Changes In Purinergic Signaling Research Articles

Altered extracellular atp, adp, and amp hydrolysis in blood serum of sedentary individuals after an acute, strength, exercise session

Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Different types of physical exercise promote changes in purinergic signaling. In the present study, we investigated the activities of soluble nucleotidases in the blood serum of sedentary young male adults before and after a resistance exercise session. In addition, we evaluated how this type of exercise could influence the concentrations of adenine nucleotides in the blood serum. The individuals were submitted to a strength exercise session, consisting of 8 exercises. Blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. Our results demonstrate that a resistance exercise session modifies ATP metabolism, being one more form of exercise that can positively influence cardiovascular and metabolic health.

Changes in P2Y6 receptor-mediated vasoreactivity following focal and global ischemia.

Ischemia, both in the form of focal thromboembolic stroke and following subarachnoid hemorrhage (SAH), causes upregulation of vasoconstrictive receptor systems within the cerebral vasculature. Descriptions regarding changes in purinergic signaling following ischemia are lacking, especially when the importance of purinergic signaling in regulating vascular tone is taken into consideration. This prompted us to evaluate changes in P2Y6‐mediated vasomotor reactivity in two different stroke models in rat. We used wire myography to measure changes in cerebral vasoreactivity to the P2Y6 agonist UDP‐β‐S following either experimental SAH or transient middle cerebral artery occlusion. Changes in receptor localization or receptor expression were evaluated using immunohistochemistry and quantitative flow cytometry. Transient middle cerebral artery occlusion caused an increase in Emax when compared to sham (233.6 [206.1–258.5]% vs. 161.1 [147.1–242.6]%, p = 0.0365). No such change was seen following SAH. Both stroke models were associated with increased levels of P2Y6 receptor expression in the vascular smooth muscle cells (90.94 [86.99–99.15]% and 93.79 [89.96–96.39]% vs. 80.31 [70.80–80.86]%, p = 0.021) and p = 0.039 respectively. There was no change in receptor localization in either of the stroke models. Based on these findings, we conclude that focal ischemic stroke increases vascular sensitivity to UDP‐β‐S by upregulating P2Y6 receptors on vascular smooth muscle cells while experimental SAH did not induce changes in vasoreactivity in spite of increased P2Y6 receptor expression.

High levels of extracellular ATP lead to different inflammatory responses in COVID-19 patients according to the severity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly impacted the world and has driven many researchers into the pathophysiology of COVID-19. In the findings, there is a close association between purinergic signaling and the immune response. Then, this study aimed to evaluate alterations in the purinergic signaling in COVID-19 patients according to range severity. We divided the COVID-19 patients into moderate and severe cases following the guideless of NIH and WHO, together with clinical characteristics. The blood samples were collected to obtain PBMCs and platelets. We analyzed the ectonucleotidase activities through ATP, ADP, AMP, Ado hydrolysis, E-NTPDase1 (CD39), and 5′-NT (CD73) expression by flow cytometry in total leukocytes. The extracellular ATP was measured by bioluminescence, and cytokines were analyzed by flow cytometry. We observed a decrease in ATP hydrolysis and increased AMP hydrolysis in PBMCs for both groups. In severe cases, ATP hydrolysis was raised for the platelets, while ADP and AMP hydrolysis have risen significantly in both groups. Additionally, there was a significant increase in ADP hydrolysis in severe cases compared to moderate cases. In addition, we observed an increase in the ADA activity in platelets of moderate patients. Moderate and severe cases showed increased expression of CD39 and CD73 in total leukocytes. To finalize the purinergic signaling, extracellular ATP was increased in both groups. Furthermore, there was an increase in IL-2, IL-6, IL-10, and IL-17 in moderate and severe groups. Thus, for the first time, our findings confirm the changes in purinergic signaling and immune response in COVID-19, in addition to making it more evident that the severity range directly impacts these changes. Therefore, the therapeutic potential of the purinergic system must be highlighted and studied as a possible target for the treatment of SARS-CoV-2 disease.Key messages COVID-19 patients exhibit alterations in purinergic system and immune response.High levels of extracellular ATP lead to different inflammatory responses.CD39 and CD73 expression were increased in COVID-19 patients.Cytokines IL-2, IL-6, IL-10, and IL-17 also were altered in these patients.The purinergic system may be a possibility target to SARS-CoV-2 treatments. Graphical abstract

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood-brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X7 and A2A density of purine receptors. RSV reduced P2X7 and A2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.

Post-weaning social isolation impairs purinergic signaling in rat brain

Early life stressors, such as social isolation (SI), can disrupt brain development contributing to behavioral and neurochemical alterations in adulthood. Purinergic receptors and ectonucleotidases are key regulators of brain development in embryonic and postnatal periods, and they are involved in several psychiatric disorders, including schizophrenia. The extracellular ATP drives purinergic signaling by activating P2X and P2Y receptors and it is hydrolyzed by ectonucleotidases in adenosine, which activates P1 receptors. The purpose of this study was to investigate if SI, a rodent model used to replicate abnormal behavior relevant to schizophrenia, impacts purinergic signaling. Male Wistar rats were reared from weaning in group-housed or SI conditions for 8 weeks. SI rats exhibited impairment in prepulse inhibition and social interaction. SI presented increased ADP levels in cerebrospinal fluid and ADP hydrolysis in the hippocampus and striatum synaptosomes. Purinergic receptor expressions were upregulated in the prefrontal cortex and downregulated in the hippocampus and striatum. A2A receptors were differentially expressed in SI prefrontal cortex and the striatum, suggesting distinct roles in these brain structures. SI also presented decreased ADP, adenosine, and guanosine levels in the cerebrospinal fluid in response to D-amphetamine. Like patients with schizophrenia, uric acid levels were prominently increased in SI rats after D-amphetamine challenge. We suggest that the SI-induced deficits in prepulse inhibition might be related to the SI-induced changes in purinergic signaling. We provide new evidence that purinergic signaling is markedly affected in a rat model relevant to schizophrenia, pointing out the importance of purinergic system in psychiatry conditions.

Purinergic Signaling and Related Biomarkers in Depression.

It is established that purinergic signaling can shape a wide range of physiological functions, including neurotransmission and neuromodulation. The purinergic system may play a role in the pathophysiology of mood disorders, influencing neurotransmitter systems and hormonal pathways of the hypothalamic-pituitary-adrenal axis. Treatment with mood stabilizers and antidepressants can lead to changes in purinergic signaling. In this overview, we describe the biological background on the possible link between the purinergic system and depression, possibly involving changes in adenosine- and ATP-mediated signaling at P1 and P2 receptors, respectively. Furthermore, evidence on the possible antidepressive effects of non-selective adenosine antagonist caffeine and other purinergic modulators is reviewed. In particular, A2A and P2X7 receptors have been identified as potential targets for depression treatment. Preclinical studies highlight that both selective A2A and P2X7 antagonists may have antidepressant effects and potentiate responses to antidepressant treatments. Consistently, recent studies feature the possible role of the purinergic system peripheral metabolites as possible biomarkers of depression. In particular, variations of serum uric acid, as the end product of purinergic metabolism, have been found in depression. Although several open questions remain, the purinergic system represents a promising research area for insights into the molecular basis of depression.

Resveratrol-mediated reversal of changes in purinergic signaling and immune response induced by Toxoplasma gondii infection of neural progenitor cells.

The effects of Toxoplasma gondii during embryonic development have not been explored despite the predilection of this parasite for neurons and glial cells. Here, we investigated the activation of the purinergic system and proinflammatory responses during congenital infection by T. gondii. Moreover, neuroprotective and neuromodulatory properties of resveratrol (RSV), a polyphenolic natural compound, were studied in infected neuronal progenitor cells (NPCs). For this study, NPCs were isolated from the telencephalon of infected mouse embryos and subjected to neurosphere culture in the presence of EGF and FGF2. ATP hydrolysis and adenosine deamination by adenosine deaminase activity were altered in conditions of T. gondii infection. P2X7 and adenosine A2A receptor expression rates were augmented in infected NPCs together with an increase of proinflammatory (INF-γ and TNF-α) and anti-inflammatory (IL-10) cytokine gene expression. Our results confirm that RSV counteracted T. gondii-promoted effects on enzymes hydrolyzing extracellular nucleotides and nucleosides and also upregulated P2X7 and A2A receptor expression and activity, modulating INF-γ, TNF-α, and IL-10 cytokine production, which plays an integral role in the immune response against T. gondii.

Caffeine modulates brain purinergic signaling in Nile tilapia (Oreochromis niloticus) under hypoxia conditions: improvement of immune and inflammatory responses.

Purinergic signaling is linked to neurodegenerative and proinflammatory damage during pathological conditions such as hypoxia, but involvement of this pathway in brain damage in fish exposed to environmental hypoxia remains unknown, and we propose dietary supplementation with caffeine in order to improve the immune response. Therefore, the aim of the study was to evaluate whether the enzymatic purinergic signaling pathway is associated with inflammatory brain damage in Nile tilapia (Oreochromis niloticus) exposed to environmental hypoxia and whether dietary supplementation with caffeine (5% and 8%) can prevent these changes in purinergic signaling. Animals were randomly divided into six groups (A-F, n = 6 per group, in triplicate), as follows: groups A-C were submitted to normoxia, while groups D-F were submitted to hypoxia. Groups A and D received the basal diet, while groups B and D and groups C and F received a diet containing 5% and 8% caffeine, respectively, and fed with their respective diets for 21days. After 21days, aeration was disconnected (groups D-F) and the dissolved oxygen levels were maintained as follows: group A (6.55 ± 0.23mg/L), group B (6.51 ± 0.24mg/L), group C (6.58 ± 0.22mg/L), group D (1.23 ± 0.11mg/L), group E (1.20 ± 0.15mg/L), and group F (1.18 ± 0.13mg/L). Cerebral triphosphate diphosphohydrolase (NTPDase) using adenosine triphosphate (ATP) as a substrate and 5'-nucleotidase activities decreased in fish exposed to 72h of hypoxia compared with the normoxia group, while adenosine deaminase (ADA) activity and levels of nitric oxide (NOx) metabolites were higher. Dietary supplementation with 5% and 8% caffeine prevented all alterations elicited by hypoxia, with the exception of ADA activity in the case of 5% caffeine. Based on this evidence, our findings reveal that nucleotide/nucleoside hydrolysis is modified in the brains of fish exposed to 72h of hypoxia, contributing to inflammatory damage, which apparently is mediated by excessive ATP content in the extracellular medium and by excessive NOx production. Also, the use of a diet containing 5% and 8% caffeine prevented these alterations (except 5% of dietary caffeine on ADA activity) and can be considered an interesting approach to preventing the impairment of immune and inflammatory responses elicited by hypoxia, principally the inclusion of 8% caffeine.

Amyotrophic Lateral Sclerosis (ALS) and Adenosine Receptors.

In the present review we discuss the potential involvement of adenosinergic signaling, in particular the role of adenosine receptors, in amyotrophic lateral sclerosis (ALS). Though the literature on this topic is not abundant, the information so far available on adenosine receptors in animal models of ALS highlights the interest to continue to explore the role of these receptors in this neurodegenerative disease. Indeed, all motor neurons affected in ALS are responsive to adenosine receptor ligands but interestingly, there are alterations in pre-symptomatic or early symptomatic stages that mirror those in advanced disease stages. Information starts to emerge pointing toward a beneficial role of A2A receptors (A2AR), most probably at early disease states, and a detrimental role of caffeine, in clear contrast with what occurs in other neurodegenerative diseases. However, some evidence also exists on a beneficial action of A2AR antagonists. It may happen that there are time windows where A2AR prove beneficial and others where their blockade is required. Furthermore, the same changes may not occur simultaneously at the different synapses. In line with this, it is not fully understood if ALS is a dying back disease or if it propagates in a centrifugal way. It thus seems crucial to understand how motor neuron dysfunction occurs, how adenosine receptors are involved in those dysfunctions and whether the early changes in purinergic signaling are compensatory or triggers for the disease. Getting this information is crucial before starting the design of purinergic based strategies to halt or delay disease progression.

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.

Changes in Adenosine Triphosphate-stimulated ATP Release Suggest Association Between Cytokine and Purinergic Signaling in Bladder Urothelial Cells

To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUCs) and/or whether antagonizing EGF activity or blocking adenosine triphosphate (ATP)-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells. IC and normal BUCs were obtained from patients' bladder biopsy specimens. IC BUCs were treated with genistein, which antagonizes EGF's activity, and normal BUCs were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat the APF-treated normal BUCs. ATP release was determined by stimulating the BUCs with 30 microM ATP and then collecting the supernatant for a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. Purinergic receptor P2X, ligand-gated ion channel, 3 (P2X3) expression on BUCs was determined by fluorescence-activated cell sorting. Genistein treatment of IC BUCs resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUCs treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUCs to an IC phenotype. Also, suramin treatment of APF-treated normal BUCs significantly reduced ATP release. Genistein and suramin reversed the augmented ATP release in IC BUCs and APF-treated normal BUCs, respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUCs, as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUCs that should be explored further.

Changes in purinergic signaling after cerebral injury – involvement of glutamatergic mechanisms?

Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y1 receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15-min sample whereas glutamate returned to a basal concentration not before a 90-min period had elapsed. We suggest, that the acute injury-evoked stimulation of P2 receptors contributes to glutamate-mediated excitotoxicity. Second, the expression of P2Y1 receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non-treated and mechanically injured animals after 4 days. The number of P2Y1-positive cells was significantly increased after injury. Furthermore, P2Y1 receptor-labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co-expression of the P2Y1 receptor on VGLUT3-immunopositive cells in the NAc was observed. No VGLUT1-, 2- and 3-immunoreactivity was found on P2Y1-positive glial fibrillary acidic protein-immunopositive astrocytes at both conditions.Our data suggest that the expression of P2Y1 receptors at neurons and astrocytes is modulated in response to cerebral injury. It can be assumed, that the enhanced sensitivity of neurons to purinergic signaling may be related directly or indirectly to changes of the glutamatergic transmission.