ADAR-Mediated A>I(G) RNA Editing in the Genotoxic Drug Response of Breast Cancer.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.

8-Aminopurines in the Cardiovascular and Renal Systems and Beyond.

Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.

8-Aminoguanine and Its Actions on Renal Excretory Function.

BACKGROUND: The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. METHODS: Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. RESULTS: Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. CONCLUSIONS: 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Protective Effects of Inosine on Memory Consolidation in a Rat Model of Scopolamine-Induced Cognitive Impairment: Involvement of Cholinergic Signaling, Redox Status, and Ion Pump Activities.

This study investigated the effects of inosine on memory acquisition and consolidation, cholinesterases activities, redox status and Na+, K+-ATPase activity in a rat model of scopolamine-induced cognitive impairment. Adult male rats were divided into four groups: control (saline), scopolamine (1 mg/kg), scopolamine plus inosine (50 mg/kg), and scopolamine plus inosine (100 mg/kg). Inosine was pre-administered for 7 days, intraperitoneally. On day 8, scopolamine was administered pre (memory acquisition protocol) or post training (memory consolidation protocol) on inhibitory avoidance tasks. The animals were subjected to the step-down inhibitory avoidance task 24 hours after the training. Scopolamine induced impairment in the acquisition and consolidation phases; however, inosine was able to prevent only the impairment in memory consolidation. Also, scopolamine increased the activity of acetylcholinesterase and reduced the activity of Na+, K+-ATPase and the treatment with inosine protected against these alterations in consolidation protocol. In the animals treated with scopolamine, inosine improved the redox status by reducing the levels of reactive oxygen species and thiobarbituric acid reactive substances and restoring the activity of the antioxidant enzymes, superoxide dismutase and catalase. Our findings suggest that inosine may offer protection against scopolamine-induced memory consolidation impairment by modulating brain redox status, cholinergic signaling and ion pump activity. This compound may provide an interesting approach in pharmacotherapy and as a prophylactic against neurodegenerative mechanisms involved in Alzheimer's disease.

Investigating the Effect of Inosine on Brain Purinergic Receptors and Neurotrophic and Neuroinflammatory Parameters in an Experimental Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by progressive impairment of memory, associated with neurochemical alterations and limited therapy. The aim of this study was to evaluate the effects of inosine on memory, neuroinflammatory cytokines, neurotrophic factors, expression of purinergic receptors, and morphological changes in the hippocampus and cerebral cortex of the rats with AD induced by streptozotocin (STZ). Male rats were divided into four groups: I, control; II, STZ; III, STZ plus inosine (50 mg/kg); and IV, STZ plus inosine (100 mg/kg). The animals received intracerebroventricular injections of STZ or buffer. Three days after the surgical procedure, animals were treated with inosine (50 mg/kg or 100 mg/kg) for 25 days. Inosine was able to prevent memory deficits and decreased the immunoreactivity of the brain A2A adenosine receptor induced by STZ. Inosine also increased the levels of brain anti-inflammatory cytokines (IL-4 and IL-10) and the expression of brain-derived neurotrophic factor and its receptor. Changes induced by STZ in the molecular layer of the hippocampus were attenuated by treatment with inosine. Inosine also protected against the reduction of immunoreactivity for synaptophysin induced by STZ in CA3 hippocampus region. However, inosine did not prevent the increase in GFAP in animals exposed to STZ. In conclusion, our findings suggest that inosine has therapeutic potential for AD through the modulation of different brain mechanisms involved in neuroprotection.

The role of peripheral adenosine receptors in glutamate-induced pain nociceptive behavior.

The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 µmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 µg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 µg/paw) significantly blocked the anti-nociceptive effect of CHA (1 µg/paw) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.

Inosine, an endogenous purine nucleoside, avoids early stages of atherosclerosis development associated to eNOS activation and p38 MAPK/NF-kB inhibition in rats.

Atherosclerosis is a multifactorial chronic disease, initiated by an endothelial dysfunction. Adenosine and its analogs can change a variety of inflammatory diseases and has shown important effects at different disease models. Inosine is a stable analogous of adenosine, but its effects in inflammatory diseases, like atherosclerosis, have not yet been studied. The aim of this study was to evaluate the pharmacological properties of inosine, administered sub chronically in a hypercholesterolemic model. Male Wistar rats were divided into four groups: control group (C) and control + inosine (C + INO) received standard chow, hypercholesterolemic diet group (HCD) and HCD + inosine (HCD + INO) were fed a hypercholesterolemic diet. At 31st experimentation day, the treatment with inosine was performed for C + INO and HCD + INO groups once daily in the last 15 days. We observed that the hypercholesterolemic diet promoted an increase in lipid levels and inflammatory cytokines production, while inosine treatment strongly decreased these effects. Additionally, HCD group presented a decrease in maximum relaxation acetylcholine induced and an increase in contractile response phenylephrine induced when compared to the control group, as well as it has presented an enhancement in collagen and ADP-induced platelet aggregation. On the other hand, inosine treatment promoted a decrease in contractile response to phenylephrine, evoked an improvement in endothelium-dependent vasorelaxant response and presented antiplatelet properties. Moreover, inosine activated eNOS and reduced p38 MAPK/NF-κB pathway in aortic tissues. Taken together, the present results indicate inosine as a potential drug for the treatment of cardiovascular disorders such as atherosclerosis.

Inosine prevents hyperlocomotion in a ketamine-induced model of mania in rats.

Bipolar Disorder is a disorder characterized by alternating episodes of depression, mania or hypomania, or even mixed episodes. The treatment consists on the use of mood stabilizers, which imply serious adverse effects. Therefore, it is necessary to identify new therapeutic targets to prevent or avoid new episodes. Evidence shows that individuals in manic episodes present a purinergic system dysfunction. In this scenario, inosine is a purine nucleoside known to act as an agonist of A1 and A2A adenosine receptors. Thus, we aimed to elucidate the preventive effect of inosine on locomotor activity, changes in purine levels, and adenosine receptors density in a ketamine-induced model of mania in rats. Inosine pretreatment (25 mg/kg, oral route) prevented the hyperlocomotion induced by ketamine (25 mg/kg, intraperitoneal route) in the open-field test; however, there was no difference in hippocampal density of A1 and A2A receptors, where ketamine, as well as inosine, were not able to promote changes in immunocontent of the adenosine receptors. Likewise, no effects of inosine pretreatments or ketamine treatment were observed for purine and metabolic residue levels evaluated. In this sense, we suggest further investigation of signaling pathways involving purinergic receptors, using pharmacological strategies to better elucidate the action mechanisms of inosine on bipolar disorder. Despite the limitations, inosine administration could be a promising candidate for bipolar disorder treatment, especially by attenuating maniac phase symptoms, once it was able to prevent the hyperlocomotion induced by ketamine in rats.

Inosine protects against impairment of memory induced by experimental model of Alzheimer disease: a nucleoside with multitarget brain actions.

RATIONALE: Inosine is a naturally occurring purine nucleoside formed by adenosine breakdown. This nucleoside is reported to exert potent effects on memory and learning, possibly through its antioxidant and anti-inflammatory actions. OBJECTIVE: The objective is to evaluate the effects of inosine on the behavioral and neurochemical parameters in a rat model of Alzheimer's disease (AD) induced by streptozotocin (STZ). METHODS: Adult male rats were divided into four groups: control (saline), STZ, STZ plus inosine (50 mg/kg), and STZ plus inosine (100 mg/kg). STZ (3 mg/kg) was administered by bilateral intracerebroventricular injection. The animals were treated intraperitoneally with inosine for 25 days. Memory, oxidative stress, ion pump activities, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT) activities and expression were evaluated in the cerebral cortex and hippocampus. RESULTS: The memory impairment induced by STZ was prevented by inosine. An increase in the Na+, K+-ATPase, and Mg-ATPase activities and a decrease in the Ca2+-ATPase activity were induced by STZ in the hippocampus and cerebral cortex, and inosine could prevent these alterations in ion pump activities. Inosine also prevented the increase in AChE activity and the alterations in AChE and ChAT expression induced by STZ. STZ increased the reactive oxygen species, nitrite levels, and superoxide dismutase activity and decreased the catalase and glutathione peroxidase activities. Inosine treatment conferred protection from these oxidative alterations in the brain. CONCLUSIONS: Our findings demonstrate that inosine affects brain multiple targets suggesting that this molecule may have therapeutic potential against cognitive deficit and tissue damage in AD.

Inosine Accelerates the Regeneration and Anticipates the Functional Recovery after Sciatic Nerve Crush Injury in Mice.

Trauma to the peripheral nervous system (PNS) results in loss of motor and sensory functions. After an injury, a complex series of events begins, allowing axonal regeneration and target reinnervation. However, this regenerative potential is limited by several factors such as age, distance from the lesion site to the target and severity of lesion. Many studies look for ways to overcome these limitations. Inosine, a purine nucleoside derived from adenosine, emerges as a potential treatment, due to its capacity to regulate axonal growth, neuroprotection and immunomodulation, contributing to motor recovery. However, no studies demonstrated their effects on PNS. C57/Black6 mice were submitted to sciatic nerve crush and received intraperitoneal injections of saline or inosine (70 mg/kg), one hour after injury and daily for one week. To evaluate axonal regeneration and functional recovery, electroneuromyography, Sciatic Function Index (SFI), rotarod and pinprick tests were performed. Our results showed that the inosine group presented a higher number of myelinated fibers and a large amount of fibers within the ideal G-ratio. In addition, the results of electroneuromyography showed greater amplitude of the compound muscle action potentials in the first and second weeks, suggesting anticipation of regeneration in the inosine group. We also observed in the inosine group, motor and sensory neurons survival, reduction in the number of macrophages and myelin ovoids in the sciatic nerves, and an early recovery of motor and sensory functions. Thus, we conclude that the use of inosine accelerates axonal regeneration promoting an early recovery of motor and sensory functions.

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleoside selectivity of PNPs.

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.

Evidence of a General Acid-Base Catalysis Mechanism in the 8-17 DNAzyme.

DNAzymes are catalytic DNA molecules that can perform a variety of reactions. Although advances have been made in obtaining DNAzymes via in vitro selection and many of them have been developed into sensors and imaging agents for metal ions, bacteria, and other molecules, the structural features responsible for these enzymatic reactions are still not well understood. Previous studies of the 8-17 DNAzyme have suggested conserved guanines close to the phosphodiester transfer site may play a role in the catalytic reaction. To identify the specific guanine and functional group of the guanine responsible for the reaction, we herein report the effects of replacing G1.1 and G14 (G; p Ka,N1 = 9.4) with analogues with a different p Ka at the N1 position, such as inosine (G14I; p Ka,N1 = 8.7), 2,6-diaminopurine (G14diAP; p Ka,N1 = 5.6), and 2-aminopurine (G14AP; p Ka,N1 = 3.8) on pH-dependent reaction rates. A comparison of the pH dependence of the reaction rates of these DNAzymes demonstrated that G14 in the bulge loop next to the cleavage site, is involved in proton transfer at the catalytic site. In contrast, we did not find any evidence of G1.1 being involved in acid-base catalysis. These results support general acid-base catalysis as a feasible strategy used in DNA catalysis, as in RNA and protein enzymes.

Glutamatergic system and mTOR-signaling pathway participate in the antidepressant-like effect of inosine in the tail suspension test.

Glutamatergic system and mTOR signaling pathway have been proposed to be important targets for pharmacological treatment of major depressive disorder. Previous studies have shown that inosine, an endogenous purine, is able to exert a remarkable antidepressant-like effect in mice. Nevertheless, the role of glutamatergic system and mTOR in this effect was not previously determined. This study was designed to investigate the possible modulation of NMDA receptors (NMDAR), AMPA receptors (AMPAR) and mTOR complex 1 (mTORC1) signaling pathway in the inosine anti-immobility effect in the tail suspension test (TST) in mice. Pre-treatment of mice with NMDA (0.1 pmol/mouse, NMDAR agonist, i.c.v.) and D-serine (30 µg/mouse, NMDAR co-agonist, i.c.v.) prevented inosine (10 mg/kg, i.p.) anti-immobility effect in the TST. In addition, a synergistic antidepressant-like effect was observed when a sub-effective dose of inosine (0.1 mg/kg, i.p.) was combined with sub-effective doses of NMDAR antagonists MK-801 (0.001 mg/kg, p.o.) or ketamine (0.1 mg/kg, i.p.). Conversely, the antidepressant-like effect elicited by inosine was not altered by pre-treatment with AMPAR antagonist, DNQX (2.5 µg/mouse, i.c.v.). The mTORC1 inhibitor rapamycin (0.2 nmol/mouse, i.c.v.) prevented the inosine anti-immobility effect in the TST. Noteworthy, inosine treatment did not change the immunocontent of the synaptic proteins PSD95, GluA1 and synapsin I. Mice locomotor activity assessed by open-field test, was not altered by treatments. Taken together, this study shows a pivotal role of NMDAR inhibition and mTORC1 activation for inosine antidepressant-like effect and extends the knowledge concerning the molecular mechanism and potential of inosine for antidepressant strategies.

Signaling pathways underlying the antidepressant-like effect of inosine in mice.

Inosine is a purine nucleoside formed by the breakdown of adenosine that elicits an antidepressant-like effect in mice through activation of adenosine A1 and A2A receptors. However, the signaling pathways underlying this effect are largely unknown. To address this issue, the present study investigated the influence of extracellular-regulated protein kinase (ERK)1/2, Ca2+/calmoduline-dependent protein kinase (CaMKII), protein kinase A (PKA), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase 3beta (GSK-3ß) modulation in the antiimmobility effect of inosine in the tail suspension test (TST) in mice. In addition, we attempted to verify if inosine treatment was capable of altering the immunocontent and phosphorylation of the transcription factor cyclic adenosine monophosphatate (cAMP) response-binding element protein (CREB) in mouse prefrontal cortex and hippocampus. Intracerebroventricular administration of U0126 (5 µg/mouse, MEK1/2 inhibitor), KN-62 (1 µg/mouse, CaMKII inhibitor), H-89 (1 µg/mouse, PKA inhibitor), and wortmannin (0.1 µg/mouse, PI3K inhibitor) prevented the antiimmobility effect of inosine (10 mg/kg, intraperitoneal (i.p.)) in the TST. Also, administration of a sub-effective dose of inosine (0.1 mg/kg, i.p.) in combination with a sub-effective dose of AR-A014418 (0.001 µg/mouse, GSK-3ß inhibitor) induced a synergic antidepressant-like effect. None of the treatments altered locomotor activity of mice. Moreover, 24 h after a single administration of inosine (10 mg/kg, i.p.), CREB phosphorylation was increased in the hippocampus. Our findings provided new evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK1/2, and CaMKII and the inhibition of GSK-3ß. These results contribute to the comprehension of the mechanisms underlying the purinergic system modulation and indicate the intracellular signaling pathways involved in the antidepressant-like effect of inosine in a preclinical test of depression.

Inosine, an Endogenous Purine Nucleoside, Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor.

Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes were blocked by inosine treatment. Additionally, inosine consistently inhibited IL-17 levels in peripheral lymphoid tissue, as well as IL-4 levels and A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. EAE: experimental autoimmune encephalomyelitis; MS: multiple sclerosis; A2AR: adenosine A2A receptor; IL-17: interleukin-17; IL-4: interleukin-4.

Mechanisms involved in the antinociception induced by spinal administration of inosine or guanine in mice.

It is well known that adenine-based purines exert multiple effects on pain transmission. Recently, we have demonstrated that guanine-based purines may produce some antinociceptive effects against chemical and thermal pain in mice. The present study was designed to investigate the antinociceptive effects of intrathecal (i.t.) administration of inosine or guanine in mice. Additionally, investigation into the mechanisms of action of these purines, their general toxicity and measurements of CSF purine levels were performed. Animals received an i.t. injection of vehicle (30mN NaOH), inosine or guanine (up to 600nmol) and submitted to several pain models and behavioural paradigms. Guanine and inosine produced dose-dependent antinociceptive effects in the tail-flick, hot-plate, intraplantar (i.pl.) glutamate, i.pl. capsaicin and acetic acid pain models. Additionally, i.t. inosine inhibited the biting behaviour induced by spinal injection of capsaicin and i.t. guanine reduced the biting behaviour induced by spinal injection of glutamate or AMPA. Intrathecal administration of inosine (200nmol) induced an approximately 115-fold increase on CSF inosine levels. This study provides new evidence on the mechanism of action of extracellular guanine and inosine presenting antinociceptive effects following spinal administration. These effects seem to be related, at least partially, to the modulation of A1 adenosine receptors.

Adenosine A1 receptor-dependent antinociception induced by inosine in mice: pharmacological, genetic and biochemical aspects.

Inosine is an endogenous nucleoside that has anti-inflammatory and antinociceptive properties. Inosine is a metabolite of adenosine, and some of its actions suggest the involvement of adenosine A1 receptors (A1Rs). The purpose of this study was to better understand mechanisms of inosine-induced antinociception by investigating the role of A1Rs and purine metabolism inhibitors. Inosine antinociception was evaluated using the formalin test in mice. An A1R-selective antagonist (DPCPX), A1R knockout mice (gene deletion) and mice with A1R reduced expression (antisense oligonucleotides) were used to assess the role of A1Rs in the antinociceptive action of inosine. Binding assays were performed to compare the affinity of inosine and adenosine for A1Rs. Finally, the role of adenosine and inosine breakdown was assessed using deoxycoformycin (DCF) and forodesine (FDS) as enzymatic inhibitors of adenosine deaminase and purine nucleoside phosphorylase, respectively. Inosine induced antinociception in the formalin test when given by systemic, spinal and peripheral routes. Systemically, inosine exhibited a potency similar to adenosine, and its effects were inhibited by DPCPX. Inosine did not induce antinociception in A1R knockout mice or in mice with reduced A1R expression. In binding studies, inosine bound to A1Rs with an affinity similar to adenosine. DCF had no effect on inosine actions. FDS augmented the antinociceptive effect of a low systemic dose of inosine and, at a higher dose, induced antinociception by itself. Collectively, these data indicate that inosine is an agonist for A1Rs with antinociceptive properties and a potency similar to adenosine and can be considered another endogenous ligand for this receptor.

Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

BACKGROUND: The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. OBJECTIVES: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. RESULTS: Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. CONCLUSION: Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Assistência fisioterapêutica na atenção primária à saúde infantil: uma revisão das experiências / Physical therapy in pediatric primary care: a review of experiences

OBJETIVO: Revisar as experiências de atenção fisioterapêutica dirigidas à população pediátrica descritas na literatura e analisar a produção de conhecimento sobre fisioterapia no contexto da atenção primária à saúde infantil (APSI). MÉTODOS: Foi realizada uma revisão sistemática conforme PRISMA, com busca nas seguintes bases de dados: MEDLINE, LILACS, SciELO, PubMed, Scopus, Cochrane; banco de teses da CAPES; e System for Information on Grey Literature in Europe (SIGLE). Foram utilizados os descritores "atenção primária à saúde", "fisioterapia", "lactente ou criança" e seus correspondentes na língua inglesa e espanhola, sem restrição de ano de publicação. RESULTADOS: Analisamos 13 artigos de seis países, reunidos em três eixos temáticos: dilemas profissionais (três artigos), competências e habilidades específicas para a APSI (sete artigos) e relatos de prática (quatro artigos). Os dilemas profissionais mencionados foram a ampliação do papel do fisioterapeuta para incluir ambientes comunitários, compartilhando a tomada de decisão com as famílias, e o trabalho em colaboração com outros serviços de saúde para identificar as necessidades da criança. As competências e habilidades citadas foram a identificação de sintomas clínicos e socioculturais para além das condições musculoesqueléticas, o diagnóstico fisioterapêutico precoce, a prevenção contra o uso excessivo de medicamentos e a capacidade de trabalhar em equipe. Os relatos de prática discorreram sobre estimulação em crianças com quadros neurológicos, tratamento respiratório e grupos com mães de crianças com esses acometimentos. CONCLUSÕES: O baixo número de estudos sugere desconhecimento quanto ao modo como a fisioterapia se insere na APSI e, provavelmente, quanto às habilidades profissionais necessárias nesse ambiente. Assim, são necessários mais estudos para fornecer dados sobre a área e um esforço de qualificação continuada por parte dos fisioterapeutas.

OBJECTIVE: To review pediatric physical therapy experiences described in the literature and to analyze the production of knowledge on physical therapy in the context of pediatric primary health care (PPHC). METHODS: A systematic review was conducted according to the PRISMA criteria. The following databases were searched: MEDLINE, LILACS, SciELO, PubMed, Scopus and Cochrane; Brazilian Ministry of Health's CAPES doctoral dissertations database; and System for Information on Grey Literature in Europe (SIGLE). The following search terms were used: ["primary health care" and ("physical therapy" or "physiotherapy") and ("child" or "infant")] and equivalent terms in Portuguese and Spanish, with no restriction on publication year. RESULTS: Thirteen articles from six countries were analyzed and grouped into three main themes: professional dilemmas (three articles), specific competencies and skills required in a PPHC setting (seven articles), and practice reports (four articles). Professional dilemmas involved expanding the role of physical therapists to encompass community environments and sharing the decision-making process with the family, as well as collaborative work with other health services to identify the needs of children. The competencies and skills mentioned in the literature related to the identification of clinical and sociocultural symptoms that go beyond musculoskeletal conditions, the establishment of early physical therapy diagnoses, prevention of overmedication, and the ability to work as team players. Practice reports addressed stimulation in children with neurological diseases, respiratory treatment, and establishing groups with mothers of children with these conditions. CONCLUSIONS: The small number of studies identified in this review suggests that there is little knowledge regarding the roles of physical therapists in PPHC and possibly regarding the professional abilities required in this setting. Therefore, further studies are required to provide data on the field, along with a continuing education effort on the part of physical therapists.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction.

BACKGROUND AND PURPOSE: The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. EXPERIMENTAL APPROACH: End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. KEY RESULTS: Inosine (100 µM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. CONCLUSION AND IMPLICATIONS: Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.