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.

Estrogen destabilizes microtubules through an ion-conductivity-independent TRPV1 pathway.

Recently, we described estrogen and agonists of the G-protein coupled estrogen receptor GPR30 to induce protein kinase C (PKC)ε-dependent pain sensitization. PKCε phosphorylates the ion channel transient receptor potential, vanilloid subclass I (TRPV1) close to a novel microtubule-TRPV1 binding site. We now modeled the binding of tubulin to the TRPV1 C-terminus. The model suggests PKCε phosphorylation of TRPV1-S800 to abolish the tubulin-TRPV1 interaction. Indeed, in vitro PKCε phosphorylation of TRPV1 hindered tubulin-binding to TRPV1. In vivo, treatment of sensory neurons and F-11 cells with estrogen and the GPR30 agonist, G-1, resulted in microtubule destabilization and retraction of microtubules from filopodial structures. We found estrogen and G-1 to regulate the stability of the microtubular network via PKC phosphorylation of the PKCε-phosphorylation site TRPV1-S800. Microtubule disassembly was not, however, dependent on TRPV1 ion conductivity. TRPV1 knock-down in rats inverted the effect of the microtubule-modulating drugs, Taxol and Nocodazole, on estrogen-induced and PKCε-dependent mechanical pain sensitization. Thus, we suggest the C-terminus of TRPV1 to be a signaling intermediate downstream of estrogen and PKCε, regulating microtubule-stability and microtubule-dependent pain sensitization.

Enhanced cytokine-induced mechanical hyperalgesia in skeletal muscle produced by a novel mechanism in rats exposed to unpredictable sound stress.

Stress exacerbates both experimental and clinical pain, most well-characterized in irritable bowel and fibromyalgia syndromes. Since it has been hypothesized that cytokines play an etiopathogenic role in fibromyalgia and other chronic widespread pain conditions, we investigated the relationship between stress and cytokines in a model of stress-induced chronic somatic pain. A series of experiments were performed to evaluate the impact of stress on the hyperalgesia-induced by endotoxin (lipopolysaccharide, LPS) and the role of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis α (TNFα). Fourteen days after exposure to a 4-day protocol of unpredictable sound stress, the ability of systemic LPS (100 µg/kg, i.p) to elicit cytokine-mediated mechanical hyperalgesia was measured in gastrocnemius muscle. LPS-induced hyperalgesia was significantly greater in stressed rats, but when rats were treated intrathecally with antisense oligodeoxynucleotide (ODN), to decrease either the gp130 subunit of the IL-6 receptor or the TNFα receptor, in nociceptors, skeletal muscle hyperalgesia in sound stressed, but not control, rats was prevented. These data suggest that chronic stress alters signaling in the primary afferent nociceptor for the hyperalgesia induced by endogenously produced pro-inflammatory cytokines.

Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization.

BACKGROUND: Dorsal root ganglia (DRG)-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM) for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base.The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. RESULTS: We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+)- and IB4(-)-neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked PGE2-induced sensitization. CONCLUSIONS: QuAM is a suitable if not necessary tool to analyze activation of endogenous signalling in heterogeneous cultures. NGF, GDNF and EGF stimulation of DRG-neurons shows differential Erk1/2 activation responses and a corresponding differential behavioral phenotype. Thus, in addition to expression-markers also signalling-activity can be taken for functional subgroup differentiation and as predictor of behavioral outcome. The anti-nociceptive function of EGF is an intriguing result in the context of tissue damage but also for understanding pain resulting from EGF-receptor block during cancer therapy.

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.

Sound stress-induced long-term enhancement of mechanical hyperalgesia in rats is maintained by sympathoadrenal catecholamines.

UNLABELLED: Although stress plays an important role in chronic widespread pain syndromes, such as fibromyalgia, the underlying mechanism has remained elusive. We have recently demonstrated, in a model of chronic widespread pain, that prolonged enhancement of immune mediator hyperalgesia, induced by unpredictable sound stress, requires a contribution of both the sympathoadrenal (epinephrine) and the hypothalamic-pituitary adrenal (corticosterone) neuroendocrine stress axes. Because this stress protocol produced sustained elevation of plasma epinephrine, in the current study we tested the hypothesis that the sympathoadrenal axis also plays a role in maintenance of symptoms in this model of chronic widespread pain. After establishment, adrenal medullectomy abolished the enhancement of epinephrine-induced cutaneous and muscle hyperalgesia. Administration of stress levels of epinephrine to adrenal medullectomized rats reconstituted the pain phenotype. These observations suggest that the sympathoadrenal stress axis plays a major role in the induction as well as maintenance of stress-induced enhancement of mechanical hyperalgesia, mediated by prolonged elevation of circulating epinephrine. PERSPECTIVE: We present data showing mechanical hyperalgesia persisting for up to 28 days after exposure to sound stress, with evidence that the sympathoadrenal axis mediator epinephrine plays a major role. These findings could have clinical implications with regard to novel potential treatments for chronic widespread pain syndromes, such as fibromyalgia.

TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization.

The transient receptor potential vanilloid 4 (TRPV4) contributes to mechanical hyperalgesia of diverse etiologies, presumably as part of a mechanoreceptor signaling complex (Alessandri-Haber et al., 2008). To investigate the hypothesis that a functional interaction between TRPV4 and stretch-activated ion channels (SACs) is involved in this mechanical transduction mechanism, we used a selective SACs inhibitor, GsMTx-4. Intradermal injection of GsMTx-4 in the rat hindpaw reversed the mechanical hyperalgesia induced by intradermal injection of inflammatory mediators. In vivo single fiber recordings showed that GsMTx-4 reversed inflammatory mediator-induced decrease in mechanical threshold in half of sensitized C-fibers. Furthermore, GsMTx-4 reduced hyperalgesia to both mechanical and hypotonic stimuli in different models of inflammatory and neuropathic pain, although it had no effect on baseline mechanical nociceptive thresholds. TRPC1 and TRPC6, two GsMTx-4-sensitive SACs, are expressed in dorsal root ganglion (DRG) neurons. Single-cell reverse transcription-PCR showed that messenger RNAs for TRPV4, TRPC1, and TRPC6 are frequently coexpressed in DRG neurons. Spinal intrathecal administration of oligodeoxynucleotides antisense to TRPC1 and TRPC6, like that to TRPV4, reversed the hyperalgesia to mechanical and hypotonic stimuli induced by inflammatory mediators without affecting baseline mechanical nociceptive threshold. However, antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4-selective agonist 4alpha-PDD (4 alpha-phorbol 12,13-didecanoate). We conclude that TRPC1 and TRPC6 channels cooperate with TRPV4 channels to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization, although they may have distinctive roles.

Neurotoxic catecholamine metabolite in nociceptors contributes to painful peripheral neuropathy.

The neurotoxic effects of catecholamine metabolites have been implicated in neurodegenerative diseases. As some sensory neurons express tyrosine hydroxylase and monoamine oxidase (MAO), we investigated the potential contribution of catecholamine metabolites to neuropathic pain in a model of alcoholic neuropathy. The presence of catecholamines in sensory neurons is supported by capsaicin-stimulated epinephrine release, an effect enhanced in ethanol-fed rats. mRNA for enzymes in dorsal root ganglia involved in catecholamine uptake and metabolism, dopamine beta-hydroxylase and MAO-A, were decreased by neonatal administration of capsaicin. Ethanol-induced hyperalgesia was attenuated by systemic and local peripheral administration of inhibitors of MAO-A, reduction of norepinephrine transporter (NET) in sensory neurons and a NET inhibitor. Finally, intradermal injection of 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), a neurotoxic MAO-A catecholamine metabolite, produced robust mechanical hyperalgesia. These observations suggest that catecholamines in nociceptors are metabolized to neurotoxic products by MAO-A, which can cause neuronal dysfunction underlying neuropathic pain.

Stress induces a switch of intracellular signaling in sensory neurons in a model of generalized pain.

Stress dramatically exacerbates pain in diseases such as fibromyalgia and rheumatoid arthritis, but the underlying mechanisms are unknown. We tested the hypothesis that stress causes generalized hyperalgesia by enhancing pronociceptive effects of immune mediators. Rats exposed to nonhabituating sound stress exhibited no change in mechanical nociceptive threshold, but showed a marked increase in hyperalgesia evoked by local injections of prostaglandin E(2) or epinephrine. This enhancement, which developed more than a week after exposure to stress, required concerted action of glucocorticoids and catecholamines at receptors located in the periphery on sensory afferents. The altered response to pronociceptive mediators involved a switch in coupling of their receptors from predominantly stimulatory to inhibitory G-proteins (G(s) to G(i)), and for prostaglandin E(2), emergence of novel dependence on protein kinase C epsilon. Thus, an important mechanism in generalized pain syndromes may be stress-induced coactivation of the hypothalamo-pituitary-adrenal and sympathoadrenal axes, causing a long-lasting alteration in intracellular signaling pathways, enabling normally innocuous levels of immune mediators to produce chronic hyperalgesia.

GPR30 estrogen receptor agonists induce mechanical hyperalgesia in the rat.

We evaluated the signalling pathway by which estrogen acts in peripheral tissue to produce protein kinase Cepsilon (PKCepsilon)-dependent mechanical hyperalgesia. Specific agonists for the classical estrogen receptors (ER), ERalpha and ERbeta, did not result in activation of PKCepsilon in neurons of dissociated rat dorsal root ganglia. In contrast, G-1, a specific agonist of the recently identified G-protein-coupled estrogen receptor, GPR30, induced PKCepsilon translocation. Involvement of GPR30 and independence of ERalpha and ERbeta was confirmed using the GPR30 agonist and simultaneous ERalpha and ERbeta antagonist ICI 182,780 (fulvestrant). The GPR30 transcript could be amplified from dorsal root ganglia tissue. We found estrogen-induced as well as GPR30-agonist-induced PKCepsilon translocation to be restricted to the subgroup of nociceptive neurons positive for isolectin IB4 from Bandeiraea simplicifolia. Corroborating the cellular results, both GPR30 agonists, G-1 as well as ICI 182,780, resulted in the onset of PKCepsilon-dependent mechanical hyperalgesia if injected into paws of adult rats. We therefore suggest that estrogen acts acutely at GPR30 in nociceptors to produce mechanical hyperalgesia.

Muscle inflammation induces a protein kinase Cepsilon-dependent chronic-latent muscle pain.

UNLABELLED: Skeletal muscle injuries can induce chronic pain, but the underlying mechanism is unknown. One possible cause has been suggested to be an increased sensitivity to inflammatory mediators. We demonstrate that self-limited inflammatory hyperalgesia induced by intramuscular carrageenan (lasting approximately 5 days) results in a state of chronic-latent hyperalgesia, revealed by injection of prostaglandin E(2) (PGE(2)) 10 days after carrageenan at the same site. In carrageenan-pretreated muscle, PGE(2) produced hyperalgesia that was unattenuated even 14 days after injection, markedly longer than the 4-hour hyperalgesia induced by PGE(2) in naive rats. This chronic-latent hyperalgesia was reversed as well as prevented by spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cepsilon, a second messenger implicated in long-lasting plasticity in cutaneous nociceptors. PERSPECTIVE: We describe a novel experimental model for chronic muscle pain, produced by mild acute muscle inflammation, that has clinical significance since it has the potential to reveal cellular processes by which acute inflammation or muscle trauma underlies chronic muscle pain.

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.

Alcohol-induced stress in painful alcoholic neuropathy.

Chronic alcohol consumption induces a painful small-fiber peripheral neuropathy, the severity of which increases during alcohol withdrawal. Chronic alcohol consumption also produces a sustained increase in stress hormones, epinephrine and corticosterone, that is exacerbated during alcohol withdrawal. We report that adrenal medullectomy and administration of a glucocorticoid receptor antagonist, mifepristone (RU 38486), both prevented and reversed a model of painful peripheral neuropathy in alcohol binge-drinking rats. Chronic administration of stress levels of epinephrine to rats that had undergone adrenal medullectomy and were being fed the alcohol diet reconstituted this phenotype. Intrathecal administration of oligodeoxynucleotides antisense to the beta(2)-adrenergic- or glucocorticoid-receptor also prevented and reversed the pro-nociceptive effects of ethanol. Our results suggest a convergence of the effects of mediators of the hypothalamic-pituitary- and sympathoadrenal-stress axes on sensory neurons in the induction and maintenance of alcohol-induced painful peripheral neuropathy.

Proinflammatory cytokines mediating burn-injury pain.

Thermal burns induce pain at the site of injury, mechanical hyperalgesia, associated with a complex time-dependent inflammatory response. To determine the contribution of inflammatory mediators to burn injury-induced mechanical hyperalgesia, we measured dynamic changes in the levels of three potent hyperalgesic cytokines, interleukin IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNFalpha), in skin of the rat, following a partial-thickness burn injury. Only IL-6 demonstrated a sustained increase ipsilateral but not contralateral to the burn, correlating with the prolonged ipsilateral mechanical hyperalgesia. Spinal intrathecal injection of oligodeoxynucleotides antisense for gp130, a receptor subunit shared by members of the IL-6 family of cytokines, attenuated both burn- and intradermal IL-6-induced hyperalgesia, as did intradermal injection of anti-IL-6 function blocking antibodies. These studies suggest that IL-6 is an important mediator of burn-injury pain.

Enhanced inflammatory hyperalgesia after recovery from burn injury.

Severe burn induces severe pain. While chronic as well as acute pain syndromes are reported, the peripheral mechanisms of burn-induced chronic pain syndromes have not been studied. We tested the hypothesis that burn induces plastic changes in primary afferent nociceptors that predispose to chronic pain states. Mechanical nociceptive thresholds were measured using the Randall-Selitto paw-withdrawal test in male Sprague-Dawley rats, before and following a small (<1% total body surface area) partial-thickness thermal injury to the dorsal surface of one hind paw. This burn induced mechanical hyperalgesia, which lasted over 2 weeks. After recovery, local injection of prostaglandin E2 (PGE2), to mimic re-injury, induced an enhanced and markedly prolonged mechanical hyperalgesia compared to the hyperalgesic effect of PGE2 in the control contralateral paw. This prolonged PGE2-induced hyperalgesia was reversed by a selective inhibitor of protein kinase C-epsilon (PKCepsilon). Our findings suggest PKCepsilon as a peripheral mechanism for burn-induced chronic pain syndromes.

TrkA and PKC-epsilon in thermal burn-induced mechanical hyperalgesia in the rat.

UNLABELLED: Although mechanical hyperalgesia associated with medical procedures is the major source of severe pain in burn-injured patients, little is known about its underlying mechanism. One reason for this has been the lack of a model for mechanical hyperalgesia at the site of injury. We have modified an established partial-thickness burn model in the rat to produce long-lasting primary mechanical hyperalgesia, which is present from the first measurement at 0.5 h, reaches a maximum at 3 days, and is still significant after 7 days. Because nerve growth factor (NGF), which is elevated in burn-injured tissue, produces mechanical hyperalgesia and activates protein kinase C (PKC)-epsilon, a key mediator in inflammatory and neuropathic pain, we used this model to evaluate the role of the NGF receptor, tyrosine-receptor kinase A (TrkA), and PKC-epsilon in burn-induced primary mechanical hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides to TrkA and PKC-epsilon, starting 3 days before inducing a burn injury, caused dose-related decrease of burn-induced primary mechanical hyperalgesia. In addition, intradermal injection of a PKC-epsilon-selective inhibitor eliminated hyperalgesia. Our model provides a method to elucidate the underlying mechanism of burn-injury pain as well as to screen for targets for novel analgesic treatments of this important clinical condition. PERSPECTIVE: This manuscript presents the first model of thermal injury-induced mechanical hyperalgesia which mimics prolonged duration of clinical burn injury pain. We also perform proof of concept experiments demonstrating that our model provides a method to elucidate the mechanism of this important clinical condition.

Estrogen controls PKCepsilon-dependent mechanical hyperalgesia through direct action on nociceptive neurons.

Protein kinase C epsilon (PKCepsilon) is an important intracellular signaling molecule in primary afferent nociceptors, implicated in acute and chronic inflammatory as well as neuropathic pain. In behavioral experiments inflammatory mediators produce PKCepsilon-dependent hyperalgesia only in male rats. The mechanism underlying this sexual dimorphism is unknown. We show that the hormone environment of female rats changes the nociceptive signaling in the peripheral sensory neuron. This change is maintained in culture also in the absence of a gender-simulating environment. Stimulation of beta(2)-adrenergic receptors (beta(2)-AR) leads to PKCepsilon activation in cultured dorsal root ganglia (DRG) neurons derived from male but not from female rats. Addition of estrogen to male DRG neurons produces a switch to the female phenotype, namely abrogation of beta(2)-AR-initiated activation of PKCepsilon. Estrogen interferes downstream of the beta(2)-AR with the signaling pathway leading from exchange protein activated by cAMP (Epac) to PKCepsilon. The interfering action is fast indicating a transcriptional-independent mechanism. Estrogen has a dual effect on PKCepsilon. If applied before beta(2)-AR or Epac stimulation, estrogen abrogates the activation of PKCepsilon. In contrast, estrogen applied alone leads to a brief translocation of PKCepsilon. Also in vivo the activity of estrogen depends on the stimulation context. In male rats, intradermal injection of an Epac activator or estrogen alone induces mechanical hyperalgesia through a PKCepsilon-dependent mechanism. In contrast, injection of estrogen preceding the activation of Epac completely abrogates the Epac-induced mechanical hyperalgesia. Our results suggest that gender differences in nociception do not reflect the use of generally different mechanisms. Instead, a common set of signaling pathways can be modulated by hormones.

Ethanol withdrawal induces hyperalgesia mediated by PKCepsilon.

Symptoms of ethanol withdrawal include heightened responses to sensory stimuli, as well as tremors and convulsions. We tested the hypothesis that repeated episodes of ethanol intake and withdrawal exacerbate the symptoms of alcohol-induced peripheral neuropathy. In contrast to the hyperalgesia produced when an alcohol (6.5%)-containing diet was fed continuously to male rats which took 4 weeks to develop (Dina et al., 2000), feeding alcohol (6.5%) in repeated cycles of 4 days of alcohol followed by 3 days without alcohol resulted in a withdrawal-induced hyperalgesia that began at the end of one weekly cycle and reached a maximum during the fourth cycle. For ethanol withdrawal to produce hyperalgesia, ethanol consumption needed to be terminated for a period of 2 days. Paradoxically, as the amount of alcohol consumed decreased, the hyperalgesia induced by withdrawal developed more rapidly, being maximal between 1.4 and 1.6% ethanol. These results suggest that continued exposure to ethanol also has a neuroprotective effect. Withdrawal-induced hyperalgesia, similar to the hyperalgesia induced by continuous, chronic alcohol intake, was inhibited reversibly by intrathecal administration of an antisense oligodeoxynucleotide to protein kinase C (PKC)epsilon.

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.

TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator.

The ligand-gated ion channel, TRPV4, functions as a transducer of hypotonic stimuli in primary afferent nociceptive neurons and contributes to inflammatory and neuropathic pain. Hypertonic saline also stimulates primary afferent nociceptors and the injection of mild hypertonic saline (2-5%) is widely used as an experimental model of pain in humans. Therefore, we tested whether TRPV4 participates in the transduction of hypertonic stimuli. Intradermal injection of 2% (607 mOsm) or 10% (3,250 mOsm) saline solution in the hind paw of rats induced a concentration-dependent pain-related behavior, flinching. Sensitization with prostaglandin E(2) (PGE(2)) caused a 7-fold increase in the number of flinches induced by 2% saline but failed to increase those caused by 10% saline. Spinal administration of antisense oligodeoxynucleotides to TRPV4 caused a 46% decrease in the number of flinches induced by 2% saline, but there was no change in flinching induced by 10% saline. Similarly, only the nociceptive behavior caused by 2% saline was reduced in TRPV4(-/-) knockout mice. The TRPV4-mediated nociceptive behaviors induced by hyper- and hypotonic stimuli were dependent on Src tyrosine kinase. We suggest TRPV4 is a transducer in primary afferents that mediates nociceptive behavior induced by small increases or decreases in osmolarity. Such changes in osmolarity might contribute to pain in inflammatory and neuropathic states.