Paul G. Green

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.

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.

Role of interleukin-6 in chronic muscle hyperalgesic priming

After recovery from acute muscle pain even minor subsequent muscle use can initiate recurrence of the same mechanical hyperalgesia months or years after the initial injury. We have recently developed a model of this chronic latent hyperalgesia in the rat. In this study, we have examined the possibility that interleukin-6 (IL-6), an inflammatory mediator produced during acute muscle inflammation, can mediate the production of this chronic latent hyperalgesic state in which subsequent exposure to inflammatory mediators produces a markedly prolonged mechanical hyperalgesia. We now report that i.m. injection of IL-6 produced mechanical hyperalgesia, lasting several hours, that was prevented by intrathecal injection of antisense to glycoprotein 130 (gp130), an IL-6 receptor subunit. Furthermore, following complete recovery from i.m. IL-6-induced hyperalgesia, i.m. prostaglandin E(2) produced a mechanical hyperalgesia that was remarkably prolonged compared with naïve controls, indicating the presence of chronic latent hyperalgesia. This ability of IL-6 to produce chronic latent hyperalgesia was prevented by intrathecal administration of antisense for gp130. Furthermore, gp130 antisense also prevented chronic latent hyperalgesia produced by i.m. injection of the inflammogen, carrageenan. These results identify a role for IL-6 in acute inflammatory muscle pain and as a potential target against which therapies might be directed to treat chronic muscle pain.

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.

Sensory neuropeptide interactions in the production of plasma extravasation in the rat

We used an experimental model of neurogenic inflammation to study the contribution of the primary afferent peptides substance P, calcitonin gene-related peptide, galanin and somatostatin to plasma extravasation in rat synovium. Perfusion of the C-fiber excitotoxin, capsaicin (1.6 mM), through the knee joint of the pentobarbital anesthetized rat, increased plasma extravasation transiently (< 30 min). Perfusion of substance P (1 microM) or calcitonin gene-related peptide (100 nM), two primary afferent neuropeptides that are released by acute capsaicin administration, had no significant effect on plasma extravasation. Co-perfusion of these two neuropeptides, however, evoked an increase in plasma extravasation that was greater than that produced by capsaicin remaining above 250% of the baseline level by the end of the perfusion period (55 min). Capsaicin co-perfused with either galanin (100 nM) or somatostatin (1 microM) failed to increase plasma extravasation. Neither galanin nor somatostatin significantly affected increase in plasma extravasation induced by co-perfusion of substance P plus calcitonin gene-related peptide. Therefore, we suggest that galanin and somatostatin inhibit, presynaptically, the release of substance P and calcitonin gene-related peptide from primary afferent terminals. The interactions among these four neuropeptides provide a novel mechanism for the regulation of primary afferent neurogenic inflammation.

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.

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.

Dynorphin A-(1-13) attenuates withdrawal in morphine-dependent rats: effect of route of administration

Rats were made tolerant to morphine by subcutaneous implantation of morphine alkaloid pellets. Three days after pellet implantation, withdrawal was induced by pellet removal and was assessed 6 h later. Immediately prior to withdrawal assessment, rats were injected with dynorphin A-(1-13) either i.th. (via a catheter), i.c.v. (via a cannula) or i.v. (via the tail vein). When administered i.th. in the dose range 1.25-5 nmol/rat, dynorphin A-(1-13) attenuated withdrawal over the 40 min observation period. Similarly, dynorphin A-(1-13) administered i.v. (37.5-150 nmol/kg) attenuated withdrawal, though only over the first 20 min following administration. Dynorphin A-(1-13) up to 10 nmol/rat had no effect on withdrawal scores. These data indicate that dynorphin acts at spinal sites to suppress withdrawal in morphine-dependent rats and may play a role in tolerance and dependence mechanisms.

Early-life stress produces muscle hyperalgesia and nociceptor sensitization in the adult rat

Summary Neonatal stress induces a persistent decrease in skeletal muscle, but not cutaneous, mechanical nociceptive threshold and a PKCε‐dependent hyperalgesic priming in muscle as well as skin. ABSTRACT Chronic pain in adults has been associated with early‐life stress. To examine the pronociceptive effect of early‐life stress, we evaluated cutaneous and muscle nociception and activity in muscle nociceptors in an animal model of neonatal stress, limited bedding, in the rat. In this neonatal limited bedding (NLB) model, litters are exposed to limited bedding between postnatal days 2 and 9, and controls to standard bedding. In adult NLB‐treated rats, mechanical nociceptive threshold in skeletal muscle was significantly lower (∼22%) than in controls. Furthermore, administration of prostaglandin E2 in skin as well as muscle produced markedly prolonged hyperalgesia, an effect prevented by spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cε (PKCε), a second messenger in nociceptors that has been implicated in the induction and maintenance of chronic pain. In electrophysiological studies, mechanical threshold of muscle nociceptors was reduced by ∼31% and conduction velocity significantly increased (∼28%). These findings indicate that neonatal stress induces a persistent hyperalgesia and nociceptor sensitization manifest in the adult and that the second messenger PKCε may be a target against which therapies might be directed to treat a chronic pain syndrome that is associated with early‐life traumatic stress.