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The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.

Background. We previously observed that adenosine A1 receptor (A1AR) had a protective role in proximal tubular megalin loss associated with albuminuria in diabetic nephropathy (DN). In this study, we aimed to explore the role of A1AR in the fibrosis progression of DN. Methods. We collected DN patients’ samples and established a streptozotocin-induced diabetes model in wild-type (WT) and A1AR-deficient (A1AR-/-) mice. The location and expression of CD34, PDGFRβ, and A1AR were detected in kidney tissue samples from DN patients by immunofluorescent and immunohistochemical staining. We also analyzed the expression of TGFβ, collagen (I, III, and IV), α-SMA, and PDGFRβ using immunohistochemistry in WT and A1AR-/- mice. CD34 and podoplanin expression were analyzed by Western blotting and immunohistochemical staining in mice, respectively. Human renal proximal tubular epithelial cells (HK2) were cultured in medium containing high glucose and A1AR agonist as well as antagonist. Results. In DN patients, the expression of PDGFRβ was higher with the loss of CD34. The location of PDGFRβ and TGFβ was near to each other. The A1AR, which was colocalized with CD34 partly, was also upregulated in DN patients. In WT-DN mice, obvious albuminuria and renal pathological leisure were observed. In A1AR-/- DN mice, more severe renal tubular interstitial fibrosis and more extracellular matrix deposition were observed, with lower CD34 expression and pronounced increase of PDGFRβ. In HK2 cells, high glucose stimulated the epithelial-mesenchymal transition (EMT) process, which was inhibited by A1AR agonist. Conclusion. A1AR played a critical role in protecting the tubulointerstitial fibrosis process in DN by regulation of the peritubular microenvironment.

The mechanisms and signaling pathways of the neuroprotective effects of hypercapnia and its combination with hypoxia are not studied sufficiently. The study aims to test the hypothesis of the potentiating effect of hypercapnia on the systems of adaptation to hypoxia, directly associated with A1-adenosine receptors and mitochondrial ATP-dependent K+ -channels (mitoK+ATP-channels). We evaluated the relative number of A1-adenosine receptors and mitoK+ATP-channels in astrocytes obtained from male Wistar rats exposed to various respiratory conditions (15 times of hypoxia and/or hypercapnia). In addition, the relative number of these molecules in astrocytes was evaluated on an in vitro model of chemical hypoxia, as well as in the cerebral cortex after photothrombotic damage. This study indicates an increase in the relative number of A1-adenosine receptors in astrocytes and in cells next to the stroke region of the cerebral cortex in rats exposed to hypoxia and hypercapnic hypoxia, but not hypercapnia alone. Hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels in astrocytes and in cells of the peri-infarct region of the cerebral cortex in rats. In an in vitro study, hypercapnia mitigates the effects of acute chemical hypoxia observed in astrocytes for A1-adenosine receptors and mitoK+ATP-channels. Hypercapnia, unlike hypoxia, does not affect the relative number of A1 receptors to adenosine. At the same time, both hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels, which can potentiate their protective effects with combined exposure.

Diabetes through adenosine A1 receptor (A1R) and P2 receptors (P2Rs) may lead to disturbances in renal microvasculature. We investigated the renal microvascular response to Ap4A, an agonist of P2Rs, in streptozotocin-induced diabetic rats. Using laser Doppler flowmetry, renal blood perfusion (RBP) was measured during infusion of Ap4A alone or in the presence of A1R antagonist, either DPCPX (8-cyclopentyl-1,3-dipropylxanthine) or 8-cyclopentyltheophylline (CPT). Ap4A induced a biphasic response in RBP: a phase of rapid decrease was followed by a rapid increase, which was transient in diabetic rats but extended for 30 min in nondiabetic rats. Phase of decreased RBP was not affected by DPCPX or CPT in either group. Early and extended increases in RBP were prevented by DPCPX and CPT in nondiabetic rats, while in diabetic rats, the early increase in RBP was not affected by these antagonists. A1R mRNA and protein levels were increased in isolated glomeruli of diabetic rats, but no changes were detected in P2Y1R and P2Y2R mRNA. Presence of unblocked A1R is a prerequisite for the P2R-mediated relaxing effect of Ap4A in nondiabetic conditions, but influence of A1R on P2R-mediated renal vasorelaxation is abolished under diabetic conditions.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

Epilepsy is a chronic nervous system disease. Excessive increase of the excitatory neurotransmitter glutamate in the body results in an imbalance of neurotransmitters and excessive excitation of neurons, leading to epileptic seizures. Long-term recurrent seizures lead to behavior and cognitive changes, and even increase the risk of death by 2- to 3-fold relative to the general population. Adenosine A1 receptor (A1R), a member of the adenosine system, has notable anticonvulsant effects, and adenosine levels are controlled by the type 1 equilibrative nucleoside transporter (ENT1); in addition the p38 MAPK signaling pathway is involved in the regulation of ENT1, although the effect of its inhibitors on the expression levels of A1R and ENT1 is unclear. Therefore, in the present study, SB203580 was used to inhibit the p38 MAPK signaling pathway in rats, and the expression levels of A1R and ENT1 in the brain tissue of rats with acute LiCl-pilocarpine-induced status epilepticus was detected. SB203580 decreased pathological damage of hippocampal neurons, prolonged seizure latency, reduced the frequency of seizures, and decreased levels of A1R and ENT1 protein in rats.

It is essential to elucidate brain-adipocyte interactions in order to tackle obesity and its comorbidities, as the precise control of brain-adipose tissue cross-talk is crucial for energy and glucose homeostasis. Recent studies show that in the peripheral adipose tissue, adenosine induces adipogenesis through peripheral adenosine A1 receptor (pADORA1) signaling; however, it remains unclear whether systemic and adipose tissue metabolism would also be under the control of central (c) ADORA1 signaling. Here, we use tissue-specific pharmacology and metabolic tools to clarify the roles of cADORA1 signaling in energy and adipocyte physiology. We found that cADORA1 signaling reduces body weight while also inducing adipose tissue lipolysis. cADORA1 signaling also increases adipose tissue sympathetic norepinephrine content. In contrast, pADORA1 signaling facilitates a high-fat diet-induced obesity (DIO). We propose here a novel mechanism in which cADORA1 and pADORA1 signaling hinder and aggravate DIO, respectively.

Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode’s-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, K i =56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α 1 -adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α 1 -adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α 1 -adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A 1 -adenosine receptors, which are known to participate in coincident signaling with α 1 -adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α 1 -adrenoceptor signaling via a mechanism involving A 1 receptor signaling.

The function of zfp91 is mainly studied in vitro, but there is no study in vivo. Accumulative data suggest that zfp91 may be an important gene to regulate all aspects of human response. However, there are no data to date about the function of zfp91 on cardiac homeostasis. Thus, we aimed to observe the role of zfp91 gene in mouse cardiomyocytes on myocardial homeostasis and related mechanisms under pressure overload. In the study, zfp91 mRNA and protein levels were significantly reduced in TAC‐operated WT mice as compared with controls. Genetic ablation of zfp91 dramatically led to pathological cardiac dysfunction and hypertrophy after transverse aortic constriction (TAC). Adenosine A1 receptor (Adora1) mRNA and protein expressions were significantly down‐regulated in the heart of zfp91‐deletion mice with TAC. Zfp91 overexpression reversed isoproterenol‐induced cardiomyocyte hypertrophy, which was abolished by selective Adora1 antagonist. Dual‐luciferase reporter and ChIP‐qPCR assays indicated that zfp91 acted on Adora1 promoter through its binding site. Last, Adora1 agonist rescued heart dysfunction and cardiac hypertrophy in zfp91 loss mice after TAC. Zfp91 may transcriptionally regulate Adora1 expression in the heart, which mainly maintained cardiac homeostasis under pressure overload status. It will provide a new approach to treat cardiac hypertrophy.

Astrocytes are fundamental building blocks of the central nervous system. Their dysfunction has been implicated in many psychiatric disorders, including alcohol use disorder, yet our understanding of their functional role in ethanol intoxication and consumption is very limited. Astrocytes regulate behavior through multiple intracellular signaling pathways, including G-protein coupled-receptor (GPCR)-mediated calcium signals. To test the hypothesis that GPCR-induced calcium signaling is also involved in the behavioral effects of ethanol, we expressed astrocyte-specific excitatory DREADDs in the prefrontal cortex (PFC) of mice. Activating Gq-GPCR signaling in PFC astrocytes increased drinking in ethanol-naïve mice, but not in mice with a history of ethanol drinking. In contrast, reducing calcium signaling with an astrocyte-specific calcium extruder reduced ethanol intake. Cortical astrocyte calcium signaling also altered the acute stimulatory and sedative-hypnotic effects of ethanol. Astrocyte-specific Gq-DREADD activation increased both the locomotor-activating effects of low dose ethanol and the sedative-hypnotic effects of a high dose, while reduced astrocyte calcium signaling diminished sensitivity to the hypnotic effects. In addition, we found that adenosine A1 receptors were required for astrocyte calcium activation to increase ethanol sedation. These results support integral roles for PFC astrocytes in the behavioral actions of ethanol that are due, at least in part, to adenosine receptor activation.

Objective The "adenosine hypothesis of SUDEP" (sudden unexpected death in epilepsy) predicts that a seizure-induced adenosine surge combined with impaired metabolic clearance can foster lethal apnea or cardiac arrest. Changes in adenosine receptor density and adenosine kinase (ADK) occur in surgical epilepsy patients. Our aim was to correlate the distribution of ADK and adenosine A2A and A1 receptors (A2A R and A1 R) in surgical tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE/HS) with SUDEP risk factors. Methods In 75 cases, patients were stratified into high-risk (n = 16), medium-risk (n = 11) and low-risk (n = 48) categories according to the frequency of generalized seizures before surgery. Using whole-slide scanning Definiens image analysis we quantified the labeling index (LI) for ADK, A2A R, and A1 R in seven regions of interest: temporal cortex, temporal lobe white matter, CA1, CA4, dentate gyrus, subiculum, and amygdala and relative to glial and neuronal densities with glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN). Results A1 R showed predominant neuronal, A2A R astroglial, and ADK nuclear labeling in all regions but with significant variation. Compared with the low-risk group, the high-risk group had significantly lower A2A R LI in the temporal cortex. In HS cases with severe neuronal cell loss and gliosis predominantly in the CA1 and CA4 regions, significantly higher A1 R was present in the amygdala in high-risk than in low-risk cases. There was no significant difference in neuronal loss or gliosis between the risk groups or differences for ADK labeling. Significance Reduced cortical A2A R suggests glial dysfunction and impaired adenosine modulation in response to seizures in patients at higher risk for SUDEP. Increased neuronal A1 R in the high-risk group could contribute to periictal amygdala dysfunction in SUDEP.

Diabetes mellitus affects the colonic motility developing gastrointestinal symptoms, such as constipation. The aim of the study was to examine the role of intracellular signaling pathways contributing to colonic dysmotility in diabetes mellitus. To generate diabetes mellitus, the rats were injected by a single high dose of streptozotocin (65 mg/kg) intraperitoneally. The proximal colons from both normal and diabetic rats were contracted by applying an electrical field stimulation with pulse voltage of 40 V in amplitude and pulse duration of 1 ms at frequencies of 1, 2, 4, and 6 Hz. The muscle strips from both normal rats and rats with diabetes mellitus were pretreated with different antagonists and inhibitors. Rats with diabetes mellitus had lower motility than the control group. There were significant differences in the percentage of inhibition of contraction between normal rats and rats with diabetes mellitus after the incubation of tetrodotoxin (neuronal blocker), atropine (muscarinic receptor antagonist), prazosin (α1 adrenergic receptor antagonist), DPCPX (adenosine A1 receptor antagonist), verapamil (L-type Ca2+ channel blocker), U73122 (PLC inhibitor), ML-9 (MLCK inhibitor), udenafil (PDE5 inhibitor), and methylene blue (guanylate cyclase inhibitor). The protein expression of p-MLC and PDE5 were decreased in the diabetic group compared to the normal group. These results showed that the reduced colonic contractility resulted from the impaired neuronal conduction and decreased muscarinic receptor sensitivity, which resulted in decreased phosphorylation of MLC via MLCK, and cGMP activity through PDE5.

The primary cause of harmaline tremor, which is a model of essential tremor (ET) in animals, is excessive activation of olivocerebellar glutamatergic climbing fibers. Our recent study indicated that 5′-chloro-5′-deoxy-(±)-N6-(±)-(endo-norborn-2-yl)adenosine (5′Cl5′d-(±)-ENBA), a potent and selective adenosine A1 receptor (A1) agonist, inhibited harmaline tremor. The present study was aimed to evaluate the role of glutamatergic transmission system in 5′Cl5′d-(±)-ENBA tremorolytic action in the harmaline model in rats, by analyzing glutamate release in the motor nuclei of the thalamus and mRNA expression of glutamatergic neuron markers (vGlut1/2) in reference to the general neuronal activity marker (zif-268) in different brain structures. The extracellular glutamate level in the motor thalamus was evaluated by in vivo microdialysis and the vGlut1/vGlut2 and zif-268 mRNA expression was analyzed by in situ hybridization. The intensity of tremor was measured automatically using Force Plate Actimeters (FPAs). 5′Cl5′d-(±)-ENBA (0.5 mg/kg) given 30 min before harmaline (30 mg/kg) decreased the harmaline-induced excessive glutamate release in the motor thalamus and reversed harmaline - induced molecular effects, such as elevation of the vGlut1 mRNA expression in the inferior olive (IO) and decrease in the motor cortex, as well as an increase of the zif-268 mRNA expression in the IO, motor thalamus and motor cortex. Moreover, 5′Cl5′d-(±)-ENBA reduced harmaline tremor by lowering its power in 9–15 Hz frequency band. Our findings show that A1 stimulation decreases glutamate release in the motor thalamic nuclei in the harmaline model of ET, suggesting that A1 receptors, especially in this structure, may be a potential therapeutic target in this disorder.

Aim of Study To investigate podocyte density in aging diabetic Ins2± and Ins2±, A1AR-/- mouse models in C57Bl/6 background. Methods Ins2± mice and especially Ins2±, adenosine A1 receptor knockout mice (Ins2±, A1AR-/-) are mouse models with a phenotype of diabetic nephropathy. Aged mice (at ~40 weeks) were assessed for glomerular filtration barrier function by measuring albuminuria, glomerular filtration, glomerular damage by electron microscopy, and podocyte numbers by Wilms Tumor protein (WT-1) staining. Results Compared to healthy wild-type mice, both diabetic mouse models developed diabetic nephropathy, including hyperfiltration (p

Febrile seizures are one of the most frequent childhood neurological disorders; they are classified into simple and prolonged, depending on their duration. Prolonged FS lasts more than 15 min and may evoke neurological sequelae in a process in which molecular alterations seem to play an important role. Adenosine is a purine nucleoside that exerts anticonvulsant effects through binding to adenosine A1 receptor (A1 R). This receptor belongs to the GPCR superfamily and is negatively coupled to adenylyl cyclase (AC) activity through Gi proteins. In the present study, we analyzed the functionality of A1 R, measured as the inhibition of forskolin-stimulated AC activity, 48 hr after hyperthermia-induced seizures (HIS). Surprisingly, the results obtained show that the activation of A1 R increased forskolin-stimulated cAMP production instead of decreasing it. This alteration was not accompanied by changes in αG protein levels. The functionality of A1 R remained altered two months after HIS. However, this alteration was abolished when AC assays were carried out in the presence of anti αGs subunit-specific antibody, suggesting that HIS can switch A1 R coupling from Gi to Gs proteins. Finally, radioligand binding assays revealed that density and affinity of A1 R were not significantly altered by HIS. In summary, the results obtained show that HIS induces long-term changes in the A1 R/AC signaling pathway in rat brain cortex.

Hibernation is a seasonal phenomenon characterized by a drop in metabolic rate and body temperature. Adenosine A1 receptor agonists promote hibernation in different mammalian species, and the understanding of the mechanism inducing hibernation will inform clinical strategies to manipulate metabolic demand that are fundamental to conditions such as obesity, metabolic syndrome, and therapeutic hypothermia. Adenosine A1 receptor agonist-induced hibernation in Arctic ground squirrels is regulated by an endogenous circannual (seasonal) rhythm. This study aims to identify the neuronal mechanism underlying the seasonal difference in response to the adenosine A1 receptor agonist. Arctic ground squirrels were implanted with body temperature transmitters and housed at constant ambient temperature (2°C) and light cycle (4L:20D). We administered CHA (N6 -cyclohexyladenosine), an adenosine A1 receptor agonist in euthermic-summer phenotype and euthermic-winter phenotype and used cFos and phenotypic immunoreactivity to identify cell groups affected by season and treatment. We observed lower core and subcutaneous temperature in winter animals and CHA produced a hibernation-like response in winter, but not in summer. cFos-ir was greater in the median preoptic nucleus and the raphe pallidus in summer after CHA. CHA administration also resulted in enhanced cFos-ir in the nucleus tractus solitarius and decreased cFos-ir in the tuberomammillary nucleus in both seasons. In winter, cFos-ir was greater in the supraoptic nucleus and lower in the raphe pallidus than in summer. The seasonal decrease in the thermogenic response to CHA and the seasonal increase in vasoconstriction, assessed by subcutaneous temperature, reflect the endogenous seasonal modulation of the thermoregulatory systems necessary for CHA-induced hibernation. Cover Image for this issue: doi: 10.1111/jnc.14528.

Pulmonary hypertension (PH), remains a challenging disease with a large impact on both humans and meat-type chickens. PH is characterized by the onset of idiopathic pulmonary arterial hypertension leading to right ventricular failure. In this experiment relative gene expression of adenosine A1 receptor (ADORA1), serotonin transporter (SERT), phosphodiesterase 5A (PDE5) and troponin T2 were compared in hearts from broilers with acute right ventricular failure and from healthy birds. There were major increases in adenosine A1 receptor (177%) and serotonin transporter (475%), and more modest but significant increases in PDE5 (146%) and troponin T2 (140%) gene expressions in broilers with right ventricular failure compared to healthy birds (P

Adenosine receptors are widely expressed in the brain, and adenosine is a key bioactive substance for neuroprotection. In this article, we clarify systematically the role of adenosine A1 receptors during a range of timescales and conditions when a significant amount of adenosine is released. Using acute hippocampal slices obtained from mice that were wild type or null mutant for the adenosine A1 receptor, we quantified and characterized the impact of varying durations of experimental ischemia, hypoxia, and hypoglycemia on synaptic transmission in the CA1 subregion. In normal tissue, these three stressors rapidly and markedly reduced synaptic transmission, and only treatment of sufficient duration led to incomplete recovery. In contrast, inactivation of adenosine A1 receptors delayed and/or lessened the reduction in synaptic transmission during all three stressors and reduced the magnitude of the recovery significantly. We reproduced the responses to hypoxia and hypoglycemia by applying an adenosine A1 receptor antagonist, validating the clear effects of genetic receptor inactivation on synaptic transmission. We found activation of adenosine A1 receptor inhibited hippocampal synaptic transmission during the acute phase of ischemia, hypoxia, or hypoglycemia and caused the recovery from synaptic impairment after these three stressors using genetic mutant. These studies quantify the neuroprotective role of the adenosine A1 receptor during a variety of metabolic stresses within the same recording system. NEW & NOTEWORTHY Deprivation of oxygen and/or glucose causes a rapid adenosine A1 receptor-mediated decrease in synaptic transmission in mouse hippocampus. We quantified adenosine A1 receptor-mediated inhibition during and synaptic recovery after ischemia, hypoxia, and hypoglycemia of varying durations using a genetic mutant and confirmed these findings using pharmacology. Overall, using the same recording conditions, we found the acute response and the neuroprotective ability of the adenosine A1 receptor depended on the type and duration of deprivation event.

We previously reported a study on 288 broiler (Gallus gallus) chicks who received caffeine in water between days 3 and 42, at levels of 0, 6.25, 12.5, 25, 50 and 100 mg/kg body weight (BW)/day. In the previous report, we found that caffeine caused pulmonary hypertension (PH)-associated mortality in a significant minority (20%-30%) of birds, including right ventricular hypertrophy and ascites. We have also shown a significant upregulation of the serotonin transporter (SERT), troponin T2, adenosine A1 receptor (ADORA1) and phosphodiesterase 5A (PDE5) in chicken suffering from PH. Here, we examine the resistant (survived) chicks from the first study that had not died due to acute heart failure and did not have clinical signs of pulmonary hypertension. Our goal was to determine whether birds who lacked overt signs of disease had subclinical manifestations, including similar changes in gene expression, growth rates and altered systemic haemodynamics. We found that growth was significantly increased by caffeine consumption (p 0.05). However, SERT gene expression linearly increased with increasing caffeine dosage (p < 0.01). The impact of caffeine on ADORA1 gene expression was dose dependent and nonlinear. In conclusion, despite the significant effects of caffeine on birds' growth, no significant negative effects of caffeine were observed on the cardiovascular function of resistant chickens. This work provides valuable information for further study on different dosage of caffeine in an animal model.

The adenosine A1 receptor is important for body temperature regulation in mammals; however, little is known about its function in avian species. In this study, we investigated the effects of the adenosine A1 receptor agonist and antagonist (adenosine 5'-monophosphate [5'-AMP] and 8 p-sulfophenyl theophylline [8-SPT], respectively) on thermoregulation in chickens. Male chicks were used in this study. After administration of 5'-AMP and 8-SPT, the rectal temperature, plasma metabolites, and gene expressions in the hypothalamus and liver were measured. The rectal temperature was reduced by peripheral administration of 5'-AMP, and the hypothermic effect of 5'-AMP was attenuated by central injection of 8-SPT in chicks. In the hypothalamus, the mRNA level of the agouti-related protein (AgRP) was increased by 5'-AMP administration, whereas it was suppressed by 8-SPT. The plasma levels of free fatty acid were elevated in 5'-AMP-treated chicks and that elevation was suppressed by the 8-SPT treatment. The gene expression of proopiomelanocortin in the hypothalamus was affected by 8-SPT. Nevertheless, the gene expressions of the thermoregulation-related genes, such as the thyrotropin-releasing hormone, were not affected by 5'-AMP and 8-SPT. Hepatic gene expressions related to lipid intake and metabolism were suppressed by 5'-AMP. However, the gene expression of the uncoupling protein was upregulated by 5'-AMP. Based on these results, birds, like mammals, will undergo adenosine A1 receptor-induced hypothermia. In conclusion, it is suggested that 5'-AMP-mediated hypothermia via the adenosine A1 receptor may affect the central melanocortin system and suppress hepatic lipid metabolism in chickens.

Quercetin is a polyphenolic flavonoid that has reported to block the binding of adenosine to A1 receptors at central nervous system and increase calcium release from the sarcoplasmic reticulum at skeletal muscle. The aim of the present study was to investigate the acute effect of quercetin ingestion on motor unit activation and muscle contractile properties. High-density surface electromyography during submaximal contractions and electrically elicited contraction torque in knee extensor muscles were measured before (PRE) and 60 min after (POST) quercetin glycosides or placebo ingestions in 13 young males. Individual motor units of the vastus lateralis muscle were identified from high-density surface electromyography by the Convolution Kernel Compensation technique. Firing rates of motor units recruited at 30–50% of the maximal voluntary contraction torque (MVC) were increased from PRE to POST only with quercetin (9.0 ± 2.3 to 10.5 ± 2.0 pps, p = 0.034). Twitch torque during doublet stimulation was decreased from PRE to POST with placebo (77.1 ± 17.1 to 73.9 ± 17.6 Nm, p = 0.005), but not with quercetin (p > 0.05). For motor units recruited at p = 0.002) but increased with placebo (1.61 ± 0.32 to 1.57 ± 0.31%MVC/pps, p = 0.005). These results suggest that ingested quercetin has the functional roles to: mitigate reduction in the muscle contractile properties, enhance activations of relatively higher recruitment threshold motor units, and inhibit activation of relatively lower recruitment threshold motor units.

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

Adenosine A(l) receptors (A(l)ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A(l)ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A(1)ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [F-18]CPFPX and immunohistochemistry (IHC). In addition, the role of AIARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [F-18]DPA-714 (TSPO) and [F-18]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [F-18]CPFPX signal and IHC showed the overexpression of A(l)ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the AAR agonist ENBA showed a significant decrease in both [F-18]DPA-714 and [F-18]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A(l)AR promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A(l)AR expression after cerebral ischemia in rats and the application of [F-18]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A(l)AR playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats. The authors would like to thank A. Leukona and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-201722412, SAF2016-75292-R, SAF2017-87670-R and PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Juan Jose Gutierrez acknowledges funding from Euskampus Fundazioa. Jordi Llop also acknowledges The Spanish Ministry of Economy and Competitiveness (Grant CTQ2017-87637-R). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720).

Neurogenic fever (NF) after subarachnoid hemorrhage (SAH) is a major cause of morbidity that is associated with poor outcomes and prolonged stay in the neurointensive care unit (NICU). Though SAH is a much more common cause of fever than sepsis in the NICU, it is often a diagnosis of exclusion, requiring significant effort to rule out an infectious source. NF does not respond to standard anti-pyretic medications such as COX inhibitors, and lack of good medical therapy has led to the introduction of external cooling systems that have their own associated problems. In a rodent model of SAH, we measured the effects of injecting whole blood, blood plasma, or erythrocytes on the sympathetic nerve activity to brown adipose tissue and on febrile thermogenesis. We demonstrate that following SAH the acute activation of brown adipose tissue leading to NF, is not dependent on PGE2, that subarachnoid space injection of whole blood or erythrocytes, but not plasma alone, is sufficient to trigger brown adipose tissue thermogenesis, and that activation of adenosine A1 receptors in the CNS can block the brown adipose tissue thermogenic component contributing to NF after SAH. These findings point to a distinct thermogenic mechanism for generating NF, compared to those due to infectious causes, and will hopefully lead to new therapies.

Immunolabeling for adenosine A1 receptors (A1Rs) is high in hippocampal area CA2 in adult rats, and the potentiating effects of caffeine or other A1R-selective antagonists on synaptic responses are particularly robust at Schaffer collateral synapses in CA2. Interestingly, the pronounced staining for A1Rs in CA2 is not apparent until rats are 4 weeks old, suggesting that developmental changes other than receptor distribution underlie the sensitivity of CA2 synapses to A1R antagonists in young animals. To evaluate the role of A1R-mediated postsynaptic signals at these synapses, we tested whether A1R agonists regulate synaptic transmission at Schaffer collateral inputs to CA2 and CA1. We found that the selective A1R agonist CCPA caused a lasting depression of synaptic responses in both CA2 and CA1 neurons in slices obtained from juvenile rats (P14), but that the effect was observed only in CA2 in slices prepared from adult animals (~P70). Interestingly, blocking phosphodiesterase activity with rolipram inhibited the CCPA-induced depression in CA1, but not in CA2, indicative of robust phosphodiesterase activity in CA1 neurons. Likewise, synaptic responses in CA2 and CA1 differed in their sensitivity to the adenylyl cyclase activator, forskolin, in that it increased synaptic transmission in CA2, but had little effect in CA1. These findings suggest that the A1R-mediated synaptic depression tracks the postnatal development of immunolabeling for A1Rs and that the enhanced sensitivity to antagonists in CA2 at young ages is likely due to robust adenylyl cyclase activity and weak phosphodiesterase activity rather than to enrichment of A1Rs.

The adenosine (Ado) system may participate in regulation of kidney function in diabetes mellitus (DM), therefore we explored its role and interrelation with NO in the control of renal circulation and excretion in normoglycemic (NG) and streptozotocin-diabetic (DM) rats. Effects of theophylline (Theo), a non-selective Ado receptor antagonist, were examined in anaesthetized NG or in streptozotocin induced diabetic (DM) rats, untreated or after blockade of NO synthesis with l-NAME. We measured arterial blood pressure (MABP), whole kidney blood flow and renal regional flows: cortical and outer- and inner-medullary (IMBF), determined as laser-Doppler fluxes. Renal excretion of water, total solutes and sodium and in situ renal tissue NO signal (selective electrodes) were also determined. Theo experiments disclosed minor baseline vasoconstrictor and vasodilator tone in the kidney of NG and DM rats, respectively. NO blockade increased baseline MABP and decreased renal haemodynamics, similarly in NG and DM rats, indicating comparable vasodilator influence of NO in the two groups. Unexpectedly, in all rats with intact NO synthesis, Ado receptor blockade increased kidney tissue NO. In NO-deficient NG and DM rats, Ado receptor blockade induced comparable renal vasodilatation, suggesting similar vasoconstrictor influence of the Ado system. However, DM rats showed an unexplained association of decreased MABP and IMBF and increased NO signal. Higher baseline renal excretion in DM rats indicated inhibition of renal tubular reabsorption due to the prevalence of natriuretic A2 over antinatriuretic A1 receptors. In conclusion, the experiments provided new insights in functional interrelation of adenosine and NO in normoglycaemia and streptozotocin-diabetes.

Objective The adenosine A1 receptor is a Gαi/o protein‐coupled receptor and inhibits upon activation cAMP formation and protein kinase A (PKA) activity. As a widely expressed receptor in the mammalian brain, A1 receptors are implicated in the modulation of a variety of neuronal and synaptic activities. In this study, we investigated the role of A1 receptors in the regulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in the adult rat brain in vivo. Methods Adult male Wistar rats were used in this study. After a systemic injection of the A1 antagonist DPCPX, rats were sacrificed and several forebrain regions were collected for assessing changes in phosphorylation of AMPA receptors using Western blots. Results A systemic injection of the A1 antagonist DPCPX induced an increase in phosphorylation of AMPA receptor GluA1 subunits at a PKA‐dependent site, serine 845 (S845), in the two subdivisions of the striatum, the caudate putamen, and nucleus accumbens. DPCPX also increased S845 phosphorylation in the medial prefrontal cortex (mPFC) and hippocampus. The DPCPX‐stimulated S845 phosphorylation was a transient and reversible event. Blockade of Gαs/olf‐coupled dopamine D1 receptors with a D1 antagonist SCH23390 abolished the responses of S845 phosphorylation to DPCPX in the striatum, mPFC, and hippocampus. DPCPX had no significant impact on phosphorylation of GluA1 at serine 831 and on expression of total GluA1 proteins in all forebrain regions surveyed. Conclusion These data demonstrate that adenosine A1 receptors maintain an inhibitory tone on GluA1 S845 phosphorylation under normal conditions. Blocking this inhibitory tone leads to the upregulation of GluA1 S845 phosphorylation in the striatum, mPFC, and hippocampus via a D1‐dependent manner., Our data demonstrate that adenosine A1 receptors maintain an inhibitory tone on GluA1 S845 phosphorylation under normal conditions. Blocking this inhibitory tone leads to the upregulation of GluA1 S845 phosphorylation in the striatum, mPFC, and hippocampus via a D1‐dependent manner.

Translational Psychiatry 10(1), 406 (2020). doi:10.1038/s41398-020-01085-w, Published by Nature Publishing Group, London

Resilience to stress is critical for the development of depression. Enhanced adenosine A1 receptor (A1R) signaling mediates the antidepressant effects of acute sleep deprivation (SD). However, chronic SD causes long-lasting upregulation of brain A1R and increases the risk of depression. To investigate the effects of A1R on mood, we utilized two transgenic mouse lines with inducible A1R overexpression in forebrain neurons. These two lines have identical levels of A1R increase in the cortex, but differ in the transgenic A1R expression in the hippocampus. Switching on the transgene promotes robust antidepressant and anxiolytic effects in both lines. The mice of the line without transgenic A1R overexpression in the hippocampus (A1Hipp-) show very strong resistance towards development of stress-induced chronic depression-like behavior. In contrast, the mice of the line in which A1R upregulation extends to the hippocampus (A1Hipp+), exhibit decreased resilience to depression as compared to A1Hipp-. Similarly, automatic analysis of reward behavior of the two lines reveals that depression resistant A1Hipp-transgenic mice exhibit high sucrose preference, while mice of the vulnerable A1Hipp + line developed stress-induced anhedonic phenotype. The A1Hipp + mice have increased Homer1a expression in hippocampus, correlating with impaired long-term potentiation in the CA1 region, mimicking the stressed mice. Furthermore, virus-mediated overexpression of Homer1a in the hippocampus decreases stress resilience. Taken together our data indicate for first time that increased expression of A1R and Homer1a in the hippocampus modulates the resilience to stress-induced depression and thus might potentially mediate the detrimental effects of chronic sleep restriction on mood.

The present study examines the effect of three adenosine receptor antagonists on tremulous jaw movements (TJMs), an animal model of tremor. Forty-five rats were pre-treated with one adenosine antagonist: caffeine (0.0, 5.0, or 10.0 mg/kg; non-selective adenosine receptor antagonist), 8-cyclopentyltheophylline (CPT; 0.0, 5.0, or 10.0 mg/kg; selective adenosine A1 receptor antagonist), or SCH 58261 (0.0 or 8.0 mg/kg; selective adenosine A2A receptor antagonist) followed by TJM induction with tacrine (0.0, 0.75, or 2.5 mg/kg; acetylcholinesterase inhibitor). CPT and SCH 58261 both significantly reduced TJMs while caffeine did not. Unexpectedly, both SCH 58261 and CPT reduced TJMs even in the absence of tacrine. Also, CPT showed a robust reduction of TJMs, achieved at both (5.0 mg/kg) and (10.0 mg/kg) doses and regardless of tacrine dose. In conclusion, this study shows adenosine receptor antagonism to generally suppress low-dose tacrine-induced TJMs. In concert with two prior studies, these results are suggestive of behavioral evidence for a biphasic effect of adenosine A2A receptor antagonists (caffeine and SCH 58261) that is modulated by tacrine, and a model of this effect is proposed.

Chronic constriction injury- (CCI-) induced neuropathic pain is the most similar model to hyperalgesia in clinical observation. Neuropathic pain is a neuronal dysfunction in the somatosensory system that may lead to spontaneous pain. In this study, electroacupuncture (EA) was applied at bilateral L4 and L6 of Hua Tuo Jia Ji points (EX-B2) for relieving neuropathic pain in rats. Eighteen Sprague-Dawley rats were randomly assigned to three groups: sham, 2-Hz EA, and 15-Hz EA groups. Following von Frey and cold plate tests, both the 2- and the 15-Hz EA groups had significantly lower mechanical and thermal hyperalgesia than the sham group. Western blot analysis results showed that γ-aminobutyric acid A (GABAA), adenosine A1 receptor (A1R), transient receptor potential cation channel subfamily V member 1 (TRPV1), TRPV4, and metabotropic glutamate receptor 3 (mGluR3) were similar in the dorsal root ganglion of all three groups. Furthermore, levels of GABAA receptors were higher in the spinal cord of rats in the 2- and 15-Hz EA groups compared with the sham control group. This was not observed for A1R, TRPV1, TRPV4, or mGluR3 receptors. In addition, all the aforementioned receptors were unchanged in the somatosensory cortex of the study rats, suggesting a central spinal effect. The study results provide evidence to support the clinical use of EA for specifically alleviating neuropathic pain.

Activation of adenosine receptors has been implicated in several biological functions, including cardiovascular and renal function. Diabetes causes morphological and functional changes in the vasculature, resulting in abnormal responses to various stimuli. Recent studies have suggested that adenosine receptor expression and signaling are altered in disease states such as hypertension, diabetes. Using a streptozotocin (STZ) mouse model of type I diabetes (T1D), we investigated the functional changes in aorta and resistance mesenteric arteries to adenosine receptor agonist activation in T1D. Organ baths and DMT wire myographs were used for muscle tension measurements in isolated vascular rings, and western blotting was used for protein analysis. Concentration response curves to selective adenosine receptor agonists, including CCPA (A1 receptor agonist), Cl-IBMECA (A3 receptor agonist), CGS-21680 (A2A receptor agonist), and BAY 60-6583 (A2B receptor agonist), were performed. We found that diabetes did not affect adenosine receptor agonist-mediated relaxation or contraction in mesenteric arteries. However, aortas from diabetic mice exhibited a significant decrease (P

The positron emission tomography (PET) radioligand for adenosine A1 receptor (A1R) [1-methyl-11C] 8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPDX) has recently been developed for human brain imaging. In the present study, we evaluated the alteration of the A1R in patients with diffuse axonal injury (DAI) in chronic stage in vivo. Ten patients with DAI (7 men and 3 women) were included in this study. Three PET examinations were sequentially performed to measure A1R binding with 11C-MPDX, glucose metabolism with 18F-fluorodeoxyglucose (FDG), and central benzodiazepine receptor binding with 11C-flumazenil (FMZ), and decreases of 11C-FMZ uptake indicate neuronal loss. 11C- MPDX did not depict any lesion with significantly decreased nondisplaceable binding potential (BPND) in comparison to healthy controls (14 men) in region of interest (ROI) analysis. Instead, it showed a significant increase of BPND in the lower frontal and posterior cingulate cortexes and rolandic area (p

Hepatic insulin resistance (IR) and enhanced hepatic glucose production (HGP) are key features of type 2 diabetes (T2D), contributing to fasting hyperglycemia. Adenosine receptors (ARs) are G protein-coupled and expressed in hepatocytes. Here, we explored the role of hepatic G(i/o)-coupled A(1)AR on insulin resistance and glucose fluxes associated with obesity. We generated a mouse model with hepatocyte-specific deletion of A(1)AR (A1L(Δ/Δ)), which was compared with whole body knockout of A(1)AR or A(1)AR/A(3)AR (both G(i)-coupled). Selective deletion of hepatic A(1)AR resulted in a modest improvement in insulin sensitivity. In addition, HFD A1L(Δ/Δ) mice showed decreased fasting glucose levels. Hyperinsulinemic-euglycemic clamp studies demonstrated enhanced insulin sensitivity with no change in HGP in HFD A1L(Δ/Δ) mice. Similar to A1L(Δ/Δ), fasting blood glucose levels were significantly reduced in whole body A1(Δ/Δ) and A1(Δ/Δ)A3(Δ/Δ) compared to wild-type mice. Taken together, our data support the concept that blocking hepatic A(1)AR may decrease fasting blood glucose levels without directly affecting hepatocyte glucose metabolism and insulin sensitivity.

Febrile seizures (FS) are one of the most common types of convulsive disorder of early childhood and they can be classified into simple and prolonged depending on the duration. Nowadays, simple FS have a good prognosis but there is controversy about the outcome of prolonged FS. In a previous work using an animal model of prolonged FS, we showed that hiperthermia-induced seizures (HIS) evoked fine motor coordination impairment and gait disturbances in adolescent rats in a process in which seemed to be involved modulation of the cerebellar adenosinergic system. The aim of the present work was to verify whether the effect was maintained in adulthood. To this end, neonatal rats (PD 12) were exposed to HIS and after 48 days (PD 60) they were assayed on balance beam and footprint tests. Animals were sacrificed 53 days after HIS and adenosine A1 and A2A receptor signalling pathways were studied in cerebellar plasma membranes by using radioligand binding assays and by measuring the activities of 5´-nucleotidase and adenylyl cyclase. Results obtained revealed that adult rats exposed to HIS showed gait disturbances and motor impairments. Besides, animals exposed to hyperthermic insult showed an increase in adenosine A2A receptor functionality and 5´-nucleotidase activity. Surprisingly, the functionality of the adenosine A1 receptor resulted significantly changed causing stimulation instead of inhibition of adenylate cyclase activity. These results showed that the effect of prolonged FS at the early age also persist in adulthood suggesting on must pay attention to FS in children.

Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca2+concentration ([Ca2+]i) elevation in smooth muscle cells (SMCs) via activation of its cognate A1receptors (A1Rs). Mechanisms that underlie A1R-dependent [Ca2+]ielevation in renal vascular SMCs are not fully resolved. Whether A1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of A1Rs by 2-chloro- N6-cyclopentyladenosine (CCPA) does not stimulate store-operated Ca2+entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca2+]ielevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca2+]iwas unchanged in afferent arterioles isolated from newborn (0-day-old) pigs compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca2+]iin afferent arterioles from 20-day-old pigs. A1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0- vs. 20-day-old pigs. However, the TRPC3 channel protein expression level was ~92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of A1Rs elicits receptor-operated Ca2+entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of A1Rs.

Background/Aims: The adenosine A1 receptor (A1R) has been reported to be involved in the pathogenesis of various cancers, and the effects of A1R on different cancers are pleiotropic. However, the role of A1R in renal cell carcinoma (RCC) remains not well-known. Methods: The expression of A1R in RCC cells was detected by quantitative real-time PCR and Western blotting analysis. Cell proliferation was detected using an MTT assay and a colony formation assay. Tumor growth was also evaluated in nude mice. Cell invasion and migration were evaluated using a wound healing assay and a transwell assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Results: A1R was the main subtype of ARs and was up-regulated in 786-O and ACHN cells. Functionally, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an A1R antagonist, inhibited RCC cell proliferation in vitro and tumor growth in vivo. Furthermore, DPCPX inhibited RCC cell migration, while N6-Cyclopentyladenosine (CPA), a selective A1 agonist, was able to rescue RCC cell migration. In addition, DPCPX promoted 786-O and ACHN cell apoptosis and induced an S phase cell cycle arrest. Finally, we demonstrated that DPCPX inhibited tumor progression in part via the ERK/JNK pathway. Conclusion: These findings suggest the potentially important role of DPCPX in the control of RCC cell proliferation and migration by regulating the ERK/JNK signaling pathway.

The direct detrusor relaxant effect of β3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1receptors to explain inhibition of cholinergic activity by β3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (−47 ± 5%) and rat (−38 ± 1%) detrusor strips. Mirabegron (0.1 µM, −53 ± 8%) and CL316,243 (1 µM, −37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking β3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1–1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by β3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.

Ischemic tolerance reduces brain damage and neurological dysfunction after brain ischemia. A ketogenic diet (KD) has disease-modifying effects in several neurodegenerative disorders. In this study, we fed mice with a KD for three weeks and performed reversible middle cerebral artery occlusion (MCAO) in the animals. KD-fed mice had a significantly reduced infarct volume, increased regional cerebral blood flow (rCBF) and extracellular adenosine levels in both the ischemic and the reperfusion phases. In vitro and in vivo experiments revealed that the KD-induced neuroprotection was mediated through the adenosine A1 receptor. The KD increased Akt and ERK1/2 phosphorylation via A1R activation. Besides, the KD also upregulated robustly HIF-1α/HIF-2α and HIF regulated genes, such as VEGF and EPO. A three-week preconditioning period with a KD improved ischemic tolerance in mice with MCAO. The underlying mechanisms might include elevated extracellular adenosine levels, and increased Akt and ERK1/2 phosphorylation via A1 adenosine receptor activation, together with upregulated HIFs and HIF-regulated genes.

Regulation of blood flow depends on systemic and local release of vasoactive molecules such as insulin and adenosine. These molecules cause vasodilation by activation of plasma membrane receptors at the vascular endothelium. Adenosine activates at least four subtypes of adenosine receptors (A(1)AR, A(2A)AR, A(2B)AR, A(3)AR), of which A(2A)AR and A(2B)AR activation leads to increased cAMP level, generation of nitric oxide, and relaxation of the underlying smooth muscle cell layer. Vasodilation caused by adenosine also depends on plasma membrane hyperpolarization due to either activation of intermediate-conductance Ca2+-activated K+ channels in vascular smooth muscle or activation of ATP-activated K+ channels in the endothelium. Adenosine also causes vasoconstriction via a mechanism involving A(1)AR activation resulting in lower cAMP level and increased thromboxane release. Insulin has also a dual effect causing NO-dependent vasodilation, but also sympathetic activity-and increased endothelin 1 release-dependent vasoconstriction. Interestingly, insulin effects require or are increased by activation or inactivation of adenosine receptors. This is phenomenon described for D-glucose and L-arginine transport where A(2A)AR and A(2B)AR play a major role. Other studies show that A(1)AR activation could reduce insulin release from pancreatic beta-cells. Whether adenosine modulation of insulin biological effect is a phenomenon that depends on co-localization of adenosine receptors and insulin receptors, and adenosine plasma membrane transporters is something still unclear. This review summarizes findings addressing potential involvement of adenosine receptors to modulate insulin effect via insulin receptors with emphasis in the human vasculature. (C) 2016 Elsevier Ltd. All rights reserved.

Adenosine as well as agonists and antagonists for the four adenosine receptor subtypes (A1R, A2AR, A2BR and A3R) play a role in several key physiological and pathophysiological processes, including the regulation of vascular tone, thrombosis, immune response, inflammation, and angiogenesis. This review focuses on the adenosine-mediated regulation of lipid availability in the cell and in the systemic circulation as well in humans and animal models. Therefore, adenosine, mainly by acting on A1R, inhibits lipolysis activity, leading to reduction of the circulating fatty acid levels. This nucleoside can also participate in the early development of atherosclerosis by inhibiting the formation of foam cells via stimulation of cholesterol efflux through A2AR expressed on macrophages and reduction of the inflammatory process by activating A2AR and A2BR. Adenosine also appears to modulate intracellular cholesterol availability in Niemann-Pick type C1 disease and Alzheimer disease via A2AR and A3, respectively. Remarkably, the role of adenosine receptors in the regulation of plasma total cholesterol and triglyceride levels has been studied in animal models. Thus, an anti-atherogenic role for A2BR as well as a pro-atherogenic role of A2AR and A1 have been proposed; A3R has not been shown to participate in the control of lipid levels or the development of atherosclerosis. Surprisingly, and despite the role of A2A in the inhibition of foam cell formation among isolated cells, this receptor appears to be pro-atherogenic in mice. Remarkably, the role of adenosine receptors in human dyslipidaemia and atherosclerosis must to be elucidated. Additionally, it has been reported that increased lipid levels impair the effects of adenosine/adenosine receptors in controlling vascular tone, and we speculate on the possibility that this impairment could be due to alterations in the composition of the membrane microdomains where the adenosine receptors are located. Finally, a possible role for adenosine/adenosine receptors in the phenomena of dyslipidaemia in pregnancy has been proposed.

Aim The mechanisms underlying the inhibitory effects of oxytocin (OT) on colon tone are not totally understood. We explore the mechanisms of OT on spontaneous contractility in rat distal colon, and identify the mediators involved in this action. Methods In rat distal colon strips, mechanical activity was analyzed and the production of nitric oxide (NO) in tissue loaded with the fluorochrome DAF-FM was visualized by confocal microscopy. OT receptor (OTR) expression was determined by western blotting and immunofluorescence. Results In rat distal colon, OT produced a concentration-dependent reduction of the spontaneous contraction, which was abolished by the OTR antagonist atosiban, the neural blocker tetrodotoxin and the inhibitor of neuronal nitric oxide synthase (nNOS) NPLA. The inhibitory effects of OT were not affected by propranolol, atropine, the nicotinic cholinoceptor blocker hexamethonium, the vasoactive intestinal peptide receptor antagonist VIPHyb, the P2 purinoceptor antagonist PPADS, the adenosine A1 receptors antagonist DPCPX and the prostacyclin receptor antagonist Ro1138452. The soluble guanylyl cyclase (sGC) inhibitor ODQ and the small conductance Ca2+-activated K+ (CaK+) channels blocker apamin significantly reduced the relaxation induced by OT, nicotine, sodium nitroprusside and the sGC activator BAY 41-2272. The neural release of NO elicited by OT was prevented by NPLA, tetrodotoxin, and atosiban. The presence of the OTR and its co-localization with nNOS was detected by immunohistochemistry and western blotting experiments. Conclusion These results demonstrate the NO release from enteric neurons induced by activation of OTR mediates distal colon relaxation. sGC and small conductance CaK+ channels are involved in this relaxation. This article is protected by copyright. All rights reserved.

SIRS is associated with lymphopenia, and prolonged lymphopenia of septic patients has been associated with increased mortality risk. We hypothesize that elevated adenosine during SIRS down-regulates Gi-coupled A1R, which signals an effect that sensitizes a cAMP-dependent lymphotoxic response. In this study, we evaluate the role of adenosine in SIRS-mediated lymphopenia and impaired IL-15 production. Cecal ligation and puncture was used to induce sepsis-associated SIRS in mice. BMDCs were cultured and used to measure the effect of adenosine on IL-15. We found that A1R mRNA levels were significantly down-regulated and A1R-dependent Gi activity was abolished in T cells of septic mice. In accordance, cAMP was elevated in isolated T cells from cecal ligation and puncture compared with sham-treated mice. Similar to septic mice, leukopenia was evident in sham A1R-KO mice, after treatment with the A1R antagonist (8-cyclopentyl-1,3-dipropylxanthine), or after A1R desensitization. In contrast, A2AR-KO mice were protected from leukopenia. In addition, we observed that septic A1R-KO mice exhibited low IL-15 levels. Cultured BMDC agonists of A2AR and A2BR inhibited IL-15 production and adenosine blocked IL-15–dependent proliferation of cytotoxic T cells that were cocultured with stimulated BMDCs. To conclude, we suggest that SIRS-associated lymphopenia is initiated by A1R desensitization and adenosine-mediated inhibition of IL-15 production is part of the mechanism that accounts for the delay in leukopenia recovery in patients with severe sepsis. Interference with adenosine signaling may thus be potentially beneficial for septic patients with leukopenia.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressedas prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additionalpathway including an autocrine mechanism at the plasma membrane. The autocrine mechanisminvolves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanismsinvolved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still producedhyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming.PerspectiveThis study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Mice enter bouts of daily torpor, drastically reducing metabolic rate, core body temperature (T b), and heart rate (HR), in response to reduced caloric intake. Because central adenosine activation has been shown to induce a torpor-like state in the arctic ground squirrel, and blocking the adenosine-1 (A1) receptor prevents daily torpor, we hypothesized that central activation of the A1 adenosine receptors would induce a bout of natural torpor in mice. To test the hypothesis, mice were subjected to four different hypothermia bouts: natural torpor, forced hypothermia (FH), isoflurane-anesthesia, and an intracerebroventricular injection of the selective A1 receptor agonist N6-cyclohexyladenosine (CHA). All conditions induced profound hypothermia. T b fell more rapidly in the FH, isoflurane-anesthesia, and CHA conditions compared to torpor, while mice treated with CHA recovered at half the rate of torpid mice. FH, isoflurane-anesthesia, and CHA-treated mice exhibited a diminished drop in HR during entry into hypothermia as compared to torpor. Mice in all conditions except CHA shivered while recovering from hypothermia, and only FH mice shivered substantially while entering hypothermia. Circulating lactate during the hypothermic bouts was not significantly different between the CHA and torpor conditions, both of which had lower than baseline lactate levels. Arrhythmias were largely absent in the FH and isoflurane-anesthesia conditions, while skipped beats were observed in natural torpor and periodic extended (>1 s) HR pauses in the CHA condition. Lastly, the hypothermic bouts showed distinct patterns of gene expression, with torpor characterized by elevated hepatic and cardiac Txnip expression and all other hypothermic states characterized by elevated c-Fos and Egr-1 expression. We conclude that CHA-induced hypothermia and natural torpor are largely different physiological states.

The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A(2A) and A(2B) adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Accumulating evidence suggests that the anti-diabetic drug, metformin, exerts anti-proliferative effects in many types of cancers. However, the function and mechanisms of metformin in human colorectal cancer (CRC) remain unknown. Here, we show that metformin induces growth inhibition and apoptosis through activating AMPK-mTOR pathway in human colorectal cancer cells. Notably, metformin treatment significantly up-regulated adenosine A1 receptor (ADORA1) expression in human colorectal cancer cells, while suppression of ADORA1 activity by its specific inhibitor rescued the growth inhibition induced by metformin. Moreover, ADORA1-mediated growth inhibition and apoptosis induced by metformin is AMPK-mTOR pathway dependent in human colorectal cancer cells. Taken together, these results indicate that metformin suppresses human colorectal cancer growth by inducing apoptosis via ADORA1, which provide evidence the anti-neoplastic effects of metformin in the treatment of human colorectal cancer.

Caffeine promotes memory consolidation. Memory consolidation is thought to depend at least in part on hippocampal sharp waves (SWs). In the present study, we investigated the effect of bath-application of caffeine in spontaneously occurring SWs in mouse acute hippocampal slices. Caffeine induced an about 100% increase in the event frequency of SWs at concentrations of 60 and 200 µM. The effect of caffeine was reversible after washout of caffeine and was mimicked by an adenosine A1 receptor antagonist, but not by an A2A receptor antagonist. Caffeine increased SWs even in dentate-CA3 mini-slices without the CA2 regions, in which adenosine A1 receptors are abundantly expressed in the hippocampus. Thus, caffeine facilitates SWs by inhibiting adenosine A1 receptors in the hippocampal CA3 region or the dentate gyrus.

Central failure of respiration during a seizure is one possible mechanism for sudden unexpected death in epilepsy (SUDEP). Neuroimaging studies indicate volume loss in the medulla in SUDEP and a post mortem study has shown reduction in neuromodulatory neuropeptidergic and monoaminergic neurones in medullary respiratory nuclear groups. Specialised glial cells identified in the medulla are considered essential for normal respiratory regulation including astrocytes with pacemaker properties in the pre-Botzinger complex and populations of subpial and perivascular astrocytes, sensitive to increased pCO(2), that excite respiratory neurones. Our aim was to explore niches of medullary astrocytes in SUDEP cases compared to controls. In 48 brainstems from three groups, SUDEP (20), epilepsy controls (10) and non-epilepsy controls (18), sections through the medulla were labelled for GFAP, vimentin and functional markers, astrocytic gap junction protein connexin43 (Cx43) and adenosine A1 receptor (A1R). Regions including the ventro-lateral medulla (VLM; for the pre-Bötzinger complex), Median Raphe (MR) and lateral medullary subpial layer (MSPL) were quantified using image analysis for glial cell populations and compared between groups. Findings included morphologically and regionally distinct vimentin/Cx34-positive glial cells in the VLM and MR in close proximity to neurones. We noted a reduction of vimentin-positive glia in the VLM and MSPL and Cx43 glia in the MR in SUDEP cases compared to control groups (p