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NATURE NEUROSCIENCE VOLUME 13 | NUMBER 7 | JULY 2010 883
ARTICLES
Acupuncture is a procedure in which fine needles are inserted into
an individual at discrete points and then manipulated, with the
intent of relieving pain. Since its development in China around 2,000
B.C., acupuncture has become worldwide in its practice1. Although
We st er n m e di c in e h as t r ea t ed a c up u nc tu r e wi t h c on si d er a bl e s ke p -
ticism2, a broader worldwide population has granted it acceptance.
For instance, the World Health Organization endorses acupuncture
for at least two dozen conditions3 and the US National Institutes of
Health issued a consensus statement proposing acupuncture as a
therapeutic intervention for complementary medicine. Perhaps most
tellingly, the U.S. Internal Revenue Service approved acupuncture as
a deductible medical expense in 1973.
Although the analgesic effect of acupuncture is well documented,
little is understood about its biological basis. Insertion of the acupunc-
ture needles in itself is not sufficient to relieve pain4. An acupuncture
session typically lasts for 30 min, during which the needles are inter-
mittently rotated, electrically stimulated or, in some cases, heated. The
pain threshold is reported to slowly increase and to outlast the treat-
ment4. The primary mechanism implicated in the anti-nociceptive
effect of acupuncture involves release of opioid peptides in the CNS
in response to the long-lasting activation of ascending sensory tracks
during the intermittent stimulation4–6. However, a centrally acting
agent cannot explain why acupuncture is conventionally applied in
close proximity to the locus of pain and why the analgesic effects of
acupuncture are restricted to the ipsilateral side7,8.
RESULTS
Acupuncture triggers adenosine and ATP metabolites release
ATP is released in response to either mechanical and electrical stimu-
lation or heat. Once released, ATP acts as a transmitter that binds
to purinergic receptors, including P2X and P2Y receptors9,10. ATP
cannot be transported back into the cell but is rapidly degraded to
adenosine by several ectonucleotidases before re-uptake10. Thus,
adenosine acts as an analgesic agent that suppresses pain through
Gi-coupled A1-adenosine receptors11–13. To determine whether
adenosine is involved in the anti-nociceptive effects of acupuncture,
we first asked whether the extracellular concentration of adenosine
increases during acupuncture.
We collected samples of interstitial fluid by a microdialysis probe
implanted in the tibialis anterior muscle/subcutis of adult mice at a
distance of 0.4–0.6 mm from the ‘Zusanli point’, which is located 3–4 mm
below and 1–2 mm lateral for the midline of the knee4. Adenine nucleo-
tides and adenosine were quantified using high-performance liquid
chromatography (HPLC) with ultraviolet absorbance before, during
and after acupuncture (Fig. 1a)14,15. At baseline, the concentrations
of ATP, ADP, AMP and adenosine were in the low nanomolar range
(Fig. 1b), consistent with previous reports16,17. Acupuncture applied
by gentle manual rotation of the acupuncture needle every 5 min for a
total of 30 min sharply increased the extracellular concentrations of all
purines detected (Fig. 1b). Adenosine concentration increased ~24-fold
(253.5 q 81.1 nM from a baseline of 10.6 q 6.7 nM) during the 30-min
acupuncture session (Fig. 1c). The extracellular concentration of ATP
returned to baseline after acupuncture, whereas adenosine, AMP and
ADP remained significantly elevated (adenosine and AMP, P < 0.01;
ADP, P < 0.05, paired t test compared to 0 min) at 60 min (Fig. 1c).
Notably, previous studies have shown that deep brain stimulation is also
associated with a severalfold increase in extracellular ATP and adenos-
ine. Similar to electroacupuncture and transcutaneous electrical nerve
stimulation, deep brain stimulation delivers electrical stimulation that
triggers an increase in extracellular adenosine concentration18.
1Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, New York, USA. 2Department of Neurology, Boston University School of
Medicine, Boston, Massachusetts, USA. 3National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland,
USA. 4These authors contributed equally to this work. Correspondence should be addressed to M.N. (nedergaard@urmc.rochester.edu).
Received 16 March; accepted 27 April; published online 30 May 2010; doi:10.1038/nn.2562
Adenosine A1 receptors mediate local anti-nociceptive
effects of acupuncture
Nanna Goldman1,4, Michael Chen1,4, Takumi Fujita1,4, Qiwu Xu1, Weiguo Peng1, Wei Liu1, Tina K Jensen1,
Yo n g Pe i 1, Fushun Wang1, Xiaoning Han1, Jiang-Fan Chen2, Jurgen Schnermann3, Takahiro Takano1,
Lane Bekar1, Kim Tieu1 & Maiken Nedergaard1
Acupuncture is an invasive procedure commonly used to relieve pain. Acupuncture is practiced worldwide, despite difficulties
in reconciling its principles with evidence-based medicine. We found that adenosine, a neuromodulator with anti-nociceptive
properties, was released during acupuncture in mice and that its anti-nociceptive actions required adenosine A1 receptor
expression. Direct injection of an adenosine A1 receptor agonist replicated the analgesic effect of acupuncture. Inhibition of
enzymes involved in adenosine degradation potentiated the acupuncture-elicited increase in adenosine, as well as its anti-
nociceptive effect. These observations indicate that adenosine mediates the effects of acupuncture and that interfering with
adenosine metabolism may prolong the clinical benefit of acupuncture.
© 2010 Nature America, Inc. All rights reserved.
884 VOLUME 13 | NUMBER 7 | JULY 2010 NATURE NEUROSCIENCE
ARTICLES
Effect of local application of A1 receptor agonist
Having established that adenosine is released during acupuncture,
we next asked whether adenosine is critical for the anti-nociceptive
effects of acupuncture. We tested the effect of the selective A1 receptor
agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA)19, in two
mouse models of chronic pain. Inflammatory pain was evoked by
injection of complete Freund’s adjuvant (CFA) into the right paw20.
Following injection of CFA, the mice developed mechanical allodynia
to innocuous stimulation with Von Frey filaments of the ipsilateral
paw peaking at day 4 to 5. The mice also developed thermal allodynia,
as defined by a substantial decrease in withdrawal latency to heat.
Administration of CCPA (0.1 mM, 20 Nl) in the ipsilateral Zusanli
point (ST36) evoked a sharp increase in the threshold to touch (Fig. 2a)
and thermal pain (Fig. 2b). Touch sensitivity, defined as the per-
cent of negative responses, improved from 35.0 q 4.3 to 78.3 q 4.8%
(P < 0.01, Tukey-Kramer). Thermal sensitivity was almost abolished,
with paw withdrawal time increasing from 3.0 q 0.2 to 13.1 q 1.7 s
(P < 0.01) following administration of CCPA. Similarly, mechanical
allodynia was sharply reduced by CCPA in mice with neuropathic
pain (touch sensitivity improved from 25.0 q 2.2 to 83.3 q 4.9%,
P < 0.01), concurrent with a reduction of the sensitivity to thermal
pain (withdrawal of foot increased from 2.9 q 0.3 to 16.2 q 2.4 s,
P < 0.01). To address whether A1 receptor signaling was sufficient
for the anti-nociceptive effect of CCPA, we examined the effects of
CCPA in mice lacking adenosine receptor A1 (ref. 21) and found that
A1 receptor expression was necessary for CCPA-mediated pain sup-
pression. Although CCPA effectively reduced mechanical and thermal
hypersensitivity in wild-type mice, CCPA had no clinical benefit in
mice lacking A1 receptors (Fig. 2a,b). Thus, the anti-nociceptive
effects of CCPA require adenosine A1 receptor expression.
We next modeled neuropathic pain by spared injury of the
sciatic nerve22, in which pain peaked 5–7 d after nerve ligation. CCPA
injected in the Zusanli point of the ipsilateral leg reduced neuropathic
pain with an efficacy that was comparable to its suppression of inflam-
matory pain (Fig. 2c,d). In each model, the anti-nociceptive effect of
CCPA was transient and did not alter sensitivity to painful stimulation
in the contralateral leg (Supplementary Fig. 1). In addition, injection
of CCPA into the contralateral leg did not alter the pain threshold
of the ipsilateral leg, suggesting that the action of CCPA is medi-
ated by activation of local A1 receptors (Supplementary Fig. 2).
Substituting CCPA injection in the ipsilateral leg with an equal volume
5 nA
Standards
AMP
a
b
c
Acupuncture
Microdialysate (nM)
Microdialysate (nM)
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Adenosine
Time (min)
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AMP
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acupuncture
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120 min
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Adenosine
AMP
ADP
ATP
ADP
Adenosine
ATP
30 min 60 min 90 min 120 min
30 60 90120 0 30 60 90120 0 30 60 90 120 0 30 60 90120
Figure 1 Acupuncture triggers an increase in the extracellular
concentration of ATP, ADP, AMP and adenosine. (a) Representative HPLC
chromatograms before, during and after acupuncture. The samples
were collected by a microdialysis probe implanted in close proximity to
the Zusanli point. Standards of adenosine, AMP, ADP and ATP (0.3 NM
each) are shown on top. (b) Time course of purine release in response
to acupuncture. (c) Histogram summarizing the mean concentrations of
adenosine, AMP, ADP and ATP during baseline nonstimulated conditions,
as well as during and following acupuncture (*P < 0.05, **P < 0.01,
paired t test compared to 0 min, n = 8). Error bars indicate s.e.m.
Thermal test (s)
a b
c
e
d
Touch test (%)
Thermal test (s)
Touch test (%)
CFA
Ligation
0
5
10
15
20
WT
0
20
40
60
80
100
A1RKO
WT
A1RKO
Before
CCPA
CCPA CCPA
##
**
**
##
##
*
*
*
*
*
##
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
–18 –12 –6 0 6 12 18 24 30
Normalized fEPSP amplitude
WT (ipsilateral CCPA)
A1RKO (ipsilateral CCPA)
min
WT (contralateral CCPA)
0
20
40
60
80
100
0
5
10
15
20
**
**
After Day
after
Before
CFA
Before
CCPA
After Day
after
Before
CFA
Before After Day
after
Before
ligation
Before
ligation
Before After Day
after
Figure 2 Anti-nociceptive effects of adenosine A1 receptors. (a,b)
Comparison of the effect of CCPA on mechanical allodynia (touch test, a)
and thermal hyperalgesia (thermal test, b) in wild-type (WT, black) and A1
receptor knockout (A1RKO, gray) mice. CFA was administered in the right
paw at day 0. The adenosine A1 receptor agonist CCPA (0.1 mM, 20 Nl)
was injected into the ipsilateral (right) Zusanli point (ST36) at day 4. All
of the mice were evaluated ~10 min after the CCPA injection (**P < 0.01,
Tukey-Kramer test compared with before CCPA; ##P < 0.01, comparison
of wild type and A1RKO at each time point; n = 5–9). (c,d) Effect of
CCPA on mechanical (c) and thermal hypersensitivity (d) in wild-type and
A1RKO mice with neuropathic pain evoked by partial ligation of the right
leg ischias nerve at day 0 and CCPA administered at day 6 (n = 6). The
sensitivity of the contralateral (control) leg to mechanical and thermal
stimulation from these experiments is shown in Supplementary Figure 1.
(e) The amplitude of fEPSP in left anterior cingulate cortex evoked by
painful stimulation in the right foot. The effect of CCPA (0.1 mM,
20 Nl, at 0 min) injected in the ipsilateral (right) or the contralateral
(left) Zusanli point on fEPSP is plotted as a function of time in wild-type
and A1RKO mice. (*P < 0.01, Tukey-Kramer test compared with the
−18- to −12-min time point, n = 4–12). Error bars indicate s.e.m.
© 2010 Nature America, Inc. All rights reserved.
NATURE NEUROSCIENCE VOLUME 13 | NUMBER 7 | JULY 2010 885
ARTICLES
of saline (control vehicle) failed to change the threshold to either thermal
or mechanically induced pain (Supplementary Fig. 2).
To u nd e rs t an d h ow C CP A r e du c ed s e ns i ti v it y t o pa i nf u l st i mu -
lation and to specifically address whether CCPA acted directly on
ascending nerve tracks, we recorded in vivo responses of the left
anterior cingulate cortex (ACC) to painful stimulation of the right
foot (Fig. 2e). The ACC is important for perception of pain23 and
painful electrical nerve stimulation in humans is linked to its acti-
vation24. We found that high-intensity stimulation (10 mA, 20 ms)
evoked consistent field excitatory postsynaptic potentials (fEPSPs)
in the ACC with a latency of ~40 ms, reflecting the involvement
of a polysynaptic pathway, which includes primary afferents and
spinothalamic and thalamocortical tracts. Lower stimulation intensi-
ties evoked either no or variable responses, consistent with the idea
that that ACC neurons respond primarily to painful stimuli24. After
recording the responses to foot shock during baseline conditions for
20 min, we injected CCPA (0.1 mM, 20 Nl) into the Zusanli point
of the left leg, that is, contralateral to the foot receiving the painful
stimuli. CCPA administered contralateral to the painful stimulation
had no effect on fEPSPs, excluding the possibility that CCPA acted
centrally (Fig. 2e). In contrast, CCPA injected in the Zusanli point in
the right leg, ipsilateral to the painful stimulation, induced a marked
decrease in the fEPSP amplitude. The decline of the fEPSP amplitude
was observed as soon as 6 min after the CCPA injection, at which point
the fEPSP amplitude fell from an average of ~0.65 mV before injection
to ~0.22 mV within 20 min, representing a drop to 26.6 q 11.0%
of baseline values. Together, these data suggest that CCPA acts
locally, probably on unmyelinated C fibers in the superficial peroneal
nerve, which travels in close proximity to the Zusanli points. It has
previously been documented that dorsal root ganglion cells express
high levels of A1 receptors in their afferent terminals in the foot13,25
and in their presynaptic terminals in the substantia gelatinosa26.
However, it seems unlikely that CCPA can diffuse over a distance of
1.8–2.0 mm and bind to A1 receptors in the foot or to the presynap-
tic terminals in the spinal cord located more than 3.0–3.2 mm from
the Zusanli point in 6 min13,25,26. In sharp contrast with the potent
depression of the amplitude of fEPSP in wild-type mice, mice with
deletion of A1 receptors failed to respond to CCPA injection (Fig. 2e).
Together, these studies suggest that CCPA reduced painful stimulation
by activating adenosine A1 receptors on unmyelinated C fibers, and
possibly Av fibers, in the superficial peroneal nerve.
Anti-nociceptive effect of acupuncture requires A1 receptors
We next asked whether adenosine released during acupuncture
mediates the anti-nociceptive effects of acupuncture by evaluating
the effects of acupuncture on inflammatory and neuropathic pain.
A needle was gently inserted 1.5 mm deep in the ipsilateral Zusanli point
and rotated once every 5 min for 30 min to mimic a typical acupuncture
session. Animals with inflammatory pain clearly benefited from the
acupuncture treatment; touch sensitivity rose from 22.2 q 3.6 to 71.1 q
3.5% (P < 0.01, Tukey-Kramer), whereas the threshold to thermal pain
increased from 3.9 q 0.4 to 10.6 q 0.8 s (P < 0.01) (Fig. 3a,b). Similarly,
acupuncture sharply reduced mechanical allodynia in mice suffering
from neuropathic pain, with touch sensitivity improving from 26.7 q
4.9 to 71.7 q 4.8% (P < 0.01, Tukey-Kramer), and reduced sensitivity
to thermal pain (withdrawal of foot increased from 3.1 q 0.5 to
11.4 q 0.9 s, P < 0.0 1, Tu key -Kr ame r; Fig. 3c,d). As with CCPA injection
(Fig. 2) and consistent with earlier publications4, acupuncture-
mediated pain suppression was transient in that hypersensitivity to
both tactile and thermal stimulation returned to pre-acupuncture
levels by the following day. Notably, acupuncture failed to reduce pain
in A1 knockout mice. Hypersensitivity to either mechanical or thermal
pain persisted in mice with deletion of adenosine A1 receptors in
contrast with their littermate wild-type controls, which clearly benefitted
from acupuncture (Fig. 3a–d). Acupuncture did not alter the pain
sensitivity of the contralateral leg (Supplementary Fig. 1). Moreover,
needle insertion without intermittent rotation stimulation failed to
reduce pain sensitivity in both legs (Supplementary Fig. 3).
A remaining question is whether adenosine released during acu-
puncture, similar to CCPA, reduces input to the ACC in response to
painful stimulation. To this end, we used a similar strategy as above
(Fig. 2) to assess the effect of acupuncture on the amplitude of fEPSPs
recorded in the left ACC that were evoked by painful stimulation
e
Touch test (%)
Thermal test (s)
a
c d
b
Touch test (%)
Thermal test (s)
0
5
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20
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40
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100
Before
CFA
Before
acupuncture
Before
ligation
Before
ligation
**********
Normalized fEPSP amplitude
CFA
Ligation
–18 –12 12 18 24 30 36 42 48 54 60–6 60 min
0
0.2
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WT
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** **
After Day after
Before
acupuncture
After Day after Before
acupuncture
After Day after
Before
CFA
Before
acupuncture
After Day after
Figure 3 Acupuncture fails to suppress pain in mice lacking adenosine
A1 receptors. (a,b) Acupuncture reduced sensitivity to both mechanical (a)
and thermal (b) stimulation in wild-type mice suffering from inflammatory
pain after injection of CFA in the right paw, but not in A1RKO littermates
tested at day 4. All of the mice were evaluated ~10 min after acupuncture
(**P < 0.01, Tukey-Kramer test compared with before acupuncture;
##P < 0.01, comparison of wild type and A1RKO at each time point,
n = 5–9). (c,d) Acupuncture suppressed mechanical allodynia (c) and
thermal hyperalgesia (d) in wild-type mice, but not in A1RKO mice,
suffering from neuropathic pain. Neuropathic pain was induced by
partial ligation of the right leg ischias nerve and the clinical effect of
acupuncture tested at day 6 (n = 6). The sensitivity of the contralateral
(control) leg to mechanical and thermal stimulation from these
experiments is shown in Supplementary Figure 1. (e) The effect of
acupuncture (0–30 min) on fEPSP amplitude in the left anterior cingulate
cortex evoked by painful stimulation in the right leg. fEPSP amplitude is
plotted as a function of time in wild-type and A1RKO mice (*P < 0.01,
Tukey-Kramer test compared with the −18- to −12-min time points,
n = 3–8). Error bars indicate s.e.m.
© 2010 Nature America, Inc. All rights reserved.
886 VOLUME 13 | NUMBER 7 | JULY 2010 NATURE NEUROSCIENCE
ARTICLES
in the right leg (Fig. 3e). Similar to CCPA injection, acupuncture
in the left Zusanli point (contralateral to the stimulation) had no
effect on the fEPSP in response to painful stimulation (data not
shown). However, acupuncture in the right Zusanli point (ipsilateral
to the stimulation) suppressed fEPSP and the inhibition continued
to increase in potency during the observation period. fEPSP ampli-
tude was maximally reduced to 53.7 q 7.2% (P < 0.01) of baseline at
60 min (Fig. 3e). On the other hand, acupuncture did not alter fEPSP
in mice with deletion of A1 receptors. Combined, these observations
provide direct evidence for a role of adenosine in acupuncture-
mediated anti-nociceptive effects in models of inflammatory and
neuropathic pain. The relatively slow time course of fEPSP depres-
sion, compared with the sharp decrease in fEPSP amplitude following
CCPA injection, suggests that adenosine slowly accumulated in the
extracellular space during acupuncture (Figs. 2e and 3e). In addition,
pathways other than A1 receptors may contribute to acupuncture-
mediated pain suppression. These mechanisms could include direct
action of ATP on P2X receptors, as has been recently proposed, or
central release of opioid peptides4,27. The lack of pro-nociceptive,
hyperalgesic effects of A2A and A3 receptor activation (except for the
immediate pain felt during the needle insertion) can be ascribed to the
low expression of these receptors in muscle28. Mice with deletion of
A2A receptors exhibited no benefit of either CCPA injection or acu-
puncture (Supplementary Fig. 4) compared with wild-type controls
(Figs. 2 and 3)29.
Manipulation of AMP metabolism prolongs acupuncture effect
Similar to other types of tissue injury, accumulation of nucleotides in
the interstitial space during acupuncture is probably a consequence of
unspecific membrane damage or opening of stress-activated channels9.
The relatively high extracellular concentration of ATP metabolites
compared with ATP (Fig. 1c) likely reflects the rapid enzymatic degra-
dation of ATP by ectonucleotidases, as ATP is present in the cytosol of
skeletal muscles, fibroblasts and fat cells in a concentration of 4–8 mM,
or about 100-fold higher than AMP and adenosine30. Thus, it may be
possible to potentiate acupuncture-induced increases in the adenosine
by manipulating the enzymatic pathways involved in catabolism of
extracellular ATP. We asked which catabolic step is rate-limiting for
the extracellular degradation of ATP to adenosine. Using sections of
tissue (muscle and subcutis) harvested at the Zusanli point, we found
that phosphate was generated at a rate of 0.428 q 0.046 NM mg−1 min−1
when 1 mM ATP was added as substrate, whereas addition of AMP
(1 mM) produced phosphate at a rate of only 0.043 q 0.005 NM
mg−1 min
−1 (n = 3, P < 0.01, Student’s t test). The slow kinetics
of the latter reaction indicates that AMP dephosphorylation is the
rate-limiting step in adenosine production (Supplementary Fig. 5).
A detailed analysis suggested that multiple enzymes, including pro-
static acid phosphatase31,32, as well as not yet identified phosphatases,
contributed to extracellular formation of adenosine in muscle/subcutis
(Supplementary Fig. 5). However, AMP is not necessarily degraded
to adenosine, as AMP in skeletal muscles also can be deaminated
to inosine monophosphate (IMP) by AMP deaminase33 (Fig. 4a).
Consistent with a prior report33, we found that IMP was generated in
much larger quantities than adenosine when AMP was added as the
substrate (Fig. 4b). AMP deaminase functions as an enzymatic shuttle
for degradation of AMP that bypasses adenosine production (Fig. 4a).
On the basis of this observation, we asked whether it is possible to
increase and/or prolong acupuncture-induced increases in adenosine
by inhibiting AMP deaminase activity. We used the nucleoside analog
deoxycoformycin, which inhibits AMP deaminase and adenosine
deaminase34 and thereby suppresses the two major pathways involved
in elimination of extracellular adenosine (Fig. 4a). Consistent with an
inhibitory effect of deoxycoformycin on both deaminases, we found
that deoxycoformycin significantly increased the accumulation of
adenosine (2.9-fold increase, P = 0.025) and suppressed IMP (0.08-
fold decrease, P = 0.023) and inosine (0.24-fold decrease, P = 0.005)
production in isolated samples of muscle/subcutis harvested at the
Zusanli point (Fig. 4b).
0
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*
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Touch test (%)Touch test (%)
Thermal test (s)Thermal test (s)
Before acupuncture
Before ligation
Control
Deoxycoformycin
gf
e
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Deoxycoformycin
0.5 1.0 2.0 3.0 4.0 h1.5 2.5 3.5
0.5 1.0 2.0 3.0 4.0 4.5 h1.5 2.5 3.5 0.5 1.0 2.0 3.0 4.0 4.5 h1.5 2.5 3.5
0.5 1.0 2.0 3.0 4.0 h1.5 2.5 3.5
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Before acupuncture
Before CFA
Before acupuncture
Before CFA
Deoxy-
coformycin
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CFA
Ligation
AMP
Inosine
Adenosine
AMPD
ADA
DeoxycoformycinControl
0
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0 0.5 h 1 h 2 h1.5 h 0 0.5 h 1 h 2 h1.5 h
0
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25
Adenosine
Adenosine / Inosine
(nmol)
Inosine
0
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700
IMP
IMP (nmol)
IMP
Figure 4 Pharmacological inhibition of deaminase activity enhances
increases in adenosine and prolongs anti-nociception actions of acupuncture.
(a) Schematic diagram outlining the two major pathways involved in
extracellular enzymatic degradation of AMP. The nucleoside analog
deoxycoformycin inhibits both AMP deaminase (AMPD) and adenosine
deaminase (ADA). (b) Histogram comparing the production of adenosine,
IMP and inosine when tissue sections harvested close to the Zusanli
point were incubated in 1 mM AMP and an inhibitor of adenosine uptake,
nitrobenzylthioinosine (100 NM), for 45 min. Deoxycoformycin (500 NM)
increased accumulation of adenosine while inhibiting the production of IMP
and inosine (*P < 0.05, **P < 0. 01, t test comparison between control and
deoxycoformycin, n = 5). (c) Analysis of microdialysis samples collected
close to the Zusanli point in mice treated with deoxycoformycin (50 mg per kg,
intraperitoneal) or vehicle (saline). Deoxycoformycin increased accumulation
of adenosine, while inhibiting the production of IMP in vivo during and after
acupuncture (n = 6–8). (d,e) Deoxycoformycin (50 mg per kg, intraperitoneal)
prolonged the anti-nociceptive effect of acupuncture in wild-type mice
with inflammatory pain in response to mechanical (d) and thermal (e)
stimulation (##P < 0.01, Tukey-Kramer test compared with before
acupuncture, n = 6–10). (f,g) Deoxycoformycin prolonged the anti-nociceptive
effect of acupuncture in wild-type mice with neuropathic pain induced by
partial ligation of the ischias nerve to mechanical (f) and to thermal (g)
stimulation (#P < 0.05, ##P < 0.01, Tukey-Kramer test compared with
before acupuncture, n = 5). The sensitivity of the contralateral (control) leg
to mechanical and thermal stimulation from these experiments is shown in
Supplementary Figure 1. Error bars indicate s.e.m.
© 2010 Nature America, Inc. All rights reserved.
NATURE NEUROSCIENCE VOLUME 13 | NUMBER 7 | JULY 2010 887
ARTICLES
To assess the clinical potential of systemic administration of deoxy-
coformycin as an adjuvant to acupuncture, we treated mice with
deoxycoformycin (50 mg per kg of body weight, intraperitoneal) and
collected microdialysis samples 0.4–0.6 mm from the Zusanli point.
Mice receiving deoxycoformycin exhibited a sharp increase in the
extracellular accumulation of adenosine during acupuncture (3.68-
fold increase, P = 0.0081; Fig. 4c). Moreover, deoxycoformycin pro-
longed the accumulation of adenosine, which remained significantly
elevated (0.5, 1 and 2 h, P < 0.01; 1.5 h, P < 0.05, t test) for the duration
of the experiment (Fig. 4c). The increases in acupuncture-induced
adenosine accumulation was, similar to the ex vivo observations, mir-
rored by a decrease of IMP consistent with an inhibitory effect of
deoxycoformycin on AMP deaminase (Fig. 4c).
The fact that deoxycoformycin potentiated and prolonged adeno-
sine increases induced by acupuncture raises the question of whether
deoxycoformycin can be used as an adjuvant to acupuncture, which
potentiate the anti-nociception. To address this point, we compared the
anti-nociceptive effect of acupuncture in mice to that received deoxy-
coformycin (50 mg per kg, intraperitoneal) versus vehicle (phosphate-
buffered saline). The 30-min acupuncture session (needle rotated
twice every 5 min for a total of 30 min) reduced pain for a duration
of ~1.0–1.5 h in mice that received vehicle (Fig. 4d–g). Notably, mice
pretreated with deoxycoformycin exhibited a significant prolongation
(P < 0.05, Tukey-Kramer test compared with before acupuncture) of
anti-nociception. Mechanical allodynia and thermal pain sensitivity
were suppressed for ~3.0–3.5 h in mice suffering from either inflam-
matory or neurogenic pain, when deoxycoformycin was added as an
adjuvant to acupuncture (Fig. 4d–g). Deoxycoformycin did not alter
the pain sensitivity of the contralateral leg (Supplementary Fig. 1).
In addition, deoxycoformycin had no effect on either the tactile or
thermal sensitivity when it was not combined with acupuncture in
the two models of chronic pain (Supplementary Fig. 6) consistent
with the fact that deoxycoformycin failed to significantly increase
(P = 0.1728, t test comparison between control and deoxycoformycin)
the resting, unstimulated concentration of adenosine. These data sug-
gest that suppression of deaminase activity can be used as an adjuvant
to acupuncture, which effectively increases its clinical benefits.
Deoxycoformycin (Pentostatin) is an antimicrobial nucleoside analog
that inhibits DNA synthesis and is approved by the Food and Drug
Administration for treatment of leukemia35.
DISCUSSION
Although acupuncture has been practiced for over 4,000 years, it has
been difficult to establish its biological basis. Our findings indicate
that adenosine is central to the mechanistic actions of acupuncture.
We found that insertion and manual rotation of acupuncture needles
triggered a general increase in the extracellular concentration of
purines, including the transmitter adenosine (Fig. 1), which is con-
sistent with the observation that tissue damage is associated with
an increase in extracellular nucleotides and adenosine36. Because
the anti-nociceptive effects of peripheral, spinal and supraspinal
adenosine A1 receptors are well established37,38, we asked whether
peripheral injection of an A1 receptor agonist suppressed hyper-
algesia25,37 (Fig. 2). We found that the A1 receptor agonist CCPA
sharply reduced inflammatory and neurogenic pain and that sup-
pression of pain mediated by acupuncture required adenosine A1
receptor expression (Fig. 3). These findings suggest that A1 receptor
activation is both necessary and sufficient for the clinical benefits of
acupunctures. To the best of our knowledge, adenosine A1 receptors
have not previously been implicated in the anti-nociceptive actions
of acupuncture.
One may speculate that other non-allopathic treatments of chronic
pain, such as chiropractic manipulations and massage, modalities that
involve the mechanical manipulation of joints and muscles, might
also be associated with an efflux of cytosolic ATP that is sufficient to
elevate extracellular adenosine. As in acupuncture, adenosine may
accumulate during these treatments and dampen pain in part by the
activation of A1 receptors on sensory afferents of ascending nerve
tracks. Notably, needle penetration has been reported to not confer
an analgesic advantage over nonpenetrating (placebo) needle applica-
tion39, as opposed to our observations (Supplementary Figs. 2 and 3)
and those of others40,41. However, it is possible that ATP release from
keratinocytes in response to mechanical stimulation of the skin results
in an accumulation of adenosine that transiently reduces pain, as A1
receptors are probably expressed by nociceptive axon terminal in epi-
dermis37. In fact, vibratory stimulation applied to the skin depressed
the activity of nociceptive neurons in the lower lumbar segments of
cats by release of adenosine42. However, this effect differs from the
anti-nociceptive effect of acupuncture, which does not depend on the
afferent inner vation of the skin4. Acupuncture is typically applied to
deep tissue, including muscle and connective tissue, and acupoints
may better overlap with their proximity to ascending nerve tracks
than to the density of cutaneous afferents.
Most patients have reported that acupuncture in itself is not a
painful procedure, except for a pinching sensation in tissue below the
acupuncture needle. Because ATP is released during acupuncture
(Fig. 1), the pinching sensation may be mediated by nociceptive P2X3
receptors, which are expressed by small-diameter, primary afferent
neurons, some of which are sensitive to capsaicin10. The most likely
explanation for the lack of direct pain during acupuncture is that extra-
cellular ATP does not reach high enough concentrations to activate
P2X3 and other nociceptive P2X receptors because of its rapid degra-
dation (Fig. 1). However, activation of P2X receptors may nonetheless
contribute to the anti-analgesic effects of acupuncture, as was recently
suggested27, perhaps by subthreshold-activating P2X receptors or by
more complex mechanisms involving dimerization of P2X and A1
receptors43. In addition to the use of acupuncture for treatment of
chronic pain, acupuncture is also frequently employed in diseases with
a local inflammatory component, such as arthritis and tendinitis44.
Adenosine has anti-inflammatory properties and we found that acu-
puncture increased extracellular adenosine36 (Fig. 1).
Quantification of extracellular purines in microdialysis samples
collected nearby the acupuncture point revealed that the extracellular
adenosine concentration rose following the release of ATP, which
was dephosphorylated to ADP, AMP and adenosine by potent ecto-
nucleotidases, and that AMP dephosphorylation represents the
rate-limiting step in this reaction (Supplementary Fig. 5). As with
most other transmitters, adenosine has a short lifespan in the extracel-
lular space as a result of facilitated uptake by nucleoside transporters
and concurrent degradation to inosine33. After reuptake, adenosine is
quickly converted to AMP by cytosolic adenosine kinase (Km ~20 nM),
thereby facilitating the rapid clearance of adenosine in the extra-
cellular space36 and shortening the anti-nociceptive effects of acu-
puncture. Moreover, our analysis confirmed the prior observation that
AMP deaminase activity is high in muscle/subcutis33 and that only
a fraction of AMP is dephosphorylated to adenosine. AMP deami-
nases constitute the primary enzymatic pathway for elimination
of extracellular AMP and this pathway bypasses adenosine. Thus,
acupuncture combined with pharmacological suppression of AMP
deaminase activity should theoretically increase the availability of
adenosine and thereby enhance the clinical benefits of acupuncture.
As a proof of principle, we found that mice treated with a Food and
© 2010 Nature America, Inc. All rights reserved.
888 VOLUME 13 | NUMBER 7 | JULY 2010 NATURE NEUROSCIENCE
ARTICLES
Drug Administration approved deaminase inhibitor, deoxycofor-
mycin, exhibited more potent increases in adenosine and benefitted
from a longer-lasting suppression of chronic pain following acu-
puncture. In summary, we found that the anti-nociceptive action of
acupuncture is mediated by activation of A1 receptors located on
ascending nerves. Thus, medications that interfere with A1 recep-
tors or adenosine metabolism may improve the clinical benefit
of acupuncture.
METHODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplement ary infor mation is available o n the Nature Neuroscience website.
ACKNOWLEDGMENTS
This work was supported by a grant from the US National Institutes of Health to
M.N. and K.T.
AUTHOR CONTRIBUTIONS
All authors contributed to experimental design and execution and manuscript
preparation.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprintsandpermissions/.
1. NIH Consensus Conference. Acupuncture. J. Am. Med. Assoc. 280, 1518–1524
(1998).
2. Culliton, B.J. Acupuncture: fertile ground for faddists and serious NIH research.
Science 177, 592–594 (1972).
3. Bonafede, M., Dick, A., Noyes, K., Klein, J.D. & Brown, T. The effect of acupuncture
utilization on healthcare utilization. Med. Care 46, 41–48 (2008).
4. Zhao, Z.Q. Neural mechanism underlying acupuncture analgesia. Prog. Neurobiol.
85, 355–375 (2008).
5. Han, J.S. Acupuncture and endorphins. Neurosci. Lett. 361, 258–261 (2004).
6. Huang, C., Wang, Y., Han, J.S. & Wan, Y. Characteristics of electroacupuncture-
induced analgesia in mice: variation with strain, frequency, intensity and opioid
involvement. Brain Res. 945, 20–25 (2002).
7. Lao, L. et al. A parametric study of electroacupuncture on persistent hyperalgesia
and Fos protein expression in rats. Brain Res. 1020, 18–29 (2004).
8. Li, W.M. et al. Analgesic effect of electroacupuncture on complete Freund’s
adjuvant–induced inflammatory pain in mice: a model of antipain treatment by
acupuncture in mice. Jpn. J. Physiol. 55, 339–344 (2005).
9. Abbracchio, M.P., Burnstock, G., Verkhratsky, A. & Zimmermann, H. Purinergic
signaling in the nervous system: an overview. Tr en ds N eu ro sci . 32, 19–29 (2009).
10. Burnstock, G. Physiology and pathophysiology of purinergic neurotransmission.
Physiol. Rev. 87, 659–797 (2007).
11. Sjölund, K.F., Segerdahl, M. & Sollevi, A. Adenosine reduces secondary hyperalgesia
in two human models of cutaneous inflammatory pain. Anesth. Analg. 88, 605–610
(1999).
12. Maione, S. et al. The antinociceptive effect of 2-chloro-2b-C-methyl-N6-
cyclopentyladenosine (2b-Me-CCPA), a highly selective adenosine A1 receptor
agonist, in the rat. Pain 131, 281–292 (2007).
13. Sawynok, J., Reid, A. & Poon, A. Peripheral antinociceptive effect of an adenosine
kinase inhibitor, with augmentation by an adenosine deaminase inhibitor, in the rat
formalin test. Pain 74, 75–81 (1998).
14. Cui, M., Tang, X., Christian, W.V., Yoon, Y. & Tieu, K. Perturbations in mitochondrial
dynamics induced by human mutant PINK1 can be rescued by the Mitochondrial
Division Inhibitor mdivi-1. J. Biol. Chem. 285, 11740–11752 (2010).
15. Volonté, M.G., Yuln, G., Quiroga, P. & Consolini, A.E. Development of an HPLC
method for determination of metabolic compounds in myocardial tissue. J. Pharm.
Biomed. Anal. 35, 647–653 (2004).
16. Li, J., King, N.C. & Sinoway, L.I. Interstitial ATP and norepinephrine concentrations
in active muscle. Circulation 111, 2748–2751 (2005).
17. Li, J., King, N.C. & Sinoway, L.I. ATP concentrations and muscle tension increase
linearly with muscle contraction. J. Appl. Physiol. 95, 577–583 (2003).
18. Bekar, L. et al. Adenosine is crucial for deep brain stimulation-mediated attenuation
of tremor. Nat. Med. 14, 75–80 (2008).
19. Lohse, M.J. et al. 2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1
adenosine receptors. Naunyn Schmiedebergs Arch. Pharmacol. 337, 687–689 (1988).
20. Raghavendra, V., Tanga, F.Y. & DeLeo, J.A. Complete Freunds adjuvant–induced
peripheral inflammation evokes glial activation and proinflammatory cytokine
expression in the CNS. Eur. J. Neurosci. 20, 467–473 (2004).
21. Sun, D. et al. Mediation of tubuloglomerular feedback by adenosine: evidence from mice
lacking adenosine 1 receptors. Proc. Natl. Acad. Sci. USA 98, 9983–9988 (2001).
22. Vadakkan, K.I., Jia, Y.H. & Zhuo, M. A behavioral model of neuropathic pain
induced by ligation of the common peroneal nerve in mice. J. Pain 6, 747–756
(2005).
23. Wei, F. & Zhuo, M. Potentiation of sensory responses in the anterior cingulate cortex
following digit amputation in the anaesthetized rat. J. Physiol. (Lond.) 532,
823–833 (2001).
24. Davis, K.D., Taylor, S.J., Crawley, A.P., Wood, M.L. & Mikulis, D.J. Functional MRI
of pain- and attention-related activations in the human cingulate cortex.
J. Neurophysiol. 77, 3370–3380 (1997).
25. Karlsten, R., Gordh, T. & Post, C. Local antinociceptive and hyperalgesic effects in
the formalin test after peripheral administration of adenosine analogues in mice.
Pharmacol. Toxicol. 70, 434–438 (1992).
26. Reeve, A.J. & Dickenson, A.H. Electrophysiological study on spinal antinociceptive
interactions between adenosine and morphine in the dorsal horn of the rat. Neurosci.
Lett. 194, 81–84 (1995).
27. Burnstock, G. Acupuncture: a novel hypothesis for the involvement of purinergic
signaling. Med. Hypotheses 73, 470–472 (2009).
28. Fredholm, B.B., IJzerman, A.P., Jacobson, K.A., Klotz, K.N. & Linden, J.
International Union of Pharmacology. XXV. Nomenclature and classification of
adenosine receptors. Pharmacol. Rev. 53, 527–552 (2001).
29. Chen, J.F. et al. A(2A) adenosine receptor deficiency attenuates brain injury induced
by transient focal ischemia in mice. J. Neurosci. 19, 9192–9200 (1999).
30. Poortmans, J. Principles of Exercise Biochemistry (Karger, Brussels, 2003).
31. Quintero, I.B. et al. Prostatic acid phosphatase is not a prostate-specific target.
Cancer Res. 67, 6549–6554 (2007).
32. Zylka, M.J. et al. Prostatic acid phosphatase is an ectonucleotidase and suppresses
pain by generating adenosine. Neuron 60, 111–122 (2008).
33. Cunha, R.A. & Sebastiao, A.M. Extracellular metabolism of adenine nucleotides
and adenosine in the innervated skeletal muscle of the frog. Eur. J. Pharmacol.
197, 83–92 (1991).
34. Golembiowska, K., White, T.D. & Sawynok, J. Modulation of adenosine release from
rat spinal cord by adenosine deaminase and adenosine kinase inhibitors. Brain Res.
699, 315–320 (1995).
35. Lamanna, N. & Kay, N.E. Pentostatin treatment combinations in chronic lymphocytic
leukemia. Clin. Adv. Hematol. Oncol. 7, 386–392 (2009).
36. Fredholm, B.B. Adenosine, an endogenous distress signal, modulates tissue damage
and repair. Cell Death Differ. 14, 1315–1323 (2007).
37. Sawynok, J. Adenosine receptor activation and nociception. Eur. J. Pharmacol. 347,
1–11 (1998).
38. Eisenach, J.C. et al. Intrathecal but not intravenous opioids release adenosine from
the spinal cord. J. Pain 5, 64–68 (2004).
39. Takakura, N. & Yajima, H. Analgesic effect of acupuncture needle penetration: a
double-blind crossover study. Open Med. 3, e54–61 (2009).
40. Weidenhammer, W., Linde, K., Streng, A., Hoppe, A. & Melchart, D. Acupuncture
for chronic low back pain in routine care: a multicenter observational study. Clin.
J. Pain 23, 128–135 (2007).
41. Kelly, R.B. Acupuncture for pain. Am. Fam. Physician 80, 481–484 (2009).
42. Salter, M.W. & Henry, J.L. Evidence that adenosine mediates the depression of
spinal dorsal horn neurons induced by peripheral vibration in the cat. Neuroscience
22, 631–650 (1987).
43. Sichardt, K. & Nieber, K. Adenosine A(1) receptor: functional receptor-receptor
interactions in the brain. Purinergic Signal. 3, 285–298 (2007).
44. Zijlstra, F.J., van den Berg-de Lange, I., Huygen, F.J. & Klein, J. Anti-inflammatory
actions of acupuncture. Mediators Inflamm. 12, 59–69 (2003).
© 2010 Nature America, Inc. All rights reserved.
NATURE NEUROSCIENCE
doi:10.1038/nn.2562
ONLINE METHODS
Surgery, experimental models, behavioral assessment, CCPA administration,
acupuncture and immunohistochemistry. The Institutional Animal Care and
Use Committee at University of Rochester approved all of the procedures used
in this study. As per the direction of the International Society for the Study of
Pain guidelines, we attempted to use the minimum number of mice necessary
to achieve statistical significance. C57BL/6J male mice (8–14 weeks old, Jackson
Labs) were used in all experiments. A1 receptor knockout mice21, A2A receptor
knockout mice29, and CD73 knockout mice45 were on C57BL/6 genetic back-
ground and wild-type littermate used as controls.
Peripheral inflammation was induced by injection of CFA (mixed with an
equal amount of oil, total volume 0.1 ml) in the plantar surface of the right hind
paw of mice20. An equal amount of saline (0.1 ml) was injected in the left hind
paw as a control. Neuropathic pain was induced by ligation of the right leg sciatic
nerve with 4.0 polypropylene suture in mice anesthetized with ketamine (60 mg
per kg, intraperitoneal) and xylazine (10 mg per kg, intraperitoneal)46.
Mechanical allodynia was evaluated using repeated stimulations with a Von
Frey filament exerting 0.02 g of force onto the plantar surface. The percentage of
negative responses of a total of ten trials was calculated for each foot. Thermal
hyperalgesia was assessed using an Analgesymeter (Ugo Basile). A mobile radi-
ant heat source was focused on the hind paw and the paw withdrawal latencies
were defined as the time taken by the mouse to remove its hind paw from the
heat source (maximum of 20 s to avoid tissue damage). The paw withdrawal
was repeated three times for each foot and the average calculated. To avoid con-
ditioning to stimulation, we interposed a 5-min rest period between each trial
in both thermal and mechanical tests. Behavioral parameters were evaluated
before intraplantar injection of CFA or nerve ligation (that is, day 0), on day 3–4
in mice receiving the CFA injection, and at day 5–7 in mice with nerve ligation,
unless otherwise noted. Prior to injection of CCPA (0.1 mM, 20 Nl), saline or
acupuncture, the mice were restrained under light isoflurane anesthesia (~1%).
All the mice were evaluated ~10 min after the CCPA injection or acupuncture.
The total duration of anesthesia was ~2 min and mice with inflammatory or
neurogenic pain and their controls were treated similarly. For acupuncture, a
small acupuncture needle, 0.16 × 13 mm (08-02, Lhass Medical), was gently
inserted in a depth of 1.5 mm in the Zusanli point (ST36) located 3–4 mm below
and lateral 1–2 mm for the midline of the knee4. The needle was slowly rotated
every 5 min for a total of 30 min during an acupuncture session. We chose to study
the effect of acupuncture on chronic pain applied in the acupoint Zusanli because
it is one of the most effective points in traditional Chinese medicine and its anti-
nociceptive effects in rodent models of chronic pain are well-established4. For
HPLC analysis of purine releases in sections of muscle/subcutis, a microdialysis
probe (MD-2211, Bioanalytical Systems) was implanted 1.5 h before collection of
microdialysis samples. The microdialysis probe was implanted 0.4–0.6 mm from
the Zusanli point. The microdialysis probe was perfused with Ringer’s solution
at a rate of 1 Nl per min. The microdialysates were collected on ice over a
30-min period (30 Nl) and were immediately frozen at −80 °C until HPLC
analysis. Vehicle (saline, intraperitoneal) or deoxycoformycin (50 mg per kg, intra-
peritoneal) was administered 30 min before acupuncture. Immunohistochemistry
was performed as previously described18.
In viv o ele ctr ophy siol og y. Mice were anesthetized with 2–3% isoflurane (vol/vol),
intubated and artificially ventilated with a small animal ventilator (SAAR-830,
CWE). Body temperature was monitored by a rectal probe and maintained at
37 °C by a heating blanket (BS4, Harvard Apparatus). A craniotomy (1–1.5 mm
in diameter), centered 0.1 mm anterior to the bregma and 1.5 mm lateral from
midline, was made over the left ACC. A custom-made metal plate was glued to the
skull with dental acrylic cement. The mice were maintained at 2 vol% isoflurane for
the remainder of the experiment. Local field potential recordings were obtained
from layer 4 of ACC, 0.8 mm below the pial surface by a patch pipette (TW100F-4,
WPI; outer diameter, 1.0 mm; inner diameter, 0.75 mm; tip diameter, 1–2 Nm).
Local field potential signals were amplified, bandpass filtered at (1–100 Hz)
and digitized at 10 kHz as described previously18. Dura matter was kept intact.
A custom-made bipolar electrode was inserted subcutaneously into the right
hindpaw. High-intensity stimulation (10 mA, 20 ms) was evoked every 120 s.
Lower stimulation intensities evoked either no or variable responses, consistent
with the idea that that ACC neurons respond primarily to painful stimuli23. fEPSP
amplitude was measured using pCLAMP 9.2 program (Axon Instruments). We
chose to record fEPSPs evoked by painful stimulation in the ACC rather than
in the substantia gelatinosa of the spinal cord to detect systemic effect of CCPA
injected in the contralateral (left) leg.
HPLC ana lysi s of p urin es. The analysis of enzymatic degradation of purines was
based on sections (~4 mm) of skeletal muscles with overlying subcutis harvested
from tissue below the Zusanli point. The sections were incubated with O2-bubbled
buffer (125 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM
CaCl2, 10 mM glucose and 25 mM NaHCO3, pH 7.3, gassed with 95% O2 and
5% CO2) for 30 min. Each section per well was placed into a 6-well plate in
3 ml Hanks’ balanced salt solution with 15 mM HEPES (pH 7.3) containing
1 mM AMP with or without 500 NM deoxycoformycin (Tocris Bioscience).
The adenosine transport inhibitor nitrobenzylthioinosine (Sigma) was added
to all samples to reduce uptake of adenosine. The samples were collected from
each well at 0 and 45 min and stored at −80 °C for HPLC analysis. The analyses
were carried out using CoulArray 5600A system (ESA) and an ESA model 526
ultraviolet detector as described previously14,15. Chromatographic separation
was achieved by using a reverse-phase column (Lichrospher 100 RP-18, 5 Nm,
250 mm × 3 mm, Merck). For measurements of microdialysates, we used a mobile
phase consisting of 215 mM KH2PO4, 2.3 mM tetrabutylammonium bisulfate,
3.2% acetonitrile (vol/vol, HPLC-grade water), pH 6.2. For measurements of
samples prepared from muscle/subcutis sections, we used a mobile phase consist-
ing 215 mM KH2PO4, 1.2 mM tetrabutylammonium bisulfate, 1% acetonitrile,
pH 6.0. The flow rate was maintained at 0.25 ml min−1. Daily calibration curves
were prepared by a four-point standard (3, 1, 0.3 or 0.1 NM) of ATP, ADP, AMP,
adenosine, inosine and IMP in 0.4 M perchloric acid, respectively. Eluted purines
were detected at 260 nm and the chromatographic peaks were integrated using
CoulArray software. Notably, deoxycoformycin-induced changes in inosine
concentration could not be evaluated in microdialysis samples, as the peak of
deoxycoformycin overlapped with inosine. Pharmacological analysis of enzymes
involved in extracellular dephosphorylation of AMP or ATP was measured using
the Malachite Green Phosphate Detection Kit (Sigma) in samples collected from
sections incubated in a phosphate-free Ringer solution. All measurements were
normalized to wet weight in milligrams.
Statistical analysis. Statistical analyses were carried out with ANOVA with
Tu ke y - Kr a me r post hoc test. Where only two groups were compared, Student’s
t test was used.
45. Castrop, H. et al. Impairment of tubuloglomerular feedback regulation of GFR in
ecto-5b-nucleotidase/CD73-deficient mice. J. Clin. Invest. 114, 634–642 (2004).
46. Martucci, C. et al. The purinergic antagonist PPADS reduces pain related behaviors
and interleukin-1 beta, interleukin-6, iNOS and nNOS overproduction in central
and peripheral nervous system after peripheral neuropathy in mice. Pain 137,
81–95 (2008).
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