Purinergic Signaling in Non-Parenchymal Liver Cells.

Purinergic signaling has emerged as an important paracrine-autocrine intercellular system that regulates physiological and pathological processes in practically all organs of the body. Although this system has been thoroughly defined since the nineties, recent research has made substantial advances regarding its role in aspects of liver physiology. However, most studies have mainly targeted the entire organ, 70% of which is made up of parenchymal cells or hepatocytes. Because of its physiological role, the liver is exposed to toxic metabolites, such as xenobiotics, drugs, and fatty acids, as well as to pathogens such as viruses and bacteria. Under injury conditions, all cell types within the liver undergo adaptive changes. In this context, the concentration of extracellular ATP has the potential to increase dramatically. Indeed, this purinergic response has not been studied in sufficient detail in non-parenchymal liver cells. In the present review, we systematize the physiopathological adaptations related to the purinergic system in chronic liver diseases of non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, and cholangiocytes. The role played by non-parenchymal liver cells in these circumstances will undoubtedly be strategic in understanding the regenerative activities that support the viability of this organ under stressful conditions.

Activation of Intra-nodose Ganglion P2X7 Receptors Elicit Increases in Neuronal Activity.

Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.

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.

Differential involvement of the canonical and noncanonical inflammasomes in the immune response against infection by the periodontal bacteria Porphyromonas gingivalis and Fusobacterium nucleatum.

We examined the involvement of the P2 × 7 receptor and the canonical and noncanonical inflammasomes in the control of single-species or dual-species infection by the periodontal bacteria Porphyromonas gingivalis and Fusobacterium nucleatum in cells and mice. Stimulation of the P2 × 7 receptor leads to activation of the canonical NLRP3 inflammasome and activation of caspase-1, which leads to cleavage of pro-IL-1ß to IL-1ß, a key cytokine in the host inflammatory response in periodontal disease. The non-canonical inflammasome pathway involves caspase-11. Thus, wildtype (WT), P2 × 7-/-, caspase-11-/- and caspase-1/11-/- mice were co-infected with both bacterial species. In parallel, bone marrow-derived macrophages (BMDMs) from WT mice and the different knockout mice were infected with P. gingivalis and/or F. nucleatum, and treated or not with extracellular ATP, which is recognized by P2 × 7. F. nucleatum infection alone promoted secretion of IL-1ß in BMDMs. Conversely, the canonical pathway involving P2 × 7 and caspase-1 was necessary for secretion of IL-1ß in BMDMs infected with P. gingivalis and in the mandible of mice coinfected with P. gingivalis and F. nucleatum. The P2 × 7 pathway can limit bacterial load in single-species and dual-species infection with P. gingivalis and F. nucleatum in BMDMs and in mice. The non-canonical pathway involving caspase-11 was required for secretion of IL-1ß induced by F. nucleatum infection in BMDMs, without treatment with ATP. Caspase-11 was also required for induction of cell death during infection with F. nucleatum and contributed to limiting bacterial load during F. nucleatum infection in BMDMs and in the gingival tissue of mice coinfected with P. gingivalis and F. nucleatum. Together, these data suggest that the P2 × 7-caspase-1 and caspase-11 pathways are involved in the immune response against infection by P. gingivalis and F. nucleatum, respectively.

Photobiomodulation inhibits inflammation in the temporomandibular joint of rats.

Photobiomodulation (PBM) has been applied as a non-invasive technique for treating temporomandibular joint symptoms, especially on painful condition's relief, however the anti-inflammatory mechanism underlying the effect of PBM remains uncertain. This study aims to evaluate the mechanisms of action of PBM (808 nm) in a carrageenan-induced inflammation on temporomandibular joint (TMJ) of rats. In this study male Wistar rats were pre-treated with irradiation of a low-power diode laser for 15 s on TMJ (infra-red 808 nm, 100 mW, 50 J/cm2 and 1.5 J) 15 min prior an injection in the temporomandibular joint of carrageenan (100 µg/TMJ). 1 h after the TMJ treatments, the rats were terminally anesthetized for joint cavity wash and periarticular tissues collect. Samples analysis demonstrated that PBM inhibit leukocytes chemotaxis in the TMJ and significantly reduces amounts of TNF-α, IL-1ß and CINC-1. In addition, Western blotting analysis demonstrated that PBM significantly decreased the protein levels of P2X3 and P2X7 receptors in the periarticular tissues. On the other hand, PBM was able to increase protein level of IL-10 (anti-inflammatory cytokine). In summary, it is possible to suggest that PBM inhibit inflammatory chemotaxis, modulation the balance of the pro- and anti-inflammatory characteristics of inflammatory cells.

Restraint of Human Skin Fibroblast Motility, Migration, and Cell Surface Actin Dynamics, by Pannexin 1 and P2X7 Receptor Signaling.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Purinergic Receptors in Basal Ganglia Diseases: Shared Molecular Mechanisms between Huntington's and Parkinson's Disease.

Huntington's (HD) and Parkinson's diseases (PD) are neurodegenerative disorders caused by the death of GABAergic and dopaminergic neurons in the basal ganglia leading to hyperkinetic and hypokinetic symptoms, respectively. We review here the participation of purinergic receptors through intracellular Ca2+ signaling in these neurodegenerative diseases. The adenosine A2A receptor stimulates striatopallidal GABAergic neurons, resulting in inhibitory actions on GABAergic neurons of the globus pallidus. A2A and dopamine D2 receptors form functional heteromeric complexes inducing allosteric inhibition, and A2A receptor activation results in motor inhibition. Furthermore, the A2A receptor physically and functionally interacts with glutamate receptors, mainly with the mGlu5 receptor subtype. This interaction facilitates glutamate release, resulting in NMDA glutamate receptor activation and an increase of Ca2+ influx. P2X7 receptor activation also promotes glutamate release and neuronal damage. Thus, modulation of purinergic receptor activity, such as A2A and P2X7 receptors, and subsequent aberrant Ca2+ signaling, might present interesting therapeutic potential for HD and PD.

Oleic and Linoleic Acids Induce the Release of Neutrophil Extracellular Traps via Pannexin 1-Dependent ATP Release and P2X1 Receptor Activation.

Non-esterified fatty acids (NEFAs) such as oleic acid (OA) and linoleic acid (LA) are associated with a higher incidence of infectious diseases such as metritis and mastitis during the bovine peripartum. Fatty acids can induce an increase in the release of ATP, and changes in the expression levels of purinergic receptors in bovine polymorphonuclears (PMN) during peripartum have also been reported. PMN respond to inflammatory processes with production of ROS, release of proteolytic and bactericidal proteins, and formation of neutrophil extracellular traps (NETs). NETs formation is known to require ATP production through glycolysis. Studies have shown that the above-mentioned metabolic changes alter innate immune responses, particularly in PMN. We hypothesized that NEFAs induce the formation of NETs through ATP release by Pannexin 1 and activation of purinergic receptors. In this study, we found that OA and LA induce NET formation and extracellular ATP release. Carbenoxolone, a pannexin-1 (PANX1) inhibitor, reduced OA- and LA-induced ATP release. We also found that P2X1, P2X4, P2X5, P2X7, and PANX1 were expressed at the mRNA level in bovine PMN. Additionally, NEFA-induced NET formation was completely abolished with exposure to NF449, a P2X1 antagonist, and partially inhibited by treatment with etomoxir, an inhibitor of fatty acid oxidation (FAO). Our results suggest that OA and LA induce NET formation and ATP release via PANX1 and activation of P2X1. These new data contribute to explaining the effects of NEFA high concentrations during the transition period of dairy cattle and further understanding of pro-inflammatory effects and outcome of postpartum diseases.

The role of purinergic P2Y12 and P2Y13 receptors in ADPßS-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats.

ATP is a cotransmitter released with other neurotransmitters from sympathetic nerves, where it stimulates purinergic receptors. Purinergic adenosine P1 receptors (coupled to Gi/o proteins) produce sympatho-inhibition in several autonomic effectors by prejunctional inhibition of neurotransmitter release. Similarly, signalling through P2Y12 and P2Y13 receptors coupled to Gi/o proteins is initiated by the ATP breakdown product ADP. Hence, this study has pharmacologically investigated a possible role of ADP-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats, using a stable ADP analogue (ADPßS) and selective antagonists for the purinergic P2Y1, P2Y12 and P2Y13 receptors. Accordingly, male Wistar rats were pithed and: (i) pretreated i.v. with gallamine (25 mg/kg) and desipramine (50 µg/kg) for preganglionic spinal (C7-T1) stimulation of the cardioaccelerator sympathetic drive (n = 78); or (ii) prepared for receiving i.v. injections of exogenous noradrenaline (n = 12). The i.v. continuous infusions of ADPßS (10 and 30 µg/kg/min) dose-dependently inhibited the tachycardic responses to electrical sympathetic stimulation, but not those to exogenous noradrenaline. The cardiac sympatho-inhibition produced by 30 µg/kg/min ADPßS was (after i.v. administration of compounds) (i) unchanged by 1-ml/kg bidistilled water or 300-µg/kg MRS 2500 (P2Y1 receptor antagonist), (ii) abolished by 300-µg/kg PSB 0739 (P2Y12 receptor antagonist) and (iii) partially blocked by 3000-µg/kg MRS 2211 (P2Y13 receptor antagonist). Our results suggest that ADPßS induces a cardiac sympatho-inhibition that mainly involves the P2Y12 receptor subtype and, probably to a lesser extent, the P2Y13 receptor subtype. These receptors may represent therapeutic targets for treating cardiovascular pathologies, including stroke and myocardial infarctions.

Using RNA Interference for Purinoceptor Knockdown In Vivo.

RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

Short-Term Hyperprolactinemia Reduces the Expression of Purinergic P2X7 Receptors during Allergic Inflammatory Response of the Lungs.

PURPOSE: We have previously shown that domperidone-induced short-term hyperprolactinemia reduces the lung's allergic inflammatory response in an ovalbumin antigenic challenge model. Since purinergic receptor P2X7R activity leads to proinflammatory cytokine release and is possibly related to the pathogenesis of allergic respiratory conditions, the present study was designed to investigate a possible involvement of purinergic and prolactin receptors in this phenomenon. METHODS: To induce hyperprolactinemia, domperidone was injected intraperitoneally in rats at a dose of 5.1 mg × kg-1 per day for 5 days. P2X7 expression was evaluated by lung immunohistochemistry while prolactin receptor expression in bronchoalveolar lavage leukocytes was analyzed through flow cytometry. RESULTS: Previous reports demonstrated that rats subjected to short-term hyperprolactinemia exhibited a decrease in leukocyte counts in bronchoalveolar lavage, especially granulocytes. Here, it is revealed that hyperprolactinemia promotes an increased expression of prolactin receptors in granulocytes. Also, increased expression of purinergic P2X7R observed in allergic animals was significantly reduced by hyperprolactinemia. CONCLUSIONS: Both purinergic and prolactin receptor expression changes occur during the anti-asthmatic effect of hyperprolactinemia.

P2X7 receptor and klotho expressions in diabetic nephropathy progression.

Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X7), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X7 and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X7 and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X7 and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X7 and klotho expressions were similar up to the 4th week; then, P2X7 expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X7 and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

POM-1 inhibits P2 receptors and exhibits anti-inflammatory effects in macrophages.

Extracellular nucleotides can modulate the immunological response by activating purinergic receptors (P2Rs) on the cell surface of macrophages, dendritic, and other immune cells. In particular, the activation of P2X7R can induce release of cytokines and cell death as well as the uptake of large molecules through the cell membrane by a mechanism still poorly understood. Polyoxotungstate-1 (POM-1) has been proposed as a potent inhibitor of ecto-nucleotidases, enzymes that hydrolyze extracellular nucleotides, regulating the activity of P2Rs. However, the potential impact of POM-1 on P2Rs has not been evaluated. Here, we used fluorescent dye uptake, cytoplasmic free Ca2+ concentration measurement, patch-clamp recordings, scanning electron microscopy, and quantification of inflammatory mediators to investigate the effects of POM-1 on P2Rs of murine macrophages. We observed that POM-1 blocks the P2YR-dependent cytoplasmic Ca2+ increase and has partial effects on the cytoplasmic Ca2+, increasing dependence on P2XRs. POM-1 can inhibit the events related with ATP-dependent inflammasome activation, anionic dye uptake, and also the opening of large conductance channels, which are associated with P2X7R-dependent pannexin-1 activation. On the other hand, this compound has no effects on cationic fluorescent dye uptake, apoptosis, and bleb formation, also dependent on P2X7R. Moreover, POM-1 can be considered an anti-inflammatory compound, because it prevents TNF-α and nitric oxide release from LPS-treated macrophages.

P2 purinergic receptor antagonists disrupt maternal behavior in lactating rats.

The involvement of purinergic signaling in several brain functions has been recognized, but the modulation on maternal behavior by the purinergic system is not established, even though there are functional interactions between the purinergic and oxytocinergic systems. Therefore, the aim of our study was to investigate whether central administration of P2 receptor antagonists affected the maternal behavior of lactating rats and c-Fos immunoreactivity in the forebrain. On day 7 of lactation, female rats were treated with vehicle (5µL; i.c.v.), suramin (9.4-75.0µg/5µL; i.c.v.) or PPADS (9.4-75.0µg/5µL; i.c.v.) 30min before the experiment began. The maternal behavior was evaluated during the 30min following suramin or PPADS administration. In addition, c-Fos-positive nuclei were counted in the medial preoptic area (MPOA) and in the bed nucleus of the stria terminalis (BNST), and neurons that were double-labeled for c-Fos/OT were counted in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus of lactating rats. The results show that P2 receptor antagonists decreased maternal care and decreased neuronal activation in the MPOA and BNST and activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the purinergic system modulates maternal behavior and neuronal activation induced by suckling during lactation.

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

P2Y2 purinergic receptors are highly expressed in cardiac and diaphragm muscles of mdx mice, and their expression is decreased by suramin.

INTRODUCTION: In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to increased calcium influx and muscle necrosis. Patients suffer progressive muscle loss, and cardiomyopathy is an important determinant of morbidity. P2 purinergic receptors participate in the increased calcium levels in dystrophic skeletal muscles. METHODS: In this study, we evaluated whether P2 receptors are involved in cardiomyopathy in mdx mice at later stages of the disease. RESULTS: Western blotting revealed that P2Y2 receptor levels were upregulated (54%) in dystrophic heart compared with a normal heart. Suramin reduced the levels of P2Y2 to almost normal values. Suramin also decreased heart necrosis (reduced CK-MB) and the expression of the stretch-activated calcium channel TRPC1. CONCLUSIONS: This study suggests that P2Y2 may participate in cardiomyopathy in mdx mice. P2-selective drugs with specific actions in the dystrophic heart may ameliorate cardiomyopathy in dystrophinopathies. Muscle Nerve 55: 116-121, 2017.

Purinergic receptors in tubulointerstitial inflammatory cells: a pathophysiological mechanism of salt-sensitive hypertension.

Recent studies have suggested that both the tubulointerstitial inflammatory cells and the activation of purinergic receptors integrate common mechanisms that result in salt-sensitive hypertension. The basis of this hypothesis is that renal endothelial cells release ATP in response to shear stress in the setting of hypertension. It has been demonstrated that the over-expression and activation of the P2X7, P2Y12 and P2X1 receptors favour the elevation of blood pressure induced by high-salt intake. In addition, the release of interleukins and inflammatory mediators in the tubulointerstitial area appears to be related to the activation of these receptors. Renal vasoconstriction and tubulointerstitial injury develop as a result, which increase sodium reabsorption by epithelial cells. Consistent with these effects, the reduction of tubulointerstitial inflammation caused by immunosuppressants, such as mycophenolate mofetil, prevents the development of salt-sensitive hypertension. Also, P2X7-receptor knockout mice develop minor renal injury when hypertension is induced via the administration of deoxycorticosterone acetate and a high-salt diet. In the setting of angiotensin II-induced hypertension, which is an early stage in the development of salt-sensitive hypertension, an acute blockade with the specific, non-selective P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid prevented the renal vasoconstriction induced by angiotensin II. In addition, it normalized glomerular haemodynamics and restored sodium excretion to control values. These findings suggest that chronic administration of P2 purinergic antagonists may prevent the deleterious effects of purinergic receptors during the development of salt-sensitive hypertension.

ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway.

The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100µM ATP, ATPγS and UTP, but not ADP, ADPßS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100µM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2.

BACKGROUND: Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. METHODS: J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. RESULTS: ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. CONCLUSIONS: Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. GENERAL SIGNIFICANCE: ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.