Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

Peripheral transient receptor potential vanilloid type 4 hypersensitivity contributes to chronic sickle cell disease pain.

ABSTRACT: Debilitating pain affects the lives of patients with sickle cell disease (SCD). Current pain treatment for patients with SCD fail to completely resolve acute or chronic SCD pain. Previous research indicates that the cation channel transient receptor potential vanilloid type 4 (TRPV4) mediates peripheral hypersensitivity in various inflammatory and neuropathic pain conditions that may share similar pathophysiology with SCD, but this channel's role in chronic SCD pain remains unknown. Thus, the current experiments examined whether TRPV4 regulates hyperalgesia in transgenic mouse models of SCD. Acute blockade of TRPV4 alleviated evoked behavioral hypersensitivity to punctate, but not dynamic, mechanical stimuli in mice with SCD. TRPV4 blockade also reduced the mechanical sensitivity of small, but not large, dorsal root ganglia neurons from mice with SCD. Furthermore, keratinocytes from mice with SCD showed sensitized TRPV4-dependent calcium responses. These results shed new light on the role of TRPV4 in SCD chronic pain and are the first to suggest a role for epidermal keratinocytes in the heightened sensitivity observed in SCD.

Use the force, fluke: Ligand-independent gating of Schistosoma mansoni ion channel TRPMPZQ.

The parasitic flatworm ion channel, TRPMPZQ, is a non-selective cation channel that mediates Ca2+ entry and membrane depolarization when activated by the anthelmintic drug, praziquantel (PZQ). TRPMPZQ is conserved in all platyhelminth genomes scrutinized to date, with the sensitivity of TRPMPZQ in any particular flatworm correlating with the overall sensitivity of the worm to PZQ. Conservation of this channel suggests it plays a role in flatworm physiology, but the nature of the endogenous cues that activate this channel are currently unknown. Here, we demonstrate that TRPMPZQ is activated in a ligand-independent manner by membrane stretch, with the electrophysiological signature of channel opening events being identical whether evoked by negative pressure, or by PZQ. TRPMPZQ is therefore a multimodal ion channel gated by both physical and chemical cues. The mechanosensitivity of TRPMPZQ is one route for endogenous activation of this ion channel that holds relevance for schistosome physiology given the persistent pressures and mechanical cues experienced throughout the parasite life cycle.

Fabry disease pain: patient and preclinical parallels.

ABSTRACT: Severe neuropathic pain is a hallmark of Fabry disease, a genetic disorder caused by a deficiency in lysosomal α-galactosidase A. Pain experienced by these patients significantly impacts their quality of life and ability to perform everyday tasks. Patients with Fabry disease suffer from peripheral neuropathy, sensory abnormalities, acute pain crises, and lifelong ongoing pain. Although treatment of pain through medication and enzyme replacement therapy exists, pain persists in many of these patients. Some has been learned in the past decades regarding clinical manifestations of pain in Fabry disease and the pathological effects of α-galactosidase A insufficiency in neurons. Still, it is unclear how pain and sensory abnormalities arise in patients with Fabry disease and how these can be targeted with therapeutics. Our knowledge is limited in part due to the lack of adequate preclinical models to study the disease. This review will detail the types of pain, sensory abnormalities, influence of demographics on pain, and current strategies to treat pain experienced by patients with Fabry disease. In addition, we discuss the current knowledge of Fabry pain pathogenesis and which aspects of the disease preclinical models accurately recapitulate. Understanding the commonalities and divergences between humans and preclinical models can be used to further interrogate mechanisms causing the pain and sensory abnormalities as well as advance development of the next generation of therapeutics to treat pain in patients with Fabry disease.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

NOD-like receptor protein 3 inflammasome drives postoperative mechanical pain in a sex-dependent manner.

Postoperative pain management continues to be suboptimal because of the lack of effective nonopioid therapies and absence of understanding of sex-driven differences. Here, we asked how the NLRP3 inflammasome contributes to postoperative pain. Inflammasomes are mediators of the innate immune system that are responsible for activation and secretion of IL-1ß upon stimulation by specific molecular signals. Peripheral IL-1ß is known to contribute to the mechanical sensitization induced by surgical incision. However, it is not known which inflammasome mediates the IL-1ß release after surgical incision. Among the 9 known inflammasomes, the NLRP3 inflammasome is ideally positioned to drive postoperative pain through IL-1ß production because NLRP3 can be activated by factors that are released by incision. Here, we show that male mice that lack NLRP3 (NLRP3) recover from surgery-induced behavioral and neuronal mechanical sensitization faster and display less surgical site inflammation than mice expressing NLRP3 (wild-type). By contrast, female NLRP3 mice exhibit minimal attenuation of the postoperative mechanical hypersensitivity and no change in postoperative inflammation compared with wild-type controls. Sensory neuron-specific deletion of NLRP3 revealed that in males, NLRP3 expressed in non-neuronal cells and potentially sensory neurons drives postoperative pain. However, in females, only the NLRP3 that may be expressed in sensory neurons contributes to postoperative pain where the non-neuronal cell contribution is NLRP3 independent. This is the first evidence of a key role for NLRP3 in postoperative pain and reveals immune-mediated sex differences in postoperative pain.

Chemokine (c-c motif) receptor 2 mediates mechanical and cold hypersensitivity in sickle cell disease mice.

Approximately one-third of individuals with sickle cell disease (SCD) develop chronic pain. This debilitating pain is inadequately treated because the underlying mechanisms driving the pain are poorly understood. In addition to persistent pain, patients with SCD are also in a tonically proinflammatory state. Previous studies have revealed that there are elevated plasma levels of many inflammatory mediators including chemokine (c-c motif) ligand 2 (CCL2) in individuals with SCD. Using a transgenic mouse model of SCD, we investigated the contributions of CCL2 signaling to SCD-related pain. Inhibition of chemokine receptor 2 (CCR2), but not CCR4, alleviated the behavioral mechanical and cold hypersensitivity in SCD. Furthermore, acute CCR2 blockade reversed both the behavioral and the in vitro responsiveness of sensory neurons to an agonist of TRPV1, a neuronal ion channel previously implicated in SCD pain. These results provide insight into the immune-mediated regulation of hypersensitivity in SCD and could inform future development of analgesics or therapeutic measures to prevent chronic pain.

Neuropathic pain in a Fabry disease rat model.

Fabry disease, the most common lysosomal storage disease, affects multiple organs and results in a shortened life span. This disease is caused by a deficiency of the lysosomal enzyme α-galactosidase A, which leads to glycosphingolipid accumulation in many cell types. Neuropathic pain is an early and severely debilitating symptom in patients with Fabry disease, but the cellular and molecular mechanisms that cause the pain are unknown. We generated a rat model of Fabry disease, the first nonmouse model to our knowledge. Fabry rats had substantial serum and tissue accumulation of α-galactosyl glycosphingolipids and had pronounced mechanical pain behavior. Additionally, Fabry rat dorsal root ganglia displayed global N-glycan alterations, sensory neurons were laden with inclusions, and sensory neuron somata exhibited prominent sensitization to mechanical force. We found that the cation channel transient receptor potential ankyrin 1 (TRPA1) is sensitized in Fabry rat sensory neurons and that TRPA1 antagonism reversed the behavioral mechanical sensitization. This study points toward TRPA1 as a potentially novel target to treat the pain experienced by patients with Fabry disease.

Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling.

The first point of our body's contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions.

Mechanosensory and ATP Release Deficits following Keratin14-Cre-Mediated TRPA1 Deletion Despite Absence of TRPA1 in Murine Keratinocytes.

Keratinocytes are the first cells that come into direct contact with external tactile stimuli; however, their role in touch transduction in vivo is not clear. The ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) is essential for some mechanically-gated currents in sensory neurons, amplifies mechanical responses after inflammation, and has been reported to be expressed in human and mouse skin. Other reports have not detected Trpa1 mRNA transcripts in human or mouse epidermis. Therefore, we set out to determine whether selective deletion of Trpa1 from keratinocytes would impact mechanosensation. We generated K14Cre-Trpa1fl/fl mice lacking TRPA1 in K14-expressing cells, including keratinocytes. Surprisingly, Trpa1 transcripts were very poorly detected in epidermis of these mice or in controls, and detection was minimal enough to preclude observation of Trpa1 mRNA knockdown in the K14Cre-Trpa1fl/fl mice. Unexpectedly, these K14Cre-Trpa1fl/fl mice nonetheless exhibited a pronounced deficit in mechanosensitivity at the behavioral and primary afferent levels, and decreased mechanically-evoked ATP release from skin. Overall, while these data suggest that the intended targeted deletion of Trpa1 from keratin 14-expressing cells of the epidermis induces functional deficits in mechanotransduction and ATP release, these deficits are in fact likely due to factors other than reduction of Trpa1 expression in adult mouse keratinocytes because they express very little, if any, Trpa1.

Cold hypersensitivity increases with age in mice with sickle cell disease.

Sickle cell disease (SCD) is associated with acute vaso-occlusive crises that trigger painful episodes and frequently involves ongoing, chronic pain. In addition, both humans and mice with SCD experience heightened cold sensitivity. However, studies have not addressed the mechanism(s) underlying the cold sensitization or its progression with age. Here we measured thermotaxis behavior in young and aged mice with severe SCD. Sickle mice had a marked increase in cold sensitivity measured by a cold preference test. Furthermore, cold hypersensitivity worsened with advanced age. We assessed whether enhanced peripheral input contributes to the chronic cold pain behavior by recording from C fibers, many of which are cold sensitive, in skin-nerve preparations. We observed that C fibers from sickle mice displayed a shift to warmer (more sensitive) cold detection thresholds. To address mechanisms underlying the cold sensitization in primary afferent neurons, we quantified mRNA expression levels for ion channels thought to be involved in cold detection. These included the transient receptor potential melastatin 8 (Trpm8) and transient receptor potential ankyrin 1 (Trpa1) channels, as well as the 2-pore domain potassium channels, TREK-1 (Kcnk2), TREK-2 (Kcnk10), and TRAAK (Kcnk4). Surprisingly, transcript expression levels of all of these channels were comparable between sickle and control mice. We further examined transcript expression of 83 additional pain-related genes, and found increased mRNA levels for endothelin 1 and tachykinin receptor 1. These factors may contribute to hypersensitivity in sickle mice at both the afferent and behavioral levels.

Mechanical sensitization of cutaneous sensory fibers in the spared nerve injury mouse model.

BACKGROUND: The spared nerve injury (SNI) model of neuropathic pain produces robust and reproducible behavioral mechanical hypersensitivity. Although this rodent model of neuropathic pain has been well established and widely used, peripheral mechanisms underlying this phenotype remain incompletely understood. Here we investigated the role of cutaneous sensory fibers in the maintenance of mechanical hyperalgesia in mice post-SNI. FINDINGS: SNI produced robust, long-lasting behavioral mechanical hypersensitivity compared to sham and naïve controls beginning by post-operative day (POD) 1 and continuing through at least POD 180. We performed teased fiber recordings on single cutaneous fibers from the spared sural nerve using ex vivo skin-nerve preparations. Recordings were made between POD 16-42 after SNI or sham surgery. Aδ-mechanoreceptors (AM) and C fibers, many of which are nociceptors, from SNI mice fired significantly more action potentials in response to suprathreshold mechanical stimulation than did fibers from either sham or naïve control mice. However, there was no increase in spontaneous activity. CONCLUSIONS: To our knowledge, this is the first study evaluating the contribution of primary afferent fibers in the SNI model. These data suggest that enhanced suprathreshold firing in AM and C fibers may play a role in the marked, persistent mechanical hypersensitivity observed in this model. These results may provide insight into mechanisms underlying neuropathic pain in humans.

Spinal muscular atrophy astrocytes exhibit abnormal calcium regulation and reduced growth factor production.

Spinal muscular atrophy (SMA) is a genetic disorder caused by the deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Although motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA-induced pluripotent stem cells exhibit morphological and cellular changes indicative of activation before overt motor neuron loss. Furthermore, our in vitro studies show mis-regulation of basal calcium and decreased response to adenosine triphosphate stimulation indicating abnormal astrocyte function. Together, for the first time, these data show early disruptions in astrocytes that may contribute to SMA disease pathology.

Failure of action potential propagation in sensory neurons: mechanisms and loss of afferent filtering in C-type units after painful nerve injury.

The T-junction of sensory neurons in the dorsal root ganglion (DRG) is a potential impediment to action potential (AP) propagation towards the CNS. Using intracellular recordings from rat DRG neuronal somata during stimulation of the dorsal root, we determined that the maximal rate at which all of 20 APs in a train could successfully transit the T-junction (following frequency) was lowest in C-type units, followed by A-type units with inflected descending limbs of the AP, and highest in A-type units without inflections. In C-type units, following frequency was slower than the rate at which AP trains could be produced in either dorsal root axonal segments or in the soma alone, indicating that the T-junction is a site that acts as a low-pass filter for AP propagation. Following frequency was slower for a train of 20 APs than for two, indicating that a cumulative process leads to propagation failure. Propagation failure was accompanied by diminished somatic membrane input resistance, and was enhanced when Ca(2+)-sensitive K(+) currents were augmented or when Ca(2+)-sensitive Cl(-) currents were blocked. After peripheral nerve injury, following frequencies were increased in axotomized C-type neurons and decreased in axotomized non-inflected A-type neurons. These findings reveal that the T-junction in sensory neurons is a regulator of afferent impulse traffic. Diminished filtering of AP trains at the T-junction of C-type neurons with axotomized peripheral processes could enhance the transmission of activity that is ectopically triggered in a neuroma or the neuronal soma, possibly contributing to pain generation.

Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception.

BACKGROUND: The Transient Receptor Potential (TRP) ion channel TRPA1 is a key player in pain pathways. Irritant chemicals activate ion channel TRPA1 via covalent modification of N-terminal cysteines. We and others have shown that 15-Deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) similarly activates TRPA1 and causes channel-dependent nociception. Paradoxically, 15d-PGJ2 can also be anti-nociceptive in several pain models. Here we hypothesized that activation and subsequent desensitization of TRPA1 in dorsal root ganglion (DRG) neurons underlies the anti-nociceptive property of 15d-PGJ2. To investigate this, we utilized a battery of behavioral assays and intracellular Ca2+ imaging in DRG neurons to test if pre-treatment with 15d-PGJ2 inhibited TRPA1 to subsequent stimulation. RESULTS: Intraplantar pre-injection of 15d-PGJ2, in contrast to mustard oil (AITC), attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not capsaicin (CAP). Intraplantar 15d-PGJ2-administered after the induction of inflammation-reduced mechanical hypersensitivity in the Complete Freund's Adjuvant (CFA) model for up to 2 h post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity is dependent on TRPA1, as this effect was absent in TRPA1 knockout mice. Ca2+ imaging studies of DRG neurons demonstrated that 15d-PGJ2 pre-exposure reduced the magnitude and number of neuronal responses to AITC, but not CAP. AITC responses were not reduced when neurons were pre-exposed to 15d-PGJ2 combined with HC-030031 (TRPA1 antagonist), demonstrating that inhibitory effects of 15d-PGJ2 depend on TRPA1 activation. Single daily doses of 15d-PGJ2, administered during the course of 4 days in the CFA model, effectively reversed mechanical hypersensitivity without apparent tolerance or toxicity. CONCLUSIONS: Taken together, our data support the hypothesis that 15d-PGJ2 induces activation followed by persistent inhibition of TRPA1 channels in DRG sensory neurons in vitro and in vivo. Moreover, we demonstrate novel evidence that 15d-PGJ2 is analgesic in mouse models of pain via a TRPA1-dependent mechanism. Collectively, our studies support that TRPA1 agonists may be useful as pain therapeutics.

The dynamic TRPA1 channel: a suitable pharmacological pain target?

Acute pain detection is vital to navigate and survive in one's environment. Protection and preservation occur because primary afferent nociceptors transduce adverse environmental stimuli into electrical impulses that are transmitted to and interpreted within high levels of the central nervous system. Therefore, it is critical that the molecular mechanisms that convert noxious information into neural signals be identified, and their specific functional roles delineated in both acute and chronic pain settings. The Transient Receptor Potential (TRP) channel family member TRP ankyrin 1 (TRPA1) is an excellent candidate molecule to explore and intricately understand how single channel properties can tailor behavioral nociceptive responses. TRPA1 appears to dynamically respond to an amazingly wide range of diverse stimuli that include apparently unrelated modalities such as mechanical, chemical and thermal stimuli that activate somatosensory neurons. How such dissimilar stimuli activate TRPA1, yet result in modality-specific signals to the CNS is unclear. Furthermore, TRPA1 is also involved in persistent to chronic painful states such as inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis and emphysema. Yet how TRPA1's role changes from an acute sensor of physical stimuli to its contribution to these diseases that are concomitant with implacable, chronic pain is unknown. TRPA1's involvement in the nociceptive machinery that relays the adverse stimuli during painful disease states is of considerable interest for drug delivery and design by many pharmaceutical entities. In this review, we will assess the current knowledge base of TRPA1 in acute nociception and persistent inflammatory pain states, and explore its potential as a therapeutic pharmacological target in chronic pervasive conditions such neuropathic pain, persistent inflammation and diabetes.

Pain tests provoke modality-specific cardiovascular responses in awake, unrestrained rats.

Nociception modulates heart rate (HR) and mean arterial pressure (MAP), suggesting their use of HR and MAP as indicators of pain in animals. We explored this with telemetric recording in unrestrained control and neuropathic (spinal nerve ligation) rats. Plantar stimulation was performed emulating techniques commonly used to measure pain, specifically brush stroke, von Frey fiber application, noxious pin stimulation, acetone for cooling, and radiant heating, while recording MAP, HR, and specific evoked somatomotor behaviors (none; simple withdrawal; or sustained lifting, shaking, and grooming representing hyperalgesia). Pin produced elevations in both HR and MAP, and greater responses accompanied hyperalgesia behavior compared to simple withdrawal. Von Frey stimulation depressed MAP, and increased HR only when stimulation produced hyperalgesia behavior, suggesting that minimal nociception occurs without this behavior. Brush increased MAP even when no movement was evoked. Cold elevated both HR and MAP whether or not there was withdrawal, but MAP increased more when withdrawal was triggered. Heating, consistently depressed HR and MAP, independent of behavior. Other than a greater HR response to pin in animals made hyperalgesic by injury, cardiovascular events evoked by stimulation did not differ between control and neuropathic animals. We conclude that (a) thermoregulation rather than pain may dominate responses to heat and cooling stimuli; (b) brush and cooling stimuli may be perceived and produce cardiovascular activation without nocifensive withdrawal; (c) sensations that produce hyperalgesia behavior are accompanied by greater cardiovascular activation than those producing simple withdrawal; and (d) von Frey stimulation lacks cardiovascular evidence of nociception except when hyperalgesia behavior is evoked.

Constitutive activity at the cannabinoid CB1 receptor is required for behavioral response to noxious chemical stimulation of TRPV1: antinociceptive actions of CB1 inverse agonists.

The potential modulation of TRPV1 nociceptive activity by the CB(1) receptor was investigated here using CB(1) wild-type (WT) and knock-out (KO) mice as well as selective CB(1) inverse agonists. No significant differences were detected in baseline thermal thresholds of ICR, CB(1)WT or CB(1)KO mice. Intraplantar capsaicin produced dose- and time-related paw flinch responses in ICR and CB(1)WT mice and induced plasma extravasation yet minimal responses were seen in CB(1)KO animals with no apparent differences in TRPV1 channel expression. Capsaicin-evoked CGRP release from spinal cord tissue and capsaicin-evoked action potentials on isolated skin-nerve preparation were significantly decreased in CB(1)KO mice. Pretreatment with intraplantar galanin and bradykinin, compounds known to sensitize TRPV1 receptors, restored capsaicin-induced flinching in CB(1)KO mice. The possibility that constitutive activity at the CB(1) receptor is required to maintain the TRPV1 receptor in a "sensitized" state was tested using CB(1) inverse agonists. The CB(1) inverse agonists elicited concentration-related inhibition of capsaicin-induced calcium influx in F-11 cells and produced dose-related inhibition of capsaicin-induced flinching in ICR mice. These data suggest that constitutive activity at the CB(1) receptor maintains the TRPV1 channel in a sensitized state responsive to noxious chemical stimuli. Treatment with CB(1) inverse agonists may promote desensitization of the channel resulting in antinociceptive actions against chemical stimulus modalities. These studies propose possible therapeutic exploitation of a novel mechanism providing pain relief by CB(1) inverse agonists.

Chemical interactions between fibrosarcoma cancer cells and sensory neurons contribute to cancer pain.

In an experimental model of cancer pain, the hyperalgesia that occurs with osteolytic tumor growth is associated with the sensitization of nociceptors. We examined functional and molecular changes in small-diameter dorsal root ganglion (DRG) neurons to determine cellular mechanisms underlying this sensitization. The occurrence of a Ca2+ transient in response to either KCl (25 mM) or capsaicin (500 nM) increased in small neurons isolated from murine L3-L6 DRGs ipsilateral to fibrosarcoma cell tumors. The increased responses were associated with increased mRNA levels for the Ca2+ channel subunit alpha2delta1 and TRPV1 receptor. Pretreatment with gabapentin, an inhibitor of the alpha2delta1 subunit, blocked the increased response to KCl in vitro and the mechanical hyperalgesia in tumor-bearing mice in vivo. Similar increases in neuronal responsiveness occurred when DRG neurons from naive mice and fibrosarcoma cells were cocultured for 48 h. The CC chemokine ligand 2 (CCL2) may contribute to the tumor cell-induced sensitization because CCL2 immunoreactivity was present in tumors, high levels of CCL2 peptide were present in microperfusates from tumors, and treatment of DRG neurons in vitro with CCL2 increased the amount of mRNA for the alpha2delta1 subunit. Together, our data provide strong evidence that the chemical mediator CCL2 is released from tumor cells and evokes phenotypic changes in sensory neurons, including increases in voltage-gated Ca2+ channels that likely underlie the mechanical hyperalgesia in the fibrosarcoma cancer model. More broadly, this study provides a novel in vitro model to resolve the cellular and molecular mechanisms by which tumor cells drive functional changes in nociceptors.

The P2Y agonist UTP activates cutaneous afferent fibers.

The majority of adenosine triphosphate (ATP)-induced nociceptive transduction and pain has been attributed to ionotropic P2X3 receptors. Metabotropic P2Y receptors, some of which bind pyrimidines as well as purines, have received little attention. Here we have examined the ability of P2Y receptor signaling to evoke action potential firing in functionally identified afferent fibers using the skin nerve preparation from adult mouse. The P2Y2/P2Y4 ligand UTP activated sustained action potential firing in 54% of C fibers in a concentration-dependent manner. The effect was specific for P2Y2/P2Y4 receptors, as the P2Y6 ligand UDP never activated C fibers. In comparison to C fibers, few thinly myelinated A-mechanoreceptors (AM) (12%) were activated by UTP. The majority (70-80%) of the UTP-sensitive C and Adelta fibers responded to the algogen capsaicin with a barrage of action potentials, whereas the UTP-insensitive fibers were largely unresponsive to capsaicin. Furthermore, 86% of the UTP-sensitive C fibers and 100% of the UTP-sensitive AM fibers also responded to the P2X agonist alpha,beta-methylene ATP, indicating that P2Y and P2X receptors are widely co-expressed. Surprisingly, a significant proportion (20-40%) of low threshold slowly and rapidly adapting Abeta fibers were also activated by UTP and alpha,beta-methylene ATP. These data indicate that P2Y receptors on the terminals of capsaicin-sensitive cutaneous sensory neurons effectively evoke nociceptive transmission, and support the hypothesis that UTP may be an endogenous nociceptive messenger. Furthermore, P2Y signaling may contribute to mechanotransduction in low threshold Abeta fibers under normal or pathological conditions.