Adenosine Receptor Research Articles

The influence of temperature on the impacts of caffeine in mussels: Evaluating subcellular impacts and model predictions

In aquatic ecosystems, the presence of pharmaceuticals, particularly caffeine (CAF), has been linked to wastewater discharge, hospital waste, and the disposal of expired pharmaceutical products containing CAF. Additionally, rising temperatures due to climate change are anticipated in aquatic environments. This study aimed to assess the toxicity of various CAF concentrations under current (17 °C) and projected (21 °C) temperature conditions, using the mussel Mytilus galloprovincialis as a bioindicator species. Subcellular impacts were evaluated following 28 days of exposure to four CAF concentrations (0.5; 1.0; 5.0; 10.0 μg/L) at the control temperature (17 °C). Only effects at an environmentally relevant CAF concentration (5.0 μg/L) were assessed at the highest temperature (21 °C). The overall biochemical response of mussels was evaluated using non-metric Multidimensional Scaling (MDS) and the Integrated Biomarker Response (IBR) index, while the Independent Action (IA) model was used to compare observed and predicted responses. Results showed that at 17 °C, increased CAF concentrations were associated with higher metabolism and biotransformation capacity, accompanied by cellular damage at the highest concentration. Conversely, under warming conditions (21 °C), the induction of antioxidant enzymes was observed, although insufficient to prevent cellular damage compared to the control temperature. Regarding neurotoxicity, at 17 °C, the activity of the acetylcholinesterase enzyme was inhibited up to 5.0 μg/L; however, at 10.0 μg/L, activity increased, possibly due to CAF competition for adenosine receptors. The IA model identified a synergistic response for most parameters when CAF and warming acted together, aligning with observed results, albeit with slightly lower magnitudes.

Norepinephrine changes behavioral state via astroglial purinergic signaling.

Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.

Cardiovascular and cerebrovascular effects of caffeine maintenance in preterm infants during the transitional period.

Caffeine is routinely used for the prophylaxis of prematurity-related apnoeas. We aimed to evaluate the effect of caffeine maintenance on cardiovascular and cerebrovascular haemodynamics using a non-invasive multimodal monitoring in preterm infants during the transitional period. Infants <32 weeks' gestational age (GA) were enrolled in this observational prospective study. The following parameters were recorded before and after the administration of caffeine citrate 5 mg/kg using near-infrared spectroscopy, pulse oximetry and electrical velocimetry: heart rate, cardiac output, stroke volume, cardiac contractility, systemic vascular resistance (SVR), perfusion index, peripheral and cerebral oxygenation, cerebral fractional oxygen extraction, correlation index between cerebral oxygenation and heart rate (TOHRx, marker of cerebrovascular reactivity). Multilevel mixed-effects linear models were used to assess the impact of caffeine and of relevant clinical covariates on each parameter. Seventy-seven infants (mean GA 29.3 ± 2.5 weeks, mean birthweight 1148 ± 353 g) were included. Caffeine administration was associated with increased SVR (B = 0.623, p = 0.004) and more negative TOHRx values (B = -0.036, p = 0.022), which suggest improved cerebrovascular reactivity. Caffeine administration at maintenance dosage during postnatal transition is associated with increased systemic vascular tone and improved cerebrovascular reactivity. A possible role for caffeine-mediated inhibition of adenosine receptors may be hypothesized. This study provides a thorough and comprehensive overview of multiple cerebrovascular and cardiovascular parameters, monitored non-invasively by combining near-infrared spectroscopy, electrical velocimetry and pulse oximetry, before and after the administration of caffeine at maintenance dosage in preterm infants during postnatal transition. Caffeine was associated with an improvement in cerebrovascular reactivity and with a slight but significant increase in systemic vascular resistance, with no additional effects on other cardiovascular and cerebrovascular parameters. Our results support the safety of caffeine treatment even during a phase at risk for haemodynamic instability such as postnatal transition and suggest potential beneficial effects on cerebral haemodynamics.

Network pharmacology integrated with experimental validation to elucidate the mechanisms of action of the Guizhi-Gancao Decoction in the treatment of phenylephrine-induced cardiac hypertrophy

Context The mechanisms of Traditional Chinese Medicine (TCM) Guizhi-Gancao Decoction (GGD) remain unknown. Objective This study explores the mechanisms of GGD against cardiac hypertrophy. Materials and methods Network pharmacology analysis was carried out to identify the potential targets of GGD. In vivo experiments, C57BL/6J mice were divided into Con, phenylephrine (PE, 10 mg/kg/d), 2-chloroadenosine (CADO, the stable analogue of adenosine, 2 mg/kg/d), GGD (5.4 g/kg/d) and GGD (5.4 g/kg/d) + CGS15943 (a nonselective adenosine receptor antagonist, 4 mg/kg/d). In vitro experiments, primary neonatal rat cardiomyocytes (NRCM) were divided into Con, PE (100 µM), CADO (5 µM), GGD (10−5 g/mL) and GGD (10−5 g/mL) + CGS15943 (5 µM). Ultrasound, H&E and Masson staining, hypertrophic genes expression and cell surface area were conducted to verify the GGD efficacy. Adenosine receptors (ADORs) expression were tested via real-time polymerase chain reaction (PCR), western blotting and immunofluorescence analysis. Results Network pharmacology identified ADORs among those of the core targets of GGD. In vitro experiments demonstrated that GGD attenuated PE-induced increased surface area (with an EC50 of 5.484 × 10−6 g/mL). In vivo data shown that GGD attenuated PE-induced ventricular wall thickening. In vitro and in vivo data indicated that GGD alleviated PE-induced hypertrophic gene expression (e.g., ANP, BNP and MYH7/MYH6), A1AR over-expression and A2aAR down-expression. Moreover, CADO exerts effects similar to GGD, whereas CGS15943 eliminated most effects of GGD. Discussion and conclusions Our findings suggest the mechanism by which GGD inhibits cardiac hypertrophy, highlighting regulation of ADORs as a potential therapeutic strategy for HF.

Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

Conversion of dendritic cells into tolerogenic or inflammatory cells depends on the activation threshold and kinetics of the mTOR signaling pathway

BackgroundRestoring impaired peripheral immune tolerance is the primary challenge in treating autoimmune diseases. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs), a fraction of low molecular weight proteins, in inhibiting the progression of psoriatic arthritis, even in the presence of high levels of the proinflammatory cytokine TNFα in the bloodstream. When specifically targeting dendritic cells (DCs), SSPs transform them into tolerogenic cells, which efficiently induce the development of regulatory Foxp3+ Treg cells. In this study, we provide further insights into the mechanism of action of SSPs.ResultsWe found that SSPs stimulate the activation of the mTOR signaling pathway in dendritic cells, albeit in a different manner than the classical immunogenic stimulus LPS. While LPS-induced activation is rapid, strong, and sustained, the activity induced by SSPs is delayed, less intense, yet still significant. These distinct patterns of activation, as measured by phosphorylation of key components of the pathway are also observed in response to other immunogenic and tolerogenic stimuli such as GM-CSF + IL-4 or IL-10 and TGFβ. The disparity in mTOR activation between immunogenic and tolerogenic stimuli is quantitative rather than qualitative. In both cases, mTOR activation primarily occurs through the PI3K/Akt signaling axis and involves ERK and GSK3β kinases, with minimal involvement of AMPK or NF-kB pathways. Furthermore, in the case of SSPs, mTOR activation seems to involve adenosine receptors. Additionally, we observed that DCs treated with SSPs exhibit an energy metabolism with high plasticity, which is typical of tolerogenic cells rather than immunogenic cells.ConclusionHence, the decision whether dendritic cells enter an inflammatory or tolerogenic state seems to rely on varying activation thresholds and kinetics of the mTOR signaling pathway.

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Methods of machine learning and big data analysis to establish the molecular mechanisms of the effects of racetams on the metabolism of adipose tissue

Racetams exhibit not only nootropic effects, but also affect the metabolism of fats and carbohydrates. Experimental studies have indicated the possibility of using phenylpiracetam in the treatment of obesity. At the same time, the molecular mechanisms of this pharmacological effect of phenylpiracetam are practically unknown. The paper presents the results of a new artificial intelligence (AI) method for comparative chemoreactome analysis of fonturacetam, piracetam, aniracetam, pramiracetam and levetiracetam. Another AI method, chemoneurocytological analysis, made it possible to compare the neuroprotective effects of molecules on neurons in culture. Despite the similarity of the proteomic interaction profiles of the studied molecules, differential analysis made it possible to establish the molecular mechanisms of the effect of phenylpiracetam on weight loss. Phenylpiracetam can activate β3-adrenoceptors, adenosine, glucagon-like peptide, sphingosine phosphate, and peroxisome proliferator receptors (PPARG); specifically inhibit cannabinoid, opioid, histamine, glutamate, nociceptin, neuropeptide Y and orexin receptors, which is important for normalizing appetite and improving the metabolism of adipose tissue. The synthetic AI method - pharmacoinformatic analysis indicated the advisability of taking phenylpiracetam together with vitamins C, D and group B.

P38 Experimental evidence of ecto-5′-nucleotidase (CD73) modulation in psoriasis-like inflammation

Abstract Introduction and aims Adenosine is an endogenous purine nucleoside that, by binding to adenosine receptors, modulates inflammation and immune responses. Psoriasis is a chronic immune-mediated inflammatory skin disease characterized by keratinocyte hyperproliferation and abnormal differentiation. A2A and A2B adenosine receptors modulate keratinocyte proliferation and their expression was found to be altered in psoriatic epidermis. Ecto-5′-nucleotidase (CD73) is an enzyme that hydrolyses extracellular adenosine monophosphate and represents a key control point for extracellular adenosine accumulation. CD73 is involved in the regulation of local immune response and tissue barrier function. As changes in CD73 expression and activity have been associated with other autoimmune diseases, the aim of this study was to investigate CD73 involvement in skin inflammation using in vitro and in vivo approaches. Methods In vitro experiments were performed on human keratinocyte (HaCaT) cell lines stimulated with tumour necrosis factor-α, interleukin (IL)-17A, IL-6 and IL-1α (10 ng mL−1 each), and in vivo, on the mouse model of imiquimod-induced psoriasis-like inflammation on the right ear, which was also performed on CD73−/− mice and their wildtype counterpart. CD73 expression in cell lysates and ear homogenates were evaluated using Western blot analysis. Results We found that CD73 expression was significantly increased (N = 3 wells, P &amp;lt; 0.05) in cytokine-treated HaCaTs compared with unstimulated cells. In vivo experiments showed increased CD73 expression on psoriatic lesions (P &amp;lt; 0.001, N = 3). In CD73−/− mice, imiquimod-induced lesions were exacerbated compared with the development of lesions in wildtype mice as shown by ear thickness (0.190 ± 0.02 mm, knockout mice vs. 0.138 ± 0.02 mm, wildtype mice; P &amp;lt; 0.05, N = 5), erythema and scaling. Conclusions Our preliminary Results show involvement of CD73 in the development of psoriasis-like inflammation, suggesting that CD73 may represent a new therapeutic target in the treatment of skin diseases. Further investigations are needed to provide an in-depth study of CD73 localization in inflamed skin.

Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

Targeting the adenosinergic system in restless legs syndrome: A pilot, "proof-of-concept" placebo-controlled TMS-based protocol.

Restless Legs Syndrome (RLS) is a common sleep disorder characterized by an urge to move the legs that is responsive to movement (particularly during rest), periodic leg movements during sleep, and hyperarousal. Recent evidence suggests that the involvement of the adenosine system may establish a connection between dopamine and glutamate dysfunction in RLS. Transcranial magnetic stimulation (TMS) is a non-invasive electrophysiological technique widely applied to explore brain electrophysiology and neurochemistry under different experimental conditions. In this pilot study protocol, we aim to investigate the effects of dipyridamole (a well-known enhancer of adenosinergic transmission) and caffeine (an adenosine receptor antagonist) on measures of cortical excitation and inhibition in response to TMS in patients with primary RLS. Initially, we will assess cortical excitability using both single- and paired-pulse TMS in patients with RLS. Then, based on the measures obtained, we will explore the effects of dipyridamole and caffeine, in comparison to placebo, on various TMS parameters related to cortical excitation and inhibition. Finally, we will evaluate the psycho-cognitive performance of RLS patients to screen them for cognitive impairment and/or mood-behavioral dysfunction, thus aiming to correlate psycho-cognitive findings with TMS data. Overall, this study protocol will be the first to shed lights on the neurophysiological mechanisms of RLS involving the modulation of the adenosine system, thus potentially providing a foundation for innovative "pharmaco-TMS"-based treatments. The distinctive TMS profile observed in RLS holds indeed the potential utility for both diagnosis and treatment, as well as for patient monitoring. As such, it can be considered a target for both novel pharmacological (i.e., drug) and non-pharmacological (e.g., neuromodulatory), "TMS-guided", interventions.

Adenosine kinase inhibition protects mice from abdominal aortic aneurysm via epigenetic modulation of VSMC inflammation.

Abdominal aortic aneurysm (AAA) is a common, serious vascular disease with no effective pharmacological treatment. The nucleoside adenosine plays an important role in modulating vascular homeostasis, which prompted us to determine whether adenosine kinase (ADK), an adenosine metabolizing enzyme, modulates AAA formation via control of the intracellular adenosine level, and to investigate the underlying mechanisms. We used a combination of genetic and pharmacological approaches in murine models of AAA induced by calcium chloride (CaCl2) application or angiotensin II (Ang II) infusion to study the role of ADK in the development of AAA. In vitro functional assays were performed by knocking down ADK with adenovirus-short hairpin RNA in human vascular smooth muscle cells (VSMCs), and the molecular mechanisms underlying ADK function were investigated using RNA-sequencing, isotope tracing, and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). The heterozygous deficiency of ADK protected mice from CaCl2- and Ang II-induced AAA formation. Moreover, specific knockout of ADK in VSMCs prevented Ang II-induced AAA formation, as evidenced by reduced aortic extracellular elastin fragmentation, neovascularization, and aortic inflammation. Mechanistically, ADK knockdown in VSMCs markedly suppressed the expression of inflammatory genes associated with AAA formation, and these effects were independent of adenosine receptors. The metabolic flux and ChIP-qPCR results showed that ADK knockdown in VSMCs decreased S-adenosylmethionine (SAM)-dependent transmethylation, thereby reducing H3K4me3 binding to the promoter regions of the genes that are associated with inflammation, angiogenesis, and extracellular elastin fragmentation. Furthermore, the ADK inhibitor ABT702 protected mice from CaCl2-induced aortic inflammation, extracellular elastin fragmentation, and AAA formation. Our findings reveal a novel role for ADK inhibition in attenuating AAA via epigenetic modulation of key inflammatory genes linked to AAA pathogenesis.

Timing Matters: Time of Day Impacts the Ergogenic Effects of Caffeine—A Narrative Review

Caffeine has attracted significant attention from researchers in the sports field due to its well-documented ergogenic effects across various athletic disciplines. As research on caffeine continues to progress, there has been a growing emphasis on evaluating caffeine dosage and administration methods. However, investigations into the optimal timing of caffeine intake remain limited. Therefore, this narrative review aimed to assess the ergogenic effects of caffeine administration at different times during the morning (06:00 to 10:00) and evening (16:00 to 21:00). The review findings suggest that circadian rhythms play a substantial role in influencing sports performance, potentially contributing to a decline in morning performance. Caffeine administration has demonstrated effectiveness in mitigating this phenomenon, resulting in ergogenic effects and performance enhancement, even comparable to nighttime levels. While the specific mechanisms by which caffeine regulates circadian rhythms and influences sports performance remain unclear, this review also explores the mechanisms underlying caffeine’s ergogenic effects, including the adenosine receptor blockade, increased muscle calcium release, and modulation of catecholamines. Additionally, the narrative review underscores caffeine’s indirect impact on circadian rhythms by enhancing responsiveness to light-induced phase shifts. Although the precise mechanisms through which caffeine improves morning performance declines via circadian rhythm regulation necessitate further investigations, it is noteworthy that the timing of caffeine administration significantly affects its ergogenic effects during exercise. This emphasizes the importance of considering caffeine intake timing in future research endeavors to optimize its ergogenic potential and elucidate its mechanisms.

Purinergic regulation of pulmonary vascular tone.

Purinergic signaling is a crucial determinant in the regulation of pulmonary vascular physiology and presents a promising avenue for addressing lung diseases. This intricate signaling system encompasses two primary receptor classes: P1 and P2 receptors. P1 receptors selectively bind adenosine, while P2 receptors exhibit an affinity for ATP, ADP, UTP, and UDP. Functionally, P1 receptors are associated with vasodilation, while P2 receptors mediate vasoconstriction, particularly in basally relaxed vessels, through modulation of intracellular Ca2+ levels. The P2X subtype receptors facilitate extracellular Ca2+ influx, while the P2Y subtype receptors are linked to endoplasmic reticulum Ca2+ release. Notably, the primary receptor responsible for ATP-induced vasoconstriction is P2X1, with α,β-meATP and UDP being identified as potent vasoconstrictor agonists. Interestingly, ATP has been shown to induce endothelium-dependent vasodilation in pre-constricted vessels, associated with nitric oxide (NO) release. In the context of P1 receptors, adenosine stimulation of pulmonary vessels has been unequivocally demonstrated to induce vasodilation, with a clear dependency on the A2B receptor, as evidenced in studies involving guinea pigs and rats. Importantly, evidence strongly suggests that this vasodilation occurs independently of endothelium-mediated mechanisms. Furthermore, studies have revealed variations in the expression of purinergic receptors across different vessel sizes, with reports indicating notably higher expression of P2Y1, P2Y2, and P2Y4 receptors in small pulmonary arteries. While the existing evidence in this area is still emerging, it underscores the urgent need for a comprehensive examination of the specific characteristics of purinergic signaling in the regulation of pulmonary vascular tone, particularly focusing on the disparities observed across different intrapulmonary vessel sizes. Consequently, this review aims to meticulously explore the current evidence regarding the role of purinergic signaling in pulmonary vascular tone regulation, with a specific emphasis on the variations observed in intrapulmonary vessel sizes. This endeavor is critical, as purinergic signaling holds substantial promise in the modulation of vascular tone and in the proactive prevention and treatment of pulmonary vascular diseases.

Learning and memory function preserved by delayed A1 adenosine receptor agonist treatment following soman intoxication in rats and a humanized esterase mouse model

Exposure to organophosphorus compounds, such as soman (GD), cause widespread toxic effects, sustained status epilepticus, neuropathology, and death. The A1 adenosine receptor agonist N-bicyclo-(2.2.1)-hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA), when given 1 min after GD exposure, provides neuroprotection and prevents behavioral impairments. Here, we tested the ability of ENBA at delayed treatment times to improve behavioral outcomes via a two-way active avoidance task in two male animal models, each consisting of saline and GD exposure groups. In a rat model, animals received medical treatments (atropine sulfate [A], 2-PAM [P], and midazolam [MDZ]) or AP + MDZ + ENBA at 15 or 30 min after seizure onset and were subjected to behavioral testing for up to 14 days. In a human acetylcholinesterase knock-in serum carboxylesterase knock-out mouse model, animals received AP, AP + MDZ, AP + ENBA, or AP + MDZ + ENBA at 15 min post seizure onset and were subjected to the behavioral task on days 7 and 14. In rats, the GD/AP + MDZ + ENBA group recovered to saline-exposed avoidance levels while the GD/AP + MDZ group did not. In mice, in comparison with GD/AP + MDZ group, the GD/AP + MDZ + ENBA showed decreases in escape latency, response latency, and pre-session crossings, as well as increases in avoidances. In both models, only ENBA-treated groups showed control level inter-trial interval crossings by day 14. Our findings suggest that ENBA, alone and as an adjunct to medical treatments, can improve behavioral and cognitive outcomes when given at delayed time points after GD intoxication.

Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits.

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Post-transcriptional regulation and subcellular localization of G-protein γ7 subunit: implications for striatal function and behavioral responses to cocaine.

The striatal D1 dopamine receptor (D1R) and A2a adenosine receptor (A2aR) signaling pathways play important roles in drug-related behaviors. These receptors activate the Golf protein comprised of a specific combination of αolfβ2γ7 subunits. During assembly, the γ7 subunit sets the cellular level of the Golf protein. In turn, the amount of Golf protein determines the collective output from both D1R and A2aR signaling pathways. This study shows the Gng7 gene encodes multiple γ7 transcripts differing only in their non-coding regions. In striatum, Transcript 1 is the predominant isoform. Preferentially expressed in the neuropil, Transcript 1 is localized in dendrites where it undergoes post-transcriptional regulation mediated by regulatory elements in its 3' untranslated region that contribute to translational suppression of the γ7 protein. Earlier studies on gene-targeted mice demonstrated loss of γ7 protein disrupts assembly of the Golf protein. In the current study, morphological analysis reveals the loss of the Golf protein is associated with altered dendritic morphology of medium spiny neurons. Finally, behavioral analysis of conditional knockout mice with cell-specific deletion of the γ7 protein in distinct populations of medium spiny neurons reveals differential roles of the Golf protein in mediating behavioral responses to cocaine. Altogether, these findings provide a better understanding of the regulation of γ7 protein expression, its impact on Golf function, and point to a new potential target and mechanisms for treating addiction and related disorders.

Purinergic Signaling and its Role in the Stem Cell Differentiation.

Purinergic signaling is a mechanism in which extracellular purines and pyrimidines interact with specialized cell surface receptors known as purinergic receptors. These receptors are divided into two families of P1 and P2 receptors, each responding to different nucleosides and nucleotides. P1 receptors are activated by adenosine, while P2 receptors are activated by pyrimidine and purines. P2X receptors are ligand-gated ion channels, including seven subunits (P2X1-7). However, P2Y receptors are the G-protein coupled receptors comprising eight subtypes (P2Y1/2/4/6/11/12/13/14). The disorder in purinergic signaling leads to various health-related issues and diseases. In various aspects, it influences the activity of non-neuronal cells and neurons. The molecular mechanism of purinergic signaling provides insight into treating various human diseases. On the contrary, stem cells have been investigated for therapeutic applications. Purinergic signaling has shown promising effect in stem cell engraftment. The immune system promotes the autocrine and paracrine mechanisms and releases the significant factors essential for successful stem cell therapy. Each subtype of purinergic receptor exerts a beneficial effect on the damaged tissue. The most common effect caused by purinergic signaling is the proliferation and differentiation that treat different health-related conditions.

Acute hyperosmotic stimulus of the Organum Vasculosum of the Lamina Terminalis leads to increase in the blood pressure dependent on release of ATP into the Paraventricular Nucleus of the Hypothalamus of rats

The neural network involved in hyperosmolality-induced hypertension includes the activation of the organum vasculosum of the lamina terminalis (OVLT) with monosynaptic connections to the paraventricular nucleus of the hypothalamus (PVN) that expresses an abundance of purinergic receptors including P2 receptors. Previously, we have shown that hyperosmotic-induced sympathoexcitation at the level of the PVN depends on the P2 receptors. More recently, we have shown that hyperosmotic stimulus induces the release of ATP in the PVN, at least in part, from astrocytes. Here we hypothesize that the increase in the blood pressure (BP) induced by acute hyperosmotic stimulus in the OVLT involves the release of ATP within the PVN neurons, acting on P2 receptors. Thus, we sought to determine (1) whether P2X7-expressing OVLT and PVN neurons are activated by hyperosmotic challenge, and (2) what effects P2 receptor antagonism on changes in the BP mediated by an acute hyperosmotic stimulus in the OVLT. Adult Wistar rats (10-12 weeks old; CEUA ICB/USP: #4895140819 and #1521230221) were used in two protocols: 1) Double-labeling immunofluorescence of P2X7 (anti-P2X7 receptor, 1:500) expressing neurons with FOS (anti-cfos, 1:3000) animals received i.v administration of hypertonic (NaCl, 3M, n=4) or isotonic saline (NaCl, 0.154M, n=4), followed by evaluation of immunostaining to FOS and P2X7 receptors in the OVLT and PVN; 2) OVLT-PVN microinjections and BP monitoring: under anesthesia rats (n=5), received catheters in the femoral vein and artery for the administration of urethane (1g/kg) and BP recording signals, respectively. Microinjections of 3M NaCl (50nL) were administered into the OVLT, before and after bilateral antagonism of P2 purinergic receptors (PPADS, 10uM-100nL on each side) in the PVN with simultaneous BP recording. Data were expressed as the mean ± SD, and statistical significance level of p&lt;0.05 by Student’s T-test. Immunoreactive neurons for the P2X7 receptor subunit were observed in all compartments of the OVLT and PVN. Hyperosmotic stimulus significantly increased the number of Fos+ neurons in both the OVLT and PVN. While a small number of P2X7 receptors expressing OVLT and PVN neurons was observed in rats treated with isotonic saline, significantly more P2X7-expressing neurons expressed Fos+ in the OVLT with hypertonic saline. The stimulus of the OVLT with 3M NaCl elicited a significant increase in the BP. This effect was attenuated after bilateral injection of PPADS in the PVN (ΔMAP: NaCl OVLT:+10±1mmHg vs. NaCl OVLT post-PPADS PVN: +2.4±1.3mmHg, p&lt;0.0001). Collectively, our findings indicate that hyperosmotic stimulation activates a subset of P2X7 expressing OVLT and PVN neurons and that the elevation in the BP induced by hyperosmotic stimulus in the OVLT involves the endogenous release of ATP within the PVN, likely as part of the neural control of circulation during osmotic challenges. Financial Support: FAPESP #2019/19894-8, CAPES #88887.364432/2019-00. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Astrocytes in preoptic area regulate acute nociception-induced hypothermia through adenosine receptors.

The preoptic area (POA) of the hypothalamus, crucial in thermoregulation, has long been implicated in the pain process. However, whether nociceptive stimulation affects body temperature and its mechanism remains poorly studied. We used capsaicin, formalin, and surgery to induce acute nociceptive stimulation and monitored rectal temperature. Optical fiber recording, chemical genetics, confocal imaging, and pharmacology assays were employed to confirm the role and interaction of POA astrocytes and extracellular adenosine. Immunofluorescence was utilized for further validation. Acute nociception could activate POA astrocytes and induce a decrease in body temperature. Manipulation of astrocytes allowed bidirectional control of body temperature. Furthermore, acute nociception and astrocyte activation led to increased extracellular adenosine concentration within the POA. Activation of adenosine A1 or A2A receptors contributed to decreased body temperature, while inhibition of these receptors mitigated the thermo-lowering effect of astrocytes. Our results elucidate the interplay between acute nociception and thermoregulation, specifically highlighting POA astrocyte activation. This enriches our understanding of physiological responses to painful stimuli and contributes to the analysis of the anatomical basis involved in the process.