Involvement of peripheral adenosine 5′‐triphosphate and P2X purinoceptor in pain‐related behavior produced by orthotopic melanoma inoculation in mice

Adenosine 5′‐triphosphate (ATP) plays an important role in nociceptive processing. We used a mouse model of skin cancer pain to investigate the role of ATP in cancer pain. Orthotopic inoculation of B16‐BL6 melanoma cells into the hind paw produced spontaneous licking of the tumor‐bearing paw. Intraperitoneal injection of the P2 purinoceptor antagonist suramin suppressed spontaneous licking dose‐dependently. Two P2X purinoceptor antagonists also suppressed spontaneous licking. An intraplantar injection of ATP, which did not induce licking in the healthy paw, increased licking of the tumor‐bearing paw. Spontaneous firing of the tibial nerve was significantly increased in tumor‐bearing mice and was inhibited by suramin. Extracellular concentration of ATP was significantly increased in the tumor‐bearing paw than in the normal paw. ATP is concentrated in the culture medium of melanoma, lung cancer and breast cancer cells, but not fibroblasts. The P2X₃ receptor was expressed in about 40% of peripherin‐positive small and medium‐sized neurons in the dorsal root ganglia. P2X₃‐positive neurons were significantly increased in melanoma‐bearing mice. These results suggest that ATP and P2X, especially P2X₃, receptors are involved in skin cancer pain, due to the increased release of ATP and increased expression of P2X₃ receptors in the sensory neurons.     

Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2-3.4 Hz) in 34% of cutaneous C-fibers adjacent to the tumor (9-17 d after implantation). C-fibers in tumor-bearing mice exhibited a mean decrease in heat threshold of 3.5 +/− 0.10 °C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16-24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor-bearing mice. The development of spontaneous activity and sensitization to heat in C-fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C-fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.     

Localization of ATP-gated P2X2 and P2X3 receptor immunoreactive nerves in rat taste buds.

P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.     

Small fibers involvement in Friedreich's ataxia.

Although the involvement of large myelinated sensory fibers in Friedreich's ataxia (FA) is well documented, an impairment of unmyelinated fibers has not been described. We demonstrate an involvement of cutaneous unmyelinated sensory and autonomic nerve fibers in FA patients. We performed a morphological and functional study of cutaneous nerve fibers in 14 FA patients and in a population of control subjects. We used immunohistochemical techniques and confocal microscopy applied to punch skin biopsies from thigh, distal leg, and fingertip, and compared the density of epidermal nerve fibers (ENFs) with the results of mechanical pain sensation and thermal and tactile thresholds performed on hand dorsum, thigh, distal leg, and foot dorsum. We observed in our patients a statistically significant loss of ENFs, a reduced innervation of sweat glands, arrector pilorum muscles and arterioles, and an impairment of thermal and tactile thresholds and mechanical pain detection.     

Multiple P2Y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury

A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Among the P2Y metabotropic receptors, we reported before that the P2Y12 receptor is upregulated in microglia following nerve injury and involved in the phosphorylation of p38 MAPK, and in the development of pain behavior. In this study, we examined the expression of P2Y6, P2Y13, and P2Y14 receptors in the spinal cord and whether these receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury. We found that spared nerve injury induced a dramatic increase of not only P2Y12, but also P2Y6, 13, and 14 receptor mRNA expression in spinal microglia. The increase continued for at least 2 weeks after injury. To determine whether p38 MAPK can induce the expression of P2Y receptors, we administered intrathecally the p38 MAPK inhibitor SB203580 and found that it significantly suppressed P2Y6, P2Y13, and P2Y14 but not P2Y12 mRNAs. Intrathecal injection of the specific P2Y6 antagonist MRS2578, specific P2Y13 antagonist MRS2211 or P2Y14 antisense LNA, attenuated mechanical pain hypersensitivity. Themixture of three antagonists for P2Y6, 12, and 13 showed a longer suppressive effect on pain behavior than the individual treatments. Our data demonstrate that ATP and other nucleotides may stimulate activated microglia with the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors following nerve injury and these receptors are involved in the development of neuropathic pain. © 2012 Wiley Periodicals, Inc.     

Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice

Dorsal root ganglion (DRG) neurons respond to ATP with transient, persistent or biphasic inward currents. In contrast, the ATP responses in nodose neurons are persistent. These sustained currents are also heterogeneous, with one component being accounted for by P2X2/3 receptors, and the residual response probably mediated by P2X2 receptors, although the direct evidence for this has been lacking. In the present study, we examined the P2X receptors on DRG and nodose neurons from P2X3-deficient (P2X3-/-) mice, using whole cell voltage-clamp recording and immunohistochemistry. We found that all P2X3-/- DRG neurons lacked rapidly desensitizing response to ATP, and both DRG and nodose neurons from P2X3-null mutant mice no longer responded to alpha,beta-methylene ATP (alphabetameATP). In contrast, ATP evoked persistent inward current in 12% of DRG neurons and 84% of nodose neurons from P2X3-/- mice. This retained persistent response to ATP on nodose neurons had an EC50 for ATP of 77 microm, was antagonized by Cibacron blue and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid, potentiated by Zn2+ and acidification, but not enhanced by ivermectin or diinosine pentaphosphate. 2',3'-O-Trinitrophenyl-ATP antagonized this response with an IC50 of 8 microm. All these properties are consistent with those of recombinant P2X2 homomeric receptors. Furthermore, specific P2X2 receptor immunoreactivity detected in wild-type sensory neurons was unaltered in null mutant mice. Therefore, the alphabetameATP-insensitive persistent responses on nodose neurons are likely to be mediated by P2X2 homomers, which contribute to 60% of currents evoked by 100 microm ATP in the wild type.     

The distribution of P2X3 purine receptor subunits in the guinea pig enteric nervous system

Adenosine 5'-triphosphate (ATP) excites 70-90% of enteric neurons through P2X type purine receptors, and is likely to be an enteric neurotransmitter. Recent studies indicate that the P2X2 subunit is expressed by specific subgroups of enteric neurons, and that there are enteric neurons that are responsive to ATP but lack this subunit. In the present work, we have investigated whether the P2X3 subunit is similarly localised to specific subgroups of neurons, and whether these are different from the P2X2 subunit-expressing neurons. The P2X3 subunit was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines, and nerve cells of the submucosal ganglia in the small and large intestines of the guinea pig. All immunoreactivity was absorbed with the P2X3 receptor peptide against which the antiserum was raised. In myenteric ganglia of the ileum, P2X3 receptor immunoreactivity was in calretinin, enkephalin and nitric oxide synthase (NOS)-immunoreactive neurons. In submucosal ganglia, all calretinin-immunoreactive nerve cells were P2X3 receptor immunoreactive. In the submucosal ganglia of the ileum, 13 +/- 3% of neuropeptide Y (NPY)-immunoreactive neurons were also P2X3 receptor immunoreactive, whereas in the distal colon, almost all NPY-expressing nerve cells were P2X3 receptor immunoreactive. The localisation of the P2X3 subunit was largely distinct from that of the P2X2 subunit, although both subunits occur in some NOS neurons, where P2X2 and P2X3 subunits may form heteromeric receptors. Unlike the P2X2 subunit, the P2X3 subunit is not expressed in intrinsic sensory neurons in the ileum. It is concluded that the P2X3 receptor subunit is expressed in specific functional groups of neurons; the major types are excitatory and inhibitory muscle motor neurons, ascending interneurons and cholinergic secretomotor neurons.     

Tumor necrosis factor receptor 1 and 2 proteins are differentially regulated during Wallerian degeneration of mouse sciatic nerve

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF) is involved in injury-induced peripheral nerve pathology and in the generation of neuropathic pain. Here, we investigated local protein levels of the two known TNF receptors, TNF receptor 1 and 2 (TNFR1, TNFR2), on days 0, 1, 3, 7, 14, and 28 after unilateral crush or chronic constriction injury (CCI) of mouse sciatic nerves using enzyme-linked immunoassay. Both receptors were detectable at a low level in nerve homogenates from naive mice. After crush or CCI, TNFR1 increased by 2-fold on days 3 and day 7. Unlike TNFR1, TNFR2 was markedly upregulated already on day 1 after crush or CCI. TNFR2 increased by 7-fold on days 3 and 7, and remained elevated at a lower level until day 28 after both CCI and crush injury. These data indicate that endoneurial TNFR1 and TNFR2 proteins are differentially regulated during Wallerian degeneration.     

P2X2 and P2X3 receptor expression in the gallbladder of the guinea pig

We investigated for the first time, the distribution pattern of P2X2 and P2X3 receptors in the gallbladder of the guinea pig using immunohistochemistry. P2X2 and P2X3 receptor-immunoreactive nerve fibers were observed within the ganglia, in the interganglionic connectives, in the muscularis and in the paravascular plexus. Immunoreactivity for P2X2 and P2X3 was also observed in most neurons in the ganglionated plexus. Double-labeling studies revealed that 58.1% of all P2X2-positive neurons and 54.3% of all P2X3-positive neurons were found to display nitric oxide synthase. Over 90% of the neurons that were immunoreactive for P2X2 and P2X3 receptor were also immunoreactive for calretinin. We also found that 30.5% of P2X2- and 32.6% of P2X3-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide. No P2X2- or P2X3- immunoreactive neurons stained for calcitonin gene-related peptide; a few calcitonin gene-related peptide-immunoreactive nerve fibers also showed immunoreactivity to P2X2 or P2X3 receptors. These results further demonstrate the neurotransmitter diversity of the nerves of the gallbladder and provide an incentive for studies of the actions of these compounds in the gallbladder wall.     

Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after peripheral nerve injury

Neuropathic pain, a debilitating chronic pain following nerve damage, is a reflection of the aberrant functioning of a pathologically altered nervous system. One hallmark is abnormal pain hypersensitivity to innocuous stimuli (tactile allodynia), for which effective therapy is lacking, and the underlying mechanisms of which remain to be determined. Here we show that Lyn, a member of the Src family kinases (SFKs), plays an important role in the pathogenesis of neuropathic pain. Nerve injury, but not peripheral inflammation, increased immunoreactivity for active SFKs that were autophosphorylated in the kinase domain (phospho-SFK-IR) in spinal microglia. In spinally derived microglial cells, we identified Lyn as the predominant SFK among the five members (Src, Fyn, Yes, Lck, and Lyn) known to be expressed in the CNS. Lyn expression in the spinal cord was highly restricted to microglia, and its level was increased after nerve injury. We found that mice lacking lyn (lyn(-/-)) exhibit a striking reduction in the levels of phospho-SFK-IR and tactile allodynia after nerve injury, without any change in basal mechanical sensitivity or inflammatory pain. Importantly, lyn(-/-) mice displayed impaired upregulation of the ionotropic ATP receptor subtype P2X(4) receptors (P2X(4)R) in the spinal cord after nerve injury, which is crucial for tactile allodynia. Microglial cells from lyn(-/-) mice showed a deficit in their ability to increase P2X(4)R expression in response to fibronectin, a factor implicated as a microglial P2X(4)R upregulator in allodynia. Together, our findings suggest that Lyn may be a critical kinase mediating nerve injury-induced P2X(4)R upregulation and neuropathic pain.     

Novel purinergic sensitivity develops in injured sensory axons following sciatic nerve transection in rat

Teased fibers were made from 153 spontaneous A afferents ending in sciatic nerve end neuromas of 3-14 days standing, 21 A afferents from intact sensory endings in the contralateral sciatic nerve, and 50 intact A afferents from the sciatic nerve in intact rats. Ninety-two percent of the injured fibers responded to adenosine 5'-triphosphate (ATP) (i.v.). However, few fibers from the contralateral nerve or nerves from intact animals responded to ATP. P2 receptor antagonist suramin or reactive blue 2 blocked the ATP-induced response in 76% of the fibers tested, whereas the P1 receptor antagonist aminophylline blocked the ATP-evoked effect in only 18% of the fibers tested. Sympathectomy did not affect the ATP-induced effects in injured axons. Close-arterial injection of ATP caused similar results as i.v. injection of ATP. The present study suggests that a novel purinergic sensitivity is developed at the injury site after sciatic nerve transection in rats, which may play a role in neuropathic pain under some conditions such as sympathetic activation.     

Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor

Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP–P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP–P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP–P2X7R in brain damage in RBI, suggesting that inhibition of ATP–P2X7R axis might be a potential strategy for the treatment of patients with RBI.     

P2X receptors: targets for novel analgesics?

The ability of adenosine 5'-triphosphate (ATP) to evoke acute pain has been known for many years, but its role in nociceptive signaling is only now becoming clear. ATP acts via P2X and P2Y receptors, and of particular importance here is the P2X(3) receptor. It is expressed selectively at high levels in nociceptive sensory neurons, where it forms functional receptors on its own and in combination with the P2X(2) receptor. Recent reports using gene knockout methods; antisense oligonucleotide and small, interfering RNA technologies; and a novel, selective P2X(3) antagonist, A-317491, show that P2X(3) receptors are involved in chronic inflammatory and neuropathic pain. The mRNA for other P2X subunits is also found in sensory neurons, and there is evidence for functional P2X(1/5) or P2X(2/6) heteromers in some of these. These data support the possibility that P2X receptors, particularly the P2X(3) subtype, could be targeted in the search for new, effective analgesics.     

Diadenosine pentaphosphate is more potent than ATP at P2X receptors in isolated rat vagus nerve

The effects of diadenosine pentaphosphate (Ap5A), diadenosine tetraphosphate (Ap4A), alpha,beta-methyleneATP (alpha,beta-meATP), and ATP were studied on the excitability of unmyelinated axons in isolated rat vagus nerve by means of a computerized threshold tracking technique. All purinergic compounds produced an increase in excitability, however, only the effects of alpha,beta-meATP and of Ap5A were strongly reduced by 2'- (or 3') -O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a selective blocker for P2X1, P2X3, and heteromeric P2X2/3 receptors. The rank order of potency for TNP-ATP-sensitive excitation was determined as follows (30 microM each): alpha,beta-meATP >Ap5A > Ap4A = ATP. These data suggest that Ap5A might be an important naturally occurring agonist for P2X receptors at the axonal membrane of unmyelinated, including nociceptive, nerve fibres.     

Mechanisms of ATP- and glutamate-mediated calcium signaling in white matter astrocytes

Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura-2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA- and NMDA-type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the "gliotransmitter" ATP by a mechanism involving P2X7 receptors; propagation of the glutamate-mediated Ca2+ signal was significantly reduced in P2X7 knock-out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP-negative glia. Our data provide a model of multiphase axon-glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia.     

Changes in circadian rhythm for mRNA expression of melatonin 1A and 1B receptors in the hypothalamus under a neuropathic pain‐like state

Several clinical reports on neuropathic pain of various etiologies have shown that it significantly interferes with sleep. Inadequate sleep due to neuropathic pain may contribute to the stressful negative consequences of living with pain. It is generally recognized that melatonin (MT) system in the hypothalmus is crusial for circadian rhythm and sleep‐wake transition. However, little, if any, is known about whether neuropathic pain could affect the MT system associated with sleep disturbance. In this study, we investigated the possible changes in circadian rhythm for the expression of MT receptors, especially MT1A and MT1B receptors, in the hypothalamus of mice with sciatic nerve ligation. The samples for real‐time RT‐PCR assay were prepared at 8:00, 14:00, 20:00, and 2:00 on day 7 after sciatic nerve ligation or sham operation. The mRNA expression of MT1A and MT1B receptors at 2:00 in sciatic nerve‐ligated mice, which exhibited thermal hyperalgesia along with an increase in wakefulness and a decrease in nonrapid eye movement sleep, was significantly greater than those in sham‐operated mice, whereas the levels of both MT1A and MT1B receptors at 8:00 in sciatic nerve‐ligated mice were significantly lower than those in sham‐operated mice. These findings suggest that neuropathic pain‐like stimuli lead to sleep disturbance in parallel with changes in circadian rhythm for mRNA expression of MT 1A and 1B receptors in the hypothalamus of mice. Synapse 68:153–158, 2014. © 2014 Wiley Periodicals, Inc.     

A skin blister method to study epidermal nerves in peripheral nerve disease

Skin is a reservoir of sensory and autonomic nerve fibers that are potential indicators of peripheral nerve disease. Biopsies of skin have shown that sensory nerves in the most superficial layer of skin, the epidermal nerve fibers (ENFs), are reduced in patients with polyneuropathy. This report describes a minimally invasive skin blister method to isolate, image, and obtain quantitative analysis of ENFs. Blisters are made by applying a suction capsule to skin. The epidermal roof of the blister is excised, immunostained, whole mounted, and analyzed for ENF number and distribution. A reduction in number and abnormal distribution of ENFs are early indicators of peripheral nerve disease. Illustrations of skin blister and skin biopsy specimens from patients with different types of peripheral nerve disorders are included. These patients were chosen because their findings demonstrate the complementary information obtained by the blister and biopsy methods and the potential of the blister procedure to evaluate single nerve lesions and polyneuropathy and to follow the progress of ENF degeneration and regeneration.