Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Release of [3H]norepinephrine from synaptic vesicles isolated from rat brain after the intracisternal administration of [3H]norepinephrine: influence of nucleotides, ions and drugs, and destabilization of transmitter storage caused by acute or chronic lithium administration

Following the intracisternal administration of [³H]norepinephrine to rats pretreated with a monoamine oxidase inhibitor, synaptic vesicles containing the radio label could be isolated from isotonically prepared microsomal fractions of rat brain. Incorporation of [³H]norepinephrine into the vesicles was reduced by pretreatment of the rats with desmethylimipramine and was also reduced if the rats had not been pretreated with a monoamine oxidase inhibitor. Incorporation of the label was totally eliminated by pretreatment with reserpine. Release in vitro of [³H]norepinephrine from the labeled vesicles was monophasic with a half-time of about 12 min at 30°C. The release was slowed by addition of adenosine 5′-triphosphate plus Mg²⁺ by a mechanism different from that of the vesicular amine uptake system; this was shown by the failure of inhibitors of adenosine triphosphate-Mg²⁺-stimulated uptake (reserpine,N-ethylmaleimide, lithium) to block the effect of adenosine triphosphate plus Mg²⁺ on release. Several other nucleotides also were able to slow the release of [³H]norepinephrine. Unlike adrenomedullary vesicles, rat brain synaptic vesicles did not show enhancement of amine release by chloride in the presence or absence of adenosine triphosphate plus Mg²⁺. The yield of labeled vesicles was substantially reduced if vesicles were prepared by hypotonic lysis of synaptosomes instead of isotonically from the microsomal fraction; the isotonic preparation appears to be superior for studies of vesicle uptake and storage properties.This preparation is readily utilizable for studies of the effects of in vivo administration of drugs thought to act on vesicular storage of catecholamines, a point illustrated by the destabilization of norepinephrine storage caused by acute or chronic lithium administration.     

Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Adenosine A2A receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and "fine tuning" modulation

This review summarizes recent developments that have contributed to understand how adenosine receptors, particularly A2A receptors, modulate brain injury in various animal models of neurological disorders, including Parkinson's disease (PD), stroke, Huntington's disease (HD), multiple sclerosis, Alzheimer's disease (AD) and HIV-associated dementia. It is clear that extracellular adenosine acting at adenosine receptors influences the functional outcome in a broad spectrum of brain injuries, indicating that A2A Rs may modulate some general cellular processes to affect neuronal cells death. Pharmacological, neurochemical and molecular/genetic approaches to the complex actions of A2A receptors in different cellular elements suggest that A2A receptor activation can be detrimental or protective after brain insults, depending on the nature of brain injury and associated pathological conditions. An interesting concept that emerges from these studies is A2A R's ability to fine tune neuronal and glial functions to produce neuroprotective effects. While the data presented here clearly highlight the complexity of using adenosinergic agents therapeutically in PD and other neurodegenerative disorders and point out many areas for further inquiry, they also confirm that adenosine receptor ligands, particularly A2A receptor ligands, have many promising characteristics that encourage the pursuit of their therapeutic potential.     

The energy hypothesis of sleep revisited

One of the proposed functions of sleep is to replenish energy stores in the brain that have been depleted during wakefulness. Benington and Heller formulated a version of the energy hypothesis of sleep in terms of the metabolites adenosine and glycogen. They postulated that during wakefulness, adenosine increases and astrocytic glycogen decreases reflecting the increased energetic demand of wakefulness. We review recent studies on adenosine and glycogen stimulated by this hypothesis. We also discuss other evidence that wakefulness is an energetic challenge to the brain including the unfolded protein response, the electron transport chain, NPAS2, AMP-activated protein kinase, the astrocyte-neuron lactate shuttle, production of reactive oxygen species and uncoupling proteins. We believe the available evidence supports the notion that wakefulness is an energetic challenge to the brain, and that sleep restores energy balance in the brain, although the mechanisms by which this is accomplished are considerably more complex than envisaged by Benington and Heller.     

A transcranial magnetic stimulation study of corticospinal excitability during the observation of meaningless, goal-directed, and social behaviour

Peripheral nerve injury causes a progressive series of morphological changes in spinal microglia, and extracellular ATP stimulates proliferation of microglia and may be involved in neuropathic pain. We defined the precise expression of P2X7 in the spinal cord following peripheral nerve injury. We found that both P2X7 mRNA and protein increased in the spinal cord, with a peak at 7 d after injury. Double labeling studies revealed that cells expressing increased P2X7 mRNA and protein after nerve injury were predominantly microglia in dorsal horn. Pharmacological blockades by intrathecal administration of a P2X7 antagonist (A 438079 hydrochloride) suppressed the development of mechanical hypersensitivity. We present distinct evidence that increases in the number of P2X7 receptors in spinal microglia may play an important role in neuropathic pain.     

Ammonia metabolism,the brain and fatigue; revisiting the link

This review addresses the ammonia fatigue theory in light of new evidence from exercise and disease studies and aims to provide a view of the role of ammonia during exercise. Hyperammonemia is a condition common to pathological liver disorders and intense or exhausting exercise. In pathology, hyperammonemia is linked to impairment of normal brain function and the onset of the neurological condition, hepatic encephalopathy. Elevated blood ammonia concentrations arise due to a diminished capacity for removal via the liver and lead to increased exposure of organs, such as the brain, to the toxic effects of ammonia. High levels of brain ammonia can lead to deleterious alterations in astrocyte morphology, cerebral energy metabolism and neurotransmission, which may in turn impact on the functioning of important signalling pathways within the neuron. Such changes are believed to contribute to the disturbances in neuropsychological function, in particular the learning, memory, and motor control deficits observed in animal models of liver disease and also patients with cirrhosis. Hyperammonemia in exercise occurs as a result of an increased production by contracting muscle, through adenosine monophosphate (AMP) deamination (the purine nucleotide cycle) and branched chain amino acid (BCAA) deamination prior to oxidation. Plasma concentrations of ammonia during exercise often achieve or exceed those measured in liver disease patients, resulting in increased cerebral uptake. In this article we propose that exercise-induced hyperammonemia may lead to concomitant disturbances in brain function, potentially through similar mechanisms underpinning pathology, which may impact on performance as fatigue or reduced function, especially during extreme exercise.     

Mimicking nature's noses: from receptor deorphaning to olfactory biosensing

The way in which organisms detect specific volatile compounds within their environment, and the associated neural processing which produces perception and subsequent behavioural responses, have been of interest to scientists for decades. Initially, most olfaction research was conducted using electrophysiological techniques on whole animals. However, the discovery of genes encoding the family of human olfactory receptors (ORs) paved the way for the development of a range of cellular assays, primarily used to deorphan ORs from mammals and insects. These assays have greatly advanced our knowledge of the molecular basis of olfaction, however, while there is currently good agreement on vertebrate and nematode olfactory signalling cascades, debate still surrounds the signalling mechanisms in insects. The inherent specificity and sensitivity of ORs makes them prime candidates as biological detectors of volatile ligands within biosensor devices, which have many potential applications. In the previous decade, researchers have investigated various technologies for transducing OR:ligand interactions into a readable format and thereby produce an olfactory biosensor (or bioelectronic nose) that maintains the discriminating power of the ORs in vivo. Here we review and compare the molecular mechanisms of olfaction in vertebrates and invertebrates, and also summarise the assay technologies utilising sub-tissue level sensing elements (cells and cell extracts), which have been applied to OR deorphanization and biosensor research. Although there are currently no commercial, "field-ready" olfactory biosensors of the kind discussed here, there have been several technological proof-of-concept studies suggesting that we will see their emergence within the next decade.     

The imprint of salivary secretion in autoimmune disorders and related pathological conditions

Xerostomia is a state of oral dryness associated with salivary gland dysfunction and is induced by stress, radiation and chemical therapy, various systemic and autoimmune diseases, and specific medications. Fluid secretion is interrupted by the stimulation of neurotransmitter-induced increase in cytosolic calcium ([Ca²⁺]_i) in salivary gland acinar cells, prompting the mobilization of ion channels and their transporters. Salivary fluid and protein secretion are principally dependent on parasympathetic and sympathetic nerves. Various inflammatory cytokines allied with lymphocytic infiltration cause glandular damage and Sjogren's syndrome, an autoimmune exocrinopathy associated with hyposalivation. A defect in IP₃Rs, a major calcium release channel, prompts inadequate agonist-induced [Ca²⁺]_i in acinar cells and deters salivary flow. The store-operated calcium entry-mediated Ca²⁺ movement into the acini activates K⁺ and Cl^? channels, which further opens a water channel protein, aquaporin-5, and triggers the release of fluid secretion from the salivary glands. The cellular mechanism of salivary gland dysfunction and hyposalivation has not yet been elucidated. In this review, we focused mainly on the proteins responsible for deficient saliva, the correlation between inflammation and salivation, autoimmune disorders and other ailments or complications associated with hyposalivation.     

Role of adenosine A2A receptors in parkinsonian motor impairment and l-DOPA-induced motor complications

Adenosine A2A receptors have a unique cellular and regional distribution in the basal ganglia, being particularly concentrated in areas richly innervated by dopamine such as the caudate-putamen and the globus pallidus. Adenosine A2A receptors are selectively located on striatopallidal neurons and are capable of forming functional heteromeric complexes with dopamine D2 and metabotropic glutamate mGlu5 receptors. Based on the unique cellular and regional distribution of this receptor and in line with data showing that A2A receptor antagonists improve motor symptoms in animal models of Parkinson's disease (PD) and in initial clinical trials, A2A receptor antagonists have emerged as an attractive non-dopaminergic target to improve the motor deficits that characterize PD. Experimental data have also shown that A2A receptor antagonists do not induce neuroplasticity phenomena that complicate long-term dopaminergic treatments. The present review provides an updated summary of results reported in the literature concerning the biochemical characteristics and basal ganglia distribution of A2A receptors. We subsequently aim to examine the effects of adenosine A2A antagonists in rodent and primate models of PD and of l-DOPA-induced dyskinesia. Finally, concluding remarks are made on post-mortem human brains and on the translation of adenosine A2A receptor antagonists in the treatment of PD.     

Physiological effects of melatonin: role of melatonin receptors and signal transduction pathways

Melatonin, an endogenous signal of darkness, is an important component of the body's internal time-keeping system. As such it regulates major physiological processes including the sleep wake cycle, pubertal development and seasonal adaptation. In addition to its relevant antioxidant activity, melatonin exerts many of its physiological actions by interacting with membrane MT1 and MT2 receptors and intracellular proteins such as quinone reductase 2, calmodulin, calreticulin and tubulin. Here we review the current knowledge about the properties and signaling of melatonin receptors as well as their potential role in health and some diseases. Melatonin MT1 and MT2 receptors are G protein coupled receptors which are expressed in various parts of the CNS (suprachiasmatic nuclei, hippocampus, cerebellar cortex, prefrontal cortex, basal ganglia, substantia nigra, ventral tegmental area, nucleus accumbens and retinal horizontal, amacrine and ganglion cells) and in peripheral organs (blood vessels, mammary gland, gastrointestinal tract, liver, kidney and bladder, ovary, testis, prostate, skin and the immune system). Melatonin receptors mediate a plethora of intracellular effects depending on the cellular milieu. These effects comprise changes in intracellular cyclic nucleotides (cAMP, cGMP) and calcium levels, activation of certain protein kinase C subtypes, intracellular localization of steroid hormone receptors and regulation of G protein signaling proteins. There are circadian variations in melatonin receptors and responses. Alterations in melatonin receptor expression as well as changes in endogenous melatonin production have been shown in circadian rhythm sleep disorders, Alzheimer's and Parkinson's diseases, glaucoma, depressive disorder, breast and prostate cancer, hepatoma and melanoma. This paper reviews the evidence concerning melatonin receptors and signal transduction pathways in various organs. It further considers their relevance to circadian physiology and pathogenesis of certain human diseases, with a focus on the brain, the cardiovascular and immune systems, and cancer.     

The serotonergic system in ageing and Alzheimer's disease

Alzheimer's disease (AD) is one of the major neurodegenerative diseases that deteriorates cognitive functions and primarily affects associated brain regions involved in learning and memory, such as the neocortex and the hippocampus. Following the discovery and establishment of its role as a neurotransmitter, serotonin (5-HT), was found to be involved in a multitude of neurophysiological processes including mnesic function, through its dedicated pathways and interaction with cholinergic, glutamatergic, GABAergic and dopaminergic transmission systems. Abnormal 5-HT neurotransmission contributes to the deterioration of cognitive processes in ageing, AD and other neuropathologies, including schizophrenia, stress, mood disorders and depression. Numerous studies have confirmed the pathophysiological role of the 5-HT system in AD and that several drugs enhancing 5-HT neurotransmission are effective in treating the AD-related cognitive and behavioural deficits. Here we present a comprehensive overview of the role of serotonergic neurotransmission in brain development, maturation and ageing, discuss its role in higher brain function and provide an in depth account of pathological modifications of serotonergic transmission in neurological diseases and AD.     

Mechanisms and biomarkers of immune quiescence in kidney transplantation

This review discusses the current understanding of biomarkers of immune quiescence based on reviews of published literature in kidney transplant operational tolerance and mechanistic studies based on a better characterization of the stable, well-functioning renal allograft.     

The nociceptive and anti-nociceptive effects of bee venom injection and therapy: A double-edged sword

Bee venom injection as a therapy, like many other complementary and alternative medicine approaches, has been used for thousands of years to attempt to alleviate a range of diseases including arthritis. More recently, additional theraupeutic goals have been added to the list of diseases making this a critical time to evaluate the evidence for the beneficial and adverse effects of bee venom injection. Although reports of pain reduction (analgesic and antinociceptive) and anti-inflammatory effects of bee venom injection are accumulating in the literature, it is common knowledge that bee venom stings are painful and produce inflammation. In addition, a significant number of studies have been performed in the past decade highlighting that injection of bee venom and components of bee venom produce significant signs of pain or nociception, inflammation and many effects at multiple levels of immediate, acute and prolonged pain processes. This report reviews the extensive new data regarding the deleterious effects of bee venom injection in people and animals, our current understanding of the responsible underlying mechanisms and critical venom components, and provides a critical evaluation of reports of the beneficial effects of bee venom injection in people and animals and the proposed underlying mechanisms. Although further studies are required to make firm conclusions, therapeutic bee venom injection may be beneficial for some patients, but may also be harmful. This report highlights key patterns of results, critical shortcomings, and essential areas requiring further study.     

Apoptosis-inducing factor: a matter of neuron life and death

The mitochondrial flavoprotein apoptosis-inducing factor (AIF) is the main mediator of caspase-independent apoptosis-like programmed cell death. Upon pathological permeabilization of the outer mitochondrial membrane, AIF is translocated to the nucleus, where it participates in chromatin condensation and is associated to large-scale DNA fragmentation. Heavy down-regulation of AIF expression in mutant mice or reduced AIF expression achieved with small interfering RNA (siRNA) provides neuroprotection against acute neurodegenerative insults. Paradoxically, in addition to its pro-apoptotic function, AIF likely plays an anti-apoptotic role by regulating the production of reactive oxygen species (ROS) via its putative oxidoreductase and peroxide scavenging activities. In this review, we discuss accumulating evidence linking AIF to both acute and chronic neurodegenerative processes by emphasising mechanisms underlying the dual roles apparently played by AIF in these processes.     

l-DOPA: A scapegoat for accelerated neurodegeneration in Parkinson's disease?

There is consensus that amelioration of the motor symptoms of Parkinson's disease is most effective with L-DOPA (levodopa). However, this necessary therapeutic step is biased by an enduring belief that L-DOPA is toxic to the remaining substantia nigra dopaminergic neurons by itself, or by specific metabolites such as dopamine. The concept of L-DOPA toxicity originated from pre-clinical studies conducted mainly in cell culture, demonstrating that L-DOPA or its derivatives damage dopaminergic neurons due to oxidative stress and other mechanisms. However, the in vitro data remain controversial as some studies showed neuroprotective, rather than toxic action of the drug. The relevance of this debate needs to be considered in the context of the studies conducted on animals and in clinical trials that do not provide convincing evidence for L-DOPA toxicity in vivo. This review presents the current views on the pathophysiology of Parkinson's disease, focusing on mitochondrial dysfunction and oxidative/proteolytic stress, the factors that can be affected by L-DOPA or its metabolites. We then critically discuss the evidence supporting the two opposing views on the effects of L-DOPA in vitro, as well as the animal and human data. We also address the problem of inadequate experimental models used in these studies. L-DOPA remains the symptomatic 'hero' of Parkinson's disease. Whether it contributes to degeneration of nigral dopaminergic neurons, or is a 'scapegoat' for explaining undesirable or unexpected effects of the treatment, remains a hotly debated topic.