purinoceptors and fast purinergic transmission at neuromuscular … · 2015-12-25 · purinoceptors...

Purinoceptors and Fast Purinergic Transmission at Neuromuscular Junctions and Synapses

Prof. Geoffrey Burnstock

1The screen versions of these slides have full details of copyright and acknowledgements

1

Prof. Geoffrey Burnstock PhD, DSc, FMedSci, FRS

Director of the Autonomic Neuroscience Institute Royal Free Hospital School of Medicine, London, UK

Department of Pharmacology and TherapeuticsThe University of Melbourne, Australia

Purinoceptors and Fast Purinergic Transmission

at Neuromuscular Junctions and Synapses

2Non-synaptic autonomic neuromuscular transmission

3




4

Innervation of mast cells

Rat dura mater

Rat intestine muscularis mucosa Rabbit middle cerebral artery

5

Taenia coli

6

Taenia coli – IJP’s




7

8

9

Criteria for establishing a neurotransmitter:

1. Synthesis and storage in nerve terminals

2. Release by a Ca2+-dependent mechanism

3. Mimicry of the neurogenic response by receptor occupation

4. Inactivation by ectoenzymes and/or neuronal uptake

5. Modification of response by drugs in a comparable manner to that of the neurogenic response




10Albert Szent-Györgyi Pamela Holton

11

Myenteric neurones - quinacrine

12

Bladder - NANC




13

14

Ectonucleotidases

From Zimmermann, 2001

15




16

17

Purinergic nerve

Burnstock 1972, Pharm Revs 24: 509-581

18

“A busy bee called ATP Whose wings were full of energy, Who worked and built inside a cellEmerged to see what he could tell

To each and every other cell.”

(Part of a poem by Nomi Burnstock, reproduced from Journal of the Autonomic Nervous System, Special Issue: Purines and the autonomic nervous system: from controversy to clinic; prologue, 81: 1-2 (2000) with permission from Elsevier)




19

20

21

Vas deferens – sympathetic nerve stimulation

From Burnstock and Holman, 1960




22

Vas deferens – EJP’s

From Sneddon and Burnstock, 1984

23

Guinea pig vas deferens – spritz 20ms

24

Isolated rabbit saphenous artery




25

Rat tail artery (sympathetic cotransmission)

From Sneddon & Burnstock 1984 (A-C) and Jobling and McLachlan, 1992 (D)

D

26

Schematic of sympathetic cotransmission

From Burnstock & Verkhratsky, 2010

27

Pre- and post junctional neuromodulation by neuropeptide Y (NPY)

depends on junctional cleft width




28

29

Sympathetic cotransmission in SHR

• Vidal, Hicks & Langer (1986) Naunyn-Schmiedeberg’sArch. Pharmacol. 332: 384-390“our results suggest a significantly greater co-transmitter role for ATP with noradrenaline on tail arteries of SHR compared with control normotensive WKY rats...”

• Bullock & McGrath (1992) Br. J. Pharmacol. 107 (S): 145P“Evidence for increased purinergic contribution in hypertensive blood vessels exhibiting cotransmission”

• Brock & Van Helden (1995) Pflügers Arch. – Eur. J. Physiol. 430: 901-908“Enhanced excitatory junction potentials in mesenteric arteries from spontaneously hypertensive rats”

30

Increase in purinergic component of nerve-mediated responses of human bladder

in pathological conditions

• Interstitial cystitis (40% purinergic)Palea et al., J. Urol. 150: 2007-2012, 1993

• Outflow obstruction (25% purinergic)Smith & Chapple, Neurol. Urodyn. 13: 414-415, 1994

• Neurogenic bladder (40% purinergic)Wammack et al., Akt. Urol. 26: 16-18, 1995




31

Sensory - motor nerves

32

Quinacrine NADPH-d

From Belai and Burnstock, 1994

33- -

Gut – NANC inhibitory nerves

VIP NO ATP

ATP ADPG

VIP

NO=

== = 8-PTRB2

L-NAME

+ - -(slow)(slow) (slow)(v.slow) (fast)

FenoprofenIndomethacin




34

Skeletal neuromuscular junction

35Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, edited by R.W. Straub and L. Bolis. Raven Press, New York © 1978

AntagonistsAgonist

potencies

Changes in cAMP

production

Induction of prostaglandin

synthesis

P1 Methylxanthines AD≥AMP>ADP≥ATP YES NO

P2Quinidine

Imidazolines

2’2 Pyridilisatogen

ATP≥ADP>AMP≥AD NO YES

36Modified from Burnstock, 1972, Pharm Revs 24, 509-58136




37

Loewi-inspired experiments (1966)

● = nerve stimulation (5 Hz for 40 sec every 6 mins at 50 V and 2 msec duration)

=

=

38

P1receptor Agonists Antagonists

Transductionmechanism Patent utility

A1

CCPA > R-PIA = S-ENBACVT-510GR79236

DPCPXN-0840

MRS1754WRC-0571

Gi/o

cAMP ↓PLC ↑

Atrial fibrillationCardioprotection

DiabetesIschaemia

A2A

HENECA > CGS21680 =

CVT-3146ATL-146e

KF17837SCH 58261ZM241385KW 6002

GS

cAMP ↑IP3 ↑

InflammationAsthmaCOPD

Parkinson’s Disease

A2B Bay60-6583

PBS603MRE-2029-F20

PSB0788MRS 1706PSB 1115Alloxazine

GS

cAMP ↑PLC ↑

Bowel and bladder abnormalities

A3

IB-MECA > 2-Cl-IB-MECA

DBXRMVT160

HEMADO

MRS1220L-268605

MRS 1191 & 1334 (h)MRS 1523 (r)

VUF 8504 & 5574PSB10

Gi/o

Gq/11

cAMP ↓IP3 /DAG ↑

PLC ↑

CardioprotectionCerebrovascular

IschaemiaCancer,Allergy

39• Subtype 1 (designated P2X)

• Subtype 2 (designated P2Y)




40

Initial cloning of P2 receptors

P2YP2Y1: Webb TE, Simon J, Krishek BJ, Bateson AN, Smart TG, King BF,

Burnstock G and Barnard EA. Cloning and functional expression of a brain G-protein-coupled ATP receptor, FEBS Lett 324: 219-225, 1993

P2Y2: Lustig KD, Shiau AK, Brake AJ and Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells, Proc Nat Acad Sci USA 90: 5113-5117, 1993

P2XP2X1: Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A and Buell G.

Cloning and expression of the P2X receptor for extracellular ATP reveals a new class of ligand-gated ion channel, Nature 371: 516-519, 1994

P2X2: Brake AJ, Wagenbach MJ and Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor, Nature 371: 519-523, 1994

41From Fields & Burnstock, 2006, Nature Neuroscience

42From North, 1996

≠

≈




43From Kawate et al., 2009 – Eric Gouaux

Crystal structure of P2X4 receptor

44

NeurotransmitterReceptors

Fast ionotropic(Ligand-gated ion channels)

Slow metabotropic(G-Protein)

AchNicotinic

muscle typeneuronal type

MuscarinicM1 → M5

GABA GABA A GABAB

GlutamateAMPA

KainateNMDA

mGlu1↓

mGlu7

5-HT 5-HT35-HT1A-F, 5-HT2A-C, 5-HT4,

5-HT5A-B, 5-HT6, 5-HT7

ATP P2XP2X1→P2X7

P2YP2Y1, P2Y2, P2Y4, P2Y6, P2Y11,

P2Y12, P2Y13, P2Y14

NA ─ α1, α2, β

Glycine α1, α2, α3 ─

From Burnstock, 1996

45

Receptor Main distribution Agonists Antagonists

P2X1

Smooth muscle, platelets, cerebellum,

dorsal horn spinal neurones

L-βγ-meATP≥α,β-meATP = ATP = 2-MeSATP

PAPET-ATP(rapid desensitization)

TNP-ATP, IP5I, NF023, NF449

RO1, RO 0437626, NF279, MRS2159

P2X2

Smooth muscle, CNS, retina, chromaffincells,

autonomic and sensory ganglia

ATP≥ATPγS≥2-MeSATP>>α,β-meATP, β,γ-CF2ATP

(pH + zinc sensitive)

Suramin, RB2, NF770,isoPPADS, PSB-1011, NF778,

aminoglycoside

P2X3Sensory neurones,

NTS, some sympathetic neurones

2-MeSATP≥ATP≥α,β-meATP =Ap4A, PAPET-ATP

(rapid desensitization)

TNP-ATP, isoPPADS A317491, NF110, RO4, RN-1838,

spinorphin, AF353

P2X4 CNS, testis, colon ATP>α,β-meATP, CTPIvermectin (potentiates)

TNP-ATP & BBG (weak),BBG, Paroxetine, 5-BDBD, CORM

2, phenolphthalein

P2X5Proliferating cells in skin,

gut, bladder, thymus, spinal cord

ATPγSAp4AGTP

Suramin, PPADS,BBG

P2X6 CNS, motor neurones in spinal cord

─ (does not function as homomultimer) ─

P2X7Apoptotic cells in immune cells,

pancreas, skin etc.

BzATP>ATP≥2-MeSATP>α,β-meATP

KN62, KN04, MRS2427, BBG, o-ATP, Decavanadate, A-804598,

RN-6189, AZD-9056, AZ10606120, A740003, A-438079, GSK-1370319




46

Heteromultimer coexpression studies

• P2X2/3 - Lewis et al., 1995; Radford et al., 1997

• P2X4/6 - Lé et al., 1998

• P2X1/5 - Torres et al., 1998; Haines et al., 1999

• P2X2/6 - King et al., 2000

• P2X1/2 - Brown et al., 2002

• P2X1/4 - Nicke et al., 2005

• P2X4/7? - Guo et al., 2007

47

Receptor Main distribution Agonists Antagonists Transduction mechanisms

P2Y1

Epithelial and endothelial cells, platelets, immune cells,

osteoclasts

MRS2365=(N)-me-2-MeSADP>2-MeSADP>

ADP=ADPβS>ATP

MRS2179MRS2500

Gq/G11; PLCβactivation

P2Y2Immune,epithelial & endothelial

cells, kidney tubules, osteoblasts

UTP=ATPUTP-γ-S

INS 37217(Up4dc)

Suramin > RB2AR-C126313

Gq/G11 and possibly Gi;

PLCβ activation

P2Y4 Endothelial cells UTP≥ATPUTP-γ-S RB2 > suramin

Gq/G11 and possibly Gi;

PLCβ activation

P2Y6 Some epithelial cells, placenta, T-cells, thymus

UDP>>UTP>>ATPUDP-β-S

MRS2578 Gq/G11; PLCβactivation

P2Y11 Spleen, intestine, granulocytes ARC67085MX>BzATP≥ATPγS>ATP

Suramin > RB2NF340,

5’-AMPS

Gq/G11 and GS; PLCβ activation

P2Y12 Platelets, glial cells 2-MeSADP>ADP>>ATP

ARC69931MXCT50547, AZD6140

INS49266,MRS2395

Gi/Go; Inhibition

of adenylate cyclase

P2Y13Spleen, brain, lymph nodes,

bone marrowADP=2meSADP>>ATP

and 2meSATP MRS2211 Gi/Go

P2Y14

Placenta, adipose tissue, stomach, intestine,

discrete brain regions

UDP glucose = UDP-galactose Gq/G11

48

• Agboh, K.C., Webb, T.E., Evans, R.J. & Ennion, S.J. (2004) Functional characterization of a P2X receptor from Schistosoma mansoni,Journal of Biological Chemistry 279: 41650-41657

• Fountain, S.J., Parkinson, K., Young, M.T., Cao, L., Thompson, C.R. & North, R.A. (2007) An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum, Nature 448: 200-203

• Fountain, S.J., Cao, L., Young, M.T. & North, R.A. (2008) Permeation properties of a P2X receptor in the green algae Ostreococcus tauri, J Biol Chem. 283: 15122-15126

• Burnstock, G. & Verkhratsky, A. (2009) Evolutionary origins of the purinergic signalling system, Acta Physiologica 195: 415-447

• Fountain, S.J. & Burnstock, G. (2009)An evolutionary history of P2X receptors, Purinergic Signalling 5: 269-272




49

• Evans, R.J., Derkach, V. and Suprenant, A. (1992) ATP mediates fast synaptic transmission in mammalian neurons, Nature 357: 503-505

• Silinsky, E.M., Gerzanich, V. and Vanner, S.M. (1992)ATP mediates excitatory synaptic transmission in mammalian neurones, British Journal of Pharmacology 106: 762-763

• Edwards, F.A., Gibb, A.J. and Colquhoun, D. (1992)ATP receptor-mediated synaptic currents in the central nervous system, Nature 359: 144-146

50

Purinergic sympathetic transmission in coeliac neurons / suramin

From Evans et al., 1992

51

Intrinsic neurons in bladder and heart

From Burnstock et al., 1987




52From Galligan, 2002

Guinea-pig small intestine non-cholinergic fast excitatory synaptic pathways

Current Opinion in Pharmacology

53From Barajas-López et al., 1996

ATP activates single channel activity in membrane patches of guinea-pig myenteric neurons

54From Galligan, 2002

Guinea-pig intestine slow excitatory synaptic pathways

Current Opinion in Pharmacology




55From Barajas-López et al., 1994

Guinea-pig submucosal neurons

56From Burnstock, 2008

57

P2X3 – IR synapse; dorsal horn: C6: rat




58

Spontaneous EPSC’s in cortical neurons mediated by P2X4 receptors

Modified from Lalo et al., 2007

59

P2XR-mediated synaptic EPSC’s in cortical (a) and hippocampal (b) neurons

From Pankratov et al., 2002 (a) and 1998 (b)

a: 1 = Control EPSC’s2 = Glutamate antagonists3 = ACh (Nicotinic) antagonist4 = P2XR antagonist5 = washout

b: 1 and 2 = Glutamate antagonists3 = PPADS (P2R antagonist)

60Modified from Pankratov et al.,2009

P2XR-dependent regulation of synaptic plasticity in the CNS




61From Sawada et al., 2008

Vesicular nucleotide transporter (VNUT)

62

63

• A P2X purinoceptor expressed by a subset of sensory neurons C-Chen , AN Akopian, L Sivilotti, D Colquhoun, G Burnstock, JN Wood, Nature 1995, 377: 428-431

• Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neuronsC Lewis, S Neldhart, C Holy, RA North, A SuprenantNature, 1995 377: 432-435




64From: Burnstock, 1996 ‘A unifying purinergic hypothesis for the initiation of pain’ Lancet, 347, 1604-1605

Sources of ATP acting on nociceptive purinoreceptors at sensory nerve terminals

Sympathetic nerveTerminal varicosities

Endothelial cellsof microvessels

Tumor cellsVasodilatation

65

P2X3 receptors are expressed in GDNF but not NGF sensitive neurons

% o

f cel

ls

expr

essi

ng P

2X3

0

20

40

60

80

CGRP IB4 NF200

66 From Burnstock and Wood, 1996

Sympathetic nerve Merkel cell

Endothelial cell

Tumor cell




67

Schematic of purinergic mechano-sensory transduction

In TUBES e.g., ureter, vagina, salivary & bile ducts, gut & SACS, e.g., urinary & gall bladder, & lung

From Burnstock, 1999

68

Discharges in the pelvic nerve induced by distension of the bladder in P2X3 KO mouse and its WT control

WT mouse P2X3 knock-out mouse

68

69 Nature 2000, 407: 1011




70

Guinea-pig ureter –antagonism of distension response by TNP-ATP

From Rong & Burnstock, 2004

71

Human ureter ATP release

72

Colo-rectum: effect of ATP in sensory nerve activity

72




73

Distension of rat colo-rectum: ATP release

74

Guinea-pig colon: P2X3 immunoreactivity in a whole-mount preparation of the subepithelial nerve plexus

75

Hypothesis:Purinergic sensory pathways in gut

Powerful distensionATP release → stimulation of high threshold extrinsic sensory nociceptive fibres → pain

Subepithelial sensorynerve plexus(with P2X3 receptors)

Moderate distensionATP release → stimulation of low threshold intrinsic sensory fibres → peristaltic reflexes

Extrinsic sensory nerves

P2X3Calbindin

Intrinsic sensory nerves

DRG

P2X3IB4

ATPATP

ATPATP

ATP

ATPATP ATP

ATP

ATP

Spinal cord & brain pain centres




76

Sensory nerves in human tooth pulp and odontoblasts - P2X3

From Alavi et al., 2001

77

Innervation of neuroepithelial bodies

78

Hypothesis: purinergic signalling in acupuncture




79

Rat tongue – sensory nerve preparation

80

Rat colitis model, using trinitrobenzenesulfonic acid (TNBS) enema

<

=

81

ATP concentration in luminal fluid samples from normal and inflamed rat colo-rectum

during distension

Values are means ± SE

0.0

2.5

5.0

7.5

10.0

12.5Normal

Colitis

Pressure (mmHg)

ATP

(pm

ol/m

l)




82

83

84

Selective antagonists with drug-like properties(e.g., orally bioavailable, metabolically stable)

From Gever et al., 2006

“Drug-like” antagonists




85

Number of papers published on P2 purinergic signalling

between 1972 and end of 2012

86

Associate EditorsMolecular Biology of Purinergic ReceptorsSimon Robson, Boston, USAJean-Marie Boeynaems, Brussels, BelgiumEctoenzymesHerbert Zimmermann, Frankfurt, GermanyMedicinal Chemistry & PharmacologyKenneth A Jacobson, Bethesda, USAAd Ijzerman, Leiden, The NetherlandsPathophysiology & Clinical ImplicationsMichael F Jarvis, Chicago, USAJim Wiley, Melbourne, Australia

Development & RegenerationKazuhide Inoue, Tokyo, JapanPhysiological RolesFrancesco Di Virgilio, Ferrara, ItalyPeter Illes, Leipzig, Germany Vera Ralevic, Nottingham, UKTim Arnett, London, UKJean-Pierre Timmermans, Antwerp, BelgiumReviewsMaria Abbracchio, Milan, ItalyNick Dale, Coventry, UK

Editorial Board Jolanta Baranska, Warsaw, PolandLuiz Belardinelli, Palo Alto, USAMax Bennett, Sydney AustraliaMichael Blackburn, Houston, TexasStefan Boehm, Vienna, AustriaPier Borea, Ferrara, ItalyRichard C Boucher, Chapel Hill, USAJose Boyer, Durham, USAGloria Cristalli, Camerino, ItalyBruce Cronstein, New York City, USARodrigo Cunha, Coimbra, PortugalJeff Dixon, London, OntarioGeorge Dubyak, Cleveland, USADavid Erlinge, Lund, SwedenAnthony Ford, Palo AltoJohn Fozard, Basel, Switzerland

Christian Gachet, Strasbourg, FranceKen Harden, Chapel Hill, USA,Stephen Holgate, Southampton, UKGary Housley, Auckland, New ZealandPablo Huidobro-Toro, Santiago, ChilePaul Insel, San Diego, USACharles Kennedy, Strathclyde, UKBaljit Khakh, Cambridge, UKYong-Chul Kim, Kwangju, KoreaOleg Kristal, Kiev, UkraineEduardo Lazarowski, North Carolina, USAJoel Linden, Charlottesville, USAMaria Teresa Miras-Portugal, Madrid, SpainChrista Müller, Bonn, GermanyJoseph T Neary, Miami, USAYasunobe Okada, Okazaki, Japan

Jesus Pintor, Madrid, SpainAlex Ribeiro, Lisbon, PortugalMichael W Salter, Toronto, CanadaJana Sawynok, Halifax, CanadaAna Sebastião, Lisbon, PortugalEugene Silinsky, Chicago, USAMichail Sitkovsky, Bethesda, USABeata Sperlagh, Budapest, HungaryRandy Sprague, St. Louis, USAAnnmarie Surprenant, Sheffield, UKAlex Verkhratsky, Manchester, UKMark Voigt, St. Louis, USACinzia Volonte, Rome, ItalyGary Weisman, Missouri, USABen Yerxa, North Carolina, USA

Purinergic SignallingEditor-in-chief: Geoffrey Burnstock, London, UK

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purinoceptors and fast purinergic transmission at neuromuscular … · 2015-12-25 · purinoceptors...

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