Background
Diabetics mellitus is a debilitating chronic disease that affects ~8% of the population in the US. About ~70% of diabetic patients are reported to have various forms of nerve damage (neuropathy). The most common type of diabetic neuropathy is nerve damages in the periphery, e.g., hands, toes and feet. Peripheral neuropathy patients often experience aberrant pain sensation, including spontaneous pain, hyperalgesia (severe pain with mild painful stimuli) and allodynia (pain with innocuous stimuli, e.g. light touch) [
1‐
6]. Treatment options for these abnormal sensations have been limited, partly because of our poor understanding of the molecular mechanisms underlying the diabetes-induced neuropathic pain.
Sensitization of dorsal root ganglion neurons and their associated nerve fibers has been suggested to be a major cause of diabetes-induced abnormal pain [
4,
7]. Changes in the expression or function of T-type Ca
2+ channels [
7] and transient receptor potential vanilloid 1 (TRPV1) receptors [
6,
8] are thought to contribute to changes in the activity of sensory neurons under diabetic conditions.
P2X receptors (P2XRs) are ligand-gated cationic channels abundantly expressed in the pain processing DRG neurons [
9‐
11]. There is growing evidence that P2XR expression and function in DRGs are greatly sensitized after tissue inflammation [
12,
13] and nerve injury [
14]. Thus, P2XRs play a pivotal role in the transmission of nociceptive information under injury conditions [
9,
15,
16]. ATP and noradrenaline are co-stored and co-released from sympathetic nerves [
17]. EctoATPase, which hydrolyzes extracellular di- and triphosphate nucleotides, modulates purinergic transmission [
17]. It has been established that ATP and purinergic receptors are involved in peripheral signaling in diabetic rats. Stimulation of sympathetic nerves in those rats causes C-fiber polymodal receptor units to fire [
18]. An increase in the extracellular level of adenosine by an adenosine kinase inhibitor attenuates diabetes-induced tactile allodynia [
19]. Studying the involvement of P2XRs in painful diabetic neuropathy, Migita et al (2009) [
20] found that P2X receptor antagonists inhibit the STZ-induced mechanical allodynia in mice and the levels of P2X2R and P2X3R mRNA in DRGs are increased. The molecular mechanism underlying the change in P2XR-mediated activity, however, has not been investigated. We therefore studied P2X3R current activity of hindpaw innervated DRG neurons in STZ-induced diabetic rats. Total and membrane expressions of the P2X3R protein were also investigated. We found that following the development of diabetes, P2X3R-mediated current activity is greatly enhanced and the membrane, but not the total, expression of P2X3Rs is significantly upregulated.
Discussion
In this study, we show that a single injection of STZ can induce diabetes and mechanical allodynia in rats two weeks later. The conditions were maintained for at least another seven weeks (Figure
1). The STZ-induced mechanical allodynia is blocked by the specific P2XR antagonists PPADS and TNP-ATP (Figure
2), an observation in agreement with a previous study of P2XR involvement in a mouse model of diabetes [
20]. We further showed that the same dose of ATP evokes much larger P2X3R-mediated inward currents in Dil-labeled hindpaw-innervated DRG neurons isolated from STZ treated rats than those isolated from control rats (Figure
4). The expression P2X3Rs in the cell membrane of STZ primary sensory neurons is significantly upregulated (Figure
5). Thus, P2X3R sensitization is a major contributor to STZ-induced mechanical allodynia. To the best of our knowledge, this is the first report showing an enhancement of P2X3R trafficking to the cell membrane of hindpaw-innervated primary sensory neurons in a painful diabetic rat model.
P2XR sensitization have been implicated in abnormal pain signaling in a variety of injuries [
13,
29‐
31], including inflammation [
12], sciatic nerve ligation [
14,
32], burn injury [
33] and chronic compression of DRGs [
34]. The results of this study and of Migita et al [
20] provide the evidence that P2X3R signaling also plays a key role in chronic pain produced by diabetic neuropathy.
TNP-ATP is a potent antagonist for P2X1, P2X3 and P2X2/3 receptors. Because of the low expression of P2X1 receptors in DRGs [
13,
28,
35,
36], the complete block of P2X3R-mediated ATP currents by TNP-ATP (Figure
3) and the upregulation of cell membrane trafficking of P2X3Rs in DRG neurons (Figure
5), we conclude that P2X3-containing receptors play a critical role in the development of abnormal pain state following STZ injection. Our study concentrated on diabetes-induced changes in fast-inactivating P2X3R currents. Since P2X2R mRNA was also found to increase after STZ treatment [
20], it is of interest to determine, in the future, if slow-inactivating P2X2/3R-mediated currents in Dil-labeled DRG neurons are altered by STZ-induced diabetes. Our conclusion is consistent with the observation that the P2X3R antagonist, A-317491, applied subcutaneously, blocks the mechanical allodynia elicited by nerve injuries [
30]. The results also support our previous observation that the P2X3R participates in neuropathic pain in the periphery [
14]. In contrast, P2X4/P2X7 receptors in microglia were reported to mediate the neuropathic pain disorders in the spinal cord [
37‐
39].
We showed that TNP-ATP reversed STZ-induced mechanical allodynia partially (Figure
2). The incomplete block of the behavioral responses could be a result of less than saturating concentration of the antagonist used in these experiments. Further experiments are needed to determine the dose response and the maximal effect of the antagonist. In addition, other receptors or channels, e.g. TRPV1 receptors [
6,
8], T-type Ca
2+ channel upregulation could also participate in producing diabetic nociception. In addition to receptor sensitization, an increase in endogenous ATP after STZ treatment could also give rise to the increase in P2XR-mediated allodynia (Figure
2). Our observation that ATP is released from both DRG neurons and glial satellite cells in DRGs [
40] have led us to suggest that the origin of ATP release in the hindpaws of STZ rats stems from peripheral nerve terminals and their surrounding glial and immune cells. We measured ATP release in the rat hindpaw following spared nerve injury and found that ATP release was increased only when a stiff (13.5 g) von Frey filament was used for stimulation, but not when a low-strength filament (3.5 g) was applied [
14]. Based on the study and the use of low-strength von Frey filaments in diabetic rat experiments (Figure
2), we suggest that an increase in ATP release is not likely the major cause for the observed increase in STZ-induced allodynia. We will test if this is indeed the case in our future study of the role of ATP in diabetes.
The P2X3R expression and trafficking are of great interest in understanding the increase in receptor function under conditions of peripheral inflammation and neuropathy. Changes in the total expression of P2X receptors have been found to vary among nerve injury models. It has been reported that P2X3R expression in DRGs increases after chronic constriction injury [
41], decreases when the sciatic or spinal nerves are ligated [
42,
43], or remains unchanged in spared nerve injury model [
14]. The reason for the different observations has not been investigated. The varying findings could simply be a result of different population of neurons in a ganglion affected by different nerve injury models. Migita et al (2009) [
20] reported that expression of P2X2 and P2X3 receptor mRNA was upregulated in STZ-induced diabetic mice. They did not study the expression of these receptors at the protein level. Here, we show that STZ treatment does not alter the expression of total P2X3R protein (Figure
5A) but enhances the membrane expression of the receptor (Figure
5C) in rats. In our experiments, the large (2.2-fold) increase in P2X3R-mediated currents following STZ-induced diabetes cannot be entirely accounted for by the increase (85.7%) in P2X3R trafficking. Nevertheless, the relationship between biochemical and electrophysiological or behavioral measurements is not necessarily linear. Other factors, e.g. changes in the modulation of the receptor by protein kinases [
24] or changes in channel properties [
12], could also contribute to an increase in the P2X3R function. The mechanism underlying the increase in trafficking of P2X3Rs following diabetes has yet to be determined. Our data do indicate that mechanisms involved in P2XR-mediated neuropathic pain and inflammatory pain are distinct. Following CFA-induced peripheral inflammation, the total P2X3R expression has been found to be upregulated [
12].
In conclusion, we provide evidence that STZ-induced diabetes promotes the trafficking of P2X3Rs to the membrane of DRG neurons and thus increases P2X3R-mediated responses. The change enhances the activity of afferent neurons and contributes to abnormal diabetes-induced peripheral neuropathic pain. The results may provide promising clues for the development of new therapeutic strategies for managing intractable neuropathic pain in patients with diabetes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors have read and approved the final manuscript. GYX designed and performed experiments, analyzed data, prepared figures and the manuscript. GL and NAL performed behavioral experiments; LYMH designed experiments and prepared the manuscript.