor tissues that contained infiltrating tumor cells than in normal brain tissues (p < 0.05). The World Health Organization (WHO) grade III gliomas expressed even higher level of ADORA1 and ADORA2A. Western blot and qRT-PCR confirmed immunohistochemical data. Moreover, higher levels of ADORA1 and ADORA2A expression occurred in high-grade gliomas, in which incidence of epilepsy were lower (p < 0.05). In contrast, a lower level of ADORA1/ADORA2A expression was found in peri-tumor tissues with tumor cell presence from patients with epilepsy compared to patients without epilepsy (p < 0.05). The data from the current study indicates that dysregulation in ADORA1/ADORA2A expression was associated with glioma development, whereas low level of ADORA1/ADORA2A expression could increase susceptibility of tumor-associated epilepsy.

Effects of perinatal exposure to lead (Pb) on purine receptor expression in the brain and gliosis in rats tolerant to morphine analgesia.

The aim of the present study was to investigate the molecular effects of perinatal exposure to lead (Pb) on protein and mRNA expression of purine receptors: P2X4, P2X7, adenosine receptor A1; and astrocytes (GFAP mRNA expression) and on microglia activation (Iba1 mRNA expression) in several structures of the mesolimbic system (striatum, hippocampus, prefrontal cortex) in rats expressing tolerance to the antinociceptive effect of morphine. Rat mothers were orally treated with 0.1% lead acetate from conception, through gestation, and postnatally, as well as to offspring up to day (PND) 28; subsequently molecular studies were conducted on adult (PND 60) male rats. Morphine tolerance developed more strongly in rats perinatally exposed to Pb. The analysis revealed a significant up-regulation of protein and mRNA P2X4 receptor expression in the striatum and prefrontal cortex but not in the hippocampus; P2X7 protein and mRNA receptor expression in the striatum and hippocampus, but not in the prefrontal cortex; A1 protein receptor expression in all investigated structures and A1 mRNA expression in the striatum and hippocampus; Iba1 mRNA expression in the striatum and hippocampus; and GFAP mRNA expression in the striatum and prefrontal cortex. Immunohistochemical analysis has also revealed significant alterations. Strong expressions of P2X4, P2X7, A1 receptors, astrocytes and microglia activation were observed in the hippocampus in Pb and/or morphine treated rats. The higher expression of purine receptors and glial cell activation are important markers of neuroinflammatory processes. Therefore, we conclude that Pb-induced neuroinflammation may be responsible for the intensification of morphine tolerance in the Pb-treated rats. Additionally, the dysregulation of A1 adenosine receptors, mainly in the hippocampus, may also be involved in the intensification of morphine tolerance in Pb-treated rats. Our study demonstrates the significant participation of environmental factors in addictive process; additionally, it shows the necessity of modification of addictive disorder with neuroprotective agents.

Increased Signaling via Adenosine A1 Receptors, Sleep Deprivation, Imipramine, and Ketamine Inhibit Depressive-like Behavior via Induction of Homer1a.

Major depressive disorder is among the most commonly diagnosed disabling mental diseases. Several non-pharmacological treatments of depression upregulate adenosine concentration and/or adenosine A1 receptors (A1R) in the brain. To test whether enhanced A1R signaling mediates antidepressant effects, we generated a transgenic mouse with enhanced doxycycline-regulated A1R expression, specifically in forebrain neurons. Upregulating A1R led to pronounced acute and chronic resilience toward depressive-like behavior in various tests. Conversely, A1R knockout mice displayed an increased depressive-like behavior and were resistant to the antidepressant effects of sleep deprivation (SD). Various antidepressant treatments increase homer1a expression in medial prefrontal cortex (mPFC). Specific siRNA knockdown of homer1a in mPFC enhanced depressive-like behavior and prevented the antidepressant effects of A1R upregulation, SD, imipramine, and ketamine treatment. In contrast, viral overexpression of homer1a in the mPFC had antidepressant effects. Thus, increased expression of homer1a is a final common pathway mediating the antidepressant effects of different antidepressant treatments.

Upregulation of adenosine A1 receptors facilitates sinoatrial node dysfunction in chronic canine heart failure by exacerbating nodal conduction abnormalities revealed by novel dual-sided intramural optical mapping.

BACKGROUND: Although sinoatrial node (SAN) dysfunction is a hallmark of human heart failure (HF), the underlying mechanisms remain poorly understood. We aimed to examine the role of adenosine in SAN dysfunction and tachy-brady arrhythmias in chronic HF. METHODS AND RESULTS: We applied multiple approaches to characterize SAN structure, SAN function, and adenosine A1 receptor expression in control (n=17) and 4-month tachypacing-induced chronic HF (n=18) dogs. Novel intramural optical mapping of coronary-perfused right atrial preparations revealed that adenosine (10 µmol/L) markedly prolonged postpacing SAN conduction time in HF by 206 ± 99 milliseconds (versus 66 ± 21 milliseconds in controls; P=0.02). Adenosine induced SAN intranodal conduction block or microreentry in 6 of 8 dogs with HF versus 0 of 7 controls (P=0.007). Adenosine-induced SAN conduction abnormalities and automaticity depression caused postpacing atrial pauses in HF versus control dogs (17.1 ± 28.9 versus 1.5 ± 1.3 seconds; P<0.001). Furthermore, 10 µmol/L adenosine shortened atrial repolarization and led to pacing-induced atrial fibrillation in 6 of 7 HF versus 0 of 7 control dogs (P=0.002). Adenosine-induced SAN dysfunction and atrial fibrillation were abolished or prevented by adenosine A1 receptor antagonists (50 µmol/L theophylline/1 µmol/L 8-cyclopentyl-1,3-dipropylxanthine). Adenosine A1 receptor protein expression was significantly upregulated during HF in the SAN (by 47 ± 19%) and surrounding atrial myocardium (by 90 ± 40%). Interstitial fibrosis was significantly increased within the SAN in HF versus control dogs (38 ± 4% versus 23 ± 4%; P<0.001). CONCLUSIONS: In chronic HF, adenosine A1 receptor upregulation in SAN pacemaker and atrial cardiomyocytes may increase cardiac sensitivity to adenosine. This effect may exacerbate conduction abnormalities in the structurally impaired SAN, leading to SAN dysfunction, and potentiate atrial repolarization shortening, thereby facilitating atrial fibrillation. Atrial fibrillation may further depress SAN function and lead to tachy-brady arrhythmias in HF.

IL-6, A1 and A2aR: a crosstalk that modulates BDNF and induces neuroprotection.

Several diseases are related to retinal ganglion cell death, such as glaucoma, diabetes and other retinopathies. Many studies have attempted to identify factors that could increase neuroprotection after axotomy of these cells. Interleukin-6 has been shown to be able to increase the survival and regeneration of retinal ganglion cells (RGC) in mixed culture as well as in vivo. In this work we show that the trophic effect of IL-6 is mediated by adenosine receptor (A2aR) activation and also by the presence of extracellular BDNF. We also show that there is a complex cross-talk between IL-6, BDNF, the Adenosine A1 and A2a receptors that results in neuroprotection of retinal ganglion cells.

Adenosine A1 receptor stimulation enhances osteogenic differentiation of human dental pulp-derived mesenchymal stem cells via WNT signaling.

In this study, mesenchymal stem cells deriving from dental pulp (DPSCs) of normal human impacted third molars, previously characterized for their ability to differentiate into osteoblasts, were used. We observed that: i) DPSCs, undifferentiated or submitted to osteogenic differentiation, express all four subtypes of adenosine receptors (AR) and CD73, corresponding to 5'-ecto-nucleotidase; and ii) AR stimulation with selective agonists elicited a greater osteogenic cell differentiation consequent to A1 receptor (A1R) activation. Therefore, we focused on the activity of this AR. The addition of 15-60nM 2-chloro-N(6)-cyclopentyl-adenosine (CCPA), A1R agonist, to DPSCs at each change of the culture medium significantly increased the proliferation of cells grown in osteogenic medium after 8days in vitro (DIV) without modifying that of undifferentiated DPSCs. Better characterizing the effect of A1R stimulation on the osteogenic differentiation capability of these cells, we found that CCPA increased the: i) expression of two well known and early osteogenic markers, RUNX-2 and alkaline phosphatase (ALP), after 3 and 7DIV; ii) ALP enzyme activity at 7DIV and iii) mineralization of extracellular matrix after 21DIV. These effects, abolished by cell pre-treatment with the A1R antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX), involved the activation of the canonical Wnt signaling as, in differentiating DPSCs, CCPA significantly increased dishevelled protein and inhibited glycogen synthase kinase-3ß, both molecules being downstream of Wnt receptor signal pathway. Either siRNA of dishevelled or cell pre-treatment with Dickkopf-1, known inhibitor of Wnt signaling substantially reduced either DPSC osteogenic differentiation or its enhancement promoted by CCPA. Summarizing, our findings indicate that the stimulation of A1R may stimulate DPSC duplication enhancing their osteogenic differentiation efficiency. These effects may have clinical implications possibly facilitating bone tissue repair and remodeling.

IL-6 treatment increases the survival of retinal ganglion cells in vitro: the role of adenosine A1 receptor.

IL-6 is a pleiotropic cytokine classically denominated pro-inflammatory. It has been already demonstrated that IL-6 can increase the survival of retinal ganglion cells (RGC) in culture. In this work, we show that the trophic effect of IL-6 is mediated by adenosine receptor (A1R) activation. The neutralization of extracellular BDNF abolished the IL-6 effect and the treatment with IL-6 and CHA (an agonist of A1R) modulated BDNF expression as well as pCREB and pTrkB levels.

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A(2A) 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 A3 receptor is involved in ADP-induced microglial process extension and migration.

The extension of microglial processes toward injured sites in the brain is triggered by the stimulation of the purinergic receptor P2Y(12) by extracellular ATP. We recently showed that P2Y(12) stimulation by ATP induces microglial process extension in collagen gels. In the present study, we found that a P2Y(12) agonist, 2-methylthio-ADP (2MeSADP), failed to induce the process extension of microglia in collagen gels and that co-stimulation with adenosine, a phosphohydrolytic derivative of ATP, and 2MeSADP restored the chemotactic process extension. An adenosine A3 receptor (A3R)-selective agonist restored the chemotactic process extension, but other receptor subtype agonists did not. The removal of adenosine by adenosine deaminase and the blocking of A3R by an A3R-selective antagonist inhibited ADP-induced process extension. The A3R antagonist inhibited ADP-induced microglial migration, and an A3R agonist promoted 2MeSADP-stimulated migration. ADP and the A3R agonist activated Jun N-terminal kinase in microglia, and a Jun N-terminal kinase inhibitor inhibited the ADP-induced process extension. An RT-PCR analysis showed that A1R and A3R were expressed by microglia sorted from adult rat brains and that the A2AR expression level was very low. These results suggested that A3R signaling may be involved in the ADP-induced process extension and migration of microglia.

Maternal glutamate intake during gestation and lactation regulates adenosine A1 and A(2A) receptors in rat brain from mothers and neonates.

Pregnant rats were treated daily with 1 g/L of L-glutamate in their drinking water during pregnancy and/or lactation. The effect on adenosine A1 receptor (A1R) and A(2A) receptor (A(2A)R) in brains from both mothers and 15-day-old neonates was assayed using radioligand binding and real time PCR assays. Mothers receiving L-glutamate during gestation, lactation, and throughout gestation and lactation showed a significant decrease in total A1R number (water+water, 302±49 fmol/mg; L-glutamate+water, 109±11 fmol/mg, P<0.01; water+L-glutamate, 52±13 fmol/mg, P<0.01; L-glutamate+L-glutamate, 128±33 fmol/mg, P<0.05). No variations were detected in the Kd parameter. Concerning adenosine A(2A)R, radioligand binding assays revealed that Bmax parameter remains unaltered in maternal brain in response to glutamate exposure. However, Kd parameter was significantly decreased in all L-glutamate-treated groups (water+water, 5.3±1.3 nM; L-glutamate±water, 0.5±0.1 nM; water+L-glutamate, 0.9±0.1 nM; L-glutamate±L-glutamate, 0.7±0.1 nM, P<0.01 in all cases). In both male and female neonates, A1R was also decreased after long-term glutamate exposure during gestation, lactation, and gestation plus lactation (male neonates: water+water, 564±68 fmol/mg; L-glutamate+water, 61±8 fmol/mg; water+L-glutamate, 95±20 fmol/mg; L-glutamate+L-glutamate, 111±15 fmol/mg; P<0.01 in all cases; female neonates: water+water, 216±35 fmol/mg; L-glutamate+water, 59±9 fmol/mg; water+L-glutamate, 139±16 fmol/mg; L-glutamate+L-glutamate, 97±14 fmol/mg; P<0.01 in all cases). No variations were found in mRNA level coding adenosine A(1)R in maternal or neonatal brain. Concerning adenosine A(2A)R, radioligand binding assays revealed that Bmax parameter was significantly increased in male and female neonates exposed to L-glutamate during lactation (male neonates: water+water, 214±23 fmol/mg; water+L-glutamate, 581±49 fmol/mg; P<0.01; female neonates: water+water, 51±10 fmol/mg; water+L-glutamate, 282±52 fmol/mg; P<0.05). No variations were found in mRNA level coding adenosine A(2A)R in maternal or neonatal brain. In summary, long-term L-glutamate treatment during gestation and lactation promotes a significant down-regulation of A1R in whole brain from both mother and neonates and a significant up-regulation of A(2A)R in neonates exposed to L-glutamate during lactation.

Small-animal PET study of adenosine A(1) receptors in rat brain: blocking receptors and raising extracellular adenosine.

UNLABELLED: Activation of adenosine A(1) receptors (A(1)R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A(1)R can be visualized using 8-dicyclopropylmethyl-1-(11)C-methyl-3-propyl-xanthine ((11)C-MPDX) and PET. This study aims to test whether (11)C-MPDX can be used for quantitative studies of cerebral A(1)R in rodents. METHODS: (11)C-MPDX was injected (intravenously) into isoflurane-anesthetized male Wistar rats (300 g). A dynamic scan of the central nervous system was obtained, using a small-animal PET camera. A cannula in a femoral artery was used for blood sampling. Three groups of animals were studied: group 1, controls (saline-treated); group 2, animals pretreated with the A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 mg, intraperitoneally); and group 3, animals pretreated (intraperitoneally) with a 20% solution of ethanol in saline (2 mL) plus the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d] pyrimidine dihydrochloride (ABT-702) (1 mg). DPCPX is known to occupy cerebral A(1)R, whereas ethanol and ABT-702 increase extracellular adenosine. RESULTS: In groups 1 and 3, the brain was clearly visualized. High uptake of (11)C-MPDX was noted in striatum, hippocampus, and cerebellum. In group 2, tracer uptake was strongly suppressed and regional differences were abolished. The treatment of group 3 resulted in an unexpected 40%-45% increase of the cerebral uptake of radioactivity as indicated by increases of PET standardized uptake value, distribution volume from Logan plot, nondisplaceable binding potential from 2-tissue-compartment model fit, and standardized uptake value from a biodistribution study performed after the PET scan. The partition coefficient of the tracer (K(1)/k(2) from the model fit) was not altered under the study conditions. CONCLUSION: (11)C-MPDX shows a regional distribution in rat brain consistent with binding to A(1)R. Tracer binding is blocked by the selective A(1)R antagonist DPCPX. Pretreatment of animals with ethanol and adenosine kinase inhibitor increases (11)C-MPDX uptake. This increase may reflect an increased availability of A(1)R after acute exposure to ethanol.

Remodeling of inward rectifying K(+) currents in rat atrial myocytes by overexpression of A(1)-adenosine receptors.

In rat atrial myocytes GIRK (Kir3) channels can be activated by acetylcholine and adenosine via M(2) and A(1) receptors coupled to Pertussis-toxin-sensitive G proteins, such as M(2)R or A(1)R. Owing to the lower density of A(1)R, the amplitude of current activated by a saturating concentration (10 µM) of Ado (I(K(Ado))) amounts to about 40% of maximum I(K(ACh)). Adenovirus-driven overexpression of A(1)R results in an increase in I(K(Ado)). In a fraction of A(1)R-overexpressing cells, both ACh and Ado failed to activate GIRK channels. These cells had a large constitutive Ba(2+)-sensitive inward rectifying background K(+) current, which was insensitive to the GIRK channel inhibitor tertiapin (200 nM), suggesting this current component to be carried by I(K1) (Kir) channels. This effect of A(1)R overexpression was reduced by treatment (48 h) with the A(1)R antagonist DPCPX. siRNA-mediated knockdown of Kir2.1, simultaneously with A(1)R overexpression, substantially reduced I(K1). The mechanisms underlying the upregulation of functional I(K1) channels involve activation of the phosphatidylinositol 3-kinase (Pi3K)/Akt (protein kinase B) pathway. Kir2.1 transcripts are not increased in myocytes overexpressing A(1)R. These data demonstrate that manipulation of the expression level of a G protein-coupled receptor has unpredictable effects on functional expression of proteins that are supposed to be unrelated to the pathway controlled by that GPCR.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie.

AIM: Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. METHODS: Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. RESULTS: PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cß1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. CONCLUSIONS: These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.

Role of adenosine and wake-promoting basal forebrain in insomnia and associated sleep disruptions caused by ethanol dependence.

Insomnia is a severe symptom of alcohol withdrawal; however, the underlying neuronal mechanism is yet unknown. We hypothesized that chronic ethanol exposure will impair basal forebrain (BF) adenosinergic mechanism resulting in insomnia-like symptoms. We performed a series of experiments in Sprague-Dawley rats to test our hypothesis. We used Majchrowicz's chronic binge ethanol protocol to induce ethanol dependency. Our first experiment verified the effects of ethanol withdrawal on sleep-wakefulness. Significant increase in wakefulness was observed during ethanol withdrawal. Next, we examined c-Fos expression (marker of neuronal activation) in BF wake-promoting neurons during ethanol withdrawal. There was a significant increase in the number of BF wake-promoting neurons with c-Fos immunoreactivity. Our third experiment examined the effects of ethanol withdrawal on sleep deprivation induced increase in BF adenosine levels. Sleep deprivation did not increase BF adenosine levels in ethanol dependent rats. Our last experiment examined the effects of ethanol withdrawal on equilibrative nucleoside transporter 1 and A1 receptor expression in the BF. There was a significant reduction in A1 receptor and equilibrative nucleoside transporter 1 expression in the BF of ethanol dependent rats. Based on these results, we suggest that insomnia observed during ethanol withdrawal is caused because of impaired adenosinergic mechanism in the BF.

Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function.

Neuroprotection is one of the prominent functions of the interleukin (IL)-6-type cytokine family, for which the underlying mechanism(s) are not fully understood. We have previously shown that neuroprotection and neuromodulation mediated by IL-6 require neuronal adenosine A(1) receptor (A(1) R) function. We now have investigated whether two other IL-6-type cytokines [oncostatin M (OSM) and leukemia inhibitory factor (LIF)] use a similar mechanism. It is presented here that OSM but not LIF, enhanced the expression of A(1) Rs (both mRNA and protein levels) in cultured neurons. Whereas the neuroprotective effect of LIF was unchanged in A(1) R deficient neurons, OSM failed to protect neurons in the absence of A(1) R. In addition, OSM pre-treatment for 4 h potentiated the A(1) R-mediated inhibition of electrically evoked excitatory post-synaptic currents recorded from hippocampal slices either under normal or hypoxic conditions. No such effect was observed after LIF pre-treatment. Our findings thus strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation.

Caffeine protects against oxidative stress and Alzheimer's disease-like pathology in rabbit hippocampus induced by cholesterol-enriched diet.

Cholesterol has been linked to the pathogenesis of sporadic Alzheimer's disease (AD) as a risk factor increasing beta-amyloid (Abeta) and oxidative stress levels. Caffeine has antioxidant properties and has been demonstrated to reduce Abeta levels in transgenic mouse models of familial AD. However, the effects of caffeine on cholesterol-induced sporadic AD pathology have not been determined. In this study, we determined the effects of caffeine on Abeta levels, tau phosphorylation, oxidative stress generation, and caffeine-target receptors in rabbits fed a 2% cholesterol-enriched diet, a model system for sporadic AD. Our results showed that the cholesterol-enriched diet increased levels of Abeta, tau phosphorylation, and oxidative stress measured as increased levels of reactive oxygen species and isoprostanes, glutathione depletion, and increased levels of endoplasmic reticulum stress marker proteins. Additionally, the cholesterol-enriched diet reduced the levels of adenosine A(1) receptors (A(1)R) but not ryanodine or adenosine A(2A) receptors. Caffeine, administered at 0.5 and 30mg/day in the drinking water, reduced the cholesterol-induced increase in Abeta, phosphorylated tau, and oxidative stress levels and reversed the cholesterol-induced decrease in A(1)R levels. Our results suggest that even very low doses of caffeine might protect against sporadic AD-like pathology.