Transient expression of thyrotropin releasing hormone peptide and mRNA in the rat hippocampus following global cerebral ischemia/reperfusion injury.

INTRODUCTION: The role of extra-hypothalamic thyrotropin-releasing hormone (TRH) has been investigated by pharmacological studies using TRH or its analogues and found to produce a wide array of effects in the central nervous system. METHODS: Immunofluorescence, In situ labeling of DNA (TUNEL), in situ hybridization chain reaction and quantitative real-time polymerase chain reaction were used in this study. RESULTS: We found that the granular cells of the dentate gyrus expressed transiently a significant amount of TRH-like immunoreactivity and TRH mRNA during the 6-24 h period following global cerebral ischemia/reperfusion injury. TUNEL showed that apoptosis of neurons in the CA1 region occurred from 48 h and almost disappeared at 7 days. TRH administration 30 min before or 24 h after the injury could partially inhibit neuronal loss, and improve the survival of neurons in the CA1 region. CONCLUSION: These data suggest that endogenous TRH expressed transiently in the dentate gyrus of the hippocampus may play an important role in the survival of neurons during the early stage of ischemia/reperfusion injury and that delayed application of TRH still produced neuroprotection. This delayed application of TRH has a promising therapeutic significance for clinical situations.

Purinergic Signaling and Related Biomarkers in Depression.

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

Introduction to Purinergic Signalling in the Brain.

ATP is a cotransmitter with glutamate, noradrenaline, GABA, acetylcholine and dopamine in the brain. There is a widespread presence of both adenosine (P1) and P2 nucleotide receptors in the brain on both neurons and glial cells. Adenosine receptors play a major role in presynaptic neuromodulation, while P2X ionotropic receptors are involved in fast synaptic transmission and synaptic plasticity. P2Y G protein-coupled receptors are largely involved in presynaptic activities, as well as mediating long-term (trophic) signalling in cell proliferation, differentiation and death during development and regeneration. Both P1 and P2 receptors participate in neuron-glial interactions. Purinergic signalling is involved in control of cerebral vascular tone and remodelling and has been implicated in learning and memory, locomotor and feeding behaviour and sleep. There is increasing interest in the involvement of purinergic signalling in the pathophysiology of the CNS, including trauma, ischaemia, epilepsy, neurodegenerative diseases, neuropsychiatric and mood disorders, and cancer, including gliomas.

Introduction to Purinergic Signaling.

Purinergic signaling was proposed in 1972, after it was demonstrated that adenosine 5'-triphosphate (ATP) was a transmitter in nonadrenergic, noncholinergic inhibitory nerves supplying the guinea-pig taenia coli. Later, ATP was identified as an excitatory cotransmitter in sympathetic and parasympathetic nerves, and it is now apparent that ATP acts as a cotransmitter in most, if not all, nerves in both the peripheral nervous system and central nervous system (CNS). ATP acts as a short-term signaling molecule in neurotransmission, neuromodulation, and neurosecretion. It also has potent, long-term (trophic) roles in cell proliferation, differentiation, and death in development and regeneration. Receptors to purines and pyrimidines have been cloned and characterized: P1 adenosine receptors (with four subtypes), P2X ionotropic nucleotide receptors (seven subtypes) and P2Y metabotropic nucleotide receptors (eight subtypes). ATP is released from different cell types by mechanical deformation, and after release, it is rapidly broken down by ectonucleotidases. Purinergic receptors were expressed early in evolution and are widely distributed on many different nonneuronal cell types as well as neurons. Purinergic signaling is involved in embryonic development and in the activities of stem cells. There is a growing understanding about the pathophysiology of purinergic signaling and there are therapeutic developments for a variety of diseases, including stroke and thrombosis, osteoporosis, pain, chronic cough, kidney failure, bladder incontinence, cystic fibrosis, dry eye, cancer, and disorders of the CNS, including Alzheimer's, Parkinson's. and Huntington's disease, multiple sclerosis, epilepsy, migraine, and neuropsychiatric and mood disorders.

Expression of P2X receptors in the rat anterior pituitary.

In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling immunofluorescence, combined method of immunofluorescence and in situ hybridization, and Western blot. The results showed that the expression level of the P2X4 receptor protein was highest, followed by P2X5, P2X3, P2X2, P2X6, and P2X7 receptor proteins, but no P2X1 receptor protein was detected. Strong P2X4 receptor-immunoreactivity was detected in almost all the anterior pituitary cells. Different combinations of P2X receptors were detected in each individual cell type of the rat anterior pituitary. Gonadotrophs express P2X4, P2X5, and P2X6 receptors. Corticotrophs express P2X3 and P2X4 receptors. Folliculo-stellate cells express P2X2 and P2X4 receptors, and somatotrophs, lactotrophs, and thyrotrophs express only P2X4 receptors. The macrophages with Iba-1-ir expressed P2X7 receptors. The possible functions of these P2X receptors in each individual cell type of the rat anterior pituitary are discussed.

Astroglia-Derived ATP Modulates CNS Neuronal Circuits.

It is broadly recognized that ATP not only supports energy storage within cells but is also a transmitter/signaling molecule that serves intercellular communication. Whereas the fast (co)transmitter function of ATP in the peripheral nervous system has been convincingly documented, in the central nervous system (CNS) ATP appears to be primarily a slow transmitter/modulator. Data discussed in the present review suggest that the slow modulatory effects of ATP arise as a result of its vesicular/nonvesicular release from astrocytes. ATP acts together with other glial signaling molecules such as cytokines, chemokines, and free radicals to modulate neuronal circuits. Hence, astrocytes are positioned at the crossroads of the neuron-glia-neuron communication pathway.

DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites. METHODS: Experiments using receptor-binding functional assays, rat nodose ganglionic cells, perfused innervated guinea pig lung preparation ex vivo, and anesthetized and conscious guinea pigs in vivo were performed. RESULTS: DT-0111 acted as a selective and effective antagonist at P2X2/3R, that is, it did not activate or block P2YR; markedly inhibited the activation by ATP of nodose pulmonary vagal afferents in vitro; and, given as an aerosol, inhibited aerosolized ATP-induced bronchoconstriction and cough in vivo. CONCLUSIONS: These results indicate that DT-0111 is an attractive drug-candidate for the treatment of COPD and chronic cough, both of which still constitute major unmet clinical needs. The reviews of this paper are available via the supplementary material section.

Myocardial metabolism in heart failure: Purinergic signalling and other metabolic concepts.

Despite significant therapeutic advances in heart failure (HF) therapy, the morbidity and mortality associated with this disease remains unacceptably high. The concept of metabolic dysfunction as an important underlying mechanism in HF is well established. Cardiac function is inextricably linked to metabolism, with dysregulation of cardiac metabolism pathways implicated in a range of cardiac complications, including HF. Modulation of cardiac metabolism has therefore become an attractive clinical target. Cardiac metabolism is based on the integration of adenosine triphosphate (ATP) production and utilization pathways. ATP itself impacts the heart not only by providing energy, but also represents a central element in the purinergic signaling pathway, which has received considerable attention in recent years. Furthermore, novel drugs that have received interest in HF include angiotensin receptor blocker-neprilysin inhibitor (ARNi) and sodium glucose cotransporter 2 (SGLT-2) inhibitors, whose favorable cardiovascular profile has been at least partly attributed to their effects on metabolism. This review, describes the major metabolic pathways and concepts of the healthy heart (including fatty acid oxidation, glycolysis, Krebs cycle, Randle cycle, and purinergic signaling) and their dysregulation in the progression to HF (including ketone and amino acid metabolism). The cardiac implications of HF comorbidities, including metabolic syndrome, diabetes mellitus and cachexia are also discussed. Finally, the impact of current HF and diabetes therapies on cardiac metabolism pathways and the relevance of this knowledge for current clinical practice is discussed. Targeting cardiac metabolism may have utility for the future treatment of patients with HF, complementing current approaches.

The involvement of purinergic signalling in obesity.

Obesity is a growing worldwide health problem, with an alarming increasing prevalence in developed countries, caused by a dysregulation of energy balance. Currently, no wholly successful pharmacological treatments are available for obesity and related adverse consequences. In recent years, hints obtained from several experimental animal models support the notion that purinergic signalling, acting through ATP-gated ion channels (P2X), G protein-coupled receptors (P2Y) and adenosine receptors (P1), is involved in obesity, both at peripheral and central levels. This review has drawn together, for the first time, the evidence for a promising, much needed novel therapeutic purinergic signalling approach for the treatment of obesity with a 'proof of concept' that hopefully could lead to further investigations and clinical trials for the management of obesity.

Purine and purinergic receptors.

Adenosine 5'-triphosphate acts as an extracellular signalling molecule (purinergic signalling), as well as an intracellular energy source. Adenosine 5'-triphosphate receptors have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of adenosine 5'-triphosphate after degradation by ectonucleotidases. Four subtypes are recognised, A1, A2A, A2B and A3 receptors. P2 receptors are activated by purine and by pyrimidine nucleotides. P2X receptors are ligand-gated ion channel receptors (seven subunits (P2X1-7)), which form trimers as both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors (eight subtypes (P2Y1/2/4/6/11/12/13/14)). There is both purinergic short-term signalling and long-term (trophic) signalling. The cloning of P2X-like receptors in primitive invertebrates suggests that adenosine 5'-triphosphate is an early evolutionary extracellular signalling molecule. Selective purinoceptor agonists and antagonists with therapeutic potential have been developed for a wide range of diseases, including thrombosis and stroke, dry eye, atherosclerosis, kidney failure, osteoporosis, bladder incontinence, colitis, neurodegenerative diseases and cancer.

The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression.

Seven P2X ion channel nucleotide receptor subtypes have been cloned and characterised. P2X7 receptors (P2X7R) are unusual in that there are extra amino acids in the intracellular C terminus. Low concentrations of ATP open cation channels sometimes leading to cell proliferation, whereas high concentrations of ATP open large pores that release inflammatory cytokines and can lead to apoptotic cell death. Since many diseases involve inflammation and immune responses, and the P2X7R regulates inflammation, there has been recent interest in the pathophysiological roles of P2X7R and the potential of P2X7R antagonists to treat a variety of diseases. These include neurodegenerative diseases, psychiatric disorders, epilepsy and a number of diseases of peripheral organs, including the cardiovascular, airways, kidney, liver, bladder, skin and musculoskeletal. The potential of P2X7R drugs to treat tumour progression is discussed.

The therapeutic potential of purinergic signalling.

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y12 receptor antagonists for stroke and thrombosis, P2Y2 receptor agonists for dry eye, and A1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles.

Purinergic Signalling: Therapeutic Developments.

Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990's when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine) receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A2A receptor antagonists are promising for the treatment of Parkinson's disease. Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer.

Cell culture: complications due to mechanical release of ATP and activation of purinoceptors.

There is abundant evidence that ATP (adenosine 5'-triphosphate) is released from a variety of cultured cells in response to mechanical stimulation. The release mechanism involved appears to be a combination of vesicular exocytosis and connexin and pannexin hemichannels. Purinergic receptors on cultured cells mediate both short-term purinergic signalling of secretion and long-term (trophic) signalling such as proliferation, migration, differentiation and apoptosis. We aim in this review to bring to the attention of non-purinergic researchers using tissue culture that the release of ATP in response to mechanical stress evoked by the unavoidable movement of the cells acting on functional purinergic receptors on the culture cells is likely to complicate the interpretation of their data.

Inhibitory effect of estrogen receptor beta on P2X3 receptors during inflammation in rats.

Estrogen receptor beta (ERß) has been shown to play a therapeutic role in inflammatory bowel disease (IBD). However, the mechanism underlying how ERß exerts therapeutic effects and its relationship with P2X3 receptors (P2X3R) in rats with inflammation is not known. In our study, animal behavior tests, visceromotor reflex recording, and Western blotting were used to determine whether the therapeutic effect of ERß in rats with inflammation was related with P2X3R. In complete Freund adjuvant (CFA)-induced chronic inflammation in rats, paw withdrawal threshold was significantly decreased which were then reversed by systemic injection of ERß agonists, DPN or ERB-041. In 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, weight loss, higher DAI scores, increased visceromotor responses, and inflammatory responses were reversed by application of DPN or ERB-041. The higher expressions of P2X3R in dorsal root ganglia (DRG) of CFA-treated rats and those in rectocolon and DRG of TNBS-treated rats were all decreased by injection of DPN or ERB-041. DPN application also inhibited P2X3R-evoked inward currents in DRG neurons from TNBS rats. Mechanical hyperalgesia and increased P2X3 expression in ovariectomized (OVX) CFA-treated rats were reversed by estrogen replacements. Furthermore, the expressions of extracellular signal-regulated kinase (ERK) in DRG and spinal cord dorsal horn (SCDH) and c-fos in SCDH were significantly decreased after estrogen replacement compared with those of OVX rats. The ERK antagonist U0126 significantly reversed mechanical hyperalgesia in the OVX rats. These results suggest that estrogen may play an important therapeutic role in inflammation through down-regulation of P2X3R in peripheral tissues and the nervous system, probably via ERß, suggesting a novel therapeutic strategy for clinical treatment of inflammation.

Purinergic Signalling and Neurological Diseases: An Update.

Purinergic signalling, i.e. ATP as an extracellular signalling molecule and cotransmitter in both peripheral and central neurons, is involved in the physiology of neurotransmission and neuromodulation. Receptors for purines have been cloned and characterised, including 4 subtypes of the P1(adenosine) receptor family, 7 subtypes of the P2X ion channel nucleotide receptor family and 8 subtypes of the P2Y G protein-coupled nucleotide receptor family. The roles of purinergic signalling in diseases of the central nervous system and the potential use of purinergic compounds for their treatment are attracting increasing attention. In this review, the focus is on the findings reported in recent papers and reviews to update knowledge in this field about the involvement of purinergic signalling in Alzheimer's, Parkinson's and Huntington's diseases, multiple sclerosis, amyotrophic lateral sclerosis, degeneration and regeneration after brain injury, stroke, ischaemia, inflammation, migraine, epilepsy, psychiatric disorders, schizophrenia, bipolar disorder, autism, addiction, sleep disorders and brain tumours. The use in particular of P2X7 receptor antagonists for the treatment of neurodegenerative diseases, cancer, depression, stroke and ischaemia, A2A receptor antagonists for Parkinson's disease and agonists for brain injury and depression and P2X3 receptor antagonists for migraine and seizures has been recommended. P2Y receptors have also been claimed to be involved in some central nervous disorders.

Purinergic Signalling in the Gut.

The article will begin with the discovery of purinergic inhibitory neuromuscular transmission in the 1960s/1970s, the proposal for purinergic cotransmission in 1976 and the recognition that sympathetic nerves release adenosine 5'-triphosphate (ATP), noradrenaline and neuropeptide Y, while non-adrenergic, non-cholinergic inhibitory nerve cotransmitters are ATP, nitric oxide and vasoactive intestinal polypeptide in variable proportions in different regions of the gut. Later, purinergic synaptic transmission in the myenteric and submucosal plexuses was established and purinergic receptors expressed by both glial and interstitial cells. The focus will then be on purinergic mechanosensory transduction involving release of ATP from mucosal epithelial cells during distension to activate P2X3 receptors on submucosal sensory nerve endings. The responses of low threshold fibres mediate enteric reflex activity via intrinsic sensory nerves, while high threshold fibres initiate pain via extrinsic sensory nerves. Finally, the involvement of purinergic signalling in an animal model of colitis will be presented, showing that during distension there is increased ATP release, increased P2X3 receptor expression on calcitonin gene-related peptide-labelled sensory neurons and increased sensory nerve activity.

Short- and long-term (trophic) purinergic signalling.

There is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body, in addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion. It is not always easy to distinguish between short- and long-term signalling. For example, adenosine triphosphate (ATP) can sometimes act as a short-term trigger for long-term trophic events that become evident days or even weeks after the original challenge. Examples of short-term purinergic signalling during sympathetic, parasympathetic and enteric neuromuscular transmission and in synaptic transmission in ganglia and in the central nervous system are described, as well as in neuromodulation and secretion. Long-term trophic signalling is described in the immune/defence system, stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption and in cancer. It is likely that the increase in intracellular Ca(2+) in response to both P2X and P2Y purinoceptor activation participates in many short- and long-term physiological effects.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Purinergic Mechanisms and Pain.

There is a brief introductory summary of purinergic signaling involving ATP storage, release, and ectoenzymatic breakdown, and the current classification of receptor subtypes for purines and pyrimidines. The review then describes purinergic mechanosensory transduction involved in visceral, cutaneous, and musculoskeletal nociception and on the roles played by receptor subtypes in neuropathic and inflammatory pain. Multiple purinoceptor subtypes are involved in pain pathways both as an initiator and modulator. Activation of homomeric P2X3 receptors contributes to acute nociception and activation of heteromeric P2X2/3 receptors appears to modulate longer-lasting nociceptive sensitivity associated with nerve injury or chronic inflammation. In neuropathic pain activation of P2X4, P2X7, and P2Y12 receptors on microglia may serve to maintain nociceptive sensitivity through complex neural-glial cell interactions and antagonists to these receptors reduce neuropathic pain. Potential therapeutic approaches involving purinergic mechanisms will be discussed.

An introduction to the roles of purinergic signalling in neurodegeneration, neuroprotection and neuroregeneration.

Purinergic signalling appears to play important roles in neurodegeneration, neuroprotection and neuroregeneration. Initially there is a brief summary of the background of purinergic signalling, including release of purines and pyrimidines from neural and non-neural cells and their ectoenzymatic degradation, and the current characterisation of P1 (adenosine), and P2X (ion channel) and P2Y (G protein-coupled) nucleotide receptor subtypes. There is also coverage of the localization and roles of purinoceptors in the healthy central nervous system. The focus is then on the roles of purinergic signalling in trauma, ischaemia, stroke and in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, as well as multiple sclerosis and amyotrophic lateral sclerosis. Neuroprotective mechanisms involving purinergic signalling are considered and its involvement in neuroregeneration, including the role of adult neural stem/progenitor cells. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.