cost-b30 2010 kiev olegkrishtal

8/8/2019 COST-B30 2010 Kiev OlegKrishtal

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Unusual properties of purinoreceptors: implications for

the antinociceptive

pharmacology

Oleg Krishtal

Bogomoletz Institute of Physiology, Kiev, Ukraine

Ionic channels

Voltage gated Ligand (or physical parameter) gated

Nerve impulse

Generator potential

sec/min

ACTIVATED BY:

ATP Heat Protons

Also present:TRPM8 (noxious cold)TRPA1 (cold)Nociceptive mechanoreception:ENIGMATIC

Nociception: the ligands areubiquitous

and ATP is one of them

Mice: P2X3-/-

R educed pain-related behaviour, urinary bladder hyporeflexia(Cockayne et at., 2000)Formalin-induced pain behaviour is reducedUnable to code thermal stimuli(Souslova et al., 2000)

Enhanced thermal avoidanceEnhanced induction of spinal c-FOS under heating(Shimizu et al., 2005)

Mice: P2X2/P2X3(Dbl-/-)

Important contribution of heteromeric P2X2/3 receptors tonociceptive responses and mechanosensory transduction, especiallywithin the urinary bladder (Cockayne et al., 2005)

X b130ms

ATP 10 QM

X b 25ms100 ms

ATP 10 Q M

100 ms

ATP 10 Q M

100 ms

0,5 nA

ATP 10 Q M

100 ms

P2X2/3

P2X3+P2X2/3

Physiology and Pathophysiology of Purinergic Neurotransmission

GEOFFREY BURNSTOCK 2007

Visualization of ATP release of fromNHEKs

Biochem. J. (2004) 380, 329Biochem. J. (2004) 380, 329- -338 338

Dynamic communication between NHEKs and DRG neurons mediated b y extracellular ATP

Biochem. J. (2004) 380, 329Biochem. J. (2004) 380, 329- -338 338

Chizmakov et al. , Eur J Neur osc i. 9

Severe cancer bone pain is oftenSevere cancer bone pain is oftenrefractory to opioid treatment.refractory to opioid treatment.

What could be the effect of boneWhat could be the effect of bonecancer cells on the opioid control of cancer cells on the opioid control of

P2X nociceptors?P2X nociceptors?

C ontrol: effect of opioids does not depend onthe current kinetics

C o-culture: the slower is the currentkinetics, the smaller is effect of opioids

Chizmakov et al. ,Eur J Neur osc i. 9

R epresentation of slow responsesincreases with time in co-culture

Co-culturing sensory neurons with tumor

cells impairs opioid control over P2X2/3receptors

ATP 10 QM

35'2 '

Rec ov ery of P2X3 recept ors f r om dese s itiza tion

Pratt at al. J Neurosci. 2005 Aug 10;25(32):7359Pratt at al. J Neurosci. 2005 Aug 10;25(32):7359- -65.65.

U se-depe nde nt inhibi tion of Xrecept ors by nanomola r agoni st

High affinity bin ding st a te

Open Desensitized

Enigmas of P2X3 receptors:

Ultra-slow desensitization removal (20-30 min)

Desensitization by nM ATP concentrations (IC50=0.5nM)

How can these receptors function in sensorysignaling?

Mice: P2X3-/-

R educed pain-related behaviour, urinary bladder hyporeflexia(Cockayne et at., 2000)Formalin-induced pain behaviour is reducedUnable to code thermal stimuli(Souslova et al., 2000)

Enhanced thermal avoidanceEnhanced induction of spinal c-FOS under heating(Shimizu et al., 2005)

Mice: P2X2/P2X3(Dbl-/-)

Important contribution of heteromeric P2X2/3 receptors tonociceptive responses and mechanosensory transduction, especiallywithin the urinary bladder (Cockayne et al., 2005)

Khmyz et al., Eur. J. Physiol. (2008)Khmyz et al., Eur. J. Physiol. (2008)

AT -induced dep ola r iza tion an da ct ion pote ntial fir ing

V ( m v )

0 ,1 1 1 0 100

IC5 03 5 o C

= 9.98 ± 0 .07 nMIC5 03 5 o C

= 3 .2 ± 0 .3 nMIC5 02 5 o C

= 0 .7 9 ± 0 .0 9 nM

3 5 o C , 1 0 min3 5 o C , 2 min25 o C , 2 min

I / I m a x

A T (nM)

AAt mammalian temperatures thet mammalian temperatures the

P2X3 receptors are not tonicallyP2X3 receptors are not tonicallydesensitiseddesensitised

Natural toxins modulating major nociceptors:

ASIC1a: PcTx1 (tarantula) - inhibitor ASIC3: APETx2 (sea anemone) - inhibitor TR PV1: AG489 (spider) - inhibitor

R esiniferatoxin (plant) - agonist NEW:

P2X3: purotoxin1 (spider) - inhibitor

Isolation of PT1 from the venom of

the spider Geolycosa sp. (a) Size-exclusion chromatography of thecrude venom on a TSK 2000SWcolumn. (b) R everse-phase HPLCof the fraction I from (a) on a

Vydac 218TP54 C18 column. The bar indicates the active fraction. I nset shows the final step purification (rechromatography of fraction 1) using the same Vydac

column, the active fractioncorresponding to pure PT1 isindicated with a line.

Ann. of neurol 2010, inpress

Purotoxin-1 structure . (a) PT1 amino acid sequence alignment with other known spider toxins.Cysteine residues are shaded dark grey, residues similar to those in PT1, light grey. Toxin namesare presented in the left column together with the corresponding UniProt accession numbers (in

parentheses). The lengths of the peptides (in residues) are given in the right column, number of cysteine residues in parentheses. (b,c) Stereo view of PT1 spatial structure determined by NM R

spectroscopy. R ibbon representation of the peptide with disulfide bonds is shown in (b) . Peptide¶ssurface painted according to the electrostatic potential is illustrated in (c) . Dark regions denote sidechains of charged residues. ( Ann of Neurol 2010)

(Ann of Neurol 2010)

P T e ff ect on rec ombinan t human P2 X recept ors e xpressed in HEK2 9 ce lls . (A) Exe mplary curre nt tr a ces . The st a tist ics de mon str a ted in (B ) indica te a dist inct inhibi tory e ff ect of P T on human P2 X recept ors ( n=6 ),tho ugh it is abo ut .5 times we ak er t han in the neur on s f r om r a t DRG .

Ann. of Neurol. 2010

High affinity bin ding st a te

Open Desensitized

PT1 dramatically decreases the rate of desensitization removal

Going in vivo only after successful

in vitro

Effect of PT1 (0.5 nmol, intraplantar injections) on thermal hyperalgesia and nociceptive behavior invarious pain models. (a) PT1 reduced thermal hyperalgesia induced by carrageenan (left diagram) and

CFA (right diagram). (b) PT1 reduced capsaicin- and formalin-induced nociceptive behavior (for formalin at second phase only). ***P<0.0001, **P<0.001, *P<0.05 vs. control group, ##P<0.001 vs.inflammation. Values are mean ± S.E.M. (n = 6 per each group).

PT1 is the first natural inhibitor of P2X3 receptors.PT1 is the first natural inhibitor of P2X3 receptors.

It represents a highly specific molecular motif for It represents a highly specific molecular motif for developing new analgesic drugsdeveloping new analgesic drugs

c A MP -induced inhibi tion of A TP -a ct iva tedcurre nts

100 ms

100 Q M A Tontrol 30 QM

200 ms

400 p A

100 QM A TP

C ontrol 10 Q M cAMP Wash

10 -6 10 -5 10 -40

cAMP ( M )Q

I/I max

A cta P hys iol (Oxf ). 2010

P uta tive e ff ect of c A MP

c A MP

G-pr ote in

High affini ty bin ding st a te

Desensitized P2X3 receptor: sensitivetarget for pharmacology and regulation

Bogomoletz Institute of Physiology,Ukrainian Academy of Sciences,Kyiv, Ukraine

Ganna A. Savchenko,Yaroslav A. Boychuk,Viacheslav Y. Viatchenko-Karpinski,

Nana V. Voitenko,Oleg A. Krishtal,

Igor V. Chizmakov,Mykola V. Mamenko,Volodymyr V. Khmyz,Olexandr P. Maximyuk,

University of ManchesterAlex Verkhratsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,R ussian Academy of Sciences,

Moscow, R ussia

Eugene V. Grishin,

Alexander A. Vassilevski,Yuliya V. Korolkova,Kirill D. Nadezhdin,Alexander S. Arseniev,Kirill A. Pluzhnikov

Toxinology in collaboration with

P2X receptors comprise a set of sensorymechanisms capable of producing either short,

time -calibrated or prolonged generator potentials that can match a variety of sensory

modalities including nociception.

Inferences obtained in vitro are targeting invivo approaches and allow to minimize them

Chizmakov et al. , Eur J Neur osc i. 200 9

The effect of intraplantar administration of PT1 on the thermal hyperalgesia and inflammatory nocifensive behaviors.(A,B) Paw withdrawal latency (PWL) to heat stimulus (plantar test, Hargreaves method) measured in carrageenan (A) andCFA (B) models of inflammation. Graphs represent time-response curves of PWL in control, inflamed and PT1-treated (0.5and 2 nmol) rats. PT1 was injected at the beginning of either experiment. Each point represents the mean (±SEM) for 6animals.(C,D) Nocifensive behavior of rats after injection of 50 µl ml-1 formalin (C) or 1 mg ml-1 capsaicin (D) into the hindpaw of rats. Graphs represent time-response curves of the jerking behavior in control and PT1-injected rats. In the formalin test, PT1(0.5 nmol) was injected either simultaneously with formalin (at 0 min) or at the beginning of the second (tonic) phase (7 minafter the injection of formalin). In the capsaicin test PT1 was injected simultaneously with capsaicin. Control rats wereinjected with saline at the same time points. Each point in the graphs represents the mean (±SEM) number of jerks per 2 minfor 6 animals.

Note that the effect of PT1 was longer lasting in the models of carrageenan, CFA and capsaicin hyperalgesia as compared tochemogenic hyperalgesia induced by formalin. Moreover, the effect of PT1 was observed in the formalin test only if PT1 was

injected at the beginning of the second phase. This could be due to specific interaction of PT1 with formalin, which couldinactivate the peptide molecule.

0 ,1 1 10 100

0 , ¡

0 , ¢

0 , £

IC5 03 5

= 3 . 0 .3 IC5 02 5

= 0 .7 0 .09 3 5

oC , 2 i2 5

oC , 2 i

I / I m

ATP (nM)

Eff ect of P T1 on the curre nts med ia ted by P2 X2 an d P2 X2 / recept ors . In order t o discr imina te t he a ct ivities of P2 X2 an d P2 X2 / recept ors we used t he ir d iff ere ntial se ns itivity to ATP an d , - me ATP: while P2 X2 / recept orshav e s imila r se ns itivity to bo th agoni sts, P2 X2 recept ors a re vis ibly a ct iva ted a t , - me ATP con ce ntr a tionse xceed ing 100 µM22 . Equ al con ce ntr a tions of bo th agoni sts were a pp lied t o the ce lls de mon str a ting s lowresp on ses . In s om e ce lls t he resp on se t o ATP wa s ma r ked ly lar ger t han the resp on se t o , -m e ATP indica ting a pred ominan t c on tr ibution of P2 X2 recept ors (A). In o ther ce lls t he resp on ses c ould be almo st equ al. Evide ntly theye xpressed pred ominan tly P2 X2 / recept ors (B). P T1 wa s ine ff ect ive again st t he resp on se t o e ither of the agoni sts(n=6 ). Howe ver, t he resp on ses t o e ither of the agoni sts were als o unaff ected by P T1 (n=6 ). C onclusion : P T1 doesno t aff ect P2 X2 an d P2 X2 / recept ors

Ann. of Neurol. 2010

cost-b30 2010 kiev olegkrishtal

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