Purinergic Signalling: Therapeutic Developments.

Purinergic signalling plays an important role in a variety of gut activities ( Liñán-Rico et al., 2015 ; Burnstock, 2016d ; Chaudhury et al., 2016 ). ATP is a cotransmitter in non-adrenergic, non-cholinergic inhibitory nerves involved in peristalsis, a synaptic transmitter in submucosal and myenteric ganglia, it mediates mucosal secretion and vascular control of the gastrointestinal tract. Both interstitial cells of Cajal and enteric glial cells express P2R. Investigations of purinoceptors as therapeutic candidates for gut disorders are underway ( Antonioli et al., 2013 ; Ochoa-Cortes et al., 2014 ; Burnstock, 2016d ). Nucleotides and their receptors are involved in the pathogenesis of IBD, of which ulcerative colitis (UC) and Crohn’s disease are the main types. P2Y 6 R are highly expressed on the T cells infiltrating IBD, suggesting that P2Y 6 R may play a role in the pathogenesis of IBD. P2X7R agonists are involved in colonic motor dysfunction associated with bowel inflammation in rats ( Antonioli et al., 2014b ) and are over-expressed in gut mucosa of patients with IBD. P2X7R KO mice are protected against gut inflammation ( Figliuolo et al., 2017b ). ATP activates mast cells, which further promote the inflammatory process ( Kurashima and Kiyono, 2016 ). Oestrogen receptor β activation plays a therapeutic role in IBD by down-regulation of P2X3 and P2X7R ( Ma B. et al., 2016 ). The roles of P2X7R in IBD are discussed in reviews ( Kurashima et al., 2015 ; Diezmos et al., 2016 ). P2X7R-expressing enteric neurons are differentially affected in UC based on their chemical codes ( da Silva et al., 2015 ). In a later paper, it was shown that UC affected secretory and vasodilatory neurons, enteric sensory neurons and enteric glia of the submucosal plexus expressing P2X7R ( da Silva et al., 2017 ). The P2X7R antagonist, A438079, down-regulated the production of proinflammatory cytokines and attenuated murine colitis, indicating that P2X7R mediate inflammatory responses during UC ( Wan P. et al., 2016 ). Activation of P2X7R triggers the death of mucosal regulatory T cells ( Figliuolo et al., 2017b ). There was P2XR enhancement in an animal model of UC. MicroRNA-16 and microRNA-206 have pro-inflammatory roles in UC by down-regulating A 2A and A 3 R expression ( Tian T. et al., 2016 ; Wu et al., 2017 ). There is increased expression of P2X7R in the inflamed mucosa in Crohn’s disease, suggesting that P2X7R antagonists may be targets for treatment of Crohn’s disease ( Neves et al., 2014 ; Eser et al., 2015 ; Wan P. et al., 2016 ; Figliuolo et al., 2017b ). P2X3R antagonists are being explored as therapeutic agents against colic and UC pain ( Eser et al., 2015 ), as well as antibodies against P2X3R ( Deiteren et al., 2015 ; Diezmos et al., 2016 ; Shcherbatko et al., 2016 ). The potential of P2X3R to treat irritable bowel syndrome has been suggested. In diarrhoea-predominant irritable bowel syndrome there is increased expression of P2Y 1 and P2Y 2 R, which was associated with abdominal pain ( Luo Y. et al., 2016 ). P2X7R in DRG play a role in transmission of the nociceptive signal from the gut ( Liu S. et al., 2015 ). A review about purinergic mediators in the control of intestinal inflammation and irritable bowel syndrome is available ( Kurashima et al., 2015 ). Purinergic signalling is involved in gastrointestinal motility disorders, including diarrhoea and constipation ( Jiménez et al., 2014 ). Purinergic fast inhibitory junction potentials are impaired in Hirschsprung’s disease ( Jiménez et al., 2015 ). P2X4 and P2X7R activity was potentiated in enteric glia isolated from mice treated long-term with morphine ( Bhave et al., 2017 ). A review focuses on the pathophysiological roles of P2YR in inflammation ( Wan H.X. et al., 2016 ). Aged mice have a lower ability to deal with inflammation evoked by Candida albicans infection, due to a lower gut density of A 2A R, which reduce inflammation ( Rodrigues et al., 2016 ). Apoptosis is induced by extracellular ATP and ATP also reduced growth of primary cultures of colorectal carcinomas, possibly via P2Y 2 R (see Burnstock and Di Virgilio, 2013 ; Wan H.X. et al., 2016 ). Purinergic responses of HT-29 cells, a colonic adenocarcinoma cell line, are mediated by P2Y 2 and/or P2Y 4 R. Enhanced expression of A 2B R on colorectal cancer cells that are proliferating suggests that antagonists to A 2B R may be a promising therapeutic target for colorectal cancer ( Ma et al., 2010 ; Molck et al., 2016 ). 8-Chloro adenosine inhibited growth of colorectal cancer cell lines 80514 and HCT116 in vivo and in vitro . A phase II, multi-centre study of CF101, an A 3 R agonist, showed stabilisation of tumour in 35% of refractory metastatic colorectal cancer patients. P2X7R antagonists reduce tumour occurrence in a colitis-associated cancer mouse model ( Hofman et al., 2015 ). There is high expression of the ectonucleotidase CD39 in human rectal adenocarcinoma ( Zhang et al., 2015a ).

Reviews about purinergic signalling in the skin during health and disease have been published ( Burnstock et al., 2012b ; Geraghty et al., 2016 ). Changes in P2R subtype expression occurs in the epidermis during proliferative disorders, such as psoriasis and scleroderma, and P2Y 2 R may be a novel target to treat these disorders. ATP plays an important role in wound healing, the defence response, innate immunity and inflammation in the skin and might be an important therapeutically in psoriasis and scleroderma. Psoriasis is a chronic skin disease involving epidermal hyperproliferation. ATP and parathyroid hormone-related protein increased proliferation in HaCaT cells and may account for the hyperproliferation that occurs in psoriasis. P2Y 2 R contribute to epidermal homeostasis and indicate a possible therapy for psoriasis. P2Y 11 R mediate IL-6 production in human keratinocytes, which is important in psoriasis ( Ishimaru et al., 2013 ). P2X7R signalling induces inflammation leading to differentiation of Th17 lymphocytes, which are involved in the pathogenesis and potential treatment of psoriasis ( Killeen et al., 2013 ). P2X7R play a role in shaping the inflammatory microenvironment in psoriasis ( Lioi et al., 2015 ). Strong P2X7R expression is confined to the basal layer cell membrane, while P2Y 1 R were expressed all through the psoriatic epidermis. In the blood of psoriasis patients, there are high levels of adenosine. Adenosine raised cyclic AMP levels in lesions of epidermis from psoriasis patients. There is a defective purine nucleotide synthetic pathway in patients with psoriasis and nucleotide metabolism is altered in psoriatic keratinocytes. In psoriasis patients, ADA levels were significantly elevated in both serum and epidermis. It was concluded from a clinical trial that caffeine, a P1R antagonist, is an inexpensive, safe and effective treatment for psoriasis. The role of adenosine as an endogenous mediator of the pathogenesis of psoriasis has been reviewed ( Festugato, 2015 ) and clinical trials for the use of A 3 R agonists for the treatment of psoriasis described ( Borea et al., 2015 ; Kofoed et al., 2015 ). A 2A R are upregulated in psoriasis and A 2A R agonists may counteract inflammation in this disease ( Merighi et al., 2017 ). A 2B R are also expressed by human epidermal keratinocytes and their expression is reduced in psoriasis ( Andrés et al., 2017 ). Scleroderma encompasses a spectrum of disorders that cause dermal fibrosis and systemic sclerosis (SSc). SSc is a severe disease of the connective tissue of affected organs, including the skin. Adenosine A 2A R contribute to the pathogenesis of dermal fibrosis and may be a therapeutic target to treat and prevent dermal fibrosis in scleroderma ( Chan and Cronstein, 2010 ). Fibroblasts from SSc patients had a high rate of spontaneous IL-6 release, which was increased by ATP stimulation. SSc fibroblasts expressed mRNA for P2X3, P2X4, P2X7, P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 , suggesting a possible therapeutic role for P2R antagonists in SSc patients via modulation of fibroblast function. The skin is an immune defence organ. Chemical, immune-specific or physical insults evoke increased expression of proinflammatory mediators, in particular keratinocytes release chemokines. Release of ATP is associated with inflammation of the skin, as well as increase in expression of, in particular, P2X7 and perhaps P2Y 1 R, and subsequent release of proinflammatory cytokines. Skin inflammation is reduced by P2X7R antagonists. In ectonucleotidase (CD39) KO mice, rapid ATP release from keratinocytes is triggered by irritant chemicals causing exacerbated skin inflammation. Ultraviolet (UV) radiation evokes ATP release from keratinocytes and P2Y 6 R mediate UV radiation-induced inflammatory responses ( Takai et al., 2011 ). ATP, when released after trauma or infection, may enhance immunoresponses and P2R agonists may increase vaccine efficacy. A 1 and A 2 R may function as cutaneous neurogenic pro-inflammatory mediators. Human skin keratinocytes infected by Staphylococcus aureus , which releases α-toxin, exhibited a transient reduction in cellular ATP levels ( Suriyaphol et al., 2009 ). P2X3 and P2X2/3 nociceptive receptors on sensory nerve endings are increased in inflamed skin and antagonists to these receptors have been developed as analgesics. Topically applied A 2B and particularly A 2A R agonists promote cutaneous wound healing in both healthy and diabetic conditions. A 2A R agonists also promote collagen production by dermal fibroblasts. Adenosine inhibits proliferation of vascular smooth muscle cells via A 2 R activation. A review discusses the involvement of A 2A and A 2B R signalling in wound healing and fibrosis ( Shaikh and Cronstein, 2016 ). ATP and ADP also play a role in wound healing. ATP, released by damaged cells and physiologically during gentle mechanical stimulation, contributes to wound healing, tissue repair and regeneration. Cutaneous wound healing is accelerated by Mg-ATP, probably by increasing synthesis of vascular endothelial growth factor. Release of ATP from platelets and other cells during wound healing results in an increase in [Ca 2+ ] i in keratinocytes, associated with epidermal growth and differentiation. ATP improves ischaemic skin flap survival after surgery, while P2R antagonists accelerate barrier repair. There is acute inflammation in the initial phase of wound healing. ATP is involved in the development of inflammation through P2X7R-mediated production and release of cytokines from immune cells. The presence of P2X7R on immune cells mediates killing of intracellular pathogens by stimulating apoptosis of the host macrophage, chemo-attraction and cell adhesion. In contrast, adenosine has anti-inflammatory effects. ATP released following infection and trauma acts as an endogenous adjuvant to increase the immune response and P2R agonists may enhance the efficacy of vaccines. Wound healing also involves new vessel growth (angiogenesis) and both ATP and adenosine contribute to cell proliferation and migration during angiogenesis. In cultured porcine artery smooth muscle cells, ADP and ATP stimulate DNA synthesis and cell proliferation. ATP and UTP are mitogenic in human vascular smooth muscle cells and P2Y 2 and P2Y 4 R might be involved. Wound healing is delayed in denervated wounds. In a rat model of this, P2X5, P2X7, P2Y 1 , and P2Y 2 R expression was altered in the epidermis. P2Y 1 R expression was increased in the basal proliferating layer of keratinocytes in the regenerating epidermis, while P2Y 2 R were significantly decreased. ATP and UTP, probably via P2Y 2 R, increased proliferation of the MCS-P5 murine keratinocyte cell line, and enhanced wound healing in mice ( Kehasse et al., 2013 ; Jin et al., 2014 ). ATP release and P2YR signalling mediate electric field-stimulated directional keratinocyte migration ( Riding and Pullar, 2016 ). A review discussed the clinical applications of purinergic compounds to enhance wound healing ( Gendaszewska-Darmach and Kucharska, 2011 ). Warts are caused by human papillomavirus infection of epidermis basal keratinocytes. P2X5R staining was increased in warts. P2X7R immunoreactivity was found in hyperkeratotic areas of the stratum corneum and in nuclei of koilocytes in the wart suprabasal layers. The nuclei positive for P2X7R were shrunken, showing much more intense P2X7R staining. The expression of P2X7R in the nucleus of human papillomavirus-infected cells was associated with disruption of the cellular machinery. P2X7R agonists may be used to trigger apoptosis in these virally infected cells. P2X5 and P2X7R are being explored for the treatment of warts. Contact allergen sensitisation involves immune system activation by endogenous danger signals. P2X7R KO mice exhibit resistance to contact hypersensitivity. P2X7R KO dendritic cells do not induce sensitisation in response to contact allergens or release IL-1β in response to ATP. This suggests that P2X7R are crucial for release of ATP from skin in response to contact allergens. Interference with P2X7R signalling could be a therapeutic approach to prevent allergic contact dermatitis. These are autosomal dominant skin disorders and are characterised by epidermal keratinocyte dissociation (acantholysis) at the epidermis suprabasal layer. In lesions P2Y 2 R are not localised on acantholytic cells, but P2X7R appear in the plasma membranes, potentially mediating apoptosis. The skin protects the water-rich internal organs from environmental dryness, with the stratum corneum being critical to the water-impermeable barrier. Topical application of ATP and α,β-meATP via P2XR delayed barrier recovery, damaged by surfactant, organic solvent or tape stripping. P2R antagonists, however, accelerated barrier repair. In mice skin wounds healed faster following treatment with ATP-encapsulated fusogenic lipid vesicles rather than with lipid vesicles. PPADS, a P2XR antagonist, accelerates skin barrier repair and prevented epidermal hyperplasia. There is increased concentration of adenosine in burn blister fluid, and depletion of ATP in skin after thermal injury. ATP has a protective effect against skin burns. ATP-MgCl 2 administration following burn injury reversed the damage to the intravascular clearing of lipid emulsions within the reticuloendothelial immune system. Glucose metabolism was affected by ATP in thermally injured skin. Burn injury produces severe pain and the relieving effects of tetramethylpyrazine, a Chinese medicine, are claimed to be due to it acting as a P2X3R antagonist ( Gao Y. et al., 2010 ). Skin P2X3R expression was increased on sensory nerve terminals in first and second degree burns, but, following treatment with tetramethylpyrazine, P2X3R expression was reduced. P2X3R may play a role in the short-lasting thermal hyperalgesia induced by mild heat injury ( Füredi et al., 2010 ). After a superficial skin burn, adenosine reduced the skin area showing hypersensitivity. Skin pain is associated with changes in sympathetic nerve activity and ATP released from rat sympathetic nerves as a cotransmitter with NA may modulate cutaneous nociceptors. P2X3R are expressed on sensory neurons in DRG, nodose and trigeminal ganglia that innervate cutaneous tissues. ATP and ADP stimulate skin afferent nerve terminals and skin cell damage stimulates nociceptive sensory nerves via ATP release. In rat skin with carrageenan-induced inflammation, nociceptors can be selectively activated with α,β-meATP, indicating the involvement of P2X3R. ATP contributes to the enhanced sensitivity of inflamed skin nociceptors, leading to heat hyperalgesia. P2X3R antagonists are anti-nociceptive. UV light evokes hypersensitivity of skin to ATP-induced pain. After subcutaneous bee venom injection, there is reduction of prolonged pain by PPADS, and it was suggested that stimulation of P2XR in the spinal cord contributes to cutaneous pain. The terminals of nociceptive neurons in the skin are targets for P2X3R antagonists. UTP sensitises a subpopulation of cutaneous C-fibre nociceptors to mechanical stimuli. There is increased P2X3R expression on calcitonin gene-related peptide-positive sensory fibres during the growth of tumours, mediating nociception. Increased expression of skin glial cell neurotrophic factor increases the mechanical sensitivity of nociceptive afferents expressing P2X3R. Ecto-5′-nucleotidase (CD73) is located on nociceptive terminals and epidermal keratinocytes in the epidermis, which, by hydrolysing AMP to adenosine, is suggested to be anti-nociceptive. Blockade of peripheral P2Y 1 R prevents thermal hyperalgesia ( Kwon et al., 2014 ). Mechanical allodynia is induced by α,β-meATP, which sensitises P2X3R on cutaneous nociceptive sensory fibres ( Ren et al., 2015 ). Reviews with coverage of purinergic signalling and cutaneous pain are available ( Zhu and Lu, 2010 ; Burnstock, 2016c ). In irritant dermatitis, there is a pathogenic role for keratinocyte-derived ATP. The necrosis produced by the chemical irritant croton oil was prevented by the pre-treatment with A438079, a selective P2X7R antagonist ( Zanin et al., 2015 ). Topical application of A 2A antagonists prevents radiation dermatitis ( Perez-Aso et al., 2016 ). Different subtypes of P1R and P2R are involved in skin cancer, playing roles in differentiation, apoptosis and proliferation. The roles of purinoceptors are complicated because there are multiple receptor subtypes on the same cell, which can have opposing effects. For example, P2Y 2 R mediate an increase in tumour cell numbers, while P2Y 1 , P2X5, and P2X7R mediate a decrease in cell numbers. In cancers of the skin, proliferation outweighs apoptotic cell death. UV light is a stimulus for the genesis of cutaneous cancer, including basal and squamous cell carcinoma as well as melanoma, where the UV-B component has the most severe effects. UV-B irradiation decreases the amount of P2X1, P2Y 2 , and P2X7R, which contributes to the malignant transformation of keratinocytes ( Ruzsnavszky et al., 2011 ). Basal cell and squamous cell carcinoma are tumours that generally occur after the age of 50, squamous cell carcinoma being the more common and aggressive of the two. Nucleoside analogues reduced basal cell carcinoma growth. A human cutaneous squamous cell (epidermal) carcinoma cell line, A431, express P2R that induce increases in [Ca 2+ ] i . ATP-stimulated A431 cells induced production of inositol-trisphosphate, suggesting the involvement of P2YR. P2X5 and P2YR were strongly expressed on both squamous and basal cell carcinomas. P2X7R are expressed in apoptotic cells in superficial multifocal and infiltrative cells and in the necrotic centre of nodular basal cell carcinomas. Expression of P2Y 1 R was confined to the stroma surrounding tumours. P2Y 4 R were present only in basal cell carcinomas. ATP and UTP at low concentrations induced an increase in the number of A431 cells, while high concentrations substantially decreased cell numbers. ATP via P2X7R caused apoptosis of A431 cells and UTP and adenosine (after ATP breakdown) also induced cell death ( Völkl et al., 2008 ). 2′(3′)- O -(4-Benzoylbenzoyl) ATP, a potent P2X7R agonist, reduced skin carcinoma and papilloma and formation ( Fu et al., 2009 ). ADA in saliva was shown to be a diagnostic marker of tongue squamous cell carcinoma ( Rai et al., 2011 ). Malignant melanoma, which is highly metastatic, is derived from melanocytes. ATP inhibited the growth of both human and animal melanoma cells in vivo . CD39 is over-expressed in differentiated human melanomas. Expression of P2X7R was increased in superficial spreading melanoma patients, which were later shown to be functional and may be a therapeutic target for melanoma therapy ( White et al., 2005 ). A low pH environment as seen in solid tumours induced ATP release from B16 melanoma cells, which increases proliferation via P2X7R. Oxidised ATP, a P2X7R antagonist, inhibited tumour growth ( Hattori et al., 2012 ). P2Y 1 , P2Y 2 , and P2Y 6 R mRNA and protein expression was observed in human melanomas. The presence of P2Y 1 and P2X7R, which had been suggested to be therapeutic targets for melanoma treatment using ATP, was demonstrated by immunohistochemistry ( White et al., 2009 ). The release of ATP from murine B16 melanoma cells was shown to upregulate CD39 expression on regulatory T cells. Dying tumour cells release ATP, which accumulates at high concentrations and acts as an immune danger signal, although it can also kill adjacent tumour cells directly via P2X7R ( Feng et al., 2011 ). Accelerated melanoma tumour progression in mice lacking P2X7R has been reported ( Adinolfi et al., 2015 ). γ-Irradiation that induces arrest in tumour cell growth and death, induced P2X7R-dependent release of ATP from B16 cells ( Ohshima et al., 2010 ). In an animal model of melanoma, tumour growth was reduced in CD73 KO mice ( Yegutkin et al., 2011 ). Implanting B16 cells into CD73 KO mice, thereby decreasing the production of adenosine, resulted in reduced tumour growth ( Ring et al., 2011 ). Adenosine potentiated the in vivo actions of chemotherapeutic agents. Antimetastatic therapies based on inhibition of A 1 R activation have been suggested. A 1 , A 2A , A 2B and A 3 R subtypes are expressed in the A375 human malignant melanoma cell line. A 3 R activation resulted in growth inhibition of melanoma cells ( Morello et al., 2011 ). However, another study claimed that adenosine, acting through A 3 R, induced cell proliferation of human malignant melanoma C32 cells ( Soares et al., 2012 ). A 2B R antagonists impaired IL-8 production that is raised in malignant melanoma patients, while A 3 R antagonists decreased vascular endothelial growth factor that promotes human carcinoma cell angiogenesis and metastasis ( Merighi et al., 2009 ). Adenosine is a potent immunoregulatory factor modulating cytotoxic activity and cytokine production of anti-melanoma specific T cells. In conclusion, P2Y 2 R mediate proliferation, P2X5R mediate differentiation (i.e., are antiproliferative) and P2X7R mediate cell death. Therefore, P2Y 2 R antagonists and P2X5 and P2X7R agonists have therapeutic potential for the treatment of skin cancer.

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P2X7, P2Y 1 , and P2Y 2 R on immune and inflammatory cells are important in immunomodulation and inflammation, and the purinergic contribution to neuroinflammation underlying neuropathology has been discussed in several recent reviews ( Jacob et al., 2013 ; Takenouchi et al., 2014 ; Di Virgilio and Vuerich, 2015 ; Beamer et al., 2016 ). The role of P2X7R in particular in diseases related to neuroinflammation and the use of P2X7R centrally penetrant antagonists has been highlighted ( Baudelet et al., 2015 ; Di Virgilio, 2015 ; Gentile et al., 2015 ; Burnstock, 2016b ; Danquah et al., 2016 ; Karmakar et al., 2016 ; Rech et al., 2016 ; Corrêa et al., 2017 ; Giuliani A.L. et al., 2017 ; Jiang et al., 2017 ; Zheng B. et al., 2017 ). It has been claimed that activation of P2X1R on neutrophils and platelets is involved in regulation of thrombo-inflammation ( Oury et al., 2015 ). P2Y 12 R activation modulates sepsis-induced inflammation ( Liverani et al., 2016 ). Adenosine, acting mainly via A 2A R, is also involved in neoplastic and inflammatory and immune-mediated disease states ( Antonioli et al., 2014a ; da Rocha Lapa et al., 2014 ; Cekic and Linden, 2016 ; Ingwersen et al., 2016 ; Zhang X. et al., 2016 ; Faas et al., 2017 ).

ATP released as a cotransmitter from sympathetic nerves, stimulates brown adipocytes. ATP stimulates lipogenesis in rat adipocytes, regulating fat stores not via established hormones. Adipocytes express two different P2YR subtypes and P2Y 11 R activation inhibited insulin-stimulated lepton production and lipolysis stimulation. P2Y 4 R mediate inhibition of cardiac fat formation ( Lemaire et al., 2016 ). The anti-obesity effects of sesamol, a potent anti-inflammatory compound, is mediated by AMP-activated protein kinase ( Go et al., 2017 ). Blocking P2Y 6 R activation in the CNS with the antagonist MRS2578 inhibits feeding in mice ( Steculorum et al., 2017 ). Some ATP effects are as a result of adenosine, after breakdown of ATP, which is also involved in the activities of adipocytes. Adenosine increased lipolysis and induced thermogenesis in brown adipocytes via A 2A R ( Gnad et al., 2014 ). They showed that A 2A R antagonists counteract high fat-induced obesity in mice. Administration of A 2A R agonists to obese mice caused improvements in glucose homeostasis and adipose tissue inflammation, suggesting that this may show promise for therapeutic treatment of obesity ( DeOliveira et al., 2017 ). There is disturbance of adiponectin secretion in obese patients and adiponectin release is controlled by ATP ( Yamauchi and Kadowaki, 2013 ). A combination of Ca 2+ and ATP augments vesicular release of adiponectin ( Komai et al., 2014 ). Both ATP and adenosine have roles in the regulation of leptin secretion from adipocytes ( Szkudelski, 2007 ). There is abnormal fat distribution in P2X7R KO mice ( Beaucage et al., 2014 ). Manipulation of P2XR signalling may represent a novel therapeutic target in metabolically unhealthy obesity under inflammatory conditions ( Pandolfi et al., 2016 ). There is protection of rats on high fat diet by adenosine ( Lee, 2015 ). Perivascular adipose tissue of heavy smokers has increased expression of P2X7R and inflammasome components leading to increased release of inflammatory cytokines ( Rossi et al., 2014 ).

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SS and handling Editor declared their shared affiliation.

Reviews are available about purinergic signalling in the reproductive system in both health and disease ( Burnstock, 2014c ; Gorodeski, 2015 ). Abnormalities in purinergic signalling, including impaired ATP-mediated cavernosal relaxation, may contribute to erectile dysfunction associated with prostate enlargement and diabetes and may provide a target for therapy ( Hupertan et al., 2012 ; Wen and Xia, 2012 ). Normal penile erectile function involves a fine balance between contraction and relaxation in the corpus cavernosum smooth muscle. The strong relaxation induced by ATP via P2YR on human corpus cavernosum is comparable to that produced by NO, thus ATP together with an NO donor may prove to be effective for erectile disorders ( Hupertan et al., 2012 ). ATP released from sympathetic nerves leads to relaxation of cavernosum smooth muscle via P2Y 4 R, whereas ADP, after breakdown of ATP released from endothelial cells, acts via P2Y 1 R on endothelial cells to produce relaxation via NO ( Calvert et al., 2008a ). P2XR might also be involved ( Gur et al., 2007 ; Phillips et al., 2014 ). In anaesthetised dogs, ATP and adenosine induce penile tumescence, probably via A 2 R; pelvic nerve stimulation also produced tumescence. Impaired adenosine signalling via A 1 R contributes to erectile dysfunction ( Ning et al., 2012a ). Elevated adenosine signalling, via A 2B R, may contribute to priapism, where there is persistent penile erection lasting at least 4 h without sexual excitation ( Dai et al., 2009 ; Ning et al., 2012b ). A review highlights adenosine signalling in penile tissue as a potential therapeutic target to treat erectile disorders ( Wen and Xia, 2012 ). Impaired erectile function occurs in CD73 KO mice resulting in decreased endogenous adenosine. Corpus cavernosum from men suffering from vasculogenic erectile dysfunction show lower ectonucleotidase CD39 activity resulting in ATP accumulation. Human corpus cavernosum relaxation by P2R agonists was substantially reduced in erectile dysfunction patients ( Faria et al., 2010 ). ATP release from cavernosal tissue increased in patients following prostatectomy. Activating ATP-mediated pathways may restore erectile function in diabetics where there is impaired NO bioavailability. It has been suggested that P2X3 antagonists may improve recovery of erectile function ( Li C.L. et al., 2015 ). ATP increases the fertilising potential of sperm in humans and is used to treat spermatozoa during in vitro fertilisation. In P2X1R KO mice fertility was diminished with decreased number of spermatozoa in the ejaculate ( Mulryan et al., 2000 ). This raises the possibility that P2X1R antagonists would provide a safe and effective contraceptive ( White et al., 2013 ). In rat testes, during spermatogenesis there is differential, stage-dependent immunostaining for P2XR subtypes ( Glass et al., 2001 ), suggesting purinergic targets for both fertility and contraception. ATP triggers the acrosome reaction via P2X7R in human spermatozoa ( Torres-Fuentes et al., 2015 ). Purinergic signalling plays a role in the maturation of sperm cells in the testes. When the selective P2X1 and P2X3R desensitiser, α,β-meATP, was injected into the cauda epididymis, fertility in male rats was impaired. Adenosine stimulates human sperm motility via A 2 R. Purinergic compounds have been suggested for the therapeutic treatment of benign prostatic hyperplasia ( Andersson et al., 2002 ). Injection of BTX into the prostate, which reduces ATP and ACh release, treats bladder obstruction hyperactivity, by decreasing prostate size and as such improving urine flow rate ( Chuang and Chancellor, 2006 ). Regulating the proliferation of ovarian granulosa cells as well as steroidogenesis contributes to ovarian pathophysiology, since in rats with polycystic ovarian syndrome, theca hyperplasia occurs ( Salvetti et al., 2009 ). The hydrolysis of ADP and ATP was reduced by ovariectomy and oestradiol replacement therapy ( Pochmann et al., 2004 ). Ovarian tumours arise largely from the surface of squamous-to-cuboid mesothelium covering the ovary. ATP stimulated mitogen-activated kinase in neoplastic and pre-neoplastic surface epithelium, suggesting that co-released ATP from sympathetic nerves may contribute to the regulation of cell proliferation in neoplastic epithelial cells from the surface of the ovary ( Choi et al., 2003 ). In the human fallopian tube, ATP-mediated contractions are increased during acute purulent inflammation, probably as a result of upregulation of P2X1 and P2X2R ( Ziganshin et al., 2008 ). It has been claimed that targeting P2X7R may lead to new treatments to prevent uterine contractions in preterm deliveries. A naturally occurring P2X7 splice variant, the P2X7jR, blocks P2X7R-mediated actions ( Feng et al., 2006 ). It is co-expressed with P2X7R in female reproductive tract epithelia. The P2X7j isoform hetero-oligomerises with the P2X7R and co-expression of P2X7R and P2X7jR blocks ATP-induced pore formation, and abolishes agonist-induced apoptosis. P2Y 2 R agonists may be a non-hormonal alternative therapy for treating vaginal dryness in post-menopausal women. ATP is considered as a therapeutic target to control uterine activity during difficult labours ( Zafrah and Alotaibi, 2017 ). The P2X7R contributes to the control of cervical infections. P2X7R-mediated activation of cervical epithelial cells inhibits Chlamydia and mycobacteria infection ( Darville et al., 2007 ). ATP regresses endometriosis in a rat model ( Zhang C. et al., 2016 ). Adenosine in the placenta mediates the placental disturbances induced by alcohol, perhaps contributing to the pathogenesis of foetal alcohol syndrome ( Acevedo et al., 1997 ). Adenosine protects vaginal epithelial cells from T. vaginalis cytotoxicity ( Menezes and Tasca, 2016 ). Plasma adenosine is raised in hyperemesis gravidarum (severe morning sickness) and serves as a prejunctional modulator of sympathetic neurotransmission, which limits further progression of this pregnancy-related disease ( Yoneyama et al., 2004 ). ATP infusion in pregnant rats involved an inflammatory response that occurs in preeclampsia ( Spaans et al., 2014a ). Elevated placental adenosine signalling contributes to the pathogenesis of preeclampsia ( Iriyama et al., 2015 ). Hypoxia stimulates ATP release, which is rapidly broken down to adenosine by ectonucleotidases and women with preeclampsia and their foetuses have increased circulating adenosine concentrations. There is elevation of adenosine A 2A R expression in placental biopsies, villous explants and placental microvillous membranes ( von Versen-Höynck et al., 2009 ). In preeclampsia A 2B R on microvascular endothelial cells have also been implicated ( Escudero et al., 2008 ). Reduced adenosine-mediated angiogenesis in preeclamptic pregnancies may be associated with hypertension development in the offspring ( Escudero et al., 2014 ). The interaction between A 2A R and the angiotensin system may be involved in the early growth of the placenta. A 2A R expression is raised in pre-eclampsia, perhaps as a consequence of poor placental perfusion in preeclampsia ( Kurlak et al., 2015 ). Elevation of ADA activity in women with preeclampsia may contribute to their increased levels of uric acid and pro-inflammatory immune activity ( Giorgi et al., 2016 ). Release of ATP increases in preeclampsia following hypoxia and oxidative/nitrative stress, which acts on P2X4R to influence homeostasis of the placenta ( Roberts et al., 2007 ). There is deficient spiral artery remodelling and trophoblast invasion in preeclampsia, both of which may be inhibited by ATP-induced activated macrophages ( Spaans et al., 2014b ). It was concluded from a study of the relationship between the foeto-placental adenosine release and utero-placental circulatory insufficiency in pregnancies featuring preeclampsia, that foetal plasma adenosine increases before utero-placental insufficiency induces generalised foetal hypoxia ( Yoneyama et al., 1996 ). Prostate cancer is the second most common male cancer and the third leading cause of cancer death. Prostate cancer cells are sensitive to extracellular ATP. ATP and adenosine inhibit the growth of human prostate cancer cells ( Lertsuwan et al., 2017 ), identified at that time to act via P2Y 1 , P2Y 2 and/or P2Y 4 , P2Y 6 and P2Y 11 R subtypes. Activation of P2Y 1 R inhibited growth and induced cell death of prostate cancer PC-3 cells and P2Y 1 R agonists were claimed to be therapeutically promising for prostate cancer ( Wei et al., 2011 ). However, prostate tumour cells were shown later to also express P2X4, P2X5, and P2X7R in PC-3 cells and P2X4 and P2X5R in DU145 cells. ATP inhibited tumour cell growth, but not by UTP or adenosine, while 2′(3′)- O -(4-benzoylbenzoyl) ATP increased apoptotic cell death in PC-3 cells, probably via P2X7R. CD73 KO mice resist prostate carcinogenesis and CD73 promoted de novo prostate tumorigenesis. Anti-CD73 monoclonal antibodies decreased tumour growth and metastasis in the prostate ( Stagg et al., 2012 ). There are many polymorphisms of the P2X7R ( Fuller et al., 2009 ), which, as well as resulting in loss of function, alter receptor activity. Cytolytic P2X7R expression was found in 116 prostate cancer pathology specimens ( Slater et al., 2004 ). In normal tissues from patients with no evidence of cancer, P2X7R were not expressed, suggesting that the appearance of P2X7R is an early marker of prostate cancer. Prostate tumour cell proliferation is inhibited by adenosine. A 3 R activation by IB-MECA inhibited proliferation of prostate cancer cells and induced cell cycle arrest and apoptosis ( Aghaei et al., 2012 ). Activation of A 3 R suppressed prostate cancer metastasis ( Jajoo et al., 2009 ). The ATP synthase β subunit also plays a role in prostate cancer metastasis ( Li W. et al., 2017 ). Inhibition of growth of human breast cancer cells by ATP was shown for the first time in 1993. Chemotherapeutic ATP release from breast tumour cells of mice increased tumour regression through apoptosis and it was suggested that P2Y 2 and/or P2Y 4 R were involved. Oestrogen, acting via oestrogen receptor α, promoted proliferation of breast cancer cells by down-regulating expression of P2Y 2 R and reducing P2Y 2 R-induced increase in [Ca 2+ ] i ( Li et al., 2011 ). P2Y 2 R activation by ATP released from cancer cells induces the invasion of metastatic breast cancer cells ( Eun et al., 2015 ). Up-regulation of P2Y 6 R occurs in the mesenchymal phenotype of breast cancer cells and inhibition of P2Y 6 R may be a useful therapeutic target for metastasis of breast cancer ( Azimi et al., 2016 ; Ma X. et al., 2016 ). Antibody therapy with anti-CD73 inhibited breast tumour growth and metastasis ( Stagg et al., 2010 ). Bisphosphonates are also effective inhibitors of breast cancer ( Fehm et al., 2012 ). Proteomic analysis of human breast carcinoma revealed upregulation of ATP synthase in tumours and the ATP synthase inhibitor, aurovertin B, inhibited proliferation of several breast cancer cell lines ( Huang et al., 2008 ). Malignant breast carcinoma cells release ATP making the pre-metastatic environment suitable for micro-metastasis in lymph nodes and associated afferent lymph vessels ( Kawai et al., 2008 ). In breast tumour cells ATP increased [Ca 2+ ] i and high concentrations produced apoptosis via P2X7R. Activation of P2X7R in the human breast cancer cell line, T47D, increased cell migration and development of metastases, suggesting that P2X7R antagonists may have therapeutic roles ( Xia et al., 2015 ). The role of hypoxia in the regulation of tumour progression has been debated. However, P2X7R expression is increased by hypoxia and hypoxia-driven increase in P2X7R enhances tumour cell invasion and migration. Silencing of P2X5R inhibited cell proliferation and may be a new mechanism to target cancer metastasis. A 1 and A 3 R mRNA are expressed on human breast tumours ( Panjehpour et al., 2012 ). Adenosine induces tumour cell proliferation and migration of T-47D and MCF-7 breast carcinoma cell lines ( Mujoomdar et al., 2004 ). MDA-MB-231, a human breast cancer cell line, expressed A 2B R, which mediated cell proliferation and A 2B R inhibition slowed growth of breast tumours ( Cekic et al., 2012 ). A 3 R agonists reduced bone metastasis of breast cancer, suggesting a therapeutic approach to bone-residing breast cancer ( Varani et al., 2013 ). HeLa cells from human cervical cancer are used to study purinergic signalling involvement in cancer. Activation of P2Y 2 R with UTP and ATP caused proliferation of HeLa cells. P2Y 4 and P2Y 6 R expression increased during proliferation. Stimulation of P2Y 1 R on HeLa cells triggered epidermal growth factor receptor mitogen signalling and P2Y 1 antagonists reduced proliferation. Permeabilisation of cervical cancer cells to a cytotoxin is activated by P2YR ( Bukhari et al., 2015 ). P2Y 6 R activation induces HeLa cell migration ( Gendaszewska-Darmach and Szustak, 2016 ). Oestrogen reversed the apoptotic activity mediated by P2X7R in normal human cervix, but not in cervical epithelial cancer cells. A truncated P2X7R variant (P2X7-j), expressed in cervical cancer cells, antagonised the P2X7R through hetero-oligomerisation. Gentle mechanical stimulation released ATP from HeLa cells. The presence of ectonucleotidase in human cervical cancer cell regulates the levels of nucleotides, limiting their effects ( Beckenkamp et al., 2014 ). ATP raised [Ca 2+ ] i and stimulated growth of SKOV-3 and OVCAR-3 human ovarian carcinoma cells. ATP may act as a messenger to control the ovarian epithelial cell cycle through P2Y 2 R on human ovarian cancer cells. A 2 R antagonists inhibited angiogenic activity of human ovarian cancer cells. The treatment of human ovarian carcinoma with cisplatin in the presence of ATP results in additive cytotoxicity ( Rotte et al., 2010 ). P2X7R were highly expressed in both human ovarian tumours and ovarian cancer cell lines and paracrine release of ATP acts on P2X7R to cause proliferation of ovarian cancer cells ( Vazquez-Cuevas et al., 2014 ). P2Y 2 R play a role in the control of the cell cycle and in the suppression of proliferation of human endometrial carcinoma cells. The P2X7R has been used as a biomarker for uterine epithelial cancers. There is decreased P2X7R expression on endometrial epithelium in pre-cancerous and cancer cells ( Li et al., 2007 , 2009 ). Activation of P2X7R-dependent apoptosis has a chemotherapeutic growth-preventive effect on pre-cancerous and early cancerous epithelial lesions ( Fu et al., 2009 ; Gorodeski, 2009 ). Loss of CD73 in endometrial cancer allows for tumour progression ( Bowser et al., 2016 ). A higher proportion of ADA2 ∗ 1/ ∗ 1 genotype was observed in women with uterine leiomyomas ( Gloria-Bottini et al., 2016 ).

Investigations into purinergic signalling and its roles in disorders of the CNS have been reported, for instance following surgery, stroke, accidents and ischemia, neurodegenerative diseases (such as Parkinson’s, Alzheimer’s and Huntington’s diseases), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), epilepsy and neuropsychiatric disorders (including schizophrenia, depression and anxiety). Reviews covering this topic are available ( Burnstock, 2008b ; Burnstock et al., 2011 ), including the recent attention to the development of centrally penetrant P2X7R antagonists for the treatment of CNS disorders ( Burnstock and Verkhratsky, 2012 ; Puchalowicz et al., 2014 ; Sperlagh and Illes, 2014 ; Burnstock, 2015b ; Cisneros-Mejorado et al., 2015b ). Neurodegeneration in the CNS is associated with inflammation and damage to both neurons and glia ( Rama Rao and Kielian, 2015 ). Reviews are available that include coverage of early papers concerned with neurodegenerative diseases ( Puchalowicz et al., 2014 ; Santiago et al., 2014 ; Fasullo and Endres, 2015 ; Förster and Reiser, 2015 ; Burnstock, 2016a ; Fumagalli et al., 2016 ). Recent attention has been directed toward the use of P2X7R antagonists for the treatment of neurodegenerative diseases ( Metzger et al., 2017 ). However, adenosine acting via A 2A R has also been claimed as a promising therapeutic agent for the prevention and treatment of neurodegenerative diseases ( Cunha, 2016 ; Harmse et al., 2016 ; Olatunji et al., 2016 ). There is progressive cognitive impairment in AD, with prominent deficits in short term memory. The potential of purinergic drugs for the treatment of AD has attracted much interest in recent years. Previous studies have led to the proposal that both P2X7R and P2Y 4 R antagonists are potential therapeutic targets for the treatment of AD ( Erb et al., 2015 ; Miras-Portugal et al., 2015 ; Woods et al., 2016 ). It has been suggested that the blockade of P2Y 1 R may have therapeutic potential against cognitive disturbances in AD ( Guzman and Gerevich, 2016 ). β-Amyloid increased release of ATP, which potentiated excitatory synaptic activity via P2XR, effects that were blocked by P2X antagonists ( Sáez-Orellana et al., 2016 ). The glycosylphosphatidylinositol-anchored prion protein binds to and modulates the expression of P2X4R, which may be involved in AD ( Carneiro et al., 2016 ). Recently, a potential role has been proposed for ATP-sensitive potassium channel (K ATP ) modulation as a therapeutic strategy against AD ( Salgado-Puga et al., 2017 ). Adenosine A 3 R agonists suppress amyloid-β protein precursor internalisation and amyloid-β generation ( Li S. et al., 2015 ). Hippocampal adenosine A 2A R up-regulation is necessary to trigger memory dysfunction in AD ( Cunha, 2015 ). The involvement of adenosine A 2A R in PD and their interactions with dopamine receptors has attracted most attention (see Jenner, 2014 ). A 2A R antagonists have been proposed for the treatment of PD ( Jenner, 2014 ; Mori, 2014 ; Pinna, 2014 ; Navarro et al., 2016 ). K ATP ( Dragicevic et al., 2015 ) and P2X1R ( Gan et al., 2015 ; Navarro et al., 2016 ; Yang Z. et al., 2016 ) in PD have also been reported, perhaps indicating new therapeutic targets. Clinical trials istradefylline, an A 2A R antagonist, have taken place and it may have a beneficial effect in conjunction with commonly used anti-Parkinson’s therapies ( Tao and Liang, 2015 ; Uchida et al., 2015 ; Vorovenci and Antonini, 2015 ), although in a later study, istradefylline was shown to enhance amyloid-β generation and γ-secretase activity ( Lu et al., 2016 ). Oligomerisation kinetics of A 2A and dopamine D 2 R have important implications for PD ( Casadó-Anguera et al., 2016 ; Ferré et al., 2016 ; Guixà-González et al., 2016 ). Clinical trials for A 2A antagonists for PD are assessed in a review ( Navarro et al., 2016 ). A 2A R inhibition stopped rotenone-induced motor impairment in a rat model of PD ( Fathalla et al., 2016 ). A 1 as well as A 2A R antagonists were recommended as promising candidates for treatment of PD in a recent paper ( Essawy et al., 2017 ). P2X7R antagonists have also been implicated in this disease ( Jörg et al., 2014 ; Wang et al., 2017 ). A P2X7R antagonist, brilliant blue G, was recently shown to be protective in a lipopolysaccharide (LPS) animal model of PD ( Wang et al., 2017 ). Earlier studies reported that A 2A R agonists could be therapeutically useful for HD and that P2X7R antagonists inhibited neuronal apoptosis and attenuated body weight loss and motor co-ordination deficit in HD patients. Adenosine A 2A R inhibition reversed working memory deficits of HD at early stages models and it was proposed that A 2A R antagonists may therapeutically reverse the cognitive deficits in HD patients ( Li W. et al., 2015 ). In earlier publications, A 1 R agonists were suggested to be therapeutic targets ( Ferrante et al., 2014 ; Lee and Chern, 2014 ). It has been claimed that K ATP channels may be potential targets for treatment of HD ( Gupta and Sharma, 2014 ). Inhibition of equilibrative nucleoside transporter 1 enhances the adenosine level and may be a potential therapeutic approach for treating HD ( Kao et al., 2017 ). Involvement of A 2A R, P2X4R and P2X7R in ALS has been reported ( Volonté et al., 2016 ). A 1 R have also been implicated in ALS ( Nascimento et al., 2015 ). Preconditioning with latrepirdine, an adenosine 5′-monophosphate (AMP)-activated protein kinase activator, was beneficial in the SOD1 mouse model of ALS ( Coughlan et al., 2015 ). A 2A R activation facilitated neuromuscular transmission in SOD1 mice during the pre-symptomatic but not symptomatic phase of the disease ( Nascimento et al., 2014 ). Pharmacological inhibition of A 2A R protects against degeneration of spinal motor neurons in the mouse SOD1G93A ALS model ( Ng et al., 2015 ). P2X7R expression was significantly reduced, leading to Ca 2+ disturbances in peripheral blood mononuclear cells in ALS patients ( Liu J. et al., 2016 ). Low concentrations of endogenous ATP acting on P2X7R induced motor neuron death ( Gandelman et al., 2013 ). Spinal cord pathology was reduced by P2X7R inhibitors in the mouse SOD1 ALS model ( Apolloni et al., 2014 ). P2X7R and the P2YR-like GPR17 are involved in MS ( Plemel et al., 2014 ; Burnstock, 2015c ; Ou et al., 2016 ). There is upregulation of ecto-5′-nucleotidase (CD73) in experimental autoimmune encephalomyelitis, which is an MS animal model ( Lavrnja et al., 2015 ). Genetic variants in P2X7R affect susceptibility to MS ( Gu et al., 2015 ; Sadovnick et al., 2017 ). The importance of inflammation in the responses to brain injury has been reviewed and the involvement of P1R and P2R ( Fiebich et al., 2014 ; Beamer et al., 2016 ) and of P2X7R in particular ( Burnstock, 2016b ; Giuliani A.L. et al., 2017 ). The roles of purinergic signalling in neurodegeneration as a consequence of brain injury, neuroprotection and neuroregeneration have been discussed in reviews ( Hu et al., 2014 ; Burnstock, 2016a ). Ectonucleotidase activities and nucleotide levels in serum are altered by brain stab injury ( Parabucki et al., 2014 ; Laketa et al., 2015 ). P2X7R antagonists appear to be effective as a treatment for radiation injury ( Xu et al., 2015 ). Astrocytic p -connexin 43 stimulates neuronic autophagy through P2X7R activation in the hippocampus, resulting in brain injury-induced cognitive deficit repair ( Sun L. et al., 2015 ). P2X7R antagonists have been implicated as a novel target to prevent secondary neurological injury after traumatic brain injury ( Kimbler et al., 2012 ) and after spinal cord injury ( Peng et al., 2009 ). P2X7R antagonists could be therapeutically effective to treat stroke patients ( Kuan et al., 2015 ). P2X7R stimulation in acute ischaemic stroke is beneficial by restricting early oedema formation and perhaps by modulating responses of glia ( Kaiser et al., 2016 ). Hypoxic-ischaemic brain injury increases intracellular Ca 2+ in oligodendrocytes, which is partly mediated by P2X7R ( Fern et al., 2014 ). P2X7R antagonists may provide a new target for the treatment of cerebral ischaemia ( Bai and Li, 2013 ; Yu Q. et al., 2013 ) and for the prevention of ischaemic damage to oligodendrocytes ( Domercq et al., 2010 ). P2X7 antagonists or inhibition of pannexin-1 channels reduced brain damage following ischaemia ( Cisneros-Mejorado et al., 2015a ; Mahi et al., 2015 ). Activation of upregulated P2Y 1 R results in neuroblast migration to sites of brain damage ( Cao et al., 2015 ). Microglial P2Y 12 R activation follows neuronal injury ( Swiatkowski et al., 2016 ). A 2A R activation was suggested for the treatment of brain injury and subsequent neuroinflammation ( Dai and Zhou, 2011 ). A 2A R on cells derived from bone marrow modulate white matter lesions following chronic cerebral hypoperfusion ( Ran et al., 2015 ). Diadenosine tetraphosphate (Ap 4 A) may be a good candidate for traumatic spinal cord injury treatment ( Reigada et al., 2017 ). Adenosine kinase facilitated astrogliosis after traumatic brain injury and its inhibition in reactive astrocytes ameliorated astrogliosis-induced cell death ( Jin et al., 2016 ). ATP and adenosine concentrations in the brain were significantly raised during brain ischaemia to stimulate P1R and P2R ( Cisneros-Mejorado et al., 2015b ; Pedata et al., 2016 ). P2X4R expressed by microglia are involved in post-ischaemic brain inflammation ( Cheng et al., 2014 ). P2X4R are required for neuroprotection via ischemic preconditioning ( Ozaki et al., 2016 ). Neuronal K ATP channels play a role in hypoxic preconditioning where they decrease neonatal hypoxic-ischaemic brain injury; it has been proposed that openers of K ATP channels may prove to be therapeutically beneficial ( Sun H.S. et al., 2015 ). Intranasal administration of guanosine reduced ischaemic brain damage in rats ( Ramos et al., 2016 ). Ambiguously, both A 2A R agonists and antagonists may protect against ischaemic brain injury ( Pedata et al., 2014 ). A 1 R contribute to immune responses following neonatal hypoxic ischaemic brain injury ( Winerdal et al., 2016 ). A valuable review of the involvement of P1R, P2XR and P2YR in brain ischemia is available ( Pedata et al., 2016 ). This topic is explored in recent reviews ( Rodrigues et al., 2015 ; Burnstock, 2016a ; Illes et al., 2016 ). Inhibition of P2XR and P2YR as well as activation of P1R following ATP breakdown to adenosine released from CNS cells, have been shown to be neuroprotective. Activation of the pannexin 1/P2X7R complex contribute to the neuroprotection that occurs after ischaemic pre- and post-conditioning ( Mahi et al., 2015 ). Docosahexaenoic acid in the diet is thought to be a purinergic modulator via P2XR where it protects against neurodegenerative diseases ( Molz et al., 2015 ). Neuroprotection mediated by microglia is associated with P2X7R activation and release of tumour necrosis factor-α ( Masuch et al., 2016 ). Blockade of P2X7R provides neuroprotection against stroke, traumatic brain injury and subarachnoid haemorrhage ( Zhao H. et al., 2016 ). P2X7R antagonists improve recovery after spinal cord injury ( Wang et al., 2004 ). From a recent study on P2Y 2 R knockout (KO) mice and murine cell lines, it was concluded that P2Y 2 R play a neuroprotective role in neurological disorders, especially AD ( de Diego-Garcia et al., 2017 ). Neuroprotection is claimed to be mediated by P2Y 13 nucleotide receptors in neurons ( Pérez-Sen et al., 2015 ). Activation of P2Y 2 R evokes regeneration of glial cells and nerves and the P2YR-like GPR17 evokes oligodendrocyte regeneration. Neural stem cell activation leads to neuroregeneration, probably via P2X4R and P2X7R. Reviews focused on the roles played by P2X4R and P2X7R ( Miras-Portugal et al., 2015 ) and A 2A R ( Ribeiro et al., 2016 ) in neurodegeneration and neuroprotection have been published. Several antipsychotic drugs (e.g., chlorpromazine, fluspirilene, and haloperidol) were found to antagonise responses mediated by P2XR. These antipsychotic drugs act therapeutically by inhibiting dopaminergic hyperactivity by suppressing P2X-mediated effects (see Burnstock, 2015b ). Brain penetrant P2X7R antagonists are being developed as drug targets for psychiatric diseases ( Bhattacharya and Biber, 2016 ). Reviews about purinergic signalling in psychiatric disorders, such as addiction, depression, schizophrenia, bipolar disorder and autism, have been previously published ( Yamada et al., 2014 ; Lindberg et al., 2015 ; Krügel, 2016 ). The possibility has been raised that P1R agonists might be beneficial in the therapy of psychiatric disorders ( Cieslak et al., 2016 ). Adenosine neuromodulation in schizophrenia has received the most attention (see Rial et al., 2014 ; Ciruela et al., 2015 ; Turcin et al., 2016 ). Deletion of A 2A R from astrocytes interferes with glutamate homeostasis resulting in cognitive and psychomotor impairment in schizophrenia ( Matos et al., 2015 ). It has been suggested that P2X7R and pannexin 1 channels are involved (see Burnstock and Verkhratsky, 2012 ; Avendano et al., 2015 ). P2X7R, mediating neuroinflammation via microglial activity, contribute to bipolar disorder (see Gubert et al., 2013 , 2016 ; Barron et al., 2014 ). Serum concentrations of uric acid increase in different clinical phases of bipolar disorder ( Albert et al., 2015 ) and in those patients treated with lithium ( Muti et al., 2015 ). The purinergic system may become dysregulated during manic episodes and it has been proposed that raised uric acid levels could be a useful indicator of bipolar disorder manic phases ( Bartoli et al., 2017a ). Purinergic modulators that reduce levels of uric acid may be therapeutically beneficial ( Bartoli et al., 2017b ). However, a decrease in serum adenosine levels in bipolar disorder patients was reported ( Gubert et al., 2016 ). Reviews describing the roles of both P1 (A 1 and A 2A ) and P2X7R in mood disorders are available ( Yamada et al., 2014 ; Ortiz et al., 2015 ). Antidepressant effects of P2X7 antagonists have been reported ( Pereira et al., 2013 ; Zhang K. et al., 2016 ). P2X2R in the medial prefrontal cortex mediate the antidepressant-like actions of ATP released from astrocytes ( Cao et al., 2013 ). Brilliant blue G, a P2X7R antagonist, had antidepressant and anti-inflammatory actions in mice after LPS administration ( Ma et al., 2014 ). The P2X7R antagonist, A-804598, affected neuroimmune and behavioural features of stress ( Catanzaro et al., 2014 ). Stress-related mood disorders activate the inflammasome via release of ATP and stimulation of P2X7R ( Iwata et al., 2016 ). Co-expression of wild-type P2X7R with the polymorphism variant Gln460Arg alters receptor function associated with mood disorders ( Aprile-Garcia et al., 2016 ). Increased K ATP channel activity due to a mutation led to reduced anxiety in mice ( Lahmann et al., 2014 ). K ATP channels are involved in the pathogenesis of depression and may be a therapeutic target for this disorder ( Fan et al., 2016 ). Altered levels of several ATP-dependent chromatin remodelling factors may be linked to high trait anxiety ( Wille et al., 2016 ). A 2A R antagonists were reported to have antidepressant activity ( Yamada et al., 2014 ). Creatine and ketamine had antidepressant effects, probably mediated by activation of A 1 R and A 2A R ( Cunha et al., 2015 ). Caffeine, acting as an A 2A R antagonist, prevented depression triggered by chronic stress ( Kaster et al., 2015 ; Dziubina et al., 2016 ). Striatal and extrastriatal A 2A R in the forebrain regulate fear responses in mice ( Wei et al., 2014 ). A 2A R mediated increased interleukin (IL)-1β in the brain contributed to anxiety ( Chiu et al., 2014 ). Fear and anxiety in a mouse model of post-traumatic stress disorder were alleviated by administration of a derivative of adenosine, WS0701 ( Huang et al., 2014 ). A 1 R agonists have been used to treat anxiety, but have troublesome side-effects so positive allosteric modulators have been developed as potent anxiolytic agents ( Vincenzi et al., 2016 ). An antidepressant-like effect of inosine in mice has been reported ( Gonçalves et al., 2017 ). Adenosine in addition to suramin, a non-selective ATP antagonist, are reported to improve behaviour in autistic individuals ( Masino et al., 2013 ; Naviaux et al., 2014 , 2015 ; Hamidpour et al., 2016 ). Targeting A 2A R may offer strategies for combating drug addiction. Striatopallidal A 2A R signalling in the dorsomedial striatum has been suggested as a therapeutic target in drug addiction by reducing habit formation ( Li et al., 2016 ). Caffeine potentiates the addictive effects of drugs of abuse, including cocaine and amphetamine derivatives ( Ferrée, 2016 ). Treatment of rodents with a P2X3R antagonist diminished opioid tolerance ( Tai et al., 2010 ) and tolerance to morphine-induced antinociception ( Ma et al., 2015 ). Lead-induced neuroinflammation via P2X7R may be responsible for the intensification of morphine tolerance ( Baranowska-Bosiacka et al., 2016 ). Opiate-induced changes in brain adenosine levels may be associated with opiate addiction and withdrawal ( Wu et al., 2013 ). Adenosine, acting via A 2A R, regulates addiction induced by cocaine. Women are more sensitive to cocaine and are therefore more vulnerable to becoming addicted. Adenosine antagonists may be an effective treatment ( Broderick and Malave, 2014 ). A 2A -D 2 receptor-receptor interactions in the dorsal striatum are differentially affected by cocaine, which contributes to compulsive drug seeking ( Pintsuk et al., 2016 ). Interactions between striatal A 2A and glutamate (mGlu5) receptors modulate the drug-seeking effects of methamphetamine ( Wright S.R. et al., 2016 ). Methamphetamine produces alterations in adenosine receptor expression in the nucleus accumbens ( Kavanagh et al., 2015 ). A 2A R antagonism in dorsomedial striatum reduces methamphetamine addiction ( Furlong et al., 2015 ). Adenosine A 2A R integrate the rewarding and motivational behaviours of methamphetamine ( Chesworth et al., 2016 ). Behavioural sensitisation provides a model in animals for drug craving that underlies human addiction and sensitisation to amphetamine was reduced by P2Y 1 R antagonists in the mesocortico-limbic dopaminergic system ( Krügel et al., 2013 ). A 2A R agonist treatment can help counteract nicotine addiction ( Jastrzebska et al., 2014 ). A 2A R are a potential target for the treatment of alcohol abuse ( Micioni Di Bonaventura et al., 2012 ; Houchi et al., 2013 ). Regulation in adenosine signalling in striatal circuits in alcohol addiction was reviewed ( Nam et al., 2013 ). A 1 R signalling contributes to the regulation of basolateral amygdala excitability and to the pathophysiology of alcohol addiction ( Rau et al., 2014 ). A review of alcohol addiction discusses the role of adenosine ( Michalak and Biala, 2016 ). P2X4R modulate synaptic signalling associated with alcohol addiction ( Franklin et al., 2014 ; Khoja et al., 2016 ). Early focus was on the role of P1R in epileptic seizures, but P2X4R and P2YR, but especially P2X7R antagonists have been recently explored as neuroleptic agents (see Burnstock, 2015b ; Engel et al., 2016 ; Rassendren and Audinat, 2016 ; Beamer et al., 2017 ; Cieslak et al., 2017 ). Seizure-induced increases in microglial process numbers were reduced and kainate-induced seizure behaviours were exacerbated in P2Y 12 KO mice ( Eyo et al., 2014 ). Antiepileptic consequences of deep brain stimulation may be mediated by P1R activation ( Miranda et al., 2014 ). P2X3R expression was upregulated in epileptic rats and humans and it was postulated that P2X3R antagonists might be therapeutically effective ( Zhou X. et al., 2016 ). The release of adenosine and ATP during on-going epileptiform activity was measured with microelectrode biosensors ( Frenguelli and Wall, 2016 ). ATP levels were increased during epileptic seizures, which were not of neuronal origin ( Lietsche et al., 2016 ). There is an insightful Editorial about purinergic signalling-induced neuroinflammation in epilepsy ( Engel, 2016 ). A role for post-transcriptional control of the P2X7R expression has been proposed and therapeutic targeting of microRNA-22 was suggested to prevent development of epilepsy and inflammation ( Jimenez-Mateos et al., 2015 ). Purinergic signalling, via P2X7R, regulates neonatal seizures associated with hypophosphatasia, an inherited metabolic bone disease characterised by spontaneous seizures ( Sebastián-Serrano et al., 2016 ). P2X7R antagonists were effective against hypoxia-induced neonatal seizures in mice ( Rodriguez-Alvarez et al., 2017 ). The role of ATP in migraine was initially suggested to involve vascular events (see Burnstock and Verkhratsky, 2012 ; Haanes and Edvinsson, 2014 ). Later, P2X3R in nociceptive brain areas, including the thalamus and trigeminal nucleus, were investigated and their interaction with P2Y 1 R in trigeminal neurons ( Hullugundi et al., 2014 ; Marchenkova et al., 2016 ). The therapeutic potential of antagonists to P2X7R for migraine treatment has been proposed ( Gölöncsér and Sperlágh, 2014 ), as well as P2X3R and P2X2/3R antagonists ( Kilinc et al., 2015 ; Yegutkin et al., 2016 ). Reviews have been published about the roles and therapeutic potential of purinergic signalling in the aetiology of migraine ( Cieslak et al., 2015 ; Yegutkin et al., 2016 ). P1R and P2R involvement in neuropathic pain has been discussed in reviews ( Burnstock and Sawynok, 2010 ; Burnstock et al., 2011 ; Burnstock, 2014a , 2016c ). The discovery by Inoue and colleagues that antagonists to P2X4R on microglia are effective against neuropathic pain was particularly important (see Tsuda, 2016 ). Antagonists to P2X7R and P2Y 12 R also act on microglia to reduce neuropathic pain (see Tsuda and Inoue, 2016 ; Tsuda, 2017 ). A 3 R agonists delay the development of neuropathic pain ( Janes et al., 2016 ). Glial P2Y 2 R are potential targets for the management of trigeminal-related pain ( Magni et al., 2015 ). Pannexin 1 and P2X7R interactions have been suggested to play a role in chronic pain ( Bravo et al., 2015 ). P2X7R antagonists have been recommended for the treatment of central post-stroke pain ( Kuan et al., 2015 ). A review discusses the use of P2XR subtype antagonists for the treatment of central neuropathic pain ( Kuan and Shyu, 2016 ). Purinergic signalling in the spinal dorsal horn and anterior cingulate cortex appear to be involved in neuropathic pain ( Tsuda et al., 2017 ). P2X3 and P2X2/3R blockade has also been claimed to reduce chronic pain ( Cantin et al., 2012 ; Xu et al., 2012 ; Giniatullin and Nistri, 2013 ). Neuroblastoma, a rare childhood tumour, expresses P2X7R, which mediate proliferation. The P2X7R appears to be a regulator of neuroblastoma metabolic activity, angiogenesis and growth and may be a target for neuroblastoma treatment ( Amoroso et al., 2015 ; Gomez-Villafuertes et al., 2015 ). The action of the antitumor agent, temozolomide, was increased by the antiproliferative actions of P2X7R agonists and antagonists to A 3 R and P2Y 1 R on human glioblastoma ( D’Alimonte et al., 2015 ). P2X4R may also be involved in human neuroblastoma ( Gualix et al., 2015 ). P2X7R were over-expressed in human malignant gliomas and P2X7R antagonists decreased tumour cell numbers ( Fang et al., 2015 ; Morrone et al., 2016 ; McLarnon, 2017 ). Extracellular nucleotides control glioma growth via P2X7R and P2Y 6 R activation ( Braganhol et al., 2015 ). P2X7 and A 2A R activation leads to release of cytokines by macrophages, which was prevented by antagonists to these receptors ( Bergamin et al., 2015 ). P2X7R antagonists blocked the cell cytotoxicity caused by irradiation for glioma ( Gehring et al., 2015 ). Purine nucleoside phosphorylase is released from rat C6 glioma cells, contributing to the purinergic system homeostasis and exhibiting a pathophysiological role ( Giuliani P. et al., 2017 ). It was claimed that K ATP channels are associated with tumorigenesis of human glioma ( Ru et al., 2014 ). A 3 R blockade enhances the actions of antitumour drugs used against human glioblastoma stem-like cells ( Torres A. et al., 2016 ). A 1 and A 2B R also sensitise glioblastoma stem cells to chemotherapy ( Daniele et al., 2014 ). P2Y 2 R interactions with caveolin-1 represents a novel target for human astrocytoma cells ( Martinez et al., 2016 ). P2Y 11 R appear to be associated with narcolepsy ( Kornum et al., 2011 ). Reduced endothelial dilation to ATP in cerebral arteries occurred in a rat model of obstructive sleep apnoea ( Crossland et al., 2013 ). Adenosine is a key player in the regulation and maintenance of sleep-wake dependent neural activity changes, where dysregulation can lead to sleep-wake disorders ( Holst et al., 2016 ). The A 2A R antagonist, SCH58261 , overcame the blood–brain barrier dysfunction as a result of sleep restriction ( Hurtado-Alvarado et al., 2016 ).

Purinoceptors are expressed widely by endocrine glands ( Burnstock and Knight, 2004 ). Actions of purines have been described in the pituitary gland, with implications for pathological as well as physiological states. 5′-Nucleotidase activity in platelets is changed by hyper- and hypothyroidism and could be a further mechanism by which alterations in thyroid hormones are related to vascular diseases. The role of purinergic signalling in thyroid hormone activities in health and disease has been reviewed ( Silveira et al., 2013 ). In the ovary, follicular atresia involves cellular degeneration that is due to apoptosis evoked by P2X7R activation in both somatic and germinal follicular cells. Adiponectin, secreted by adipocytes, is anti-inflammatory, protecting against fatty liver disorder, insulin-resistant type 2 diabetes and atherosclerosis. A review including the limited knowledge of purinergic signalling in the endocrine system has been published ( Burnstock, 2014b ). Fibroblast P2YR are impaired in type 2 diabetes, which leads to lower glucose uptake, indicating that P2YR could be therapeutically useful as antidiabetic drugs. Analogues of the P2Y 1 R agonist, 2-methylthio ADP, have been developed for the treatment of type 2 diabetes ( Yelovitch et al., 2012 ). P2YR activation potentiates insulin secretion making it a promising therapeutic target for type 2 diabetes ( Zhang et al., 2015b ; DeOliveira et al., 2017 ). Adenosine, acting via A 2A R, increases pancreatic β-cell proliferation, and has been suggested as a therapeutic target for diabetes ( Schulz et al., 2016 ). Uridine adenosine tetraphosphate may be a therapeutic target for diabetes ( Matsumoto et al., 2015 ). There is an enhancement of P2X7R-induced apoptosis on the retinal microvasculature in early diabetes. P2X7R located on glucagon-containing α cells in pancreatic islets in STZ-induced diabetic animals increase and they migrate centrally to replace missing insulin-containing β cells. P2X7R antagonists have been proposed as a therapeutic target to cause immunosuppression and tolerance induction in pancreatic islet transplantation ( Aikin, 2013 ; Vergani et al., 2013 ). NONRATT021972 siRNA decreases the expression of P2X7 mRNA and protein in DRG, reducing mechanical and thermal hyperalgesia in type 2 diabetic rats ( Liu et al., 2016 c). Type 1 diabetes impairs P2X7R signalling in osteocytes that affects osteoblast function and maintenance of bone health ( Seref-Ferlengez et al., 2016 ). STZ-induced type 1 diabetes was prevented in P2X7R KO mice ( Vieira et al., 2016 ). Diabetic sympathetic neuropathy in type 2 diabetic rats was reduced by decreasing the expression of P2X7R in superior cervical ganglia ( Wu et al., 2016 ). P2X7R are expressed in pancreatic cancer cells. P2X7R antagonists are likely to be effective therapeutic agents ( Giannuzzo et al., 2016 ). P2X3R antagonists have been proposed for the treatment of diabetic neuropathic pain ( Guo et al., 2015 ; Zhang et al., 2015b ; Peng et al., 2017 ; Rao et al., 2017 ). Recently P2X7R have also been claimed to be involved in painful diabetic neuropathy in rats ( Liu et al., 2017 ). P2X7R polymorphisms are associated with severe diabetic neuropathic pain scores ( Ursu et al., 2014 ). A 1 R agonists improve mechanical allodynia in a painful diabetic neuropathy mouse model ( Katz et al., 2015 ). There is up-regulation of hippocampal A 2A R in STZ-diabetes and A 2A R antagonists gave neuroprotection. A 2A R are a therapeutic target for diabetic retinopathy ( Ibrahim et al., 2011 ). Reduced expression of A 1 R in pancreatic α-cells could result in the development of type 1 diabetes ( Yip et al., 2013 ). There is increased expression of A 2B R in women with gestational diabetes mellitus ( Wojcik et al., 2014 ). In diabetic rats, A 2B R agonists improve erectile function ( Wen et al., 2015 ). It was suggested that reduction in A 3 R expression/function may slow the progression of diabetic neuropathy ( Yan H. et al., 2016 ). Blockade of ATP synthase interaction with cyclophilin D has been proposed as a promising therapeutic target to treat diabetic encephalopathy ( Yan S. et al., 2016 ). K ATP channels have neuroprotective effects in patients with type 2 diabetes ( Liu R. et al., 2016 ; Rubaiy, 2016 ). P2Y 1 and P2Y 2 R mediate relaxation of the rat corpus cavernosum and may improve erectile function in men with diabetes ( Gür et al., 2009 ). An A 2A R agonist was shown to enhance healing of chronic diabetic foot ulcers in a clinical trial ( Squadrito et al., 2014 ; Montesinos et al., 2015 ). Adenosine kinase inhibitors attenuate inflammation in diabetic retinopathy ( Elsherbiny et al., 2013a ). Retinal inflammation in diabetic retinopathy is mediated by ADA2 and the anti-inflammatory activity of A 2A R signalling is impaired with increased ADA2 activity ( Elsherbiny et al., 2013b ). Several reviews about purinergic signalling in diabetes and its therapeutic potential are available ( Burnstock and Novak, 2013 ; Cieslak and Roszek, 2014 ; Antonioli et al., 2015 ; Fotino et al., 2015 ; Kishore et al., 2015 ; Merighi et al., 2015 ; Vindeirinho et al., 2016 ). The involvement of purinergic signalling in diabetic nephropathy is discussed later in the Section on Kidney.

Reviews have been published concerned with purinergic signalling in immune cell trafficking at sites of infection ( Ferrari et al., 2016b ) and P2X7R in infectious inflammatory diseases ( Morandini et al., 2014 ). Apoptosis of macrophages via ATP-stimulated P2X7R leads to the killing of the mycobacteria they contain, which may lead to new strategies to combat bacterial infections. The cytotoxic effects of ATP on macrophages are through P2X7R, while the bactericidal actions of ATP (and UTP) may be though P2Y 2 R. There is a valuable article about purinergic signalling in infection and autoimmune diseases ( Savio and Coutinho-Silva, 2016 ). The P2X7R is a potential target for the treatment of Clostridium perfringens type C infection ( Nagahama et al., 2015 ). P2X7R activation modulates cell death during Porphyromonas gingivalis infection ( Almeida-da-Silva et al., 2016 ). P2X7 and P2X4R activation is protective during severe Escherichia coli infection ( Greve et al., 2017 ). P2X7R develop the inflammatory response associated with sepsis ( Santana et al., 2015 ) and might serve as a therapeutic target to ameliorate brain damage in sepsis ( Savio et al., 2016 ). A 2A R have also been recommended as a therapeutic target to treat sepsis ( Sivak et al., 2016 ). The role of purinergic signalling in the immune response in sepsis has been reviewed ( Ledderose et al., 2016 ). ATP protects against sepsis through P2X7R on macrophages by enhancing intracellular bacterial killing ( Csóka et al., 2015 ). Chlamydiae are intracellular bacterial pathogens and these infections are influenced by inflammasomes and purinergic signalling ( Pettengill et al., 2016 ). Purines modulate the inflammatory response in rats infected by Cryptococcus neoformans ( de Azevedo et al., 2016 ). ADP facilitates monocyte recruitment in bacterial infection ( Zhang X. et al., 2016 ). ATP synthase has been proposed as a target to kill Mycobacterium tuberculosis ( Tantry et al., 2017 ). Purinergic enzymatic activities in lymphocytes and cardiomyocytes modulated the inflammatory responses of mice infected by T. cruzi ( do Carmo et al., 2017 ). Adenosine restored LPS-inhibited chemotaxis via A 1 R, making this a promising therapeutic strategy for infectious diseases ( Xu et al., 2017 ). Infection by Schistosoma mansoni , a parasitic blood fluke, results in thymic atrophy. The cloning and characterisation of a P2XR (schP2X) from S. mansoni was the first non-vertebrate ATP-gated ion channel, which could be an alternative drug target to treat schistosomiasis (see Burnstock and Kennedy, 2011 ). Purinergic signalling influences the immune response to infection by Leishmania , a protozoan parasite ( Chaves et al., 2014 ; Figueiredo et al., 2016 ). Purinergic signalling is involved in Trichomonas vaginalis parasite infection ( Menezes and Tasca, 2016 ). The dysfunction of P1, P2Y and P2X7R and NTPDases are likely to contribute to morbidity due to human schistosomiasis ( Silva, 2016 ). P2X7R are important in parasite control as they regulate T effector cells and inflammation during L. amazonensis infection ( Figliuolo et al., 2017a ). A commentary about ATP as an initiator of immunity to parasitic infections has been published ( Nelson et al., 2017 ). P2X7R modulate the antiviral and inflammatory processes that occur during Dengue virus-2 infection ( Corrêa et al., 2016 ) and exacerbate the immune response that occurs during influenza virus infection ( Leyva-Grado et al., 2017 ). P2X4R antagonists reduce herpetic pain ( Matsumura et al., 2016 ). Both P2X4 and P2X7R are involved in hepatitis C virus infection ( Manzoor et al., 2016 ). Purinoceptors are putative targets for the treatment of HIV-1 infection ( Pacheco et al., 2014 ). Ribavirin, an adenosine analogue, exhibited potent antiviral activities ( Hao et al., 2017 ).

Clinical interventions involving purinergic signalling are just beginning. However, the beginning and future of purinergic compounds for the treatment of a wide range of diseases is described in this review. P2Y 12 R antagonists, such as clopidogrel and ticagrelor, are currently in wide use for stroke and thrombosis, as are P2Y 2 R agonists for dry eye and A 1 R agonists for supraventricular tachycardia. The use of P2X7R antagonists for the treatment of inflammatory diseases is promising, but the presence of polymorphic variations of this receptor is holding up the development of selective antagonists appropriate for each disease. P2X3R antagonists are in clinical trials for use against visceral pain, chronic cough and hypertension. A 2A R agonists are in use for the treatment of PD, and perhaps soon in wider use. P2X1R antagonists are being investigated for treatment of bladder disorders and hypertension, while P2X4, P2X7 and A 3 R antagonists are being explored for neuropathic pain. Treatments with inhalation of ectonucleotidases to alter the balance of ATP and adenosine and inhibitors of ATP release, are also a therapeutic approach being explored. The development of novel purinergic compounds by medicinal chemists that are orally available and stable in vivo would be a significant advantage in developing therapeutic approaches, including centrally penetrant P2X7R antagonists ( Able et al., 2011 ). The majority of the therapeutic approaches for many heart disorders based on purinergic signalling manipulation are not fully understood yet and strategies to overcome the side-effects of treatment need to be considered. The pathophysiological roles of purinergic signalling in blood vessels are clearer and it plays an important role in controlling vascular tone and remodelling. Immunologic factors related to purinergic signalling are attracting more attention and should be considered ( Cekic and Linden, 2016 ). Human embryonic stem cells are able to self-renew and have the potential to differentiate into different cell types, including cardiovascular progenitor cells. This system of differentiation is being investigated for cardiac regenerative therapy ( Huang et al., 2016 ). The single nucleotide polymorphisms in purinergic receptor genes and their association with diseases are being explored for potential use as diagnosis biomarkers (see Caseley et al., 2014 ). Purinoceptors modulate neural stem cell proliferation, differentiation, migration and cell death and could be therapeutic approaches for the treatment of neurological and psychiatric illnesses ( Illes and Rubini, 2017 ). MicroRNAs modulating purinergic signalling are gaining interest as potential original therapeutic targets and disease biomarkers ( Ferrari et al., 2016a ). Although still in its infancy, clinical use of purinergic compounds has started. Several relevant pharmacological interventions are currently in clinical use. The lack of more established purinergic therapies may be due to there being relatively few receptor subtype-specific agonists and antagonists that are both effective and stable in vivo (see Jacobson and Muller, 2016 ). In some situations, a degree of redundancy is present, with several different subtypes of receptor mediating similar functional effects. Purinergic signalling is implicated in multiple disorders and therefore offers many potential future therapeutic targets. It should be noted, however, that since most purinoceptors are ubiquitous, to selectively target specific cell types may prove to be a challenge. As well as the development of selective agonists and antagonists, therapeutic strategies will probably include compounds that control P2R expression, inhibitors of extracellular ATP breakdown and inhibitors or enhancers of ATP transport. Understanding the interactions of purinergic signalling with other established signalling systems will be necessary.

Purinergic signalling, i.e., nucleotides as extracellular signalling molecules, was proposed in 1972 ( Burnstock, 1972 ). However, this concept was not well accepted until the 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 eight subtypes of the P2Y G protein-coupled receptor ( Ralevic and Burnstock, 1998 ). Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling ( Burnstock, 2007 ). Adenosine 5′-triphosphate (ATP) is a cotransmitter with classical transmitters in both the peripheral and central nervous systems. In addition, purines are powerful extracellular messengers to non-neuronal cells, including secretory, exocrine and endocrine, endothelial, immune, musculo-skeletal and inflammatory cells ( Burnstock and Knight, 2004 ). Purinergic signalling is rapid in neurotransmission, neuromodulation and in secretion, but is also long-term in proliferation, differentiation, migration and death in development and regeneration ( Burnstock, 2016f ). More recently, the focus has been on the pathophysiology and therapeutic potential of both P1 ( Chen et al., 2013 ; de Lera Ruiz et al., 2014 ; Layland et al., 2014 ; Liu and Xia, 2015 ; Borea et al., 2016 ) and P2 ( Burnstock and Kennedy, 2011 ; Bartlett et al., 2014 ; Ford et al., 2015 ; Burnstock, 2016e ) receptors. Reviews focussed on different aspects of purinergic pathophysiology are also available, including inflammatory and immune disorders ( Arulkumaran et al., 2011 ; Junger, 2011 ; Hansson et al., 2016 ); cancer ( Burnstock and Di Virgilio, 2013 ; Di Virgilio and Adinolfi, 2017 ); gout and fibrosis ( Gicquel et al., 2017 ); P2X7 receptors (R) as therapeutic targets ( Romagnoli et al., 2008 ); medicinal chemistry of purinoceptors ( Jacobson and Muller, 2016 ); pain ( Burnstock and Sawynok, 2010 ; Alves et al., 2013 ; Kuan and Shyu, 2016 ; Sawynok, 2016 ) and adenosine kinase inhibitors ( Kowaluk and Jarvis, 2000 ). A number of purine-related compounds have been patented. Therapeutic developments for disorders of different systems in the body will now follow. Reviews concerned with the early literature will be quoted, so the focus of this review will be concerned largely with the most recent findings.

Reviews about this topic are available ( Erlinge and Burnstock, 2008 ; Headrick et al., 2013 ; Burnstock and Ralevic, 2014 ; Burnstock, 2015a ; Burnstock and Pelleg, 2015 ; Ralevic, 2015 ; Sousa and Diniz, 2017 ). In chronic heart failure adenosine accumulates, probably as a result of lowered adenosine deaminase (ADA) gene expression and raised CD73 activity. Adenosine therapy mediated by A 1 R and A 3 R is cardioprotective for chronic heart failure ( Greene et al., 2016 ; Voors et al., 2017 ). A 1 R agonists attenuate cardiac hypertrophy and prevent heart failure in a mouse model (left-ventricular pressure-overload) and in a rat model (neonatal cardiac myocyte) ( Chuo et al., 2016 ). A 2B R agonists exert stronger cardioprotective effects against cardiac ischaemia/reperfusion injury compared to A 2A R activation in rats ( Ke et al., 2015 ). CD73 and A 2B R agonists have been considered as therapeutic agents for myocardial ischaemia. Genetic deletion of CD39 results in increased myocardial ischaemia-reperfusion injury ( Smith et al., 2016 ). Inflammatory responses initiated during ischemia-mediated immune injury may be regulated by adenosine ( Boros et al., 2016 ). Early studies were concerned with the role of adenosine in ischaemic and reperfusion injuries. However, there is also interest in the role of ATP. Application of ATP, prior to or just after cardiac ischaemia is cardioprotective ( Ren et al., 2016 ). ATP released from the ischaemic myocardium causes reflex responses mediated by cardiac sympathetic afferent nerves ( Dong et al., 2016 ). P2YR are important therapeutic targets in myocardial protection during ischemia/reperfusion ( Djerada et al., 2017 ). P2Y 6 R could be a therapeutic target to regulate cardiac hypertrophy ( Clouet et al., 2016 ). There is increased expression of P2Y 2 and P2X1R in the hearts of rats with congestive heart failure. P2X4R are needed for neuroprotection via ischemic preconditioning ( Ozaki et al., 2016 ). P2X3R expression increased in dorsal root ganglion (DRG) and superior cervical ganglia neurons, resulting in exaggerated sympathoexcitatory reflexes. NONRATT021972 siRNA decreases the upregulation of P2X7R and improves cardiac function after myocardial ischemia ( Tu et al., 2016 ). Mitochondrial K ATP channels provide protection against myocardial ischemia/reperfusion injury ( Wang et al., 2015 ; Shimizu and Calvert, 2016 ). K ATP channels maintain high energy phosphates and myocardial perfusion in heart failure ( Jameel et al., 2016 ). A clinical trial (Acute Myocardial Infarction Study of Adenosine) concluded that infusion for 3 h of adenosine resulted in a reduction of infarct size ( Yetgin et al., 2015 ; Bulluck et al., 2016 ). Protection against myocardial infarction was mediated by A 1 R in the rabbit heart. The A 3 R agonist IB-MECA produced cardioprotection against myocardial infarction ( Tian et al., 2015 ). Microglial P2X7R in the rat hypothalamic paraventricular nuclei regulate the sympathoexcitatory responses in acute myocardial infarction ( Du D. et al., 2015 ). Loss of mouse P2Y 4 R protects against myocardial infarction ( Horckmans et al., 2015 ). CD39 reduced infarct size following ischaemia-reperfusion injury ( Smith et al., 2016 ). P2Y 12 R antagonists have been recommended for long-term protection of patients, post-myocardial infarction ( Alexopoulos et al., 2016 ). The K ATP channel opener, natakalim, improves ventricular remodelling of congestive heart failure after myocardial infarction ( Jin, 2016 ). Adenosine reduces post-operative atrial fibrillation (AF). Up-regulation of A 2A R involves abnormal calcium handling in AF. Prevention of A 2A R activation in patients with AF may sustain uniform beat-to-beat responses at higher beating frequencies ( Molina et al., 2016 ). Adenosine-guided pulmonary vein isolation following a randomised clinical trial was recommended to treat paroxysmal AF ( Macle et al., 2015 ), although this has been queried in a more recent clinical trial ( Ghanbari et al., 2016 ). ATP-induced AF has also been investigated ( Hasebe et al., 2016 ). The efficacy of the P2Y 12 antagonists ticagrelor and prasugrel are not affected in AF ( Ondrakova et al., 2016 ). The roles of adenosine and ATP in atrial arrhythmias and fibrillation have been discussed ( Jared Bunch, 2015 ; Belhassen and Michowitz, 2016 ). Acute therapy by ATP for paroxysmal supraventricular tachycardia was used in the late 1940’s. In patients with paroxysmal supraventricular tachycardia bolus injection of Adenocard (adenosine) is clinically prescribed to slow conduction time via the atrioventricular node, via A 1 R ( Sachdeva and Gupta, 2013 ). Treatment of paroxysmal supraventricular tachycardia by adenosine and ATP is discussed ( Lerman, 2015 ). To provoke vasovagal reaction in syncope patients ATP and adenosine are administered together with the head-up tilt table test ( Fragakis et al., 2015 ). Cardiomyopathy can be an inherited disease, but can occur as a result of vitamin B deficiency, amyloidosis, alcoholism or viral infections. ATP synthase disruption contributes to diabetic cardiomyopathy ( Ni et al., 2016 ). P2X7R involvement in dilated cardiomyopathy has been reported ( Martinez et al., 2015 ). P2Y 11 R agonists reduce cardiac fibrosis ( Certal et al., 2015 ). Extracellular nucleotide regulation of signalling in cardiac fibrosis has been discussed ( Novitskaya et al., 2016 ). The P2X7R antagonist, A740003, reduces experimental autoimmune myocarditis, suggesting a treatment for clinical myocarditis ( Zempo et al., 2015 ). ATP injections to treat angina pectoris associated with coronary disease were used during the 1940s, while AMP was used to treat angina. ATP treatment for patients with coronary insufficiency was also used early. Intracoronary administration of adenosine results in angina pain. Intracoronary administration of adenosine in patients with unstable angina produced decreased myonecrosis and improved coronary blood flow ( Kizilirmak et al., 2015 ). In vascular pain, which encompasses pelvic and ischaemic pain, migraine and angina, it is thought that release of ATP from endothelial cells during reactive hyperaemia after vasospasm diffuses through the microvascular wall to activate P2X3R on perivascular sensory nerves to send impulses that travel to pain centres in the brain via the spinal cord ( Joseph et al., 2015 ). P2X2/3R expressed on airway nociceptive sensory nerves mediate cardiovascular reflexes in conscious rats ( Hooper et al., 2016 ). Responses of the transplanted human heart to adenosine show supersensitivity. Protection of cardiac grafts from cold ischaemia/reperfusion injury is caused by donor pretreatment with AMP-activated protein kinase ( Yang C. et al., 2016 ). Treatment with P2XR antagonists prolongs cardiac transplant survival. It has been proposed in a recent review ( Burnstock, 2017 ) that there are five different ways that purinergic signalling can contribute to the development of hypertension: ATP released as a cotransmitter from sympathetic nerves together with noradrenaline (NA) contributes, via P2X1R, to the vasoconstriction that results from increased sympathetic vasomotor activity in hypertension. Therefore, P2X1R antagonists should be useful for the treatment of hypertension, especially since there is a substantial increase in ATP relative to NA released from sympathetic nerves in spontaneously hypertensive rats ( Brock and Van Helden, 1995 ; Goonetilleke et al., 2013 ). Release of ATP from endothelial cells by shear stress as a result of changes in blood flow acts on P2YR and P2X4R on endothelial cells to release nitric oxide (NO) resulting in vasodilation. Introduction of P2Y 1 , P2Y 2 , and P2X4 agonists would cause increased vasodilation in hypertension. Brain stem and hypothalamic neurons mediate sympathetic nerve activity. Recent studies show that P2X3R antagonists are antihypertensive, due to reduced sympathetic nerve activity as a result of increased peripheral P2X3R-mediated carotid body chemoreceptor reflexes ( Pijacka et al., 2016 ). P2X7R in the kidney contribute to the pathophysiology of hypertension and P2X7R antagonists may have promise as clinical antihypertensive agents. In hypertensive patients adenosine activates the vascular renin-angiotensin system. P1R agonists have been suggested for the treatment of hypertension ( Ho M.F. et al., 2016 ). Recently, P2Y 6 R were shown to age-dependently promote vascular remodelling in a mouse model, an effect inhibited by MRS2578, suggesting that P2Y 6 R are a therapeutic target for the prevention of age-related hypertension ( Sunggip et al., 2017 ). ATP signalling influences the development of atherosclerosis ( Burnstock, 2008a ; Ferrari et al., 2015 ). Endothelial and smooth muscle cell proliferation are promoted by adenosine and ATP in atherosclerosis via P2Y 1 , P2Y 2 , and P1R. In a human model, adenosine, via A 2A R, modulates foam cell formation ( Reiss and Cronstein, 2012 ). A 2B and A 3 antagonists reduce atherosclerotic plaque development. There are increased concentrations of circulating adenosine 5′-diphosphate (ADP) and ATP in atherosclerosis ( Jalkanen et al., 2015 ). Uridine 5′-triphosphate (UTP), via P2Y 2 R, induces expression of vascular cell adhesion molecule-1 in coronary artery endothelial cells, which leads to the monocyte recruitment associated with atherosclerosis development. Upregulated P2Y 2 R mediate intimal hyperplasia in collared rabbit carotid artery. P2Y 1 R antagonists are a therapeutic target for neointima formation ( Liu R. et al., 2015 ). Endothelial P2X4R play a more important role in intense proliferation in atherosclerosis than P2Y 2 R. ATP, by inducing leukocyte recruitment in mice, contributes to atherogenesis, via P2Y 2 , P2Y 6 , P2X4, and P2X7R. P2X7R are over-expressed in atherosclerosis and P2X7R deficiency leads to less plaque formation ( Stachon et al., 2016 ). CD39 mRNA-coated stents may be a treatment for atherosclerosis ( Abraham et al., 2015 ). Atherosclerosis of coronary vessels is called coronary artery disease or coronary artery syndrome. P2Y 12 R antagonists combined with aspirin are beneficial for patients with acute coronary syndrome ( De Luca et al., 2016 ; Rollini et al., 2016 ). A 2A R on coronary arteries have also been claimed to be involved in coronary artery disease ( Gariboldi et al., 2017 ). An initiating event in the pathogenesis of vascular diseases is often vascular injury. Injured cells release ATP, which, together with adenosine, evoke endothelial and smooth muscle cell growth, proliferation, migration and death. Adenosine, following breakdown of ATP, is protective against ischaemic injury. P2Y 12 R antagonists are prescribed as prevention against ischaemic stroke ( Liu F. et al., 2015 ). P2Y 2 R mediate regulation of endothelial inflammation and angiogenesis ( Gidlöf et al., 2015 ). Sustained control of proliferation of endothelial and smooth muscle cells as a result of P1 and P2YR activation during vascular remodelling in restenosis after angioplasty has been reported and therapeutic possibilities are being explored ( Burnstock, 2002 ). Activation of A 2B R stimulates angiogenesis in human microvascular endothelial cells ( Du X. et al., 2015 ). Adenosine, via A 2A R, stimulates wound healing and angiogenesis following tissue injury in mice. CD39 administration decreases injury-induced platelet deposition and recruitment of leukocytes and inhibits neointimal hyperplasia. A review discusses the vascular actions of P2XR in renal injury ( Howarth et al., 2015 ). Nucleotides are extracellular mediators of vascular inflammation and thrombosis. Clopidogrel, a P2Y 12 antagonist, inhibits aggregation in platelets and is widely prescribed for thrombosis and stroke ( Sarafoff et al., 2012 ). Other P2Y 12 antagonists have been developed, including ticlopidine, cangrelor, ticagrelor, prasugrel, elinogrel, BX 667, and PSB 0739. P2Y 1 R antagonists also have antithrombotic actions and have been recommended as a complement to current P2Y 12 anti-platelet strategies. The P2Y 6 R may be a therapeutic target for systemic inflammatory responses. Review articles are available about purinergic signalling in thrombosis and inflammation, including the use of different oral or intravenous P2Y 12 R antagonists ( Tang et al., 2015 ; Nylander and Schulz, 2016 ; Rollini et al., 2017 ). P2X7R are pro-thrombotic and genetic KO of the gene for the P2X7R was shown to be protective in a mouse carotid artery thrombosis model ( Furlan-Freguia et al., 2011 ). Adenosine, via A 2A R and A 3 R, had antithrombotic effects ( Cristalli et al., 1994 ; Hofer et al., 2013 ). Migraine pain involves two distinct cerebrovascular phases: an initial vasoconstriction (with no pain), followed by vasodilation (reactive hyperaemia) associated with pain. A purinergic hypothesis for migraine was proposed in 1981 ( Burnstock, 1981 ). It was suggested that after the initial vasospasm ATP and adenosine (following breakdown of ATP) may mediate the vasodilation during reactive hyperaemia associated with pain. It was also hypothesised that stimulation of P2X3R on sensory nerve terminals located on the adventitia of the cerebral microvasculature by ATP contributed to the migraine pain. Data has also been presented recently that is consistent with the purinergic hypothesis of migraine pain ( Yegutkin et al., 2016 ). P2X3R antagonists have been proposed as potential candidates for migraine treatment ( Waeber and Moskowitz, 2003 ). The non-steroidal anti-inflammatory compound, naproxen, currently in use for migraine pain, blocks P2X3R-mediated responses in trigeminal neurons of the rat. Migraine could involve a chronic disorder of the sympathetic nervous system, where increased release of the sympathetic cotransmitter ATP contributes to the initial vasospasm. Adenosine may also be involved in migraine. Adenosine infusion resulted in symptoms that were migraine-like and withdrawal of the P1R antagonists theophylline and caffeine also resulted in migraine-like symptoms. Clinical trials with the adenosine uptake inhibitor dipyridamole, that results in increased extracellular adenosine, were halted due to the increase of migraine attacks in all patients. Overactive P2YR on glial cells may contribute to pain transduction during migraine. Reviews have been published about the role of purinergic signalling in the aetiology of migraine and the potential of purinergic compounds ( Cieslak et al., 2015 ; Jacobs and Dussor, 2016 ). There is pre-junctional A 1 R-mediated sympathetic neurotransmission and ATP-mediated endothelial vasodilatation in mesenteric arteries of streptozotocin (STZ) diabetic rats ( Burnstock and Novak, 2013 ). There is enhanced A 2A R-mediated increase in coronary flow in type 1 diabetic mice ( Labazi et al., 2016 ). UTP, ATP and adenosine evoked vasodilation is reduced in the circulation of skeletal muscle of type 2 diabetic patients. Erythrocyte release of ATP is diminished in type 2 diabetics, supporting the view that a defect in the physiology of erythrocytes may contribute to diabetic vascular disease. Erythrocytes are less deformable in type 2 diabetes leading to lowered levels of deformation-induced ATP release. Low erythrocyte ATP release may contribute to the prevention and treatment of diabetic peripheral vascular disease ( Richards et al., 2015 ). P2X7R expression by monocytes might play a role in the pathological changes of type 2 diabetes mellitus ( Wu et al., 2015 ). ATP inhibits mineralisation of the aortic valve seen in calcific aortic valve disease ( Côté et al., 2012 ). Increased levels of ectonucleotidase are found in calcific aortic valve disease and inhibition of ectonucleotidase with ARL67156 prevented disease development in rats. Adenosine is a potential therapeutic target for the prevention and treatment of sickle cell disease ( Fu and Davies, 2015 ). The circulating levels of adenosine are elevated in pregnant women with sickle cell disease ( Ashimi et al., 2015 ). Amyloid-β peptide inhibits ATP release from erythrocytes, suggesting that in AD, vascular amyloid peptide may play a role.

Reviews that include discussion of the pathophysiology of musculoskeletal diseases have been published ( Burnstock et al., 2013 ; Young et al., 2013 ; Agrawal and Gartland, 2015 ; Jørgensen et al., 2015 ; Orriss et al., 2016 ). There are multiple purinoceptor subtypes expressed by bone and cartilage, which are potential targets for original therapeutic strategies to inhibit bone resorption in osteoporosis, rheumatoid arthritis (RA), periodontitis and tumour-induced osteolysis. ATP was used early to treat myopathies, although the mechanism of action was not known. Allopurinol has been used for the treatment of Duchenne muscular dystrophy (DMD). It counteracts low purine nucleotide degradation levels that occur in Duchenne muscle, but chronic administration of allopurinol failed to improve DMD symptoms. Lymphoblastoid cells from DMD patients were found to be highly sensitive to ATP stimulation. The mdx model of DMD, lacking the dystrophin protein, mimics muscle damage and subsequent regeneration. Sequential expression of P2X5, P2Y 1 and P2X2R was described during muscle regeneration in the mdx model ( Ryten et al., 2004 ). It was claimed recently that a P2Y 2 R antagonist may ameliorate cardiomyopathy in DMD ( De Oliveira Moreira et al., 2017 ). P2Y 1 R were expressed on infiltrating immune cells. A purinergic dystrophic phenotype was seen during the earliest stage of developing dystrophic muscle. Dystrophic myoblasts express P2X4 and P2X7R proteins and it was claimed that antagonists to these receptors may be of potential therapeutic benefit. ATP signalling is altered in muscular dystrophy, but was partly recovered after nifedipine treatment ( Valladares et al., 2014 ). It was claimed that dihydropyridines may be used as a therapeutical tool to reduce muscle damage observed in dystrophic muscles. It was suggested that P2XR antagonists alter the adaptive immune component in the muscle infiltrates in DMD and are a promising therapeutic approach to treat DMD ( Gazzerro et al., 2015 ). Myofascial pain is a feature of DMD and ATP stimulates myofascial nociceptors. P2X7R upregulation occurs in dystrophic mdx mouse muscles and treatment with P2X7R antagonists slows the progression of DMD ( Sinadinos et al., 2015 ). Sensitivity to ATP is higher, release of ATP is greater and expression of P2Y 2 R is increased, but P2Y 1 R expression is decreased in mdx mice. A review about purinergic receptors in DMD was published ( Krasowska et al., 2014 ). Myasthenia gravis is an autoimmune disease, which affects the neuromuscular junction. Lack of neuronal A 2A R-mediated Ca v 1 (L-type) influx causes tetanic failure in MG ( Noronha-Matos et al., 2011 ). It was suggested that the adenosinergic pathway was dysfunctional in autoimmune MG and that stimulation of CD73 activity and A 2A R may have therapeutic potential for MG ( Oliveira et al., 2015 ). Fibromyalgia (a musculoskeletal disease) is characterised by allodynia, as well as mood disorders. In fibromyalgia patients, there are decreased levels of ATP in platelets perhaps contributing to the pathogenesis of the disease ( Bazzichi et al., 2008 ). Osteoporosis, characterised by bone mass loss (bone mineral density decrease), leads to a high risk of bone fracture. The P2X7R is considered to be important in relation to the treatment of osteoporosis ( Kvist et al., 2014 ). P2X7R plays a critical role in both cortical and cancellous bone mass augmentation and were shown to stimulate cancellous and periosteal bone formation and inhibit cancellous bone resorption during growth. Single nucleotide polymorphisms of the P2X7R gene are associated with the risk of fractures, decrease in bone mineral density and osteoporosis. P2X7R are involved in the formation of human osteoclasts. P2X7R antagonists are being considered for the treatment of osteoporosis and with remodelling disorders, where bone mass is reduced ( Jørgensen et al., 2011 ). Skeletal pain accompanies osteoporosis and P2X2/3R might have a role in osteoporosis patients under a high bone turnover state ( Iba and Yamashita, 2016 ). P2X5R may be a therapeutic target for treatment for inflammatory bone loss ( Kim et al., 2017 ). Polymorphisms in the P2X4 and P2X7R genes ( Husted et al., 2013 ) and the Leu46Pro polymorphism of the human P2Y 2 R gene were linked to bone mineral density and the risk of osteoporosis in Dutch fracture patients ( Wesselius et al., 2013 ). It was suggested that clopidogrel, a P2Y 12 R antagonist used for stroke and thrombosis, increases the risk of fractures in osteoporotic patients ( Jørgensen et al., 2012 ). P2Y 13 R antagonists have also been proposed for the treatment of osteoporosis ( Wang N. et al., 2013 ). Adenosine receptors are involved in osteoporosis ( McPhee and Whiting, 1989 ). Trans -differentiation of osteoblasts to adipocytes, which involves A 2B R, may contribute to the pathogenesis of osteoporosis ( Rayalam et al., 2011 ). P1R might be targets for treating osteoporosis and other diseases characterised by excessive bone turnover. Osteoarthritis is a degenerative joint disease as a result of wear of the articular cartilage due to abnormal load to the joint or from infection, trauma or due to ageing. The pain from OA is due largely to inflammation. Increased ATP levels and 5′-nucleotidase activity are present in osteoarthritic joint synovial fluid, compared to the joints from RA patients, particularly from osteoarthritic patients where deposition of calcium-containing crystals were also present. ATP contributes to pathologic mineralisation in articular cartilage and therefore P2R antagonists might provide therapeutic tools for crystal-associated arthritis ( Costello et al., 2011 ). ATP levels in knee synovial fluid of patients with OA are related to pain intensity ( Kumahashi et al., 2011 ). P2X3 and P2X2/3R play an important role in the development of articular hyperalgesia of arthritic joints ( Teixeira et al., 2016 ). In the long-term complications following total hip arthroplasty, different polymorphic variants of the P2X7R are associated with high or reduced periprosthetic osteolysis ( Mrazek et al., 2010 ). There are elevated concentrations of ATP in the synovial fluid of dogs with OA ( Torres B.T. et al., 2016 ). Purinergic signalling, via P2R, produces calcium oscillations in migratory chondrogenic progenitor cells isolated from OA cartilage ( Matta et al., 2015 ). Platelets promote cartilage repair and chondrocyte proliferation via release of ADP in a rodent model of OA, suggesting that P2Y 1 or P2Y 12 R agonists may be useful for treatment of OA ( Zhou Q. et al., 2016 ). Treatment with the selective P2X7R antagonist, AZD9056, produced pain-relieving and anti-inflammatory effects in rats with OA ( Hu H. et al., 2016 ). Adenosine signalling is also involved in OA. Adenosine, produced following breakdown of ATP, is released from chondrocytes and contributes to tissue damage in arthritic conditions. The regulation of LPS-induced IL-6 release involves A 2A R, indicating that adenosine has a regulatory role in controlling osteoclastogenesis and inflammation. Electromagnetic field stimulation up-regulates A 2A R in synovial fibroblasts and adenosine, acting through both A 1 and A 2A R, had anti-inflammatory activity to control joint inflammation ( De Mattei et al., 2009 ). A 2A and A 3 R agonists modulate prostaglandin E 2 and cytokine release in human osteoarthritic fibroblasts ( Ongaro et al., 2012 ). Adenosine receptors also mediate regulation of inflammatory responses in human synoviocytes ( Varani et al., 2010b ). A 2A R agonists are used to reduce joint destruction due to septic arthritis ( Cohen et al., 2004 ). A 2A R deletion resulted in the development of OA in mice, suggesting that A 2A R agonists might be a target for the treatment of OA ( Corciulo et al., 2017 ). The potential involvement in RA of purinergic signalling was recognised first in the 1990’s when concentrations of adenosine, after released ATP was broken down, were reduced in synovial fluid in RA compared to OA. Increased levels of adenosine as a treatment for RA was suggested. UTP and ATP activate calcium-mobilising P2UR to synergistically act with IL-1 to stimulate release of prostaglandin E 2 from human rheumatoid synovial cells. Hypotonic stress promotes ATP release and cell proliferation via transient receptor potential vanilloid 4 activation in RA rat synovial fibroblasts ( Hu et al., 2017 ). IL-1β is a proinflammatory cytokine that substantially contributes to the progression of RA. ATP, through P2X7R, induced increased levels of IL-1β in RA patient blood samples compared to control samples. Mononuclear cells from these patients were more sensitive to stimulation by ATP, possibly because of P2X7R genetic polymorphism ( Portales-Cervantes et al., 2012 ). P2X7R are involved in the pathogenesis of RA and systemic lupus erythematosus ( Portales-Cervantes et al., 2010 ). P2X7R mRNA and protein are expressed in human rheumatoid synoviocytes. Studies of arthritis animal models suggest an in vivo role for the P2X7R in the progression of this inflammatory disease. There was a lower incidence and reduced severity of the symptoms of arthritis induced by anti-collagen treatment in P2X7R KO mice. Therefore targeting the P2X7R may be a potential treatment for RA. P2X7R antagonists in the collagen-induced animal model of RA reduced destruction of peripheral inflammatory tissue. In a later study, it was claimed that AZD9056, a P2X7R antagonist, was not effective against RA ( Keystone et al., 2012 ). P2X7R antagonists are being investigated for clinical use against inflammatory joint pain ( Beswick et al., 2010 ). A multicentre, double-blind, placebo-controlled clinical trial showed that ATP infusions reduced inflammation and disease symptoms in patients with RA ( Bours et al., 2010 ). Bovine chondrocytes express P2X1 and P2X3R and following stimulation there was release of inflammatory mediators. Therefore antagonists to these receptors may be therapeutically useful for articular cartilage resorption and diseases involving inflammation. There was an increased platelet response to ADP in RA patients ( Mac Mullan et al., 2010 ). Sympathetic nerves mediate proinflammatory responses during the initial phase of arthritis induced by type II collagen, probably via cytokines such as interferon-γ released in response to the sympathetic cotransmitters ATP and NA ( Straub et al., 2008 ). α,β-MeATP-sensitive P2XR (probably P2X3) are expressed on rat knee joint peripheral nociceptive afferent fibres and the increased ATP levels in damaged and inflamed tissues, may contribute to nociception and pain. P2X3R were found on nociceptive sensory fibres in lumber facet joints, where low back pain originates ( Ishikawa et al., 2005 ). Changes in P2X3R expression on DRG neurons that label isolectin B4 were seen following the induction of RA ( Averill et al., 2008 ). P2X3R expressed on trigeminal ganglia also contribute to orofacial pressure pain in monoarthritis of the temporomandibular joint. Plasma extravasation in the rat knee joint induced by bradykinin was enhanced by ATP, released as a cotransmitter from sympathetic nerves. Intravenous guanethidine, which inhibits release of sympathetic cotransmitters, proved to be effective in RA patients. Uncaria tomentosa extract affects the metabolism of adenine nucleotides and has been suggested as an adjuvant to treat arthritis ( Castilhos et al., 2015 ). The evidence showing a role for adenosine in RA has been reviewed ( Varani et al., 2010a ). Increased activity of ADA was seen in synovial fluid from patients with RA as well as in rheumatoid synovial fibroblasts ( Nakamachi et al., 2003 ). The ImmuKnow assay might effectively identify RA patients that are more at risk of developing infections ( Akimoto et al., 2013 ). Signalling via the A 2A R caused modification of the cytokine milieu in RA ( Masahiro et al., 2003 ). Over-expression of A 3 R was observed in peripheral blood mononuclear cells of RA patients. Patients with RA had greater expression A 3 R in the synovium ( Stamp et al., 2012 ). There are increased levels of A 2A and A 3 R on the lymphocytes and neutrophils of RA patients. In the dorsal horn of rats with induced RA, A 1 R agonists decreased activation of c-fos and astrocytes. In a later study of the adjuvant-induced monoarthritis model an A 3 specific agonist prevented bone resorption. ATP, working via A 2 R, also reduced joint injury. A phase II clinical trial provided evidence for A 3 R agonists as a treatment for RA ( Fishman et al., 2008 ; Silverman et al., 2008 ). CGS 21680, an agonist of the A 2A R, reduced progression of murine type II collagen-induced arthritis ( Mazzon et al., 2011 ). A 2A R agonists ameliorate adjuvant-induced arthritis in rats ( Vincenzi et al., 2013 ). Methotrexate (MTX) is often used to treat RA. In human joints with inflammatory disease, MTX treatment involved A 2A R. Adenosine via A 2B R prevented MTX-induced inhibition of osteoclast bone destruction in arthritis induced by adjuvant ( Teramachi et al., 2011 ). In RA patients treated with MTX, studies of polymorphisms of the genes involved in adenosine release concluded that genotyping may help identify patients who would most benefit from MTX treatment ( Wessels et al., 2006 ). Anti-tumour necrosis factor-α has also been used to treat RA, but it raises the risk of reactivating tuberculosis. ADA assay is a specific and sensitive test for the quick diagnosis of rheumatoid effusions ( Zakeri et al., 2012 ). There are many sensory nerves, originating in the trigeminal ganglia, expressing P2X3R in tooth pulp and ATP is released from odontoblasts in response to mechanical stimulation to act on these receptors, resulting in pain ( Shibukawa et al., 2015 ). LPS-induced pulp inflammation increased the expression of P2XR in trigeminal sensory nerves ( Chen et al., 2014 ). Mechanical or cold stimulation of odontoblast processes in dentin tubules, results in ATP release and dental pain ( Liu X. et al., 2015 ). Therefore P2X3R antagonists may be therapeutically useful to reduce toothache. Bone metastases, common in prostate and breast cancer patients, may cause substantial bone loss and pain. Purinergic signalling involvement in bone cancer was initially reported in the 1990’s when P2U (i.e., P2Y 2 /P2Y 4 ) receptors were cloned from osteoclastoma. The expression of P2Y 2 R from human osteoclasts from bone giant cell tumour was later reported. Butyl benzyl phthalate, which interferes with mammalian ion channel receptors, inhibited ATP-induced cell proliferation via P2XR in human osteosarcoma HOS cells ( Liu and Chen, 2010 ). ATP was used in autologous bone marrow transplantation for removing residual tumour cells. In mice, a type of human apyrase, APT102, in addition to aspirin disrupts bone metastasis ( Uluçkan et al., 2008 ). Release of ATP from tumour cells further stimulates osteoclast formation and activity, contributing to bone destruction that often happens around tumour metastases. Bisphosphonates, used to treat osteoporosis, treat bone cancer and may involve apoptosis induced by ApppI, an ATP analogue produced by bisphosphonates ( Sillero et al., 2009 ). Purinergic signalling plays a role in bone cancer pain. Bone pain can be relieved by radiation therapy, which may be related to the Ca 2+ -signalling cascade, mediated by P2X6R. In a mouse model of cancer pain, increased expression of P2X3R on calcitonin gene-related peptide immunoreactive DRG neurons during tumour growth, it was claimed that ATP had a role in cancer-related pain ( Liu M. et al., 2013 ). In rats, systemic inhibition of P2X3 and P2X2/3R with AF-353 strongly attenuated bone cancer pain-related behaviour ( Kaan et al., 2010 ). In mice, administration of A-317491, a selective P2X3 and P2X2/3R antagonist, attenuated the early stages of bone pain in cancer ( Hansen et al., 2012 ). Functional up-regulation of P2X3R has been described in DRG of bone cancer pain in a rat model ( Wu et al., 2012 ). In P2X7R KO mice, bone cancer pain-related behaviours had an earlier onset ( Hansen et al., 2011 ). Most human osteosarcomas expressed P2X7R isoforms A and B ( Giuliani et al., 2014 ) and P2X7R are involved in cancer-induced bone pain and P2X7R antagonists were suggested as a useful analgesic target ( Falk et al., 2015 ). P2Y 1 R signalling in the DRG and spinal cord may mediate pain from bone cancer ( Chen et al., 2012 ). Activation of K ATP channels at the spinal cord level reduces pain associated with bone cancer ( Xia et al., 2014 ). Stimulation by AMP-activated protein kinase suppresses neuroinflammation and reduces bone cancer pain ( Song et al., 2015 ). Multiple myeloma (cancer of plasma cells) involves osteolytic bone lesions, due largely to enhanced osteoclast activity. A 2 R may be therapeutically useful to treat and prevent multiple myeloma-induced bone disease as activation of A 2A R reduces osteoclast function, while activation of A 2B R stimulates osteoblast differentiation ( He et al., 2012 ). 8-Amino-adenosine is another possible therapeutic compound for the treatment of multiple myeloma. A major cause of severe combined immunodeficiency are genetic defects in the ADA gene. Lack of ADA causes accumulation of adenosine. Bone defects as a result of reduced osteoclastogenesis together with a defect in osteoblast function leading to low bone formation were seen in about a half of early-onset ADA-deficient patients ( Sauer et al., 2009 ). Further, the microenvironment of bone marrow in ADA KO mice had a lower ability to support haematopoiesis. In ADA KO mice, treatment with gene therapy, bone marrow transplantation or enzyme replacement, led to a full recovery. ADA-transduced hematopoietic stem cell gene therapy also enhanced the growth of children with this disease. This is a congenital dysplasia of the skeleton as a result in a mutation in the gene encoding fibroblast growth factor receptor type 3 (FGR3). Ap 4 A diminished the expression of the achondroplasic FGFR3 receptor and P2Y 1 , P2Y 2 , P2Y 6 , and P2Y 11 R are expressed by achondroplasic chondrocytes mediating the action of Ap 4 A ( Guzmán-Aránguez et al., 2008 ). Ap 4 A reversed the morphological changes supporting a therapeutic role for Ap 4 A as a possible treatment of dwarfism ( Huete et al., 2011 ). There is an increase in osteoclast numbers in Paget’s disease, leading to an increase in bone resorption and a high turnover of bone. Bisphosphonates have been employed as a treatment for Paget’s disease and P2X7 antagonists have also been considered ( Agrawal et al., 2010 ). This disease causes neurological damage as a result of ectopic bone formation in spinal ligaments. In this disease, extracellular ATP in ossification of cell cultures of the posterior longitudinal ligament of the spine (OPLL) is increased. P2Y 1 R are highly expressed in OPLL cells. Mechanical stress and ATP increase the levels of osteopontin and alkaline phosphatase mRNA in OPLL cells, effects that can be inhibited by MRS2179, a selective P2Y 1 R antagonist. Over-expression of P2Y 1 R in OPLL-induced mineralisation resulted in ectopic bone formation in the spinal ligament cells of patients with OPLL ( Tanaka et al., 2011 ).