David B. Reichling

Bradykinin-12-lipoxygenase-VR1 signaling pathway for inflammatory hyperalgesia

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain.

Transient Receptor Potential Vanilloid 4 Is Essential in Chemotherapy-Induced Neuropathic Pain in the Rat

The development of treatments for neuropathic pain has been hindered by our limited understanding of the basic mechanisms underlying abnormalities in nociceptor hyperexcitability. We recently showed that the polymodal receptor transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) family of ion channels, may play a role in inflammatory pain (Alessandri-Haber et al., 2003). The present study tested whether TRVP4 also contributes to neuropathic pain, using a rat model of Taxol-induced painful peripheral neuropathy. Taxol is the most widely used drug for the treatment of a variety of tumor types, but the dose of Taxol that can be tolerated is limited by the development of a small-fiber painful peripheral neuropathy. We found that Taxol treatment enhanced the nociceptive behavioral responses to both mechanical and hypotonic stimulation of the hind paw. Spinal administration of antisense oligodeoxynucleotides to TRPV4, which reduced the expression of TRPV4 in sensory nerve, abolished Taxol-induced mechanical hyperalgesia and attenuated hypotonic hyperalgesia by 42%. The enhancement of osmotic nociception involves sensitization of osmotransduction in primary afferents because osmotransduction was enhanced in cultured sensory neurons isolated from Taxol-treated rats. Taxol-induced TRPV4-mediated hyperalgesia and the enhanced osmotransduction in cultured nociceptors were dependent on integrin/Src tyrosine kinase signaling. These results suggest that TRPV4 plays a crucial role in a painful peripheral neuropathy, making it a very promising target for the development of a novel class of analgesics.

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 ϵ, 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.

Mechanical transduction by rat dorsal root ganglion neurons in vitro

Although it is generally presumed that mechanical sensitivity of somatosensory nerve fibers results from the activation of mechanosensitive ion channels, a mechanically-gated whole-cell current has never been demonstrated in dorsal root ganglion (DRG) neurons. We performed patch clamp experiments on rat DRG neurons in culture, and report the first mechanically-activated current in somatosensory neurons (I(mech)). This whole-cell current is observed in most dorsal root ganglion neurons but not in non-sensory sympathetic ganglion neurons. The current-voltage relation of I(mech) indicates that it is a non-selective cation current. Sensitivity of I(mech) to block by gadolinium suggests that it may be mediated by a member of a family of mechanosensitive non-selective cation channels observed in many cell types. Sensitivity to benzamil supports this idea, and further suggests that the current might be mediated by a member of the degenerin/ epithelial sodium channel (DEG/ENaC) family.

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 α2β1 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, α2 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.

Role of protein kinase Cϵ and protein kinase A in a model of paclitaxel-induced painful peripheral neuropathy in the rat

The clinical use of the antineoplastic agent paclitaxel (Taxol) is significantly limited in its effectiveness by a dose-related painful peripheral neuropathy. To evaluate underlying mechanisms, we developed a model of Taxol-induced painful peripheral neuropathy in the rat and determined the involvement of two second messengers that contribute to enhanced nociception in other models of inflammatory and neuropathic pain, protein kinase Cepsilon and protein kinase A. Taxol administered acutely, or chronically over 12 days, produced a decrease in mechanical nociceptive threshold. Acutely, Taxol induced hyperalgesia that was significant within 1 h, maximal after 6 h and resolved completely by 24 h after a single treatment. Chronically, Taxol treatment resulted in a dose (0.1-1 mg/kg/day)-dependent decrease in nociceptive threshold, measured 24 h after administration, maximal within 5 days from the commencement of Taxol administration and resolving by 2 weeks after the last dose of Taxol. Chronic Taxol treatment also increased the number of action potentials evoked by sustained (60-s) threshold and suprathreshold (10-g) stimulation of a sub-population of C-fibers in rats with Taxol-induced hyperalgesia. Mechanical allodynia and thermal hyperalgesia were also present in Taxol-treated rats. Hyperalgesia, produced by both acute and chronic Taxol, was attenuated by intradermal injection of selective second messenger antagonists for protein kinase Cepsilon and protein kinase A. These findings provide insight into the mechanism of Taxol-induced painful peripheral neuropathy that may help control side effects of chemotherapy and improve its clinical efficacy.

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 E2 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 (Gs to Gi), and for prostaglandin E2, emergence of novel dependence on protein kinase Cε. 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.

Chapter 2 Anatomy, physiology and pharmacology of the periaqueductal gray contribution to antinociceptive controls

Publisher Summary This chapter discusses the anatomy, physiology, and pharmacology of the periaqueductal gray (PAG) contribution to antinociceptive controls. Studies emphasized the multiplicity of the bulbospinal antinociceptive control systems that are activated by electrical stimulation of or microinjection of opiates into the midbrain PAG. There are important noradrenergic controls and a wide variety of peptide-containing bulbospinal neurons. The chapter summarizes the cytochemistry of the afferent and efferent connections of the PAG and some intrinsic circuits within the PAG through which the controls are activated. The complexity of antinociceptive controls exerted from the midbrain PAG is illustrated. Anatomically and functionally, the PAG is heterogeneous. There is evidence that β-endorphin, enkephalin, dynorphin, gamma-aminobutyric acid (GABA), and neurotensin neurons contribute to the controls generated from the PAG. Electrical stimulation of or microinjection of drugs into the PAG activates multiple stages of this circuitry. The complex interaction of narcotics, such as morphine, with the multiple opioid receptor sites in the PAG makes interpretation of drug injection studies equally complicated.

Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCε second messenger pathways

&NA; Toward the goal of defining new pharmacological targets for the treatment of chronic pain conditions, in previous studies we established a model, termed ‘hyperalgesic priming,’ in which an acute inflammatory stimulus causes a long‐lasting latent susceptibility to hyperalgesia induced by subsequent exposures to the inflammatory mediator, prostaglandin E2 (PGE2). Those investigations suggested the hypothesis that priming induces a novel linkage between the PGE2‐activated second messenger cascade and the epsilon isoform of protein kinase C (PKCϵ). In the present study, comparison of dose–response relations for hyperalgesia produced by PGE2, forskolin, 8‐Br‐cAMP, or the protein kinase A (PKA) catalytic subunit, in primed versus normal animals, demonstrated that priming‐induced enhancement of the PGE2‐activated second messenger cascade occurs downstream to adenylate cyclase and upstream to PKA. Therefore, PGE2‐induced hyperalgesia in the primed animal is enhanced by the recruitment of a novel cAMP/PKCϵ signaling pathway in addition to the usual cAMP/PKA pathway. These observations suggest that pharmacological disruption of the novel interaction between cAMP and PKCϵ might provide a route toward the development of highly specific methods to reverse cellular processes that underlie chronic pain states.

Transient attenuation of protein kinase Cϵ can terminate a chronic hyperalgesic state in the rat

Abstract Recently we demonstrated that a single 3-day episode of carrageenan-induced acute cutaneous inflammation can create a chronic state of increased susceptibility to inflammatory hyperalgesia. In this latent “primed” state, although there is no ongoing hyperalgesia, the hyperalgesic response to subsequent challenges with inflammatory agent (prostaglandin E 2 ; PGE 2 ) is greatly enhanced. Furthermore, the PGE 2 -induced hyperalgesia in primed skin was found to require activity of the ϵ isozyme of protein kinase C (PKCϵ), a second messenger that is not required for PGE 2 -induced hyperalgesia in control animals. In the present study we tested the hypothesis that activity of PKCϵ not only plays a critical role in the expression of primed PGE 2 -induced hyperalgesia, but also in the development and maintenance of the primed state itself. Antisense oligodeoxynucleotide was employed to produce a decrease in PKCϵ in the nerve, verified by Western blot analysis. PKCϵ was found to be essential both for the development of carrageenan-induced hyperalgesic priming, as well as for the maintenance of the primed state. Furthermore, hyperalgesic priming could be induced by an agonist of PKCϵ (pseudo-receptor octapeptide for activated PKCϵ) at a dose that itself causes no hyperalgesia. The finding that transient inhibition of PKCϵ can not only prevent the development of priming, but can also terminate a fully developed state of priming suggests the possibility that selective targeting PKCϵ might be an effective new strategy in the treatment of chronic inflammatory pain.