Oxidative testicular injury: effect of L-leucine on redox, cholinergic and purinergic dysfunctions, and dysregulated metabolic pathways.

The antioxidant and anti-proinflammatory activities of L-leucine were investigated on oxidative testicular injury, ex vivo. In vitro analysis revealed L-leucine to be a potent scavenger of free radicals, while inhibiting acetylcholinesterase activity. Oxidative injury was induced in testicular tissues using FeSO4. Treatment with L-leucine led to depletion of oxidative-induced elevated levels of NO, MDA, and myeloperoxidase activity, with concomitant elevation of reduced glutathione and non-protein thiol levels, SOD and catalase activities. L-leucine caused a significant (p < 0.05) alteration of oxidative-elevated acetylcholinesterase and chymotrypsin activities, while concomitantly elevating the activities of ATPase, ENTPDase and 5'-nucleotidase. L-leucine conferred a protective effect against oxidative induced DNA damage. Molecular docking revealed molecular interactions with COX-2, IL-1 beta and iNOS. Treatment with L-leucine led to restoration of oxidative depleted ascorbic acid-2-sulfate, with concomitant depletion of the oxidative induced metabolites: D-4-Hydroxy-2-oxoglutarate, L-cystine, adenosine triphosphate, maleylacetoacetic acid, cholesteryl ester, and 6-Hydroxy flavin adenine dinucleotide. Treatment with L-leucine reactivated glycolysis while concomitantly deactivating oxidative-induced citrate cycle and increasing the impact-fold of purine metabolism pathway. L-leucine was predicted not to be an inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, with a predicted LD50 value of 5000 mg/Kg and toxicity class of 5. Additionally, L-leucine showed little or no in vitro cytotoxicity in mammalian cells. These results suggest the therapeutic potentials of L-leucine on oxidative testicular injury, as evident by its ability to attenuate oxidative stress and proinflammation, while stalling cholinergic dysfunction and modulating nucleotide hyrolysis; as well as modulate oxidative dysregulated metabolites and their pathways.

Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.

New frontiers in probing the dynamics of purinergic transmitters in vivo.

Purinergic transmitters such as adenosine, ADP, ATP, UTP, and UDP-glucose play important roles in a wide range of physiological processes, including the sleep-wake cycle, learning and memory, cardiovascular function, and the immune response. Moreover, impaired purinergic signaling has been implicated in various pathological conditions such as pain, migraine, epilepsy, and drug addiction. Examining the function of purinergic transmission in both health and disease requires direct, sensitive, non-invasive tools for monitoring structurally similar purinergic transmitters; ideally, these tools should have high spatial and temporal resolution in in vivo applications. Here, we review the recent progress with respect to the development and application of new methods for detecting purinergic transmitters, focusing on optical tools; in addition, we provide discussion regarding future perspectives.

Purinergic signaling creates an anti-inflammatory profile in spleens of grass carp Ctenopharyngodon idella naturally infected by Saprolegnia parasitica: An attempt to prevent ATP pro-inflammatory effects.

Extracellular adenosine triphosphate (ATP) is as key mediator of immune and inflammatory responses. ATP is normally sequestered in the intracellular milieu and released by apoptotic and necrotic cells, where it acts as a pro-inflammatory mediator in the extracellular milieu. A limited number of studies have explored the involvement of purinergic signaling in oomycete infections, including Saprolegnia parasitica; this is a most destructive oomycete pathogen, associated with high mortality and severe economic losses for fish producers. The aim of this study was to determine whether purinergic signaling exerts anti- or pro-inflammatory effects in spleens of grass carp (Ctenopharyngodon idella) naturally infected by S. parasitica. Animals naturally infected with S. parasitica showed typical gross lesions characterized by cotton-wool tufts on the tail and fins, as well as severe histopathological lesions such as necrosis. Spleen ATP and metabolites of nitric oxide (NOx) levels were higher in fish naturally infected by S. parasitica compared to control on day 7 post-infection (PI). Spleen nucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) was greater in fish naturally infected by S. parasitica than in uninfected on day 7 PI, while no significant differences were observed between groups with respect to NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. Finally, adenosine deaminase (ADA) activity was lower in fish naturally infected by S. parasitica than in uninfected fish on day 7 PI. In summary, spleen tissue necrosis in the context of saprolegniosis provokes an intense release of ATP into the extracellular milieu, where it interacts with the P2X7 purine receptor and leads to a self-sustained pro-inflammatory deleterious cycle, contributing to an intense inflammatory process. In response to excessive ATP levels in the extracellular milieu, ATP and adenosine hydrolysis were modulated in an attempt to restrict the inflammatory process via upregulation of NTPDase and downregulation of ADA activities. We conclude that the purinergic signaling pathway modulates immune and inflammatory responses during natural infection with S. parasitica.

Purinergic Signaling in Pulmonary Inflammation.

Purine nucleotides and nucleosides are at the center of biologic reactions. In particular, adenosine triphosphate (ATP) is the fundamental energy currency of cellular activity and adenosine has been demonstrated to play essential roles in human physiology and pathophysiology. In this review, we examine the role of purinergic signaling in acute and chronic pulmonary inflammation, with emphasis on ATP and adenosine. ATP is released into extracellular space in response to cellular injury and necrosis. It is then metabolized to adenosine monophosphate (AMP) via ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and further hydrolyzed to adenosine via ecto-5'-nucleotidase (CD73). Adenosine signals via one of four adenosine receptors to exert pro- or anti-inflammatory effects. Adenosine signaling is terminated by intracellular transport by concentrative or equilibrative nucleoside transporters (CNTs and ENTs), deamination to inosine by adenosine deaminase (ADA), or phosphorylation back into AMP via adenosine kinase (AK). Pulmonary inflammatory and hypoxic conditions lead to increased extracellular ATP, adenosine diphosphate (ADP) and adenosine levels, which translates to increased adenosine signaling. Adenosine signaling is central to the pulmonary injury response, leading to various effects on inflammation, repair and remodeling processes that are either tissue-protective or tissue destructive. In the acute setting, particularly through activation of adenosine 2A and 2B receptors, adenosine signaling serves an anti-inflammatory, tissue-protective role. However, excessive adenosine signaling in the chronic setting promotes pro-inflammatory, tissue destructive effects in chronic pulmonary inflammation.

1st European Purine Meeting Santiago de Compostela: September: 2-6, 2019

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Purinergic Signaling and Cochlear Injury-Targeting the Immune System?

Hearing impairment is the most common sensory deficit, affecting more than 400 million people worldwide. Sensorineural hearing losses currently lack any specific or efficient pharmacotherapy largely due to the insufficient knowledge of the pathomechanism. Purinergic signaling plays a substantial role in cochlear (patho)physiology. P2 (ionotropic P2X and the metabotropic P2Y) as well as adenosine receptors expressed on cochlear sensory and non-sensory cells are involved mostly in protective mechanisms of the cochlea. They are implicated in the sensitivity adjustment of the receptor cells by a K+ shunt and can attenuate the cochlear amplification by modifying cochlear micromechanics. Cochlear blood flow is also regulated by purines. Here, we propose to comprehend this field with the purine-immune interactions in the cochlea. The role of harmful immune mechanisms in sensorineural hearing losses has been emerging in the horizon of cochlear pathologies. In addition to decreasing hearing sensitivity and increasing cochlear blood supply, influencing the immune system can be the additional avenue for pharmacological targeting of purinergic signaling in the cochlea. Elucidating this complexity of purinergic effects on cochlear functions is necessary and it can result in development of new therapeutic approaches in hearing disabilities, especially in the noise-induced ones.

Induction of antiinflammatory purinergic signaling in activated human iNKT cells.

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3-D microenvironment.

Calcific aortic valve disease is an active disease process with lipoprotein deposition, chronic inflammation, and progressive leaflet degeneration. Expression of ectonucleotidases, a group of membrane-bound enzymes that regulate the metabolism of ATP and its metabolites, may coregulate the degeneration process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the degeneration process of VICs. Ovine VICs were cultivated in vitro under different prodegenerative conditions and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)/ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5'-nucleotidase (CD73), as well as with adenosine and adenosine receptor agonists. Experiments were performed both in 2-dimensional (2-D) and 3-dimensional (3-D) cell-culture models. Our main findings were that VICs continuously release ATP. Inhibition of ATP hydrolyzing enzymes (CD39 and ENPP1) resulted in profound prodegenerative effects with a vigorous up-regulation of CD39, ENPP1, and CD73, as well as TGF-ß1 and osteopontin at the gene level. In our 3-D model, the effect was more pronounced than in 2-D monolayers. Increasing adenosine levels, as well as stimulating the adenosine receptors A2A and A2B, exhibited strong prodegenerative effects, whereas conversely, lowering adenosine levels by inhibition of CD73 resulted in protective effects against degeneration. Dysregulation of any one of these enzymes plays an important role in the degeneration process of VICs. Stimulation of ATP and adenosine has prodegenerative effects, whereas lowering the adenosine levels exerts a protective effect.-Weber, A., Barth, M., Selig, J. I., Raschke, S., Dakaras, K., Hof, A., Hesse, J., Schrader, J., Lichtenberg, A., Akhyari, P. Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3-D microenvironment.

Neuronal activity determines distinct gliotransmitter release from a single astrocyte.

Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca2+ uncaging) stimuli modulate putative single CA3-CA1 hippocampal synapses. The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses.

An autocrine purinergic signaling controls astrocyte-induced neuronal excitation.

Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. Using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a calcium-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDA receptors. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal.

Purinergic Signaling: A Common Path in the Macrophage Response against Mycobacterium tuberculosis and Toxoplasma gondii.

Immune responses are essential for the protection of the host against external dangers or infections and are normally efficient in the clearance of invading microbes. However, some intracellular pathogens have developed strategies to replicate and survive within host cells resulting in latent infection associated with strong inflammation. This excessive response can cause cell and tissue damage and lead to the release of the intracellular content, in particular the nucleotide pool, into the extracellular space. Over the last decade, new studies have implicated metabolites from the purinergic pathway in shaping the host immune response against intracellular pathogens and proved their importance in the outcome of the infection. This review aims to summarize how the immune system employs the purinergic system either to fight the pathogen, or to control collateral tissue damage. This will be achieved by focusing on the macrophage response against two intracellular pathogens, the human etiologic agent of tuberculosis, Mycobacterium tuberculosis and the protozoan parasite, Toxoplasma gondii.

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

BACKGROUND: Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. METHODS: Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. RESULTS: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. CONCLUSIONS: Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.

Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

BACKGROUND: It has recently been suggested that the adenosine A2A receptor plays a role in several animal models of depression. Additionally, A2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. METHODS: In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A2A receptors but poor A1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. RESULTS: The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. CONCLUSION: Available data support the proposition that the examined compounds with adenosine A2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents.

Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP-a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts-activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca(2+)-activated large-conductance K(+) channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis.

Purinergic signaling in scarring.

Adenosine (ADO) and nucleotides such as ATP, ADP, and uridine 5'-triphosphate (UTP), among others, may serve as extracellular signaling molecules. These mediators activate specific cell-surface receptors-namely, purinergic 1 and 2 (P1 and P2)-to modulate crucial pathophysiological responses. Regulation of this process is maintained by nucleoside and nucleotide transporters, as well as the ectonucleotidases ectonucleoside triphosphate diphosphohydrolase [ENTPD; cluster of differentiation (CD)39] and ecto-5'-nucleotidase (5'-NT; CD73), among others. Cells involved in tissue repair, healing, and scarring respond to both ADO and ATP. Our recent investigations have shown that modulation of purinergic signaling regulates matrix deposition during tissue repair and fibrosis in several organs. Cells release adenine nucleotides into the extracellular space, where these mediators are converted by CD39 and CD73 into ADO, which is anti-inflammatory in the short term but may also promote dermal, heart, liver, and lung fibrosis with repetitive signaling under defined circumstances. Extracellular ATP stimulates cardiac fibroblast proliferation, lung inflammation, and fibrosis. P2Y2 (UTP/ATP) and P2Y6 [ADP/UTP/uridine 5'-diphosphate (UDP)] have been shown to have profibrotic effects, as well. Modulation of purinergic signaling represents a novel approach to preventing or diminishing fibrosis. We provide an overview of the current understanding of purinergic signaling in scarring and discuss its potential to prevent or decrease fibrosis.

Continuous intravenous infusion of ATP in humans yields large expansions of erythrocyte ATP pools but extracellular ATP pools are elevated only at the start followed by rapid declines.

The pharmacokinetics of adenosine 5'-triphosphate (ATP) was investigated in a clinical trial that included 15 patients with advanced malignancies (solid tumors). ATP was administered by continuous intravenous infusions of 8 h once weekly for 8 weeks. Three values of blood ATP levels were determined. These were total blood (erythrocyte) and blood plasma (extracellular) ATP pools along with the initial rate of release of ATP into the blood plasma. We found that values related to erythrocyte ATP pools showed great variability (diversity) among individuals (standard deviation of about 30-40% of mean at baseline). It was discovered that erythrocyte baseline ATP pool sizes are unique to each individual and that they fall within a narrow range in each individual. At the end of an 8 h continuous intravenous infusion of ATP, intracellular erythrocyte ATP pools were increased in the range of 40-60% and extracellular ATP declined from elevated levels achieved at the beginning and middle of the infusion, to baseline levels. The ability of erythrocytes to sequester exogenously administered ATP to this degree, after its initial conversion to adenosine in the blood plasma is unexpected, considering that some of the adenosine is likely to have been degraded by in vivo catabolic activities or taken up by organs. The data suggest that administration of ATP by short-term intravenous infusions, of up to 4 h, may be a favorable way for elevating extracellular ATP pools. A large fraction of the total exogenously administered ATP is sequestered into the intracellular compartments of the erythrocytes after an 8 h intravenous infusion. Erythrocytes loaded with ATP are known to release their ATP pools by the application of previously established agents or conditions applied locally or globally to circulating erythrocytes. Rapid degradation of intravenously administered ATP to adenosine and subsequent accumulation of ATP inside erythrocytes indicate the existence of very effective mechanisms for uptake of adenosine from blood plasma. These in vivo studies offer an understanding as to how both adenosine and ATP can act as purinergic transmission signals. ATP levels in blood are always accompanied by adenosine formed by catabolism of ATP. The continuous uptake of adenosine enables both to act in transmission of sometimes opposite functions.

Purinergic signalling: from discovery to current developments.

This lecture is about the history of the purinergic signalling concept. It begins with reference to the paper by Paton & Vane published in 1963, which identified non-cholinergic relaxation in response to vagal nerve stimulation in several species, although they suggested that it might be due to sympathetic adrenergic nerves in the vagal nerve trunk. Using the sucrose gap technique for simultaneous mechanical and electrical recordings in smooth muscle (developed while in Feldberg's department in the National Institute for Medical Research) of the guinea-pig taenia coli preparation (learned when working in Edith Bülbring's smooth muscle laboratory in Oxford Pharmacology), we showed that the hyperpolarizations recorded in the presence of antagonists to the classical autonomic neurotransmitters, acetylcholine and noradrenaline, were inhibitory junction potentials in response to non-adrenergic, non-cholinergic neurotransmission, mediated by intrinsic enteric nerves controlled by vagal and sacral parasympathetic nerves. We then showed that ATP satisfied the criteria needed to identify a neurotransmitter released by these nerves. Subsequently, it was shown that ATP is a cotransmitter in all nerves in the peripheral and central nervous systems. The receptors for purines and pyrimidines were cloned and characterized in the early 1990 s, and immunostaining showed that most non-neuronal cells as well as nerve cells expressed these receptors. The physiology and pathophysiology of purinergic signalling is discussed.

Hepatic purinergic signaling gene network expression and its relationship with inflammation and oxidative stress biomarkers in blood from peripartal dairy cattle.

The liver plays a central role in allowing dairy cattle to make a successful transition into lactation. In liver, as in other tissues, extracellular nucleotides and nucleosides trigger cellular responses through adenosine and ATP receptors. Adenosine triphosphate and certain nucleotides serve as signals that can heighten purinergic receptor activation in several pathologic processes. We evaluated the mRNA expression of genes associated with the purinergic signaling network in liver tissue during the peripartal period. Seven multiparous Holstein cows were dried off at d -50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24 Mcal/kg of DM) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65 Mcal/kg of DM) until 30 DIM. Biopsies of liver were harvested at d -10, 7, and 21 for mRNA expression of 9 purinergic receptors, 7 ATP and adenosine transport channels, and 10 enzymes associated with ATP hydrolysis. Blood collected at d -21, -10, 7, 14, and 21 was used to measure concentrations of inflammation and oxidative stress biomarkers. The expression of type 1 purinergic receptors (ADORA2A and ADORA3), several nucleoside hydrolases [ectonucleoside triphosphate diphosphohydrolase 7 (ENTPD7), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), ENPP3, and adenosine deaminase (ADA)], and a type 2 purinergic receptor (P2RX7) was downregulated after calving. In contrast, the expression of type 2 purinergic receptors (P2RX4 and PR2Y11), an ATP release channel (gap junction hemichannel GJB1), and an adenosine uptake protein (SLC29A1) followed the opposite response, increasing after calving and remaining elevated through 21 d. Haptoglobin, ceruloplasmin, and reactive oxygen metabolite concentrations increased gradually from d -21 d through at least d 7. The opposite response was observed for albumin, paraoxonase, α-tocopherol, and nitric oxide, which decreased gradually to a nadir at 7 and 14 d. Our results suggest that alterations after calving of the expression of hepatic purinergic signaling genes could be functionally important because in nonruminants, they play roles in bile formation, glucose metabolism, cholesterol uptake, inflammation, and steatosis. The correlation analysis provided evidence of a link between purinergic signaling genes and biomarkers of inflammation and oxidative stress.