Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

Ca++/CaMKII switches nociceptor-sensitizing stimuli into desensitizing stimuli.

Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3-induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second 'sensitizing' stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII-dependent manner. This mechanism was also observed by in vivo C-fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.

Shared mechanisms for opioid tolerance and a transition to chronic pain.

Clinical pain conditions may remain responsive to opiate analgesics for extended periods, but such persistent acute pain can undergo a transition to an opiate-resistant chronic pain state that becomes a much more serious clinical problem. To test the hypothesis that cellular mechanisms of chronic pain in the primary afferent also contribute to the development of opiate resistance, we used a recently developed model of the transition of from acute to chronic pain, hyperalgesic priming. Repeated intradermal administration of the potent and highly selective mu-opioid agonist, [d-Ala(2),N-MePhe(4),gly-ol]-enkephalin (DAMGO), to produce tolerance for its inhibition of prostaglandin E(2) hyperalgesia, simultaneously produced hyperalgesic priming. Conversely, injection of an inflammogen, carrageenan, used to produce priming produced DAMGO tolerance. Both effects were prevented by inhibition of protein kinase Cepsilon (PKCepsilon). Carrageenan also induced opioid dependence, manifest as mu-opioid receptor antagonist (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2))-induced hyperalgesia that, like priming, was PKCepsilon and G(i) dependent. These findings suggest that the transition from acute to chronic pain, and development of mu-opioid receptor tolerance and dependence may be linked by common cellular mechanisms in the primary afferent.

Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia.

Although the transient receptor potential vanilloid 4 (TRPV4) has been implicated in the process of osmomechanical transduction, it appears to make little contribution to the normal somatosensory detection of mechanical stimuli. However, evidence suggests that it may play an important role in mechanical hyperalgesia. In the present study, we examined the common requirement for TRPV4 in mechanical hyperalgesia associated with diverse pain models and investigated whether the very close association observed between TRPV4 and mechanical hyperalgesia, regardless of etiology, reflects a close functional connection of TRPV4 with other molecules implicated in mechanical transduction. In models of painful peripheral neuropathy associated with vincristine chemotherapy, alcoholism, diabetes, and human immunodeficiency virus/acquired immune deficiency syndrome therapy, mechanical hyperalgesia was markedly reduced by spinal intrathecal administration of oligodeoxynucleotides antisense to TRPV4. Similarly, mechanical hyperalgesia induced by paclitaxel, vincristine, or diabetes was strongly reduced in TRPV4 knock-out mice. We also show that alpha2beta1 integrin and Src tyrosine kinase, which have been implicated in mechanical transduction, are important for the development of mechanical hyperalgesia, and that their contribution requires TRPV4. Furthermore, we establish a direct interaction between TRPV4, alpha2 integrin, and the Src tyrosine kinase Lyn in sensory neurons. We suggest that TRPV4 plays a role in mechanotransduction, as a component of a molecular complex that functions only in the setting of inflammation or nerve injury.

GDNF hyperalgesia is mediated by PLCgamma, MAPK/ERK, PI3K, CDK5 and Src family kinase signaling and dependent on the IB4-binding protein versican.

The function of the isolectin B4 (IB4+)-binding and GDNF-dependent Ret (Ret+)-expressing non-peptidergic subpopulation of nociceptors remain poorly understood. We demonstrate that acute administration of GDNF sensitizes nociceptors and produces mechanical hyperalgesia in the rat. Intrathecal IB4-saporin, a selective toxin for IB4+/Ret+-nociceptors, attenuates GDNF but not NGF hyperalgesia. Conversely, intrathecal antisense to Trk A attenuated NGF but not GDNF hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides targeting mRNA for versican, the molecule that renders the Ret-expressing nociceptors IB4-positive (+), also attenuated GDNF but not NGF hyperalgesia, as did ADAMTS-4, a matrix metalloprotease known to degrade versican. Finally, inhibitors for all five signaling pathways known to be activated by GDNF at GFRa1/Ret: PLCc, CDK5, PI3K,MAPK/ERK and Src family kinases, attenuated GDNF hyperalgesia. Our results demonstrate a role of the non-peptidergic nociceptors in pain produced by the neurotrophin GDNF and suggest that the IB4-binding protein versican functions in the expression of this phenotype.

Oxaliplatin acts on IB4-positive nociceptors to induce an oxidative stress-dependent acute painful peripheral neuropathy.

UNLABELLED: The toxicity profile of oxaliplatin, a platinum derivative currently used in the treatment of colorectal cancer, differs from those of the other platinum compounds, cisplatin and carboplatin. Oxaliplatin treatment induces an acute neurotoxicity characterized by a rapid onset of cold-induced distal dysesthesia and a chronic sensory peripheral neuropathy. A single intravenous dose of oxaliplatin produced a dose-dependent mechanical hyperalgesia and heat and cold allodynia; repeated administration intensified symptoms. A single intradermal dose of oxaliplatin produced a dose-dependent mechanical hyperalgesia. A single dose intravenous oxaliplatin also lowered thresholds and increased responses of C-fiber nociceptors to mechanical stimulation, confirming a peripheral site of action. Whereas peripheral administration of inhibitors of second messengers implicated in models of other painful peripheral neuropathies (PKA, PKC, NO, Ca(2+), and caspase) had no effect; both systemic and local administration of antioxidants (acetyl-L-carnitine, alpha-lipoic acid or vitamin C), all markedly inhibited oxaliplatin-induced hyperalgesia. Intrathecal administration of the neurotoxin for IB4-positive nociceptors, IB4-saporin, markedly attenuated IB4 staining in the dorsal horn of the spinal cord and completely prevented oxaliplatin-induced hyperalgesia. We suggest that oxaliplatin acts on IB4 (+)-nociceptors to induce oxidative stress-dependent acute peripheral sensory neuropathy. PERSPECTIVE: Many drugs used to treat cancer produce pain as their dose-limiting side effect. We used a model of this pain syndrome induced by oxaliplatin to demonstrate that pain is produced by action on a subset of nociceptors, the IB4-positive DRG neurons. This information could help define cellular targets against which protective therapies could be developed.

A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators.

The transient receptor potential vanilloid 4 (TRPV4) is a primary afferent transducer that plays a crucial role in neuropathic hyperalgesia for osmotic and mechanical stimuli, as well as in inflammatory mediator-induced hyperalgesia for osmotic stimuli. In view of the clinical importance of mechanical hyperalgesia in inflammatory states, the present study investigated the role of TRPV4 in mechanical hyperalgesia induced by inflammatory mediators and the second-messenger pathways involved. Intradermal injection of either the inflammogen carrageenan or a soup of inflammatory mediators enhanced the nocifensive paw-withdrawal reflex elicited by hypotonic or mechanical stimuli in rat. Spinal administration of TRPV4 antisense oligodeoxynucleotide blocked the enhancement without altering baseline nociceptive threshold. Similarly, in TRPV4(-/-) knock-out mice, inflammatory soup failed to induce any significant mechanical or osmotic hyperalgesia. In vitro investigation showed that inflammatory mediators engage the TRPV4-mediated mechanism of sensitization by direct action on dissociated primary afferent neurons. Additional behavioral observations suggested that multiple mediators are necessary to achieve sufficient activation of the cAMP pathway to engage the TRPV4-dependent mechanism of hyperalgesia. In addition, direct activation of protein kinase A or protein kinase C epsilon, two pathways that mediate inflammation-induced mechanical hyperalgesia, also induced hyperalgesia for both hypotonic and mechanical stimuli that was decreased by TRPV4 antisense and absent in TRPV4(-/-) mice. We conclude that TRPV4 plays a crucial role in the mechanical hyperalgesia that is generated by the concerted action of inflammatory mediators present in inflamed tissues.

Mitochondrial electron transport in models of neuropathic and inflammatory pain.

Although peripheral nerve function is strongly dependent on energy stores, the role of the mitochondrial electron transport chain, which drives ATP synthesis, in peripheral pain mechanisms, has not been examined. In models of HIV/AIDS therapy (dideoxycytidine), cancer chemotherapy (vincristine), and diabetes (streptozotocin)-induced neuropathy, inhibitors of mitochondrial electron transport chain complexes I, II, III, IV, and V significantly attenuated neuropathic pain-related behavior in rats. While inhibitors of all five complexes also attenuated tumor necrosis factor alpha-induced hyperalgesia, they had no effect on hyperalgesia induced by prostaglandin E2 and epinephrine. Two competitive inhibitors of ATP-dependent mechanisms, adenosine 5'-(beta,gamma-imido) triphosphate and P1,P4-di(adenosine-5') tetraphosphate, attenuated dideoxycytidine, vincristine, and streptozotocin-induced hyperalgesia. Neither of these inhibitors, however, affected tumor necrosis factor alpha, prostaglandin E2 or epinephrine hyperalgesia. These experiments demonstrate a role of the mitochondrial electron transport chain in neuropathic and some forms of inflammatory pain. The contribution of the mitochondrial electron transport chain in neuropathic pain is ATP dependent.

Hyperalgesic priming in the rat demonstrates marked sexual dimorphism.

In male rats, carrageenan (CAR)-induced inflammation or exposure to a selective protein kinase C epsilon (PKC epsilon ) agonist (psi epsilon RACK) produces prolongation of the hyperalgesia induced by a subsequent exposure to an inflammatory mediator, a phenomenon referred to as hyperalgesic priming. Since many chronic inflammatory conditions are sexually dimorphic, we tested the hypothesis that hyperalgesic priming is sexually dimorphic. Prior injection of CAR or psi epsilon RACK produced a prolongation of the hyperalgesia induced by a subsequent injection of prostaglandin E(2), from less than 3 h to greater than 24 h, but only in male rats. In ovariectomized female rats priming with CAR and psi epsilon RACK produced hyperalgesic priming effects similar to that observed in the male rat, and this effect was reversed by estrogen replacement. While gonadectomy in males had no effect on CAR and psi epsilon RACK induced hyperalgesic priming, female phenotype was observed following implantation of estrogen in males. Thus, mechanisms mediating the development of hyperalgesic priming produced by inflammation are suppressed by estrogen. This regulation of priming by estrogen appears to occur at or downstream of the activation of PKC epsilon.

Novel mechanism of enhanced nociception in a model of AIDS therapy-induced painful peripheral neuropathy in the rat.

To elucidate the underlying mechanisms involved in AIDS therapy-induced peripheral neuropathy, we have developed a model of nucleoside analog reverse transcriptase inhibitor-induced painful peripheral neuropathy in the rat, using 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI) and 2',3'-didehydro-3'-deoxythymidine (d4T), AIDS chemotherapeutic drugs that are also components of AIDS highly active anti-retroviral therapy. Administration of ddC, ddI and d4T produced dose-dependent mechanical hypersensitivity and allodynia. Peripheral administration of inhibitors of protein kinase A, protein kinase C, protein kinase G, p42/p44-mitogen-activated protein kinase (ERK1/2) and nitric oxide synthase, which have demonstrated anti-hyperalgesic effects in other models of metabolic and toxic painful peripheral neuropathies, had no effect on ddC-, ddI- and d4T-induced hypersensitivity. Since suramin, an anti-parasitic and anti-cancer drug, which shares with the anti-retroviral nucleoside analogs, mitochondrial toxicity, altered regulation of intracellular calcium, and a sensory neuropathy in humans, also produced mechanical hypersensitivity that was not sensitive to the above second messenger inhibitors we evaluated the role of intracellular calcium. Intradermal or spinal injection of intracellular calcium modulators (TMB-8 and Quin-2), which had no effect on nociception in control rats, significantly attenuated and together eliminated ddC and suramin-induced mechanical hypersensitivity. In electrophysiology experiments in ddC-treated rats, C-fibers demonstrated alterations in pattern of firing as indicated by changes in the distribution of interspike intervals to sustained suprathreshold stimuli without change in mechanical activation thresholds or in number of action potentials in response to threshold and suprathreshold stimulation. This study provides evidence for a novel, calcium-dependent, mechanism for neuropathic pain in a model of AIDS therapy-induced painful peripheral neuropathy.

ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia.

UNLABELLED: Endothelin-1 (ET-1) acts on endothelial cells to enhance mechanical stimulation-induced release of adenosine triphosphate (ATP), which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study, we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane-associated ATP synthase, ATP-binding cassette transporters, and ion channels. Inhibitors of vesicular exocytosis (ie, monensin, brefeldin A, and bafilomycin), plasma membrane-associated ATPase (ie, oligomycin and pigment epithelium-derived factor peptide 34-mer), and connexin ion channels (carbenoxolone and flufenamic acid) but not ATP-binding cassette transporter (ie, dipyridamole, nicardipine, or CFTRinh-172) attenuated SDH. This study reports a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes. PERSPECTIVE: ET-1 acts on endothelial cells to produce mechanical stimulation-induced hyperalgesia. Inhibitors of 3 different ATP release mechanisms attenuated this SDH. This study provides support for a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes.

Electrophysiological correlates of hyperalgesic priming in vitro and in vivo.

We have modeled the transition from acute to chronic pain in the rat. In this model (termed hyperalgesic priming) a chronic state develops after a prior inflammatory process or exposure to an inflammatory mediator, in which response to subsequent exposure to prostaglandin E2 (PGE2) is characterized by a protein kinase Cε-dependent marked prolongation of mechanical hyperalgesia. To assess the effect of priming on the function of the nociceptor, we have performed in vitro patch clamp and in vivo single-fiber electrophysiology studies using tumor necrosis factor α to induce priming. In vitro, the only change observed in nociceptors cultured from primed animals was a marked hyperpolarization in resting membrane potential (RMP); prolonged sensitization, measured at 60 minutes, could not be tested in vitro. However, complimentary with behavioral findings, in vivo baseline mechanical nociceptive threshold was significantly elevated compared to controls. Thirty minutes after injection of PGE2 into the peripheral receptive field, both primed and control nociceptors showed enhanced response to mechanical stimulation. However, 60 minutes after PGE2 administration, the response to mechanical stimulation was further increased in primed but not in control nociceptors. Thus, at the level of the primary afferent nociceptor, it is possible to demonstrate both altered function at baseline and prolonged PGE2-induced sensitization. Intrathecal antisense (AS) to Kv7.2, which contributes to RMP in sensory neurons, reversibly prevented the expression of priming in both behavioral and single-fiber electrophysiology experiments, implicating these channels in the expression of hyperalgesic priming.

Wound-healing growth factor, basic FGF, induces Erk1/2-dependent mechanical hyperalgesia.

UNLABELLED: Growth factors such as nerve growth factor and glial cell line-derived neurotrophic factor are known to induce pain sensitization. However, a plethora of other growth factors is released during inflammation and tissue regeneration, and many of them are essential for wound healing. Which wound-healing factors also alter the sensitivity of nociceptive neurons is not well known. We studied the wound-healing factor, basic fibroblast growth factor (bFGF), for its role in pain sensitization. Reverse transcription polymerase chain reaction showed that the receptor of bFGF, FGFR1, is expressed in lumbar rat dorsal root ganglia (DRG). We demonstrated presence of FGFR1 protein in DRG neurons by a recently introduced quantitative automated immunofluorescent microscopic technique. FGFR1 was expressed in all lumbar DRG neurons as quantified by mixture modeling. Corroborating the mRNA and protein expression data, bFGF induced Erk1/2 phosphorylation in nociceptive neurons, which could be blocked by inhibition of FGF receptors. Furthermore, bFGF activated Erk1/2 in a dose- and time-dependent manner. Using single-cell electrophysiological recordings, we found that bFGF treatment of DRG neurons increased the current-density of NaV1.8 channels. Erk1/2 inhibitors abrogated this increase. Importantly, intradermal injection of bFGF in rats induced Erk1/2-dependent mechanical hyperalgesia. PERSPECTIVE: Analyzing intracellular signaling dynamics in nociceptive neurons has proven to be a powerful approach to identify novel modulators of pain. In addition to describing a new sensitizing factor, our findings indicate the potential to investigate wound-healing factors for their role in nociception.

Sexual dimorphism in endothelin-1 induced mechanical hyperalgesia in the rat.

While the onset of mechanical hyperalgesia induced by endothelin-1 was delayed in female rats, compared to males, the duration was much longer. Given that the repeated test stimulus used to assess nociceptive threshold enhances hyperalgesia, a phenomenon we have referred to as stimulus-induced enhancement of hyperalgesia, we also evaluated for sexual dimorphism in the impact of repeated application of the mechanical test stimulus on endothelin-1 hyperalgesia. In male and female rats, endothelin-1 induced hyperalgesia is already maximal at 30 min. At this time stimulus-induced enhancement of hyperalgesia, which is observed only in male rats, persisted for 3-4h. In contrast, in females, it develops only after a very long (15 day) delay, and is still present, without attenuation, at 45 days. Ovariectomy eliminated these differences between male and female rats. These findings suggest marked, ovarian-dependent sexual dimorphism in endothelin-1 induced mechanical hyperalgesia and its enhancement by repeated mechanical stimulation.

In vivo and in vitro comparison of female and male nociceptors.

UNLABELLED: While it is generally accepted that women have lower pain thresholds for diverse forms of noxious stimuli, the mechanistic basis for this sexual dimorphism in nociceptive pain remains to be elucidated. We confirmed, in the rat, that females have lower cutaneous mechanical nociceptive thresholds and established a similar sexual dimorphism in muscle. To determine if a peripheral mechanism underlies this sexual dimorphism in pain threshold, we compared biophysical properties of cultured dorsal root ganglion (DRG) neurons that innervated the gastrocnemius muscle in female and male rats. DRG neurons from female rats, which innervated the gastrocnemius muscle, had a more hyperpolarized resting membrane potential. To determine if this was associated with a higher mechanical nociceptive threshold, in contradiction to our working hypothesis, we compared the function, in vivo, of nociceptive afferents innervating the gastrocnemius muscle in male and female rats. C-fiber nociceptors innervating muscle in female rats had higher mechanical thresholds than those in males. Other response characteristics of these nociceptors were not significantly different. Thus, both in vitro and in vivo electrophysiology experiments support the idea that lower mechanical nociceptive threshold in females may be due to sexual dimorphism in central nervous system mechanisms, a difference large enough to overcome an opposing difference in peripheral pain mechanisms. PERSPECTIVE: This article unifies in vivo and in vitro electrophysiology with behavioral data examining the differences in mechanical nociceptive threshold between male and female rats. The data provide a novel perspective on the peripheral and behavioral outcomes of noxious mechanical stimulation.

Multiple PKCε-dependent mechanisms mediating mechanical hyperalgesia.

We have recently implicated mitochondrial mechanisms in models of neuropathic and inflammatory pain, in some of which a role of protein kinase Cepsilon (PKCepsilon) has also been implicated. Since mitochondria contain several proteins that are targets of PKCepsilon, we evaluated the role of mitochondrial mechanisms in mechanical hyperalgesia induced by proinflammatory cytokines that induce PKCepsilon-dependent nociceptor sensitization, and by a direct activator of PKCepsilon (psiepsilonRACK), in the rat. Prostaglandin E(2) (PGE(2))-induced hyperalgesia is short lived in naïve rats, while it is prolonged in psiepsilonRACK pre-treated rats, a phenomenon referred to as priming. Inhibitors of two closely related mitochondrial functions, electron transport (complexes I-V) and oxidative stress (reactive oxygen species), attenuated mechanical hyperalgesia induced by intradermal injection of psiepsilonRACK. In marked contrast, in a PKCepsilon-dependent form of mechanical hyperalgesia induced by prostaglandin E(2) (PGE(2)), inhibitors of mitochondrial function failed to attenuate hyperalgesia. These studies support the suggestion that at least two downstream signaling pathways can mediate the hyperalgesia induced by activating PKCepsilon.

Mechanisms mediating vibration-induced chronic musculoskeletal pain analyzed in the rat.

UNLABELLED: While occupational exposure to vibration is a common cause of acute and chronic musculoskeletal pain, eliminating exposure produces limited symptomatic improvement, and reexposure precipitates rapid recurrence or exacerbation. To evaluate mechanisms underlying these pain syndromes, we have developed a model in the rat, in which exposure to vibration (60-80Hz) induces, in skeletal muscle, both acute mechanical hyperalgesia as well as long-term changes characterized by enhanced hyperalgesia to a proinflammatory cytokine or reexposure to vibration. Exposure of a hind limb to vibration-produced mechanical hyperalgesia measured in the gastrocnemius muscle of the exposed hind limb, which persisted for approximately 2 weeks. When nociceptive thresholds had returned to baseline, exposure to a proinflammatory cytokine or reexposure to vibration produced markedly prolonged hyperalgesia. The chronic prolongation of vibration- and cytokine-hyperalgesia was prevented by spinal intrathecal injection of oligodeoxynucleotide (ODN) antisense to protein kinase Cepsilon, a second messenger in nociceptors implicated in the induction and maintenance of chronic pain. Vibration-induced hyperalgesia was inhibited by spinal intrathecal administration of ODN antisense to receptors for the type-1 tumor necrosis factor-alpha (TNFalpha) receptor. Finally, in TNFalpha-pretreated muscle, subsequent vibration-induced hyperalgesia was markedly prolonged. PERSPECTIVE: These studies establish a model of vibration-induced acute and chronic musculoskeletal pain, and identify the proinflammatory cytokine TNFalpha and the second messenger protein kinase Cepsilon as targets against which therapies might be directed to prevent and/or treat this common and very debilitating chronic pain syndrome.

Comparison of oxaliplatin- and cisplatin-induced painful peripheral neuropathy in the rat.

UNLABELLED: Although platinum-based cancer chemotherapies produce painful peripheral neuropathy as dose-limiting side effects, there are important differences in the pain syndromes produced by members of this class of drugs. In the rat, cisplatin-induced hyperalgesia has latency to onset of 24 to 48 hours, is maximal by 72 to 96 hours, and is attenuated by inhibitors of caspase signaling but not by inhibitors of the mitochondrial electron transport chain (mETC) and antioxidants. In contrast, oxaliplatin-induced mechanical hyperalgesia is already present by 5 minutes and peaks by 20 minutes. Whereas oxaliplatin hyperalgesia persists for weeks, starting around day 10 to 15, its severity decreases to a lower 2nd plateau level. The rapid-onset 1st plateau in oxaliplatin-induced hyperalgesia was characterized by prominent cold allodynia and in contrast to cisplatin was attenuated by inhibitors of the mETC and antioxidants but not inhibitors of caspase signaling. However, tested later during the 2nd plateau, it was characterized by less intense hyperalgesia and no cold allodynia and was attenuated by inhibitors of caspase signaling as well as by inhibitors of the mETC and by antioxidants. PERSPECTIVE: The findings of this study distinguish between the neuropathic pain syndromes produced by members of a single chemical class of anticancer drugs and suggest that the underlying mechanisms of various forms of peripheral neuropathy may be different. Further, it defines the need for selective therapy for different types of neuropathy.

PLC-beta 3 signals upstream of PKC epsilon in acute and chronic inflammatory hyperalgesia.

While protein kinase C epsilon has been shown to contribute to acute and chronic mechanical hyperalgesia, its upstream signaling pathway has received little attention. Since phospholipase C can signal to PKC epsilon and has been implicated in nociceptor sensitization, we tested if it is upstream of PKC epsilon in mechanisms underlying primary mechanical hyperalgesia. In the rat, the PKC epsilon-dependent mechanical hyperalgesia and hyperalgesic priming (i.e., a form of chronic latent enhanced hyperalgesia) induced by carrageenan were attenuated by a non-selective PLC inhibitor U-73122. A lipid mediator of PLC signaling, l-alpha-lysophosphatidylcholine produced dose-dependent mechanical hyperalgesia and hyperalgesic priming, which was attenuated by EAVSLKPT, a selective PKC epsilon inhibitor. However, U-73122 did not attenuate hyperalgesia induced by psi epsilon RACK, a selective PKC epsilon activator. Antisense to PLC-beta 3 isoform, which was found in small-diameter dorsal root ganglion neurons, also attenuated carrageenan-induced acute and chronic-latent hyperalgesia. These studies support the suggestion that PLC-beta 3 is an important upstream signaling molecule for PKC epsilon-mediated acute and chronic inflammatory pain.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice.

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.