How to cite this article:WIREs Membr Transp Signal 2012, 1:116–125. doi: 10.1002/wmts.14
NON-ADRENERGIC,NON-CHOLINERGIC TRANSMISSION
The existence of transmitters released from auto-nomic nerves that were neither of the classical
neurotransmitters, acetylcholine (ACh) or nora-drenaline (NA), was discovered in 1963 wheninhibitory junction potentials were recorded in theguinea pig taenia coli in response to nerve stimu-lation in the presence of atropine and guanethidine(see Ref 1). These non-adrenergic, non-cholinergic(NANC) responses were shown to be present inintrinsic enteric neurons controlled by vagal and
∗Correspondence to: [email protected] Neuroscience Centre, University College MedicalSchool, Royal Free Campus, London NW3 2PF, UK2Faculty of Life Sciences, The University of Manchester, ManchesterM13 9PT, UK
sacral parasympathetic nerves, and NANC transmis-sion was later shown in the urinary bladder andvascular system.
ATP AS A TRANSMITTER IN NANCNERVES
The next step was to try to identify the transmitterreleased during NANC inhibitory transmission in thegut and by NANC excitatory transmission in the uri-nary bladder. Several criteria needed to be satisfiedto establish a neurotransmitter: synthesis and stor-age in nerve terminals; release by a Ca2+-dependentmechanism; mimicry of the nerve-mediated responsesby the exogenously applied transmitter; inactiva-tion by ectoenzymes and/or neuronal uptake; andparallel block or potentiation of responses to stimu-lation by nerves and exogenously applied transmitter.
WIREs Membrane Transport and Signaling Purinergic signaling
Many different substances were examined in thelate 1960s, including amino acids, monoamines, andneuropeptides, but none satisfied the criteria. A crit-ical influence, however, was made by a seminalpaper by Drury and Szent-Gyorgyi2 showing pow-erful extracellular actions of purines on heart andblood vessels, papers by Feldberg showing extracel-lular actions of ATP on autonomic ganglia,3 and apaper by Pamela Holton, published in 1959, whichshowed release of ATP during antidromic stimulationof sensory nerves supplying the rabbit ear artery.4
Thus, ATP was tested and, to the genuine surpriseof the experimenters, it perfectly satisfied all the cri-teria needed to establish it as a transmitter involvedin NANC neurotransmission.5 In 1972, an article inPharmacological Reviews6 formulating the purinergicneurotransmission hypothesis was published. Unfor-tunately, few believed this hypothesis over the next25 years and it was often ridiculed at meetings andworkshops. Resistance to this concept was perhapsunderstandable because ATP was well established asan intracellular energy source involved in the Krebscycle and other biochemical pathways, and it seemedunlikely that such a ubiquitous molecule would alsoact as an extracellular messenger. In reality of course,ATP, recognized as an early biological molecule,evolved both as an intracellular energy source andas an extracellular signaling molecule. It was not untilreceptors for ATP were cloned and characterized inthe early 1990s and neurone–neurone synaptic trans-mission identified in sympathetic ganglia and in thebrain in 1972 that the purinergic hypothesis began tobe widely accepted (see Ref 7).
PURINERGIC COTRANSMISSION
For many years, our understanding of neurotransmis-sion was dominated by the concept that one neuronreleases only a single transmitter, known as ‘Dale’sPrinciple’. This idea arose from a widely adoptedmisinterpretation of Dale’s suggestion in 19358 thatthe same neurotransmitter was stored in and releasedfrom all terminals of a single neurone, a suggestionwhich did not specifically preclude the possibility thatmore than one transmitter may be associated with thesame nerve cell.
Based on experiments that showed release ofATP with NA from sympathetic nerves9 and manyhints in the literature, the cotransmission hypothesiswas formulated in 1976.10 Purinergic cotransmissionis now well established, not only in sympatheticnerves, but also in parasympathetic, sensory-motor,enteric nerves and developing motor nerves to skeletalmuscle. More recently ATP has been shown to
TABLE 1 ATP as a Ubiquitous Cotransmitter
Cotransmitters References
Peripheral nervous system
Sympathetic nerves ATP + NA + NPY 12
Parasympathetic nerves ATP + ACh + VIP 13
Sensory motor ATP + CGRP + SP 14
NANC enteric nerves ATP + NO + VIP 15
Motor nerves (in earlydevelopment)
ATP + ACh 16
Central nervous system
Cortex, caudate nucleus ATP + ACh 17
Hypothalamus, locus coeruleus ATP + NA 18
Hypothalamus, dorsal horn,retina
ATP + GABA 19
Mesolimbic system ATP + DA 20
Hippocampus, dorsal horn,cortex
ATP + glutamate 21–24
ACh, acetylcholine; ATP, adenosine 5′-triphosphate; CGRP, calcitoningene-related peptide; DA, dopamine; GABA, γ -aminobutyric acid; NA,noradrenaline; NANC, non-adrenergic, non-cholinergic; NO, nitric oxide;NPY, neuropeptide Y; SP, substance P; VIP, vasoactive polypeptide.(Reprinted with permission from Ref 37. Copyright 2004 Elsevier)
be co-released with glutamate, γ -aminobutyric acid,dopamine, NA, 5-hydroxytryptamine, and ACh indifferent populations of nerve fibers in the centralnervous system (CNS) (see Ref 11). It is now clearthat ATP is a cotransmitter in most, if not all, nervesin the peripheral nervous system (PNS) and CNS (seeTable 1). Furthermore, purines and/or pyrimidines actas signaling molecules in virtually all nonneuronaltissues (see Tables 1–3).
RECEPTORS TO PURINESAND PYRIMIDINES
Implicit in purinergic transmission is the existence ofspecific receptors. In 1978, a basis for distinguishingtwo types of purinergic receptors, one selective toadenosine (called P1), which was antagonized bymethylxanthines, and the other selective for ATP/ADP(called P2), was proposed.84 This was a useful stepforward, explaining some of the early confusion inthe literature resulting from the rapid extracellularbreakdown of ATP to adenosine and extendedour concept of purinergic neurotransmission, byidentifying postjunctional receptors as P2, whileprejunctional P1 receptors mediated neuromodulatorynegative feedback responses or autoregulation oftransmitter release. A pharmacological basis fordistinguishing two types of P2-purinoceptors, definedas P2X and P2Y, was proposed in 1985,85 and whenP2 receptors were cloned in the early 1990s86–89
(Reprinted with permission from Ref 37. Copyright 2004 Elsevier)
and second messenger mechanisms examined, thissubclassification was consistent with P2X ion channelreceptors and P2Y G protein-coupled receptors.Currently, four subtypes of P1 receptors arerecognized, seven subtypes of P2X receptors, andeight subtypes of P2Y receptors, including someresponsive to the pyrimidines UTP and UDP.90,91
It was shown that three of the P2X receptor
subunits combine to form cation pores92 eitheras homomultimers or heteromultimers, and morerecently heterodimerization has been shown betweenP2Y receptor subtypes.25 Many non-neural as wellas neuronal cells express multiple receptors,37 andthis poses problems about how they mediateinteracting physiological events. It is becoming clearthat the purinergic signaling system has an earlyevolutionary basis (see Ref 93) with fascinating recentstudies showing cloned receptors in two primitiveinvertebrates, Dictyostelium and Schistosoma thatresemble mammalian P2X receptors94,95 and ATPsignaling in plants has also been described.96–98
PHYSIOLOGY OF PURINERGICSIGNALING
While early studies were largely focused on short-term signaling in such events as neurotransmission,neuromodulation, secretion, chemoattraction, andacute inflammation, there has been increasing inter-est in long-term (trophic) signaling involving cellproliferation, differentiation, motility and death indevelopment, regeneration, wound healing, resteno-sis, epithelial cell turnover, cancer, and aging.99,100 Forexample, in blood vessels, there is dual short-term con-trol of vascular tone by ATP released as an excitatorycotransmitter from perivascular sympathetic nervesto act on P2X receptors on smooth muscle, whileATP released from endothelial cells during changes inblood flow (shear stress) and hypoxia acts on P2X andP2Y receptors on endothelial cells leading to produc-tion of nitric oxide and relaxation.101,102 In addition,there is long-term control of cell proliferation anddifferentiation, migration and death involved neo-vascularization, restenosis following angioplasty andatherosclerosis.42
For many years, the source of ATP acting onreceptors was considered to be damaged or dyingcells, except for exocytotic vesicular release fromnerves. However, it is now known that many cell typesrelease ATP physiologically in response to mechanicaldistortion, hypoxia, or to some agents.103 The mecha-nism of ATP transport is currently being debated andincludes in addition to vesicular release, ABC trans-porters, connexin or pannexin hemi-channels, maxi-ion channels, and even P2X7 receptors.7,26 There isnow much known about the extracellular breakdownof released ATP by various types of ectonucleoti-dases, including: E-NTPDases, E-NPPS, alkaline phos-phatase, and ecto-5′-nucleotidose (see Ref 104). Thereis current interest in the roles of purinergic signal-ing in neuron–glial interactions in the CNS (see Refs27 and 105).
WIREs Membrane Transport and Signaling Purinergic signaling
TABLE 3 Physiological Roles of Purinergic Signaling in Living Tissues
Tissue Functional Role Reference
CNS Fast excitatory cotransmission in CNS, modulation of synaptic plasticity, metabotropictransmission, regulation of growth and development, chemical transmission inneuronal-astroglial networks; signaling between axons and oligodendrocytes, CO2
chemosensitivity, control of microglial motility and activation
Negative chronotropic and ionotropic effects in atria, positive chronotropic and ionotropiceffect in ventricles, regulation of cardiomyocytes Ca2+ signaling, control of excitation ofintrinsic cardiac neurones
37,42
Cardiovascular system:blood vessels
Vasodilation (P2Y-mediated) and vasoconstriction (P2X-mediated) 37,42–44
Exocrine glands Regulation of ionic permeability and Ca2+ signaling in salivary and lachrymal gland cells,induction of sweat production by sweat gland epithelial cells
37
Endocrine glands Regulation of Ca2+ signaling in pituitary and thyroid cells, regulation of cathecholaminerelease from adrenal chromaffin cells, stimulation of insulin, glucagons, and soatostatinsecretion from endocrine pancreas
37
Immune system Regulation of mitogenesis and DNA synthesis in thymocytes, regulation of activation anddeath of macrophages, aggregation of neutrophiles, regulation of secretory response inbasophiles, and chemotactic response in eosynophiles, modulation of proliferativeresponse in lympocytes, release of histamine and degranulation of mast cells, mediationof intercellular Ca2+ waves in mast cells, regulation of release of proinflammatory factors
37,45–49
Lung Bronchodilation, stimulation of surfactant release from airway epithelial cells; stimulation ofmucins secretion from goblet cells; increase in ciliary beat frequency of ciliated epithelialcells, activation of lung myeloid dendritic cells; modulation of O2 chemotransmission incells of neuroepithelial bodies; contraction/relaxation of tracheal ring
37,50–53
Gastrointestinal tract Control of mucociliary activity of esophageal epithelial cells, regulation of acid secretion ingastric mucosa, regulation of contraction/relaxation of small intestine, inhibition of AChrelease from enteric neurons, regulation of peristaltic activity of ileum and duodenum,inhibition of amino acid, sugar and ion transport in epithelial cells of small intestine,relaxation of taenia coli, control of contraction/relaxation of colon and rectum, relaxationof internal anal sphincter
37,54–58
Liver Stimulation of glycogenolysis, inhibition of glycolysis, regulation of bile formation andsecretion via stimulation of Cl− efflux, mediate chemosensitivity of cholangiocyte cilia
37,59–62
Kidney Regulation of renal blood flow, microvascular function and glomerular filtration rate,generation of prostanoids, regulation of Cl− secretion, regulation of renal Na+, glucoseand water transport, possible involvement in biosensing activity of kidney macula densacells
63–68
Bladder and urethra Control of contraction/relaxation of mammalian bladder, relaxation of mammalian urethra 37,69–72
Male genital system Regulation of penile erection, contraction of prostate smooth muscle and seminal vesicles,micturition, peristalsis of the male excurrent duct system and thus sperm transport andejaculation; control of steroid production by testis leydig cells, inhibition of spermmotility, initiation of acrosome reaction
73–76
Female genital system Regulation of myometrium contraction, modulation of ovarian function, control of bloodflow in placenta, relaxation of vaginal smooth muscle, stimulation of vaginal moistureproduction, stimulation of Cl− and mucus secretion from endocervical epithelial cells
37,77–82
Bone and cartilage Regulation of osteoclast/bone formation and resorption, formation of multinucleatedosteoclasts, stimulation of resorption in cartilage, regulation of chondrocalcinosis
37,83
Skeletal muscle Regulation of proliferation and differentiation of developing myoblasts, modulation ofcontractile response of myocytes
37
(Reprinted with permission from Ref 93. Copyright 2009 Wiley-Blackwell)
It is well known that the autonomic nervous systemshows high plasticity compared to CNS. For example,substantial changes in cotransmitter and receptorexpression occur during development and aging, inthe nerves that remain following trauma or surgeryand in disease situations.106 For example, a P2Y-likereceptor was identified in Xenopus that was tran-siently expressed in the neural plate and again laterin secondary neuralation in the tail bud, suggestinginvolvement of purinergic signaling in the develop-ment of the nervous system.107 There is transientexpression of P2X5 and P2X6 receptors during devel-opment of myotubules and of P2X2 receptors duringdevelopment of the neuromuscular junction.108 Inthe rat brain, P2X3 receptors are expressed first atembryonic day 11 (E11), P2X2 and P2X7 receptorsappear at E14, P2X4, P2X5, and P2X6 receptors atpostnatal day 1 (P1), and P2X1 receptors at P16.109
Primitive sprouting of central axons was shownin experiments in which the enteric nervous systemwas transplanted in the striatum of the brain.110 Itwas later shown that a growth factor released fromenteric glial cell acting synergistically with ATP (andits breakdown product, adenosine) and nitric oxidewas involved.111 It is suggested that similar synergis-tic activity of purines and growth factors might beinvolved in stem cell activity.112
It was established early that ATP was a major cotrans-mitter with ACh in parasympathetic nerves mediatingcontraction of the urinary bladder of rodents.113 Inhealthy human bladder, the role of ATP as a cotrans-mitter is minor. However, in pathological conditions,such as interstitial cystitis, outflow obstruction, andmost types of neurogenic bladder, the purinergic com-ponent is increased to about 40%.106,114 Similarly, inspontaneously hypertensive rats, there is a significantly
greater cotransmitter role for ATP in sympatheticnerves.115
Clopidogrel, a P2Y12 receptor antagonist, whichinhibits platelet aggregation, is a highly successful drugagainst thrombosis and stroke.116 Purinergic com-pounds are also being developed for the treatment ofhypertension and atherosclerosis (see Ref 42), inflam-matory bowel disease,54 dry eye and cystic fibrosis,117
cancer (see Refs 118,119), and for a number of otherdiseases (see Ref 106).
P2X3 receptors were cloned in 1995 and shownto be largely located in small nociceptive sensorynerves that label with isolectin B4.120,121 Central pro-jections are located in inner lamina 2 of the dorsal hornof the spinal cord and peripheral extension in skin,tongue, and visceral organs. A unifying purinergichypothesis for the initiation of pain was published122
as well as a hypothesis describing purinergicmechanosensory transduction in visceral organs,where ATP, released from lining epithelial cells dur-ing distension, acts on P2X3 and P2X2/3 receptors insubepithelial sensory nerve endings to send nociceptivemessengers via sensory ganglia to the pain centers inthe brain.123,124 Supporting evidence including epithe-lial release of ATP, immunolocalization of P2X3 recep-tors on subepithelial nerves, and activity recorded insensory nerves during distension that is mimicked byATP and reduced by P2X3 receptor antagonists hasbeen reported in the bladder,125 ureter,126 and gut.127
Purinergic mechanosensory transduction is alsoinvolved in urine voiding as evidenced in P2X3 knock-out mice.128 For neuropathic and inflammatory pain,P2X4, P2X7, and P2Y12 receptors on microglia havebeen implicated, and antagonists to these receptors arevery effective in abolishing allodynia,129,130 and thereis also strong interest in the potential roles of puriner-gic signaling in trauma and ischemia, neurodegener-ative conditions including Alzheimer’s, Parkinson’s,and Huntington’s diseases and in multiple sclero-sis and amyotrophic lateral sclerosis. There are alsostudies in progress of purinergic signaling in neuropsy-chiatric diseases, including depression, anxiety, andschizophrenia and in epileptic seizures (see Ref 131).
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FURTHER READINGBurnstock G, Fredholm BB, North RA, Verkhratsky A. The birth and postnatal development of purinergic signalling. ActaPhysiol (Oxf) 2010, 199:93–147.
