Sachia G. Khasar

A Novel Nociceptor Signaling Pathway Revealed in Protein Kinase C ε Mutant Mice

Abstract There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid–associated hyperalgesia are markedly attenuated in PKCe mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor– (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na + current (TTX-R I Na ) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCe-selective inhibitor peptide. Our findings indicate that PKCe regulates nociceptor function and suggest that PKCe inhibitors could prove useful in the treatment of pain.

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.

A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat

We report evidence for a contribution of tetrodotoxin-resistant sodium current (TTX-R INa) to prostaglandin E2 (PGE2)-induced hyperalgesia. Behavioral experiments were performed in rats chronically implanted with spinal cannulae. The study employed intrathecal administration of oligodeoxynucleotide (ODN) antisense to the recently cloned channel underlying TTX-R INa (PN3/SNS). The nociceptive flexion reflex was employed to determine changes in mechanical stimulus-induced paw-withdrawal threshold. Administration of antisense but not of sense or mismatch ODN, led to a decrease in PGE2-induced hyperalgesia. PGE2-induced hyperalgesia returned to normal 7 days after the last injection of antisense ODN. Antisense ODN selectively and significantly reduced TTX-R INa current density in cultured sensory neurons. Our observations support the hypothesis that modulation of TTX-R INa, present in peripheral terminals of primary afferent nociceptors, contributes, at least in part, to inflammatory hyperalgesia. Since TTX-R INa is found only in primary afferent nociceptors, our findings suggest TTX-R INa as a promising target for novel therapeutic interventions for the treatment of inflammatory 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 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.

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.

Peripheral nociceptive effects of α2-adrenergic receptor agonists in the rat

Abstract We have previously shown that norepinephrine can produce hyperalgesia via an α 2 -adrenergic receptor mechanism. The α 2 -adrenergic receptor agonist clonidine has, however, also been shown to produce peripheral analgesia. In view of the multiple α 2 -subtypes currently known (i.e. α 2A , α 2B andα 2C ), we evaluated the α 2 -receptor subtypes mediating norepinephrine-induced peripheral hyperalgesia and clonidine analgesia. Norepinephrine and the α 2 -adrenergic agonists clonidine and UK 14,304 (1–1000 ng), when co-injected with the calcium ionophore A23187 (1000 ng) produced dose-dependent hyperalgesia in the Randall-Selitto paw withdrawal test. Norepinephrine (100 ng) hyperalgesia was dose-dependently antagonized by α 2 -adrenergic receptor antagonists. From the estimated ID 50 , the rank order of potency was: SK&F 104856 ( α 2B ) ≅ imiloxan ( α 2B ) rauwolscine ( α 2C ) ≫ BRL 44408 ( α 2A ). Norepinephrine hyperalgesia was not significantly affected by pertussis-toxin treatment. Prostaglandin E 2 (100 ng) hyperalgesia was inhibited dose-dependently, by clonidine and UK 14,304. Rauwolscine was more potent in reversing the inhibitory effect of clonidine on prostaglandin E 2 than imiloxan while BRL 44408 was ineffective. The inhibitory effect of clonidine on prostaglandin E 2 hyperalgesia was reversed by pertussis toxin. These data suggest that α 2B -subtype receptors mediate (norepinephrine hyperalgesia while the antinociceptive effect of α 2 -agonist is mediated by the α 2C -subtype receptor. Differential coupling of these receptor subtypes to second messenger systems and location on different cell types in the rat paw may explain, at least in part, their differential responses to α 2 -agonist stimulation, leading to hyperalgesia and analgesia.

Repeated sound stress enhances inflammatory pain in the rat

&NA; While it is well established that acute stress can produce antinociception, a phenomenon referred to as stress‐induced analgesia, repeated exposure to stress can have the opposite effect. Since, chronic pain syndromes, such as fibromyalgia and rheumatoid arthritis, may be triggered and/or exacerbated by chronic stress, we have evaluated the effect of repeated stress on mechanical nociceptive threshold and inflammatory hyperalgesia. Using the Randall–Selitto paw pressure test to quantify nociceptive threshold in the rat, we found that repeated non‐habituating sound stress enhanced the mechanical hyperalgesia induced by the potent inflammatory mediator, bradykinin, which, in normal rats, produces hyperalgesia indirectly by stimulating the release of prostaglandin E2 from sympathetic nerve terminals. Hyperalgesia induced by the direct‐acting inflammatory mediator, prostaglandin E2 as well as the baseline nociceptive threshold, were not affected. Adrenal medullectomy or denervation, reversed the effect of sound stress. In sound stressed animals, bradykinin‐hyperalgesia had a more rapid latency to onset and was no longer inhibited by sympathectomy, compatible with a direct effect of bradykinin on primary afferent nociceptors. In addition, implants of epinephrine restored bradykinin‐hyperalgesia in sympathectomized non‐stressed rats, lending further support to the suggestion that increased plasma levels of epinephrine can sensitize primary afferents to bradykinin. These results suggest that stress‐induced enhancement of inflammatory hyperalgesia is associated with a change in mechanism by which bradykinin induces hyperalgesia, from being sympathetically mediated to being sympathetically independent. This sympathetic‐independent enhancement of mechanical hyperalgesia is mediated by the stress‐induced release of epinephrine from the adrenal medulla.

Proinflammatory cytokines mediating burn-injury pain

&NA; 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‐1β, IL‐6, and tumor necrosis factor‐α (TNFα), 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.

Vagal modulation of nociception is mediated by adrenomedullary epinephrine in the rat.

Vagal afferent activity modulates mechanical nociceptive threshold and inflammatory mediator‐induced hyperalgesia, effects that are mediated by the adrenal medulla. To evaluate the role of epinephrine, the major hormone released from the adrenal medulla, the β2‐adrenergic receptor antagonist ICI 118,551 was chronically administered to vagotomized rats and epinephrine to normal rats. In vagotomized rats, chronic administration of ICI 118,551 markedly attenuated vagotomy‐induced enhancement of bradykinin hyperalgesia but had no effect on nociceptive threshold. In normal rats, chronic epinephrine had the opposite effect, enhancing bradykinin hyperalgesia. Like vagotomy‐, epinephrine‐induced enhancement of hyperalgesia developed slowly, taking 14 days to reach its peak. Vagotomy induced a chronic elevation in plasma concentrations of epinephrine. We suggest that ongoing activity in vagal afferents inhibits the release of epinephrine from the adrenal medulla. Chronically elevated levels of epinephrine, occurring after vagotomy, desensitize peripheral β2‐adrenergic receptors and lead to enhancement of bradykinin hyperalgesia. The ability of prolonged elevated plasma levels of epinephrine to sensitize bradykinin receptors could contribute to chronic generalized pain syndromes.

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.