Elizabeth K. Joseph

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 4α‐phorbol 12,13‐didecanoate (4αPDD) and hypotonic solutions. PAR2‐agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cβ and protein kinases A, C and D inhibited PAR2‐induced sensitization of TRPV4 Ca2+ signals and currents. 4αPDD 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 4αPDD 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.

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.

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.

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

&NA; 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% (3250 mOsm) saline solution in the hind paw of rats induced a concentration‐dependent pain‐related behavior, flinching. Sensitization with prostaglandin E2 (PGE2) 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.

Caspase signalling in neuropathic and inflammatory pain in the rat.

Whereas small‐fibre sensory neuropathies might ultimately lead to cell death and loss of sensation, they first progress through a phase, which might last for years, characterized by the presence of analgesia‐resistant neuropathic dysesthesias and pain. Much previous research has addressed these two phases as separate phenomena mediated by presumably discrete biochemical mechanisms. We hypothesized that activity in signalling pathways that ultimately lead to apoptosis plays a critical role in the generation of neuropathic pain, before death of sensory neurons becomes apparent. We have tested the hypothesis that activator and effector caspases, defining components of programmed cell death (apoptosis) signalling pathways, also contribute to pain‐related behaviour in animals with small‐fibre peripheral neuropathies and that the death receptor ligand, tumour necrosis factor‐α, and its downstream second messenger, ceramide, also produce pain‐related behaviour via this mechanism. In two models of painful peripheral neuropathy, HIV/AIDS therapy (induced by the nucleoside reverse transcriptase inhibitor, dideoxycytidine), and cancer chemotherapy (induced by vincristine) peripheral neuropathy, and for pain‐related behaviour induced by tumour necrosis factor‐α and its second messenger, ceramide, inhibition of both activator (1, 2, 8 and 9) and effector (3) caspases attenuates neuropathic pain‐related behaviour, although has no effect in streptozotocin‐diabetic neuropathy and control rats. We conclude that during a latent phase, before apoptotic cell death is manifest, the caspase signalling pathway can contribute to pain in small‐fibre peripheral neuropathies, and that inflammatory/immune mediators also activate these pathways. This suggests that these pathways are potential targets for novel pharmacological agents for the treatment of inflammatory as well as neuropathic pain.

Tumor necrosis factor receptor type‐1 in sensory neurons contributes to induction of chronic enhancement of inflammatory hyperalgesia in rat

Carrageenan‐induced inflammatory pain lasting hours to days produces a protein kinase C epsilon (PKCɛ)‐dependent ‘primed’ state lasting several weeks, during which time injection of prostaglandin E2 induces hyperalgesia which is markedly enhanced and prolonged compared to PGE2‐induced hyperalgesia in normal ‘unprimed’ rats. In the present study, we demonstrate that while inhibition of prostaglandin synthesis and antagonism of β2‐adrenergic receptors markedly attenuated the hyperalgesia induced by carrageenan, these interventions did not affect hyperalgesic priming. Tumor necrosis factor‐α (rat recombinant; rrTNFα), another mediator of carrageenan‐induced inflammation, alone produced hyperalgesia and priming, which were attenuated and prevented, respectively, by intrathecal administration of antisense to PKCɛ. Inhibition of TNFα with thalidomide or a rat polyclonal anti‐TNFα antibody attenuated carrageenan‐induced hyperalgesia and prevented priming. Intrathecal administration of antisense to tumour necrosis factor receptor type‐1 (TNFR1) reduced the level of TNFR1 transported toward the peripheral terminals of sensory neurons, and attenuated both carrageenan‐ and rrTNFα‐induced priming. Acute hyperalgesia induced by carrageenan or rrTNFα remained intact in animals treated with TNFR1 antisense. Our results demonstrate that the generation of the primed state does not require production of hyperalgesia and that TNFα, which is generated during acute inflammation, can act on sensory neurons to induce hyperalgesic priming by activating neuronal PKCɛ.

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.

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

&NA; 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.

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.

Mitochondrial electron transport in models of neuropathic and inflammatory pain

Abstract 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 &agr;‐induced hyperalgesia, they had no effect on hyperalgesia induced by prostaglandin E2 and epinephrine. Two competitive inhibitors of ATP‐dependent mechanisms, adenosine 5′‐(&bgr;,&ggr;‐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 &agr;, 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.