Group Title: BMC Neuroscience
Title: Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin
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Title: Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin
Physical Description: Book
Language: English
Creator: Caudle, Robert
Mannes, Andrew
Keller, Jason
Perez, Federico
Suckow, Shelby
Neubert, John
Publisher: BMC Neuroscience
Publication Date: 2007
 Notes
Abstract: BACKGROUND:Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA). This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing.RESULTS:The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 µg) injected intrathecally produce thermal hyperalgesia. At higher doses (10 µg) peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons.CONCLUSION:The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury.
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Methodology article

Sensitization of spinal cord nociceptive neurons with a conjugate of
substance P and cholera toxin
Robert M Caudle*1,3, Andrew J Mannes4, Jason Keller4, Federico M Perez1,
Shelby K Suckow3 and John K Neubert2,3


Address: 'Department of Oral and Maxillofacial Surgery and Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, FL 32610,
USA, 2Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610, USA, 3Department of Neuroscience,
University of Florida College of Medicine, McKnight Brain Institute, Gainesville, FL 32610, USA and 4Pain and Neurosensory Mechanisms Branch,
National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
Email: Robert M Caudle* caudle@ufl.edu; Andrew J Mannes amannes@nidcr.nih.gov; Jason Keller jakeller@nidcr.nih.gov;
Federico M Perez fperez@dental.ufl.edu; Shelby K Suckow ssuckow@ufl.edu; John K Neubert jneubert@dental.ufl.edu
* Corresponding author



Published: 10 May 2007 Received: 21 December 2006
BMCNeuroscience 2007, 8:30 doi:10.1186/1471-2202-8-30 Accepted: 10 May 2007
This article is available from: http://www.biomedcentral.com/1471-2202/8/30
2007 Caudle et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Several investigators have coupled toxins to neuropeptides for the purpose of
lesioning specific neurons in the central nervous system. By producing deficits in function these
toxin conjugates have yielded valuable information about the role of these cells. In an effort to
specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P
to the catalytic subunit of cholera toxin (SP-CTA). This conjugate should be taken up selectively by
neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and
neuronal firing.
Results: The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected
with either the NKI or NK2 receptor, but not the NK3 receptor. We further demonstrate that
intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic
AMP response element binding protein (CREB) and also enhances the expression of the immediate
early gene c-Fos. Behaviorally, low doses of SP-CTA (I Jig) injected intrathecally produce thermal
hyperalgesia. At higher doses (10 Jig) peripheral sensitivity is suppressed suggesting that descending
inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons.
Conclusion: The finding that stimulation of adenylate cyclase in neurokinin receptor expressing
neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of
these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by
coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore,
these results demonstrate that SP-CTA can be used as a tool to study sensitization of central
neurons in vivo in the absence of an injury.



Background cific g-protein coupled receptors (GPCRs) as transporters
Several groups have developed potential therapeutics that to deliver a toxin to an intracellular target [1-91. When
produce highly selective lesions in vivo by exploiting spe- GPCRs bind a peptide agonist they are internalized by the


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cell; delivering the peptide, and any attached toxin, to the
inside of the cell [10]. The toxin is then able to act on its
intracellular target. Some of these investigators have used
lethal toxins such as saporin, diphtheria and pseu-
domonas exotoxin coupled to the neuropeptide substance
P to target the agents to cells expressing neurokinin recep-
tors [1,3,7-9,11]. These toxins produce highly specific
lesions of neurokinin receptor expressing cells while not
harming cells in the region that do not express these
receptors. The investigators have also demonstrated by
ablating these cells that neurons expressing the NK1
receptor in the spinal cord are required for central sensiti-
zation. Thus, these targeted toxins were found to be valu-
able tools for evaluating the function of neurons in the
central nervous system [2-4,12]. Moreover, it has been
suggested that these targeted toxins may have clinical util-
ity for the treatment of intractable pain.

In an effort to compliment the armamentarium of tar-
geted toxins we sought to selectively activate, rather than
kill, neurokinin receptor expressing cells by coupling
cholera toxin to the neuropeptide substance P. Cholera
toxin, unlike previously used toxins, is not universally
lethal to the cells. The toxin is useful because it ADP ribo-
sylates the g-protein Gs, which results in the uncoupling
of the protein from GPCRs and activation of the g-protein
[13-16]. Cholera toxin activation of Gs stimulates ade-
nylate cyclase activity to produce higher levels of CAMP in
the cells, altered protein kinase activity and altered ion
channel activity [13,16-21]. Thus, we hypothesized that a
conjugate of substance P and the catalytic subunit of chol-
era toxin (SP-CTA) would selectively activate neurokinin
receptor expressing neurons and would provide a novel
tool for evaluating cell function in vivo.

Results
Synthesis of SP-CTA
The neuropeptide substance P was coupled to the catalytic
subunit of cholera toxin (CTA) using the bifunctional
linking agent sulfosuccinimidyl 4-N-maleimidomethyl
cyclohexane-1-carboxylate (Sulfo-SMCC) as indicated in
figure 1A. Briefly, the Sulfo-SMCC was reacted with the N-
terminal amine of substance P to form an amide linkage
to the maleimide group. The substance P maleimide was
then conjugated to CTA through two cysteine residues in
the C-terminal region of the CTA protein. The final prod-
uct was washed and concentrated by centrifugation in
Centricon filters with a cutoff of 5 kd. The success of the
synthesis was confirmed on western blots by using anti-
bodies to both substance P and CTA. As demonstrated in
figure 1 B the final product produced bands on the western
blot with a molecular weight of approximately 30 kd that
reacted with antibodies to substance P and CTA indicating
a successful coupling of substance P to CTA (SP-CTA).
Based on protein assays the synthetic yields were quanti-


tative. In preliminary syntheses bands for substance P and
CTA in the western blots were doublets. Increasing the
concentration of substance P in the reaction produced a
single band at the higher molecular weight suggesting that
the stoichiometry of substance P to CTA in the final prod-
uct was 2:1.

In situ evaluation of SP-CTA
SP-CTA was tested on Chinese Hamster Ovary cells that
were stably transfected with the NK1 receptor [22] (CHO-
NK1). To verify selective uptake of SP-CTA by the cells, the
cells were incubated over night in either SP-CTA (1 gg/ml)
or CTA (1 gg/ml). The CHO-NK1 cells were then fixed and
prepared for immunocytochemistry with antibodies to
CTA using a rhodamine-tyramide amplification system.
As illustrated in figure 2A only the SP-CTA treated cells
demonstrated an uptake of CTA indicating that linkage of
CTA to substance P was required for the conjugate to be
internalized.

We further evaluated the functionality of the SP-CTA by
examining the ability of the conjugate to stimulate cAMP
production in CHO-NK1 cells. Figure 2B demonstrates
the concentration response relationship for SP-CTA (0.1
ng/ml (3.4 pM) to 1,000 ng/ml (34 nM)) when the SP-
CTA was applied for 1 hour and the cAMP was measured
after culturing the cells for an additional 24 hours. The
time course of SP-CTA's effect on cAMP production was
evaluated by treating the cells for 1 hour with 100 ng/ml
SP-CTA and then harvesting the cells for cAMP analysis 1,
2, 3 and 4 days following exposure to the conjugate. As
demonstrated in figure 2C, SP-CTA's effects on cAMP
peaked at 1 day in the CHO-NK1 cells and remained sig-
nificantly elevated for 3 days.

Figure 2D demonstrates that 48 hours following a 4 hour
exposure to 100 ng/ml of either substance P, CTA or SP-
CTA only SP-CTA treated CHO-NK1 cells produced an
increase in cAMP production. When 100 ng/ml of sub-
stance P, CTA or SP-CTA remained in the culture media
for the full 48 hours the substance P treated cells and the
SP-CTA treated cells had significantly elevated levels of
cAMP, whereas the CTA treated cells did not differ from
control cells (Figure 2E). The findings illustrated in figures
2D and 2E indicate that substance P activation of NK1
receptors can stimulate adenylate cyclase, but that the
effects of SP-CTA on cAMP production outlast any stimu-
lation of the NK1 receptors produce by the substance P
portion of the conjugate when the material is washed out
of the culture media. The ability of substance P to stimu-
late cAMP production was previously demonstrated in
several different cell types including CHO-NK1 cells [23-
25], which is consistent with our findings.




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+ H2N-Arg-Pro-Lys-Pro-GIn-GIn-Phe-Phe-Gly-Leu-Met-N H


o
o o


-38 kd
- 29
-20


N-Arg-Pro-Lys-Pro-GIn-GIn-Phe-Phe-Gly-Leu-Met-NH2


Substance P
Antibody


CTA
Antibody


SH SH



I!


N-Arg-Pro-Lys-Pro-GIn-GIn-Phe-Phe-Gly-Leu-Met-NH,



0N kg-Pro-Lys-Pro-Gl n-G n -Phe-Phe-Gly-Leu -Met-N H.


Figure I
Synthesis of SP-CTA. A. Schematic representation of the procedure used to synthesize SP-CTA. B. Western blots of final SP-
CTA product. SP-CTA and the filtrate from the Centricon Plus-20 concentrating tubes (Wash) were run on western blots and
probed with antibodies to substance P and the catalytic subunit of cholera toxin (CTA). The SP-CTA product reacted with
both antibodies.


To evaluate the relative selectivity of SP-CTA for NK1, NK2
and NK3 receptors, CHO cells were transfected with NK1
or NK2 plasmids and a concentration response relation-
ship was performed on SP-CTA's ability to stimulate
cAMP production. As demonstrated by figure 3, cells con-
taining NK1 or NK2 receptors demonstrated equivalent
concentration response relationships when measuring
levels of cAMP stimulation by SP-CTA. However, in CHO
cells not expressing neurokinin receptors or CHO cells sta-
bly expressing NK3 receptors SP-CTA did not stimulate
cAMP production (Figure 3).

In vivo evaluation of SP-CTA
Previous work with lethal toxins coupled to substance P
demonstrated that neurons expressing NK1 receptors are
necessary for the expression of thermal hyperalgesia
[ 1,3,7-9 ]. Therefore it was hypothesized that uptake of SP-


CTA by NK1 receptor expressing neurons in the spinal
cord would stimulate the cells and produce thermal
hyperalgesia. Initially, a group of 3 rats received intrathe-
cal injections of 10 |ig of SP-CTA. One hour later the spi-
nal cords were harvested and prepared for
immunohistochemistry to determine if the SP-CTA was
taken up by NK1 receptor expressing neurons. Figure 4A
demonstrates that the conjugate co-labels with NK1 recep-
tors in the superficial layers of the spinal cord. To test the
idea that SP-CTA would stimulate the neurons that have
taken up the conjugate rats were injected intrathecally
with SP-CTA (50 itg), CTA (50 itg) or saline (20 itl) (N =
6 per treatment group). Twenty four hours following the
injection of SP-CTA, but not CTA, the animals were agi-
tated and aggressive toward their cage mates. The animals
were euthanized by pentobarbital overdose and prepared
for immunohistochemistry of the spinal cords to evaluate


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S


li


BMC Neuroscience 2007, 8:30









BMC Neuroscience 2007, 8:30


SP-CTA


http://www.biomedcentral.com/1471-2202/8/30


CTA


Control


*










0 0.1 1.0 10 100 1,000
[SP-CTA(ng/ml)]
*


0


35-
S30-
25-
20-
15-
10-
< 5-
0-

E.
800 -
S700
S600
S500
400
, 300
200
S100
0


2
Days


Control SP CTA SP-CTA


Control SP


CTA SP-CTA


Figure 2
In situ evaluation of SP-CTA. A. CHO cells stably transfected with the NKI receptor were treated with either SP-CTA (I jig/
ml), CTA (I ig/ml) or were not exposed to any agents (Control). The cells were cultured over night in these solutions and
then immunocytochemistry was performed on the cells using an antibody to CTA and Rhodamine-tyramide amplification.
Labeling was observed only in the SP-CTA treated cells. B. Concentration response relationship for SP-CTA on cAMP produc-
tion. CHO-NKI cells were exposed to the indicated concentrations of SP-CTA for I hour. The cells were cultured for
another 24 hours following washout of the SP-CTA with fresh culture media. The cells were harvested and cAMP content was
measured using a commercial cAMP assay. (N = 5, 100 mm plates/concentration) (ANOVA F = 56.15, df = 83, p < 0.0001). C.
Time course of SP-CTA's effects on cAMP content of CHO-NKI cells. The cells were incubated for I hour with 100 ng/ml SP-
CTA and cultured in fresh media for the indicated times. The cells were then harvested and analyzed for cAMP content. (N =
5, 100 mm plates/treatment) (ANOVA F = 25.35, df = 29, p < 0.0001). D. Comparison of the effects of substance P (SP), the
catalytic subunit of cholera toxin (CTA) and SP-CTA on cAMP production in CHO-NKI cells. Cells were incubated with 100
ng/ml of CTA, SP, SP-CTA or received no treatment for 4 hours. The cells were then cultured for 48 hours in fresh media,
harvested and assayed for cAMP content. (N = 5, 100 mm plates/treatment) (ANOVA F = 5.27, df = 19, p = 0.0102) E. Further
comparison of the effects of SP, CTA and SP-CTA on cAMP production. Cells were treated as described in D except the
agents remained in the culture media for the full 48 hours. The cells were then harvested and analyzed for cAMP content. (N =
5, 100 mm plates/treatment) (ANOVA F = 23.06, df = 19, p < 0.0001). Data are expressed as means SEM. Asterisks indicate
p < 0.05 one way ANOVA followed by Dunnett's test, F = 23.06, df = 19).






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700-
600-
S500-
400-
300-
200-
| 100-
0-


C.
110-
100-
90-
80-
70-
60-
S50-
40-
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20-
10-
0-







http://www.biomedcentral.com/1471-2202/8/30


100

I 80
so

60

40
*<


Ctrl Ctrl 10 2 4 6 8 10
[SP-CTA] ng/ml


Figure 3
Relative selectivity of SP-CTA for NKI, NK2 and NK3
receptors. Concentration response relationships for SP-CTA
on the stimulation of cAMP production were performed on
CHO cells transiently transfected with NKI or NK2 recep-
tors. Non-transfected CHO cells were either not exposed to
SP-CTA (Ctrl) or treated with 10 ng/ml SP-CTA (Ctrl 10).
CHO cells stably transfected with NK3 receptors were
treated with a single concentration of 10 ng/ml SP-CTA.
(Two way ANOVA of NKI versus NK2, F = 1.7, df = I, p =
0.20: Dose, F = 54.61, df = 6, p < 0.0001). Asterisks indicate
p < 0.01 (one way ANOVA followed by Dunnett's post hoc
test, F = 56.15, df = 83) when compared to control cells not
treated with SP-CTA.


the phosphorylation of cAMP response element-binding
protein (CREB) and the expression of the immediate early
gene c-Fos. We chose to examine CREB because elevated
levels of cAMP lead to the phosphorylation of this tran-
scription factor [261. We found that SP-CTA treatment
resulted in a large increase in the phosphorylation of
CREB in the spinal cord dorsal horn (Figure 4B), while the
injection of CTA produced levels of phosphorylated CREB
that were similar to saline injected animals. In addition to
elevated levels of phosphorylated CREB we found that c-
Fos was also enhanced in SP-CTA treated animals (Figure
5).

A dose response relationship and time course was deter-
mined for SP-CTA using the hind paw thermal nocicep-
tion assay of Hargreaves et al. [271. Figure 6A
demonstrates that intrathecally administered SP-CTA (N =
10 rats per dose) has a biphasic dose response relation-
ship 24 hours following the injections with peak thermal
hyperalgesia observed at a dose of approximately 1 jig (34
pmoles). At 10 jig of SP-CTA the animals demonstrated
agitated behaviors similar to the first group that received
50 jig; therefore, no higher doses were tested. Finally, to
determine the time course of action of SP-CTA 1 jig was


injected intrathecally (N = 20 rats) and thermal nocicep-
tion was tested before injections and 1 to 4 days following
the injection. As illustrated in figure 6B the peak of ther-
mal hyperalgesia occurred 1 day following the injection.
Some recovery occurred over the next three days; however,
a complete return to baseline paw withdrawal thresholds
was not observed.

Discussion
Previously used lethal toxins targeted to GPCRs via cou-
pling to a peptide agonist were useful for evaluating the
function of the cells that expressed the GPCRs. By using
the receptor to direct the toxins to the desired cell type the
agents produced highly specific lesions, even when the
targeted cells were a minor constituent of a heterogeneous
population of cells [1-9,11,12]. However, because these
toxins kill the targeted cells, the cells' function must be
inferred by the deficit that is produced by the lesion. Ide-
ally, a method to selectively stimulate the cells could pro-
vide more information about the cells' function. To
achieve this goal we coupled the neuropeptide substance
P, which targets neurokinin receptors [2,3,8], to the cata-
lytic subunit of cholera toxin (SP-CTA). Cholera toxin is
not lethal when taken up by cells, but it does enhance the
activity of adenylate cyclase resulting in an increase in
intracellular cAMP [16]. Thus, by targeting cholera toxin
to neurokinin receptor bearing neurons with substance P
we altered the function of these cells rather than killing
them.

We found that SP-CTA produced equivalent effects on
cAMP production in cultured cells expressing NK1 or NK2
receptors, but did not influence cAMP production in cells
that expressed NK3 receptors or non-transfected cells.
These data suggest that the conjugate binds to NK1 and
NK2 receptors with approximately the same affinity.
Takeda and colleagues previously demonstrated that sub-
stance P had a ten fold higher affinity for NK1 than NK2
receptors [25]. It is possible that the presence of CTA on
the N terminus of substance P reduces the NK1 selectivity
of the peptide. Alternatively, since we used a functional
assay, any affinity differences between the receptors may
have been obscured by uptake mechanisms and by the
action of the toxin on Gs. For example, the association
rate of substance P is much higher for NK1 than NK2
receptors [25]. Because of the higher binding rate the NK1
receptors may be internalized faster than the NK2 recep-
tors. However, if the NK1 receptors do not rapidly recycle
to the cell surface the NK2 receptors will eventually bind
and internalize an equal amount of SP-CTA. Thus our
functional assay would negate the differences in binding
affinity of substance P for the NK1 and NK2 receptors.
Additionally, because cholera toxin stimulates cAMP pro-
duction and protein kinase A activity it is possible that due
to excessive phosphorylation the neurokinin receptors are


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SP-CTA


SP-CTA


CTA


Saline


Saline


*. W .N. .


Figure 4
Intrathecal administration of SP-CTA A. NKI expressing
neurons in the dorsal horn take up SP-CTA. Three rats
received intrathecal injections of 10 pg SP-CTA. One hour
following the injections the animals were euthanized and the
spinal cords were removed and labeled for NKI (green) and
CTA (red) using immunohistochemistry. Cells co-labeled for
NKI and CTA appear yellow to orange. These data indicated
that SP-CTA was taken up by the NKI receptor expressing
neurons. B. Activation of dorsal horn neurons by SP-CTA.
Rats (N = 6 per treatment group) received intrathecal injec-
tions of SP-CTA (50 rig), CTA (50 rig) or Saline (20 til). The
animals were allowed to recover for 24 hours. The animals
were euthanized and perfused as described in the methods
section. The spinal cords were removed and prepared for
immunohistochemistry using an antibody to pCREB, a sec-
ondary antibody coupled to HRP and diaminobenzidine.
Phosphorylated CREB is visible as dark staining nuclei. The
arrow points to an example of pCREB expressing nuclei. The
approximate locations of lamina 1, III and V are indicated.
These data indicate that CREB phosphorylation is stimulated
by SP-CTA, but not CTA alone.




trapped in the internalized state making the receptors
unavailable to transport more toxin. This process could
also mask any binding affinity differences.

In vivo our data demonstrate that when SP-CTA was
injected intrathecally in rats the animals became hyper-
sensitive to thermal stimuli (Figure 6A and 6B). This data
supports the previous lesion studies indicating that cen-
tral sensitization is mediated by NK1 receptor expressing
neurons in the spinal cord [1-3,6]. We further found that


Figure 5
SP-CTA stimulates the production of the immediate early
gene c-Fos in dorsal horn neurons. Sections from the same
animals as in figure 4 were used for immunohistochemistry
with antibodies to c-Fos. The sections were prepared as pre-
viously described. The c-Fos labels as dark staining nuclei.
The arrow points to an example of a c-Fos positive nucleus.
The approximate locations of lamina 1, III and V are indicated.
These data indicate that c-Fos expression is stimulated by SP-
CTA.


with higher doses of SP-CTA peripheral sensitivity was
suppressed (Figure 6A). However, the animals demon-
strated behaviors that suggested centrally mediate nocice-
ption. We hypothesize that the suppression of peripheral
hypersensitivity was due to stimulation of inhibitory
pathways by the NK1 receptor expressing neurons that
have taken up the SP-CTA. These inhibitory systems may
be previously characterized descending inhibitory path-
ways [28] or local inhibitory neurons. Further work
should clarify this finding. These data, however, support
the idea that selectively stimulating cells provides unique
information on their function that is not available when a
lesioning strategy is used. What is most notable about
these results is that the inhibition of peripheral nocicep-
tion could not have been predicted from several previous
lesioning studies [1-3,6]. However, when specifically
examining descending inhibition Suzuki and colleagues'
found that lesioning of NK1 expressing neurons in the
spinal cord suppressed peripheral afferent stimulated
descending inhibition [29], which is consistent with our
findings.

The effects of intrathecally administered SP-CTA also con-
trast with the effects produced by intrathecal wild type
cholera toxin. In a previous study wild type cholera toxin
inhibited the hyperalgesia and allodynia produced by a
variety of peripheral injuries [30]. The inhibitory effect of
the toxin was blocked by the opioid antagonist naloxone.


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S10-

, 8-

: 6-

S4-

S2-
- o


Saline 0.3 1.0 3.0 10.0
SP-CTA ug


^-*


14-
-
12-
10-
-
*- 8-

, 6-
4-

2-
0-


Figure 6
Effect of intrathecal SP-CTA on thermal nociception in rats. A. SP-CTA dose response relationship. Rats (N = I 0/dose)
received intrathecal injections of the indicated doses of SP-CTA in 20 [il of saline. Twenty four hours following the injections
the animals were tested for thermal nociception using a Hargreaves apparatus [27]. Asterisks indicate p < 0.05 one way
ANOVA (F = 5.527, df = 49) followed by Dunnett's test when compared to the saline treated animals. B. Time course of
intrathecal SP-CTA on thermal nociception. Rats (N = 20) were injected intrathecally with I [ig of SP-CTA in 20 il\ of saline.
The animals were tested for thermal nociception at the times indicated. Zero represents the day of injection. Data are pre-
sented as means SEM. Asterisk indicates p < 0.05 repeated measures ANOVA (F = 5.06, df = 99) followed by Dunnett's test
when compared to the zero time point.


The mechanism that produced this endogenous opioid
mediated inhibition of nociception was not investigated,
but work by Shen and Crain indicates that cholera toxin
can enhance the inhibitory effects of opioids on primary
afferent neurons [18]. Thus, it can be hypothesized that
wild type cholera toxin acts principally on primary affer-
ent neurons to inhibit peripherally mediated nociception;
while SP-CTA affects NK1 receptor expressing spinotha-
lamic tract neurons to generate centrally mediated nocice-
ption.

Conclusion
We have synthesized a novel tool for activating adenylate
cyclase in neurokinin receptor expressing cells to evaluate
the function of these cells in heterogeneous populations,
as are typically found in vivo. Intrathecal injections of SP-
CTA in rats demonstrated that the toxin conjugate pro-
duced thermal hyperalgesia as would be expected if NK1
receptor expressing spinothalamic tract neurons were sen-
sitized by the treatment. These data indicate that GPCRs
can be exploited to transport cholera toxin into a host of
different cell types. Because the toxin is targeted to the
GPCR by a peptide agonist it is remarkably simple to
change the peptide to direct the conjugate to receptors
other than neurokinin receptors. These targeted cholera
toxin conjugates could find utility in a number ofbiomed-
ical research endeavors.


Methods
Synthesis of SP-CTA
The A subunit of cholera toxin (CTA) was purchased from
List Biological Laboratories inc. (Cambell, CA, USA). CTA
has two cysteine residues in the C-terminal region [15,31]
therefore these cysteine residues were used to attach sub-
stance P to CTA. The synthesis of SP-CTA was accom-
plished using a modification of Pierce Biotechnology
inc.'s maleimide protein cross-linking procedure. The syn-
thesis was carried out in two stages. The first stage was to
link maleimide to the N-terminus of substance P by com-
bining a 5 fold excess of Sulfosuccinimidyl 4-N-maleimi-
domethyl cyclohexane-1-carboxylate (Sulfo-SMCC) with
substance P in phosphate buffered saline (PBS, pH 7.4).
The mixture was incubated at room temperature for 1
hour. The substance P maleimide conjugate was separated
from unreacted Sulfo-SMCC using a Sephadex G-10 (30 x
1.5 cm) column eluted with PBS. For the second phase of
the synthesis the substance P maleimide conjugate was
linked to the two cysteines on CTA by adding a 10 fold
excess of the conjugate to CTA in PBS. This mixture was
then incubated at room temperature for another hour.
The SP-CTA was separated from the unreacted substance P
maleimide conjugate, washed with PBS three times and
concentrated using Centricon Plus-20 filters. A sample of
the final product was evaluated by western blots. Briefly,
the sample was run on 4-20% polyacrylamide electro-


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phoresis gels, transferred to Polyvinylidene fluoride
(PVDF) membranes and then probed with antibodies to
either substance P or the catalytic subunit of cholera toxin.
A secondary antibody coupled to horse radish peroxidase
(HRP) and enhanced chemiluminescence were used to
visualize the bands. Figure 1 illustrates the synthetic path-
way as well as western blots of the final product.

Cell culture
Chinese Hamster Ovary cells stably expressing NK1, NK2
or NK3 receptors (a generous gift from Dr. James Krause,
Neurogen Corp. [22]) were plated on 100 mm plates for
cAMP assays or 13 mm cover slips in 24 well culture plates
for immunocytochemistry experiments. The cultures were
grown in F12K media, 10% Fetal Bovine Serum, 1% L-
glutamine, 1% penicillin-Streptomyosin, 25 mM Hepes
buffer, and G418 (500 ig/ml). The cells were cultured at
37C in a 5% C02 atmosphere. Additionally, plasmids
containing either NK1 or NK2 receptors were purchased
(UMR cDNA Resource Center, Rolla, MO, USA) and trans-
fected into CHO cells using Lipofectamine (Invitrogen,
Carlsbad, CA, USA) as per the manufacturer's instructions.
These cells were cultured as described for the stably trans-
fected cells.

cAMP assay
To assay cAMP levels in the cell cultures Sigma inc's (St.
Louis, MO, USA) Direct cAMP Enzyme Immunoassay was
used according to the manufacturer's instructions. Briefly,
the media on the cell cultures was removed and the cells
were washed once with PBS (pH 7.4). The PBS was
removed and 1 ml 0.1 M HCL was added to the cells. The
cells were scraped from the plates into the HCL solution,
sonicated and centrifuged (600 g, 10 minutes, 5 C) and
the supernatant collected. The protein in each sample was
measured using Bio-Rad's (Hercules, CA, USA) protein
assay. The cAMP was acetylated with the addition of 100
Al of the kit's acetic anhydride solution. A 100 Al sample
was then neutralized with 50 |il of the kit's neutralizing
buffer and the samples were added to the kit's 96 well
plates that were pre-absorbed with antibodies to cAMP. A
standard curve and controls were set up as suggested by
the manufacturer. A cAMP- alkaline phosphatase conju-
gate (50 |il) was added to the wells and the solution was
incubated for 2 hours. The plates were then washed 3
times and 200 |il of p-nitrophenyl phosphate solution
(substrate) was added to each well and the plates were
incubated for 1 hour. The reaction was stopped with 50 Al
0.1 M HCL and the plate was read at 405 nm. The concen-
tration of cAMP in the samples was extrapolated from the
data collected for the cAMP standards and expressed as the
number of moles of cAMP per mg protein.


Animals
Male Sprague Dawley rats (200 300 g) were housed in
pairs and supplied standard rat chow and water ad libitum
in the University of Florida's vivarium, which is an AAA-
LAC certified facility.

Intrathecal injections were performed under isoflurane
anesthesia via lumbar puncture between L4 and L5. All
animal procedures in this project were reviewed and
approved by the University of Florida's Institutional Ani-
mal Care and Use Committee.

Immunocytochemistry and immunohistochemistry
Rats were euthanized with pentobarbital and immediately
transcardially perfused with ice cold PBS and then ice cold
4% paraformaldehyde in phosphate buffered saline
(PBS)(pH 7.4). Cell cultures on cover slips were washed
with PBS and fixed with 4% paraformaldehyde in PBS.
The spinal cords were removed and post fixed overnight in
4% paraformaldehyde in PBS. The tissue was cryopro-
tected in 30% sucrose, mounted and sectioned in a cryo-
stat (-20 C)(10-20 rim) and mounted on slides. The
sections or cells were then blocked with 3% normal goat
serum for 60 minutes with 0.75% triton X-100. The pri-
mary antibody was added to the blocking solution
(1:500) and the sections were incubated for 48 hours at
4C. The sections or cells were then washed (8 x 5 mins)
in PBS. Following the wash the sections or cells were incu-
bated for 1 hour at room temperature in PBS with a sec-
ondary antibody that was coupled to horse radish
peroxidase (HRP), Alexa Fluro 594 or Alexa Fluro 488
(Molecular Probes, Boston, MA). The sections labeled
with the Alexa Fluro stains were washed (8 x 5 mins) and
viewed using fluorescence microscopy. The HRP label sec-
tions were washed similarly and treated with diami-
nobenzidine. The cultured cells, which were treated with
HRP coupled secondary antibodies, were washed simi-
larly and labeled using rhodamine labeled tyramide as
described by the manufacturer (NEN, Boston, MA, USA).
Antibodies to c-Fos were obtained from Dr. Michael lad-
arola (National Institute of Dental and Craniofacial
Research, National Institutes of Health) [32], antibodies
to CTA were purchased from Sigma (St. Louis, MO), anti-
bodies to pCREB were purchased from New England
Biolabs (Ispwich, MA) and antibodies to NK1 receptor
were purchased from Zymed (San Francisco, CA).

Thermal nociception assay
Thermal nociception was measured using the method of
Hargreaves et al. [27]. Briefly, the rats were placed on a
clear glass surface and allowed 15 minutes to accommo-
date to the enclosure. An infrared light was directed onto
a hind paw's plantar surface approximately in the middle
of the foot. The latency for the animal to remove its foot
from the path of the light was used as the dependent


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measure for thermal sensitivity. The test was performed 4
times for each time point (2 tests for each hind foot) with
each test separated by 2 minutes to prevent paw sensitiza-
tion. The withdrawal latencies of the tests were averaged
to obtain the final value for that time point.

Statistics
Data were analyzed using a one way ANOVA followed by
Dunnett's post-hoc test, one way repeated measures
ANOVA or two way ANOVA as appropriate. Significance
was assigned to p _< 0.05.


Authors' contributions
RMC was the principle investigator on the project, synthe-
sized the SP-CTA and performed behavioral experiments.
AJM, SKS and JK performed the immunohistochemistry
on the spinal cord tissue. FMP performed the cell culture
and cAMP experiments. JKN assisted in experimental
design and statistical analysis.


Acknowledgements
This work was supported by the National Institute on Drug Abuse,
National Institutes of Health, DA016562. The authors would also like to
thank Daniel Martinez and Anthony Carter for their excellent technical
assistance.

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