Trieste meeting Dec 2001

1
Why pain gets worse the molecular basis of
sensitization in nociceptors
Peter McNaughton
DBSF Università di Insubria, Varese 11 April
2006
2
Injury enhances heat pain sensation one aspect
of inflammatory hyperalgesia
Raja et al, 1984
3
Sensitization is seen in single thermal
nociceptors in vivo
Belmonte Giraldez, 1984
4
Important contributors to heat hyperalgesia
following injury, inflammation or infection are
bradykinin and NGF
What is the cellular and molecular basis of the
actions of bradykinin?
5
(No Transcript)
6
Activation of an isolated voltage-clamped
nociceptor by a noxious heat stimulus
BUT sensitization is not seen with prolonged
stimulation in vitro.
Sensitization is therefore not endogenous to the
nociceptor but results in vivo from the release
of inflammatory mediators from surrounding
tissues.
Cesare McNaughton, 1996
7
The pro-inflammatory peptide bradykinin enhances
the heat gated current and lowers the threshold
an effect which is mimicked by direct activation
of PKC
Cesare McNaughton, 1996
8
The capsaicin-gated ion channel, TRPV1 (formerly
VR1)
Caterina et al, 1997 Jordt, Tominaga Julius,
2000 Jordt Julius, 2002
9
TRPV1 is activated by heat, capsaicin, acid,
anandamide are they all sensitized by PKC
activation?
10
TRPV1 is activated by a wide range of stimuli.
In each case the activation is potentiated by
PKC. (Here expressed in HEK cells also works in
neurones)
Vellani et al, 2001
11
TRPV1
Vellani et al, 2001
12
Bradykinin activates PKC, which in turn enhances
gating of TRPV1 to all stimuli

• Which isoforms of PKC are involved?

• Which residues on TRPV1 are phosphorylated by PKC?

13
Only PKCe is translocated following exposure to
bradykinin
Cesare et al, 1999
14
Sensitization by bradykinin pathways
TRPV1
6
?
COOH
PKCe
NH2
PLCb
15
Which residues on TRPV1 are phosphorylated by
PKC??
Numazaki et al (2002) found that phosphorylation
of two serines in rat TRPV1 explains
sensitization by PKCe
T344
PKCe phosphorylation sites
Numazaki et al, 2002
16
Sensitization by bradykinin targets
TRPV1
6
COOH
PKCe
NH2
PLCb
17
Alternative mechanism of action of bradykinin
Removal of PIP2 by PLC
In a Xenopus expression system Bk enhances gating
of TRPV1
Chuang et al, Nature 411, 957, 2001
Prescott Julius, Science 300, 1284, 2003
18
However, the weight of evidence favours
phosphorylation of TRPV1 by PKCe

• TRPV1 enhancement by BK is mimicked by PKC
  activation
• totally abolished by PKC inhibition
• mimicked by active PKCe infusion
• largely abolished by specific PKCe inhibition
• totally abolished by two PKC phosphorylation
  site mutations

19
NGF is also a powerful sensitizing agent which
causes a long-lasting heat hyperalgesia.
Which signalling pathways are involved here?
20
NGF-induced sensitisation
In vivo In vitro Lewin, Rueff
Mendell, 1994 Shu Mendell, 2001
21
Sensitization is conveniently measured using
calcium imaging
22
NGF sensitisation of capsaicin-gated current is
seen only in some neurones
, (n 112)
, (n 219)
No. cells
Ratio
23
TrkA
NGF
TRPV1
P
P
PI3K
Ca2
PLCg
Sos
Ras
MEK
24
Only PI3K inhibition abolishes the effect of NGF
Cells sensitised (mean )
NGF
NGF Sos inhib peptide
NGF U0126
NGF neomycin
NGF wortmannin
Bonnington McNaughton, 2004
25
Alternative mechanism of action of NGF removal
of PIP2 by PLCg
NGF enhances TRPV1 gating
Chuang et al, 2001
Prescott Julius, 2003
26
Reconstruction of TrkA to TRPV1 signalling in
HEK293 cells
27
NGF 0 5
10 20 40 60min
Tyrosine residues on TRPV1 are phosphorylated by
NGF
IP TRPV1 IB pY
IB TRPV1
and the action of NGF is enhanced by Src and
antagonised by DN Src or the Src inhibitor PP2
28
Both tyrosine phosphorylation and functional
sensitization are abolished by a TRPV1 N-terminal
Y200F mutation
29
TRPV1
Src
?
TrkA
30
Sites on TrkA responsible for initiating TRPV1
sensitization
TrkA
31
Sites on TrkA responsible for initiating TRPV1
sensitization
TrkA
32
How is TRPV1 potentiated?

• Enhancement of channel gating?
• Insertion of new channels?

33
Activation of PKC enhances channel gating
this action probably explains the effect of
bradykinin
34
Gating enhancement is abolished by the
S502A/S801A mutation
WTcaps
S502/801Acaps
WTcaps PMA
S502/801AcapsPMA
35
BUT insertion of new channels occurs in response
to NGF
.and is abolished by the Y200 mutation
36
Insertion of TRPV1 can be visualised in DRG
neurons
37
Visualisation of membrane TRPV1 by evanescent
wave microscopy
38
Visualisation of GFP-tagged TRPV1 in the membrane
39
Summary sensitization of TRPV1 by NGF
TRPV1
NH2
SHP1
COOH
Src
PLCg
Y794
Y760
Y499
TrkA
NGF
40
Long-term effects of NGF and other growth
factors upregulation of receptors for bradykinin
41
NGF upregulates expression of B2 receptors for
bradykinin
-NGF
NGF
Lee et al 2003
42
NGF (and to a lesser extent GDNF) upregulate B2
expression
but neither changes the proportion of neurons
expressing B2 receptors
43
NGF
GDNF
TrkA, CGRP, SP
RET, CGRP-, SP-
44
B1 agonists do not cause pain in normal
animals. BUT Following inflammation B1 agonists
do produce pain in vivo

• Which neurons express B1 receptors?
• How does inflammation cause B1 upregulation?

45
Like the B2 receptor, B1 receptor
activation specifically activates PKC-e and not
PKC-d
Anti- e
Anti- d
1-3
B2 -/-
46
GDNF strongly increases the number of neurones in
which PKCe translocation is observed in response
to B1 activation
47
B1 expressing cells are identified by PKC-?
translocation following exposure to a B1
agonist. These cells are all IB4-positive.
None of them contain the neuropeptides CGRP or
substance P.
The IB4, SP - population is known to express the
RET receptor for GDNF
48
Like B2, B1 activation enhances the heat-gated
current.
The effect is much more prolonged than that of B2
activation
49
NGF
GDNF
TrkA, CGRP, SP
RET, CGRP-, SP-
50
Collaborators
NGF Ca imaging and inhibitors Jenny
Bonnington Molecular targets of NGF/TrkA Xuming
Zhang, Jiehong Huang TRPV1 single channel
gating Milena Studer B2 upregulation by NGF and
GDNF Yih-Jing Lee, Olof Zachrisson B1
upregulation by GDNF Vittorio Vellani, Olof
Zachrisson