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Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture

ABSTRACT

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.

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 Western medicine has treated acupuncture with considerable skepticism2, 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 acupuncture needles in itself is not sufficient to relieve pain4. An acupuncture session typically lasts for 30 min, during which the needles are intermittently rotated, electrically stimulated or, in some cases, heated. The pain threshold is reported to slowly increase and to outlast the treatment4. 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 stimulation 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 nucleotides 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 ± 81.1 nM from a baseline of 10.6 ± 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 adenosine. Similar to electroacupuncture and transcutaneous electrical nerve stimulation, deep brain stimulation delivers electrical stimulation that triggers an increase in extracellular adenosine concentration18.

  • Effect of local application of A1 receptor agonist
  • Anti-nociceptive effect of acupuncture requires A1 receptors
  • Manipulation of AMP metabolism prolongs acupuncture effect

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 consistent 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 suppression 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 application39, 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 epidermis37. 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 innervation 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 extracellular ATP does not reach high enough concentrations to activate P2X3 and other nociceptive P2X receptors because of its rapid degradation (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 acupuncture 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 ectonucleotidases, 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 extracellular 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 ( K m ~20 nM), thereby facilitating the rapid clearance of adenosine in the extracellular space36 and shortening the anti-nociceptive effects of acupuncture. 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 deaminases 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 Drug Administration approved deaminase inhibitor, deoxycoformycin, exhibited more potent increases in adenosine and benefitted from a longer-lasting suppression of chronic pain following acupuncture. 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 receptors or adenosine metabolism may improve the clinical benefit of acupuncture

Quelle: NIH Public Access Author Manuscript Nat Neurosci . Author manuscript; available in PMC 2012 October 10

Studienautoren:
Nanna Goldman1,4, Michael Chen1,4, Takumi Fujita1,4, Qiwu Xu1, Weiguo Peng1, Wei Liu1, Tina K Jensen1, Yong Pei1, Fushun Wang1, Xiaoning Han1, Jiang-Fan Chen2, Jurgen Schnermann3, Takahiro Takano1, Lane Bekar1, Kim Tieu1, and Maiken Nedergaard1 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

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