Input-selective adenosine A1 receptor-mediated synaptic depression of excitatory transmission in dorsal striatum.

The medial (DMS) and lateral (DLS) dorsal striatum differentially drive goal-directed and habitual/compulsive behaviors, respectively, and are implicated in a variety of neuropsychiatric disorders. These subregions receive distinct inputs from cortical and thalamic regions which uniquely determine dorsal striatal activity and function. Adenosine A1 receptors (A1Rs) are prolific within striatum and regulate excitatory glutamate transmission. Thus, A1Rs may have regionally-specific effects on neuroadaptive processes which may ultimately influence striatally-mediated behaviors. The occurrence of A1R-driven plasticity at specific excitatory inputs to dorsal striatum is currently unknown. To better understand how A1Rs may influence these behaviors, we first sought to understand how A1Rs modulate these distinct inputs. We evaluated A1R-mediated inhibition of cortico- and thalamostriatal transmission using in vitro whole-cell, patch clamp slice electrophysiology recordings in medium spiny neurons from both the DLS and DMS of C57BL/6J mice in conjunction with optogenetic approaches. In addition, conditional A1R KO mice lacking A1Rs at specific striatal inputs to DMS and DLS were generated to directly determine the role of these presynaptic A1Rs on the measured electrophysiological responses. Activation of presynaptic A1Rs produced significant and prolonged synaptic depression (A1R-SD) of excitatory transmission in the both the DLS and DMS of male and female animals. Our findings indicate that A1R-SD at corticostriatal and thalamostriatal inputs to DLS can be additive and that A1R-SD in DMS occurs primarily at thalamostriatal inputs. These findings advance the field's understanding of the functional roles of A1Rs in striatum and implicate their potential contribution to neuropsychiatric diseases.

Role of Purinergic Signaling in Acupuncture Therapeutics.

Acupuncture is a therapeutic treatment that is well recognized in many countries. However, the initiation mechanisms of acupuncture are not well understood. Purinergic signaling has been considered a key signaling pathway in acupuncture in recent years. Acupuncture-induced ATP is mainly produced by mast cells and fibroblasts, and ATP is gradually hydrolyzed into adenosine. ATP and adenosine further participate in the process of acupuncture information transmission to the nervous and immune systems through specific purine receptors. Acupuncture initiates analgesia via the down-regulation of the expression of P2 receptors or up-regulation of the expression of adenosine A1 receptors on nerve fibers. ATP also promotes the proliferation of immune cells through P2 receptors and A3 receptors, causing inflammation. In contrast, adenosine activates A2 receptors, promotes the production and infiltration of immunosuppressive cells, and causes an anti-inflammatory response. In summary, we described the role of purinergic signaling as a general signaling pathway in the initiation of acupuncture and the influence of purinergic signaling on the neuroimmune network to lay the foundation for future systematic research on the mechanisms of acupuncture therapeutics.

Role of spinal adenosine A1 receptors in the analgesic effect of electroacupuncture in a rat model of neuropathic pain.

OBJECTIVE: The aim of this study was to determine the role of spinal adenosine A1 receptors (A1Rs) in the analgesic effects of electroacupuncture (EA) for neuropathic pain. METHODS: We performed EA for 30 minutes at the zusanli acupoint in the legs of rats with previously induced chronic constriction injuries and observed the mechanical and thermal pain thresholds 1 hour later. We also examined adenosine levels by high-performance liquid chromatography and A1R expression in the L4-6 spinal cord by western blot analysis. We then injected A1R short interfering RNA (AV-shA1RNA) into the L4-6 spinal cord to downregulate A1R expression and re-examined the mechanical and thermal pain thresholds. RESULTS: Adenosine levels and A1R expression in the L4-6 spinal cord were increased at 1 hour after EA. In addition, EA exhibited an analgesic effect that was reversed by AV-shA1RNA. CONCLUSIONS: Our results suggest that EA at the zusanli acupoint elicits an analgesic effect against neuropathic pain, mediated by A1Rs in the spinal cord.

Effects of Xingnaojing Injection on Adenosinergic Transmission and Orexin Signaling in Lateral Hypothalamus of Ethanol-Induced Coma Rats.

Acute alcohol exposure induces unconscious condition such as coma whose main physical manifestation is the loss of righting reflex (LORR). Xingnaojing Injection (XNJI), which came from Chinese classic formula An Gong Niu Huang Pill, is widely used for consciousness disorders in China, such as coma. Although XNJI efficiently shortened the duration of LORR induced by acute ethanol, it remains unknown how XNJI acts on ethanol-induced coma (EIC). We performed experiments to examine the effects of XNJI on orexin and adenosine (AD) signaling in the lateral hypothalamic area (LHA) in EIC rats. Results showed that XNJI reduced the duration of LORR, which implied that XNJI promotes recovery form coma. Microdialysis data indicated that acute ethanol significantly increased AD release in the LHA but had no effect on orexin A levels. The qPCR results displayed a significant reduction in the Orexin-1 receptors (OX1R) expression with a concomitant increase in the A1 receptor (A1R) and equilibrative nucleoside transporter type 1 (ENT1) expression in EIC rats. In contrast, XNJI reduced the extracellular AD levels but orexin A levels remained unaffected. XNJI also counteracted the downregulation of the OX1R expression and upregulation of A1R and ENT1 expression caused by EIC. As for ADK expression, XNJI but not ethanol, displayed an upregulation in the LHA in EIC rats. Based on these results, we suggest that XNJI promotes arousal by inhibiting adenosine neurotransmission via reducing AD level and the expression of A1R and ENT1.

Omega-3 Polyunsaturated Fatty Acids Enhance Cisplatin Efficacy in Gastric Cancer Cells by Inducing Apoptosis via ADORA1.

It has been suggested that administration of the omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can alter the toxicity and/or activity of several anticancer drugs in in vitro and in vivo studies. Here, we investigated the ability of ω-3 PUFAs to potentiate the antineoplastic activity of cisplatin (CDDP) in gastric cancer cells. The increase in CDDP-induced growth inhibition was measured by the IC50 values obtained when the cells were incubated with CDDP alone or with CDDP plus DHA or EPA. DHA and EPA enhanced the growth-inhibition activity of increasing concentrations of CDDP. The interactions between CDDP and DHA or EPA at the cellular level were assessed through the combination index (CI) method of Chou-Talalay. The results demonstrated synergism between CDDP and DHA or EPA in MKN45 cells. Cell cycle analysis showed that the combination treatment increased G0/G1 phase and S phase arrest, and significantly increased the number of apoptotic cells. According to our previous study, ω -3 PUFAs induce apoptosis of gastric cells via ADORA1, a subtype of adenosine receptor functionally related to cell death. The ADORA1 mRNA and protein expression was higher in the combination treatment than in the individual treatments. Notable, when GC cells were pretreated with DPCPX, a selective ADORA1 antagonist, the combination treatment effect on apoptosis was significantly reduced. Our results suggest that ω-3 PUFAs enhance the antineoplastic effects of CDDP in gastric cancer cells, and the synergistic effect between ω-3 PUFAs and CDDP is partly dependent on activating the ADORA1-mediated apoptosis pathway.

The administration of Fructus Schisandrae attenuates dexamethasone-induced muscle atrophy in mice.

In the present study, we aimed to determine whether ethanol extracts of Fructus Schisandrae (FS), the dried fruit of Schizandra chinensis Baillon, mitigates the development of dexamethasone-induced muscle atrophy. Adult SPF/VAT outbred CrljOri:CD1 (ICR) mice were either treated with dexamethasone to induce muscle atrophy. Some mice were treated with various concentrations of FS or oxymetholone, a 17α-alkylated anabolic-androgenic steroid. Muscle thickness and weight, calf muscle strength, and serum creatine and creatine kinase (CK) levels were then measured. The administration of FS attenuated the decrease in calf thickness, gastrocnemius muscle thickness, muscle strength and weight, fiber diameter and serum lactate dehydrogenase levels in the gastrocnemius muscle bundles which was induced by dexamethasone in a dose-dependent manner. Treatment with FS also prevented the dexamethasone-induced increase in serum creatine and creatine kinase levels, histopathological muscle fiber microvacuolation and fibrosis, and the immunoreactivity of muscle fibers for nitrotyrosine, 4-hydroxynonenal, inducible nitric oxide synthase and myostatin. In addition, the destruction of the gastrocnemius antioxidant defense system was also inhibited by the administration of FS in a dose-dependent manner. FS downregulated the mRNA expression of atrogin-1 and muscle ring-finger protein-1 (involved in muscle protein degradation), myostatin (a potent negative regulator of muscle growth) and sirtuin 1 (a representative inhibitor of muscle regeneration), but upregulated the mRNA expression of phosphatidylinositol 3-kinase, Akt1, adenosine A1 receptor and transient receptor potential cation channel subfamily V member 4, involved in muscle growth and the activation of protein synthesis. The overall effects of treatment with 500 mg/kg FS were comparable to those observed following treatment with 50 mg/kg oxymetholone. The results from the present study support the hypothesis that FS has a favorable ameliorating effect on muscle atrophy induced by dexamethasone, by exerting anti-inflammatory and antioxidant effects on muscle fibers, which may be due to an increase in protein synthesis and a decrease in protein degradation.

Omega-3 PUFAs induce apoptosis of gastric cancer cells via ADORA1.

Omega-3 polyunsaturated fatty acids (Omega-3 PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been suggested to have anti-cancer effects by epidemiological and clinical studies. However, their underlying anti-cancer mechanisms are still unclear. In this study, we examined the influence of two Omega-3 PUFAs (DHA and EPA) on the proliferation and apoptosis of gastric cancer (GC) cells, and found that DHA and EPA reduced the viability of GC cells and induced apoptosis by activating caspase-3. Moreover, we screened the expression profile of apoptosis-related genes in GC cells upon the treatment of DHA and/or EPA, and discovered that ADORA1, one subtype of adenosine receptor functionally involved in cell death, was up-regulated in response to DHA and EPA. Importantly, when GC cells were treated with a selective ADORA1 antagonist, DPCPX, the DHA/EPA-induced apoptosis was substantially reduced. Taken together, our results suggest that the anti-cancer effect of Omega-3 PUFAs on gastric cancer is at least partly dependent on activating the ADORA1-mediated apoptosis pathway.

Adenosine A1 receptor activation as a brake on the microglial response after experimental traumatic brain injury in mice.

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

Determination of adenosine A1 receptor agonist and antagonist pharmacology using Saccharomyces cerevisiae: implications for ligand screening and functional selectivity.

The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Galpha/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A(1) receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (-)-N(6)-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Galpha(o), Gpa1/Galpha(i1/2), and Gpa1/Galpha(i3), whereas the novel compound, 5'-deoxy-N(6)-(endo-norborn-2-yl)-5'-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Galpha(i) proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or (35)S-labeled guanosine 5'-(gamma-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of low-efficacy agonists.

Eliminating the antilipolytic adenosine A1 receptor does not lead to compensatory changes in the antilipolytic actions of PGE2 and nicotinic acid.

AIM: We examined whether compensatory changes after adenosine A(1) receptor knockout [A(1)R (-/-)] eliminate the antilipolytic actions mediated by this receptor. METHODS: Lipolysis experiments were performed on adipocytes prepared from the wild type A(1)R (+/+), A(1)R (-/-) and heterozygous mice. Gene expression was assayed with cDNA microarray technique and real-time PCR; protein expression with immunoblotting. RESULTS: The A(1)R was the only adenosine receptor involved in lipolysis. The effects of adenosine deaminase and 2-chloroadenosine were abolished in A(1)R (-/-) mice. The IC(50) value of 2-chloroadenosine doubled from 16.6 to 33.6 nm when half of the A(1)Rs were eliminated. Adrenergic alpha(2) agonists had no effects on lipolysis. Prostaglandin E(2) (PGE(2)) inhibited lipolysis with an IC(50) value of 5.8 nm (4.7-7.2 nm) in the A(1)R (+/+) mice and 10.6 nm (9.0-12.6 nm) in the A(1)R (-/-) mice. Nicotinic acid inhibited lipolysis with an IC(50) value of 0.30 microm (0.19-0.46 microm) in the A(1)R (+/+) mice and 0.24 microm (0.16-0.37 microm) in the A(1)R (-/-) mice. G(i)alpha(1) mRNA was significantly up-regulated in adipose tissue from A(1)R (-/-) mice. However, immunoblotting showed that G(ialpha1) was not up-regulated at the protein level. CONCLUSION: The A(1)R mediates the antilipolytic actions of adenosine. Deletion of the A(1)R in mice does not result in compensatory increases in G-protein-mediated antilipolytic actions of PGE(2) or nicotinic acid.

A1 antagonism in asthma: better than coffee?

Adenosine is a ubiquitous biological mediator with the capacity to produce both pro- and anti-inflammatory effects in tissues. Proinflammatory and bronchoconstrictive actions of adenosine in the asthmatic lung are well recognized, with the latter being mediated, in part, through A(1) receptor activation on airway smooth muscle. In this issue of the JCI, Sun et al. report findings in adenosine deaminase-deficient mice that suggest the occurrence of anti-inflammatory actions of adenosine in the lung, mediated through A(1) adenosine receptors on macrophages. Here we discuss the history of the study of adenosine receptor ligands for asthma and how enhanced understanding of adenosine receptor biology may aid in the rational exploitation of these receptors as therapeutic targets.