REVIEW

Purinergic Signalling: Pathophysiology and Therapeutic Potential
Geoffrey Burnstock 1, 2
1Autonomic Neuroscience Centre, University College Medical School, London, UK

2Department of Pharmacology, The University of Melbourne, Melbourne, Australia(Received for publication on April 26, 2013)(Revised for publication on May 31, 2013)(Accepted for publication on June 28, 2013)

The article begins with a review of the main conceptual steps involved in the development of our understanding of purinergic signalling, including non-adrenergic, non-cholinergic (NANC) neurotransmission; identification of ATP as a NANC transmitter; purinergic cotransmission; recognition of two
families of purinoceptors [P1 (adenosine) and P2 (ATP/ADP)]; and, later, cloning and characterisationof P1 (G protein-coupled), P2X (ion channel) and P2Y (G protein-coupled) receptor subtypes. Further studies have established the involvement of ATP in synaptic neurotransmission in both ganglia
and in the central nervous system; long-term (trophic) purinergic signalling in cell proliferation, differentiation and death occurring in development and regeneration; and short-term purinergic signalling in neurotransmission, neuromodulation and secretion. ATP is released from most cell types in
response to gentle mechanical stimulation and is rapidly degraded to adenosine by ecto-nucleotidases.
This review then focuses on the pathophysiology of purinergic signalling in a wide variety of systems,
including urinogenital, cardiovascular, airway, musculoskeletal and gastrointestinal. Considerationis also given to the involvement of purinoceptors in pain, cancer and diseases of the central nervous
system. Purinergic therapeutic approaches for the treatment of some of these diseases are discussed.
(doi: 10.2302/kjm.2013-0003-RE; Keio J Med 62 (3) : 63–73, September 2013)

Keywords: ATP, bladder disorders, hypertension, pain, CNS diseases

scribed9 and applied to a guinea-pig taenia coli innervatedsmooth muscle preparation.10,11 Experiments were carriedout at Melbourne University in which neurotransmission
mediated by the classical autonomic neurotransmitters acetylcholine (ACh) and noradrenaline (NA) were blocked by atropine and bretylium. We were expecting to see direct stimulation of the smooth muscle resulting in depolarisation andcontraction. However, to our surprise and excitement, the response was hyperpolarisation to single pulses and relaxation.
Tetrodotoxin, which was known to block nerve conduction
but not affect smooth muscle activity, completely blocked thehyperpolarisations (Fig. 1), showing that they were inhibitory junction potentials in response to non-adrenergic, noncholinergic (NANC) neurotransmission.12 It was later shownthat the response to NANC nerve stimulation was mediatedIntroduction
The emphasis in this article is on the growing interestin the pathophysiology and therapeutic potential of purinergic signalling.1–8 However, it begins with a review ofthe discovery and main conceptual steps that have contributed to our current understanding of purinergic signalling.
Basic Science − Conceptual Steps
Non-adrenergic, non-cholinergic neurotransmission
The sucrose-gap technique for recording correlated mechanical and electrical activity of smooth muscle was de-Presented at the 1852nd Meeting of The Keio Medical Society in Tokyo, March 25, 2013.
Reprint requests to: Geoffrey Burnstock, PhD, Autonomic Neuroscience Centre, University College Medical School, Rowland Hill Street,
London NW3 2PF, UK, E-mail: g.burnstock@ucl.ac.uk
Copyright © 2013 by The Keio Journal of Medicine

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Purinergic cotransmission

Fig. 1 Sucrose-gap recording of membrane potential changes insmooth muscle of guinea-pig taenia coli in the presence of atropine (0.3 μM) and guanethidine (4 μM). Transmural field stimulation (0.5 ms, 0.033 Hz, 8 V) evoked transient hyperpolarisations,which were followed by rebound depolarizations. Tetrodotoxin(TTX, 3 μM) added to the superfusing Kreb’s solution (applied atarrow) rapidly abolished the response to transmural field stimulation, establishing these as inhibitory junction potentials in response to non-adrenergic, non-cholinergic neurotransmission.
(Reproduced from Burnstock98 with permission of Blackwell
Publishing.)

by intrinsic inhibitory enteric neurones that were controlled
by vagal and sacral parasympathetic nerves.13
Purinergic neurotransmission
We tried to identify the transmitter involved in NANC
inhibitory neurotransmission in the gut and NANC excitatory neurotransmission in the urinary bladder by seeing
if neuropeptides, monoamines or amino acids satisfied
the criteria proposed by Eccles, but we were not successful. However, after reading two papers, one by Drury and
Szent-Györgyi14 that described extracellular actions ofpurines on the heart and blood vessels and a later paper
by Pamela Holton15 that showed release of ATP during
antidromic stimulation of sensory nerves, in 1970, together with David Satchell, we showed that ATP satisfied the criteria for NANC neurotransmission in both gut
and bladder.16,17 In a review 2 years later, I invented theword “purinergic,” ATP being a purine nucleotide, andcame up with the purinergic hypothesis (Fig. 2).18 Unfortunately, this concept met with almost universal opposition during the following 20 years. This was perhaps
not surprising, because ATP was well established as an
intracellular energy source, and it seemed unlikely thatsuch a ubiquitous molecule would also be involved in extracellular signalling.19,20

During a sabbatical leave at UCLA, we showed that
ATP was released not only from NANC nerves in thetaenia coli but also from sympathetic nerves supplying the smooth muscle.21 This was followed by several
other papers supporting the concept that ATP might bea cotransmitter with NA from sympathetic nerves,22,23
and I published a Commentary in Neuroscience entitled:“Do some nerve cells release more than one transmitter?” which challenged what had been known as Dale’s
Principle, that one nerve only released one transmitter.24
This was later followed by convincing evidence that theexcitatory junction potentials recorded in smooth muscleof the vas deferens in response to sympathetic stimulation25,26 were due to release of ATP as a cotransmitterwith NA.27,28 Parasympathetic nerves supplying the urinary bladder were shown to utilize ATP as a cotransmitter with ACh.17,29 The cotransmitter concept was also
initially resisted, but it is now well established that every
nerve, in both the peripheral and central nervous system(CNS), utilises ATP as a cotransmitter (Table 1).30
Purinergic receptors
In 1978, I recognised from hints in the literature andsome simple experiments that there were different receptor
families for adenosine (called P1 receptors) and for ATP
and ADP (called P2 receptors).31 P1, but not P2 receptors,
were antagonised by methylxanthines. P2 receptors werelater subdivided into P2X and P2Y families, based on pharmacology.32 However, widespread acceptance of purinergic
signalling did not come until the early 1990s, when the receptors to purines and pyrimidines were cloned and characterised. First, four P1 receptor subtypes were identified: A1,
A2A, A2B and A3.33,34 In 1993, we cloned a G protein-coupled ATP receptor, which we named P2Y1,35 at about thesame time as David Julius and his colleagues in San Francisco cloned a P2Y2 receptor.36 The first two P2X ion channel receptors were cloned and characterised the following
year.37,38 Later, a formal division into P2X ionotropic andP2Y metabotropic receptor families was proposed.39
A major conceptual advance was made when the crystal structure of P2X4 receptors was presented (Fig. 3),40
thereby enabling medicinal chemists to start developmentof selective P2X receptor agonists and antagonists fortherapeutic purposes. Cloning of P2 receptors led to studies of immunohistochemical localization of purinoceptors,
and this showed that most non-neuronal cells, as well as
neurones, expressed multiple P2 receptors.41 More recent
studies have shown that P2X purinoceptors are expressedin primitive animals, such as amoeba and Schistosoma, thathave comparable molecular structures to those identified in
mammals, indicating that ATP was likely one of the earliest
extracellular messengers.42

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65

Fig. 2 Purinergic neuromuscular transmission depicting the synthesis, storage, release and inactivation of ATP. ATP stored in vesicles
in nerve varicosities is released by exocytosis to act on postjunctional receptors for ATP on smooth muscle. ATP is broken down extracellularly by ATPases and 5′-nucleotidase to adenosine, which is taken up by varicosities to be resynthesised and reincorporated
into vesicles. Adenosine is broken down further by adenosine deaminase (A.deaminase) to inosine and hypoxanthine and removedby the circulation. (Reproduced from Burnstock18 with permission from the American Society for Pharmacology and Experimental
Therapeutics.)

Table 1 Summary of cotransmitters in the peripheral and central nervous systems (Modified from Abbracchio et al99 with permission from Elsevier)
References     Cotransmitters
Peripheral nervous system
Sympathetic nerves
Parasympathetic nerves
Sensory-motor
NANC enteric nerves
Motor nerves (in early development)
Central nervous system
Cortex, caudate nucleus
Hypothalamus, locus coeruleus
Hypothalamus, dorsal horn, retina
Mesolimbic system
Hippocampus, dorsal horn     ATP + NA + NPY     ATP + ACh +VIP
     ATP + CGRP + SP
     ATP +NO +VIP
     ATP + ACh     28, 100     101     102     103,104     105,106     ATP + ACh     ATP + NA     ATP + GABA     ATP + DA     ATP + glutamate     107     108, 109     110     111     112, 113

NPY, neuropeptide Y; ACh, acetylcholine; VIP, vasoactive intestinal peptide; CGRP, calcitonin gene-related peptide; SP, substance
P; NO, nitric oxide; GABA, γ-aminobutyric acid; DA, dopamine

Purinergic synaptic transmission

Short- and long-term (trophic) purinergic signalling

Purinergic synaptic neurotransmission was reported
between neurones in ganglia43,44 and in the brain.45 This
was a conceptual step, which was particularly influential
to neuroscientists interested in the CNS.

In addition to short-term purinergic signalling in neurotransmission, neuromodulation, secretion and platelet
aggregation, it is now recognised that long-term (trophic)
purinergic signalling is involved in cell proliferation, dif-

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Burnstock G: Purinergic Signalling: Pathophysiology and Therapeutics

cal disturbance, hypoxia and some agents.53,54 Purinergic mechanosensory transduction occurs in a variety ofphysiological and pathophysiological events, including
visceral pain.55,56 The mechanism of ATP transport from
cells appears to be a combination of connexin and pannexin-1 hemichannels and vesicular exocytosis. Much
is now known about the ectoenzymes involved in the
breakdown of released ATP into ADP, AMP, adenosine,
inosine and hypoxanthine.57,58 The enzymes involved include ectonucleoside triphosphate diphosphohydrolases,
ecto-nucleotide
pyrophosphatase/phosphodiesterases,
alkaline phosphatases, 5′-nucleotidase and monoamineoxidase.
Pathophysiology and Therapeutic Potential
Bladder disorders

Fig. 3 The architecture of the P2X4 receptor. Stereoview of thehomotrimeric ΔzfP2X4 structure viewed parallel to the membrane. Each subunit is depicted in a different colour. N-acetylglucosamine and glycosylated asparagine residues are shown instick representation. The grey bars suggest the boundaries of theouter (Out) and inner (In) leaflets of the membrane bilayer. (Reproduced from Kawate et al40 with permission from the Nature
Publishing Group.)

ferentiation and death during development and regeneration,46 for example in vascular47 and bone48 remodelling,
and in cancer.49 There is evidence that ATP acts synergistically with growth factors to produce trophic signalling,for example, during neurite extension following transplantation of the myenteric plexus into the brain50 and instem cell differentiation.51,52
ATP release and breakdown
It is now recognised that ATP is released, without causing any damage, from many cell types, although for many
years it was assumed that the main source of ATP acting
on purinoceptors was damaged or dying cells. Endothelial and urothelial cells, astrocytes, macrophages, osteoblasts, odontoblasts and many other cell types have beenreported to release ATP in response to gentle mechani-

In contrast to laboratory animals, in which the purinergic component of parasympathetic cotransmission
supplying the bladder is about 50%, in healthy human
bladder it is only about 2%, even though receptors for
ATP are expressed by the smooth muscle. However, inpathological conditions such as interstitial cystitis, outflow obstruction and neurogenic bladder, the purinergic
component increases to up to 40% and is therefore atarget for therapeutic treatment.3 Botulinum neurotoxin
type A is widely used to treat bladder incontinence andacts by inhibiting the release of ATP and ACh from bothparasympathetic nerves and urothelial cells.59,60
Cardiovascular disorders
ADP acting via P2Y1 and P2Y12 receptors mediates
platelet aggregation, and so P2Y12 receptor antagonists
clopidogrel and related compounds are widely used forthe treatment of thrombosis and stroke.61,62
There are reports that in spontaneously hypertensive
rats there is a significantly greater cotransmitter role for
ATP in sympathetic nerves supplying blood vessels.4
Long-term purinergic control of proliferation of smoothmuscle and endothelial cells via P1 and P2Y receptors
during vascular remodelling in restenosis following angioplasty and in atherosclerosis has been reported, andtherapeutic possibilities are being explored.47
There is increased expression of P2X1 and P2Y2 receptors in the hearts of rats with congestive heart failure.63
Diabetes
α-Cells in pancreatic islets express P2X7 receptors. In
streptozotocin-induced diabetes, these P2X7 receptorexpressing cells replace the space no longer occupied byinsulin-containing β-cells.64 Purinergic compounds arebeing explored for the treatment of diabetes.65

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67

Osteoporosis

HIV/AIDS

The involvement of purinergic signalling in bone development and regeneration has been reported, and therapeutic approaches are being explored for osteoporosis, inparticular treatment with P2X7 receptor antagonists.47,66

Recent papers have implicated purinergic signallingin HIV/AIDS and may offer novel therapeutic approaches.81–83

Kidney failure
There is rich expression of purinoceptor subtypes in
the kidney, both in the glomerulus and tubules. Increased
expression of P2X7 receptors in diabetes, hypertension,
polycystic kidney disease and glomerulonephritis is
opening up novel purinergic possibilities for the treatment of kidney failure.67–69
Erectile dysfunction and in vitro fertilization (IVF)
Abnormalities in purinergic signalling may be involvedin the pathophysiology of erectile dysfunction associatedwith diabetes and prostate enlargement and might be atarget for therapy.70–72
Treatment of sperm with extracellular ATP improves
the in vitro fertility rate for IVF.73,74
Gut disorders
A variety of different activities in the gut involve purinergic signalling, including peristalsis, synaptic transmission in both myenteric and submucous plexuses, vasculartone, mucosal secretion and pain.75 Exploratory studies
for purinoceptors as therapeutic targets for gut disorders
are in progress,76 including P2X3 receptor antagonists for
inflammatory bowel disease.77
Cystic fibrosis
A long-lasting P2Y2 receptor agonist has been developed and has been in clinical trials for the treatment of
cystic fibrosis. It has been accepted on the Japanese drug
market for the treatment of dry eye.78
Hyperventilation
Cells of the neuroendocrine bodies, which line thelungs at intervals in the epithelium, release ATP in response to distension, which then acts via P2X3 receptors
to activate vagal sensory fibres originating in the nodose
ganglion.79,80 This appears to be a mechanism underlying
reflex responses to hyperventilation and noxious gases.

Pain
The involvement of purinergic signalling in the initiation of pain was proposed in an article in the Lancet in
1996.84 It was proposed that P2X3 receptors, expressedon nociceptive nerve endings,85 were stimulated by ATP
released as a cotransmitter from sympathetic nerves during causalgia and reflex sympathetic dystrophy. It wasalso suggested that ATP was released from endothelial
cells in the microvasculature supplying heart, skeletal
muscle and cerebral vessels during angina, ischemia andmigraine and from tumour cells during cancer pain. Later,
purinergic mechanosensory transduction was identified(Fig. 4)55 and its involvement in the initiation of visceralpain proposed.86,87 Kazu Inoue and colleagues showedthat there was increased expression of P2X4 receptors on
microglia in neuropathic pain, and this pain was reduced
by antagonists to the P2X4 receptor and in P2X4 receptor
knockout mice.88,89 Later, antagonists to P2X7 and P2Y
receptors expressed on microglia were also shown to reduce neuropathic pain.86 P2X3 and P2X7 receptors arebeing targeted for the treatment of arthritis.90,91
Disorders of the CNS
Investigations of the roles of purinergic signalling havebeen reported in disorders of the CNS, including injury
due to accidents, surgery, stroke and ischemia, neurodegenerative diseases (such as Alzheimer’s, Parkinson’sand Huntington’s diseases), multiple sclerosis, epilepsy
and neuropsychiatric disorders (including schizophrenia,
depression and anxiety).56,92,93
Cancer
It was recognised early that ATP was effective against
cancer.94 More recent studies have shown that P2Y1 andP2Y2 receptors mediate proliferation in most tumours,that P2X5 receptors mediate differentiation and therefore are anti-proliferative, and that P2X7 receptors leadto apoptotic death of tumour cells (Fig. 5).5,49 The therapeutic potential of P2Y1 and P2Y2 receptor antagonists
and P2X5 and P2X7 receptor agonists is being explored.

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Burnstock G: Purinergic Signalling: Pathophysiology and Therapeutics

Fig. 4 Schematic hypothesis for purinergic mechanosensory transduction in tubes (e.g., ureter, vagina, salivary and bile ducts, gut)and sacs (e.g., urinary and gall bladders and lung). It is proposed that distension leads to release of ATP from epithelium lining the tube
or sac, which then acts on P2X3 and/or P2X2/3 receptors on subepithelial sensory nerves to convey sensory/nociceptive information
to the CNS. (Reproduced from Burnstock55 with permission from Blackwell Publishing.)

Fig. 5 Schematic of the different mechanisms by which P2 receptor subtypes might alter cancer cell function.
P2Y1 and P2Y2 receptors could affect the rate of cell proliferation through altering the intracellular levels of cAMP by modulating adenylyl cyclase (AC) or by increasing intracellular calcium levels through the phospholipase C (PLC) pathway. P2X5 and P2Y11 receptor
activation might switch the cell cycle from proliferation into a state of differentiation. The P2X7 receptor activates the apoptotic caspase
enzyme system. PtdIns (4, 5)P2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; ER, endoplasmic reticulum. (Reproduced
from White and Burnstock49 with permission from Elsevier.)

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69

Fig. 6 Schematic hypothesis of purinergic signalling in acupuncture.
AMPA, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid; NMDA,
N-methyl-D-aspartate. (Reproduced from Burnstock95 with permission from
Elsevier.)

Diseases of the special senses
Purinergic signalling is much involved in the physiology of the eye, ear, nasal organs and tongue. Consequently,
purinergic therapeutic strategies are being developed forglaucoma, retinal detachment, diabetic retinopathy, Ménière’s disease, tinnitus, sensorineural deafness, noxious
odour damage and taste defects.1
Acupuncture
Acupuncture has been used over many years to treat awide variety of diseases and to alleviate pain. The traditional Chinese view is that manipulation of the flow of
meridian energy (qi) is the underlying mechanism for
acupuncture. However, it seems more likely that the nervous system is involved. It has been proposed that purinergic signalling is a major factor in the physiologicalmechanism responsible for the effects of acupuncture.95
It is suggested that mechanical stimulation caused bytwisting needles in the skin and tongue, heat, electri-

cal currents or laser treatment leads to release of ATP
from keratinocytes and mast cells that accumulate at
acupuncture sites. ATP then initiates activity via P2X3
receptors on sensory nerves supplying the skin that relay
messages through interneurones to the brain stem where
they modulate the activity of motor neurones that control
autonomic function (Fig. 6). Sensory messages will also
modulate the pain pathways leading to the conscious pain
centres in the cortex.
Concluding Comments
The clinical manipulation of purinergic signalling is in
its infancy. Therapeutic strategies to deal with pathologicaldisorders include the design of selective P1 and P2 receptor subtype agonists and antagonists, as well as of inhibitors of extracellular ATP breakdown and of ATP transportenhancers and inhibitors. Medicinal chemists are developing small-molecule purinergic drugs that are orally bioavailable and stable in vivo.3,6,96,97 There has been a remarkable growth in the number of papers published aboutpurinergic signalling via ATP since 1972 (Fig. 7).

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Burnstock G: Purinergic Signalling: Pathophysiology and Therapeutics

Fig. 7 Graph showing the number of papers published on P2 purinergic signalling between
1972 and the end of 2012. (Reproduced and updated from Burnstock 20 with permission
from John Wiley & Sons.)

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