A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

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 PKCepsilon 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 PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

Pain-induced analgesia mediated by mesolimbic reward circuits.

We tested the hypothesis that noxious stimuli induce pain modulation by activation of supraspinal structures. We found that intense noxious stimuli (i.e., subdermal injection of capsaicin or paw immersion in hot water) induced profound attenuation of the jaw-opening reflex in the anesthetized rat; forepaw subdermal capsaicin also elevated the mechanical hindpaw-withdrawal threshold in the awake rat. These antinociceptive effects were blocked by previous injection of either a dopamine antagonist (flupentixol) or an opioid antagonist (naloxone) into the nucleus accumbens. Additional experiments in anesthetized animals showed that the antinociceptive effect of noxious stimulation by either capsaicin (>/=100 micrograms) or hindpaw immersion in hot water (>/=45 degrees C for 4 min) correlated with the intensity of the stimulus. The maximal antinociceptive effect of capsaicin was similar in magnitude to that of a high dose of morphine (10 mg/kg) injected subcutaneously. Injection of the GABA(A)-receptor agonist muscimol, but not naloxone, into the rostroventral medulla, a major component of descending pain modulation systems, blocked capsaicin-induced antinociception. Although it is widely thought that painful stimuli may induce analgesia by activating forebrain structures, this is the first demonstration that such a mechanism exists. Furthermore, this mechanism can be engaged by naturalistic stimuli in awake animals. These observations imply that painful stimuli might under certain conditions be rewarding.

Nociceptor sensitization by extracellular signal-regulated kinases.

Inflammatory pain, characterized by a decrease in mechanical nociceptive threshold (hyperalgesia), arises through actions of inflammatory mediators, many of which sensitize primary afferent nociceptors via G-protein-coupled receptors. Two signaling pathways, one involving protein kinase A (PKA) and one involving the epsilon isozyme of protein kinase C (PKCepsilon), have been implicated in primary afferent nociceptor sensitization. Here we describe a third, independent pathway that involves activation of extracellular signal-regulated kinases (ERKs) 1 and 2. Epinephrine, which induces hyperalgesia by direct action at beta(2)-adrenergic receptors on primary afferent nociceptors, stimulated phosphorylation of ERK1/2 in cultured rat dorsal root ganglion cells. This was inhibited by a beta(2)-adrenergic receptor blocker and by an inhibitor of mitogen and extracellular signal-regulated kinase kinase (MEK), which phosphorylates and activates ERK1/2. Inhibitors of G(i/o)-proteins, Ras farnesyltransferases, and MEK decreased epinephrine-induced hyper-algesia. In a similar manner, phosphorylation of ERK1/2 was also decreased by these inhibitors. Local injection of dominant active MEK produced hyperalgesia that was unaffected by PKA or PKCepsilon inhibitors. Conversely, hyperalgesia produced by agents that activate PKA or PKCepsilon was unaffected by MEK inhibitors. We conclude that a Ras-MEK-ERK1/2 cascade acts independent of PKA or PKCepsilon as a novel signaling pathway for the production of inflammatory pain. This pathway may present a target for a new class of analgesic agents.

Different peripheral mechanisms mediate enhanced nociception in metabolic/toxic and traumatic painful peripheral neuropathies in the rat.

Mechanisms underlying neuropathic pain states are poorly understood. We have compared mechanisms mediating enhanced nociception of four established models of neuropathic pain produced by very different types of insults to the peripheral nervous system: streptozotocin-induced hyperalgesia, a model of diabetic (metabolic) peripheral neuropathy, vincristine-induced hyperalgesia, a model of chemotherapeutic agent (toxic) peripheral neuropathy, and chronic constriction injury and partial nerve ligation, models of trauma-induced painful neuropathies. All four models resulted in prolonged mechanical hyperalgesia (>30% decrease in mechanical nociceptive threshold) and allodynia (detected by 10-209-mN-intensity von Frey hairs). In vincristine- and streptozotocin-induced hyperalgesia, the protein kinase A, protein kinase C and nitric oxide second messenger pathways in the periphery contributed to the hyperalgesia, while N-methyl-D-aspartate (NMDA) receptor-mediated events were not detected. None of these second messengers nor the NMDA receptor, which can contribute to peripheral sensitization of nociceptors, contributed to chronic constriction injury- and partial nerve ligation-induced hyperalgesia. In all four models the hyperalgesia was not antagonized by peripheral administration of a mu-opioid agonist.Our findings support the presence of a common abnormality in second messenger signaling in the periphery to the maintenance of two very different models of non-traumatic neuropathic pain, not shared by models of trauma-induced neuropathic pain.

Delayed sympathectomy after a prolonged hyperalgesia results in a subsequent enhanced acute hyperalgesic response.

We report on the ability of a delayed sympathectomy after a prolonged hyperalgesia to result in a subsequent enhanced hyperalgesic response. Sympathectomy was performed one day after injection of prostaglandin E2 plus rolipram, which induces a prolonged sympathetically-maintained hyperalgesia [Aley K. O. and Levine J. D. (1995) Eur. J. Pharmac. 273, 107-112]. The duration of hyperalgesia produced by a subsequent injection of prostaglandin E2 or prostaglandin E2 plus rolipram was then assessed. Lumbar surgical sympathectomy, done on day 2 or 3, prevented prostaglandin E2 plus rolipram-induced prolonged hyperalgesia from developing when they were injected five days after surgery, similar to the previous report of the effect of prior sympathectomy to block the rolipram enhancement [Aley K. O. and Levine J. D. (1995) Eur. J. Pharmac. 273, 107-112]. Sympathectomy done five days after injection of prostaglandin E2 plus rolipram, however, paradoxically prolonged (at least 10 days) hyperalgesia induced by a subsequent prostaglandin E2 plus rolipram injection, a duration much greater than that seen after prostaglandin E2 plus rolipram in naive animals. To determine the roles of prostaglandin E2 and rolipram in the prolongation of hyperalgesic response after delayed sympathectomy, rats were treated with either prostaglandin E2 or rolipram prior to sympathectomy. Prostaglandin E2 or rolipram alone were also administered five days after the sympathectomy. It was found that sympathectomy done five days after first injecting either prostaglandin E2 or rolipram alone did not produce enhanced hyperalgesic response. These data suggest that induction of a prolonged state of mechanical hyperalgesia causes time-dependent alterations in the sympathetic control of peripheral nociceptive mechanisms such that sympathectomy can lead to enhanced hyperalgesic response. These findings may be relevant to post-sympathectomy pain, a clinical entity for which there has been no available animal models.

Vincristine hyperalgesia in the rat: a model of painful vincristine neuropathy in humans.

To investigate the mechanism of vincristine-induced pain in humans undergoing chemotherapy we have established a model of vincristine-induced hyperalgesia in rat. Vincristine (100 micrograms/kg) was administered daily over a period of two weeks. An acute dose-dependent decrease in mechanical nociceptive threshold and an increased response to non-noxious mechanical stimuli ("hyperalgesia") occurred after the second day of administration. Chronic lowered threshold and increased response to stimuli (determined 24 h after each injection) was first noted during the second week of vincristine administration. Responses gradually returned to baseline during the two weeks following discontinuation of treatment. Vincristine also increased sensitivity to heat stimulation. At a dose that produced hyperalgesia (100 micrograms/kg), vincristine did not cause a significant motor deficit. Peripheral administration of a mu-opioid agonist did not reduce vincristine-induced acute hyperalgesia. Hyperalgesia induced by vincristine in the rat provides a good model for the experimental study of painful peripheral neuropathies in human patients receiving vincristine as a chemotherapeutic agent.

Rapid onset pain induced by intravenous streptozotocin in the rat.

Pain in diabetes is a common debilitating condition for which pathophysiology remains poorly understood. To evaluate the underlying mechanisms, we used intravenous injection of streptozotocin to produce rapid (24-hour) onset of diabetes (blood glucose > 300 mg/dL and urine glucose > 2,000 mg/dL with polyuria). In this model, mechanical and thermal hyperalgesia and tactile allodynia are detectable by 48 hours after streptozotocin administration in the absence of ketonuria or physical debility. Treatment with insulin attenuated hyperglycemia and prevented the development of mechanical and thermal hyperalgesia. Direct application of streptozotocin to peripheral nerve did not produce hyperalgesia. We conclude that streptozotocin can induce pain independent of a general debility or direct toxic effect of streptozotocin on peripheral nerve and that elevated blood glucose may contribute to the enhanced nociception.

GABA-mediated modification of despair behavior in mice.

Gamma-aminobutyric acid (GABA) and GABA agonists exhibited biphasic response on forced swimming-induced despair behavior in mice; smaller doses, GABA (100 mg/kg), muscimol (0.05 and 0.1 mg/kg), baclofen (0.5 and 1 mg/kg), sodium valproate (100 mg/kg), piracetam (25 mg/kg), and fengabine (10 and 20 mg/kg) decreasing forced swimming-induced immobility period, while higher doses enhancing the immobility period. GABA, muscimol, and baclofen also reversed reserpine-induced prolongation of the forced swimming-induced immobility. When GABA was administered with antidepressant agents, it potentiated the effect of some classical antidepressants. Bicuculline and picrotoxin, the GABAergic antagonists, by themselves enhanced the forced swimming-induced immobility period, but they had no significant effect on reserpine-induced prolongation of the immobility period. When animals were chronically exposed to forced swimming, there was a gradual increase of the immobility period which was reduced on treatment with GABAergic agents. It is suggested that GABA has a modulatory role in reversing forced swimming-induced despair and also in potentiating the effect of antidepressants.

Studies on meperidine-induced catalepsy in mice.

Anticataleptic effect of different pharmacological agents was studied against meperidine-induced (50 mg/kg i.p.) catalepsy in mice. MAO (A and B) inhibitors, alpha 2-agonists in addition to dopaminergic, GABAergic and serotoninergic agents, were found to be effective in attenuating the cataleptic symptoms. However, naloxone, an opiate antagonist, failed to reverse meperidine-induced akinesia and rigidity. Clorgyline, a specific MAO-A inhibitor, was found to be more potent than the MAO-B inhibitor selegiline.

Studies on the neuropsychopharmacological profile of fengabine (SL 79229) in mice.

The effect of fengabine (a novel benzylidene derivative) on neuropsychopharmacological parameters was investigated in mice. On acute systemic administration, it showed modulatory effects on 1) forced swimming-induced immobility, 2) foot shock-induced aggression, 3) electromaximal shock-induced convulsion, 4) radiant heat-induced nociception and 5) locomotor activity. However, it has no effect on the muscle strength of the animal. The GABAA receptor antagonists reversed its effects on forced swimming-induced immobility and foot shock-induced aggression, implicating a GABAergic involvement in its mechanism of action. But these antagonists failed to reverse its potentiating effect on morphine antinociception, suggesting a possible interaction of the drug with opioid receptors. A potential clinical usefulness of the drug and of GABA agonists in general is commented upon.

GABAergic agents-induced antinociceptive effect in mice.

The effect of GABA agonists, namely gamma aminobutyric acid, muscimol, sodium valproate and baclofen was studied on radiant heat-induced nociception in mice. All of the drugs, with the exception of sodium valproate, enhanced the reaction time to radiant heat as effect per se. Concomitant administration of any of these agents with morphine showed a potentiation of morphine-induced analgesia. The GABA antagonists bicuculline and picrotoxin failed to reverse the antinociceptive effect; paradoxically both these agents showed antinociceptive effect per se and also enhanced the response due to morphine.

GABAergic agents and clonidine attenuate footshock-induced aggression in mice.

The effects of gamma aminobutyric acid (GABA), baclofen, and clonidine was studied on footshock-induced aggression in mice. A lower dose (200 mg/kg) of GABA enhanced the aggressive score, while at a higher dose (400 mg/kg) it attenuated the aggressive behavior, the latter effect being reversed both by bicuculline and picrotoxin (PTX). Clonidine (0.5 and 1 mg/kg) enhanced the aggressive score, and the effect of clonidine was reversed by idazoxan. GABA (400 mg/kg) and clonidine (1 mg/kg) significantly reduced the aggressive score in reserpinized mice. Baclofen (5 mg/kg) showed no effect per se in either reserpinized or non-reserpinized mice. On concomitant administration of clonidine (0.5 mg/kg) with a subeffective dose of either GABA, baclofen, or diazepam, there was significant reduction in the aggressive score. A modulatory role of GABAergic and noradrenergic systems in footshock-induced aggression is suggested.

Anticatatonic effect of venoruton.

The protective effect of venoruton O-(beta-hydroxy-ethyl) rutoside, HR was investigated against drug induced catatonia in rats. Perphenazine, haloperidol, reserpine and meperidine produced frank catalepsy in rats. The cataleptic effect of these neuroleptics was significantly attenuated when the animals were pretreated with venoruton. The protective effect of venoruton is speculated to be due to its inhibitory action on superoxide formation and free radicals are considered to have a role in the neurotoxicity caused by the above agents.

Baclofen analgesia in mice: a GABAB-mediated response.

The effect of baclofen, a GABAB agonist, has been studied in three antinociceptive tests (tail flick latency, hot plate method and acetic acid-induced writhing) in mice. In all three models, baclofen was found to elicit a dose-dependent antinociceptive effect. The observed antinociceptive effect was stereospecific, as the levo isomer of baclofen was found to be more potent than the racemic mixture. Baclofen also potentiated morphine analgesia. The antinociceptive effect of baclofen was reversed by both CGP 35348, a GABAB antagonist, and naloxone, an opioid antagonist, but not by bicuculline or picrotoxin, GABAA antagonists. However, in acetic acid-induced writhing, naloxone failed to reverse baclofen analgesia. The data suggest that the antinociceptive effect of baclofen is GABAB receptor-mediated and that there may be a GABAergic and opiate/or non-opiate interaction in eliciting the analgesic effect.

MPTP: a pharmacological tool to study parkinsonism.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is reported to selectively degenerate dopaminergic cells of the zona compacta of the substantia nigra and produce Parkinson's-like symptoms in man and animals. Although the exact mechanism of its neurotoxicity is not well defined, a major metabolite MPP+ may be responsible for the ultimate neuron toxicity. Several classes of drugs useful in Parkinson's disease are effective in reversing MPTP-induced neurotoxicity. Future pharmacological studies on the compound may unlock some of the mysteries of the age old disease, parkinsonism.

Altered response to GABAergic agents following electro and chemo convulsions in mice.

Effects of GABAergic agents and that of electroconvulsive shock (ECS) treatment were studied on bicuculline and picrotoxin (PTX)-induced convulsions in mice. Neither acute nor chronic ECS had any significant effect on bicuculline-induced convulsions, whereas the latency for PTX-induced convulsions was delayed by both acute and chronic ECS. Baclofen treatment delayed significantly the latency for PTX-induced convulsions in animals which were subjected to both acute and chronic ECS, whereas in bicuculline-induced convulsions, it shortened the latency of convulsions 24 hr after acute ECS. Progabide delayed the bicuculline-induced convulsions except in the case of 24 hr after acute ECS and PTX-induced convulsions except in the case of animals treated chronically with ECS. Fengabine showed no significant effect on bicuculline-induced convulsions. However, on PTX-induced convulsions, the latency was delayed in animals not subjected to ECS and in those subjected to chronic ECS. The possible explanations for the alterations in the effect of GABAergic agents following electro and chemo convulsions are (i) differences in the nature of antagonism by bicuculline and PTX, (ii) alterations in receptor sensitivity or number, and (iii) alterations in the levels of endogenous neurotransmitters, the latter two resulting as a result of acute or chronic ECS.

Effect of chronic treatment of Ro 15-1788 and its withdrawal on cortical and hippocampal EEG activity in rats.

Effect of chronic treatment with Ro 15-1788, a benzodiazepine (BZ) receptor antagonist, and its withdrawal, on the cortical and hippocampal electroencephalogram (EEG) was investigated in rats. Chronic treatment with Ro 15-1788 and its withdrawal (24 and 48 hr) were found to reduce the EEG amplitude in both cortical and hippocampal regions. This reduction in cortical and hippocampal EEG amplitude produced by chronic treatment with Ro 15-1788 and its withdrawal was reversed by gamma aminobutyric acid (GABA), pentobarbitone and picrotoxin, agents known to modulate the GABA/BZ synaptic events by acting at different sites on the complex. Baclofen a GABAB agonist and FG7142, a BZ inverse agonist were found to further reduce the EEG amplitude in the cortical and hippocampal regions of these rats, chronically treated with Ro 15-1788. Diazepam, a BZ agonist was found to have no significant effect on the alteration produced in the cortical and hippocampal EEG amplitude by chronic treatment with Ro 15-1788 or its withdrawal. It is suggested that the conformational changes produced on the GABA/BZ receptor complex by BZ receptor occupation, has a facilitatory effect on the actions of those drugs which act on the GABA/BZ receptor complex and the direction of this enhancement depended on the nature of the drug.

Multiple receptors involved in peripheral alpha 2, mu, and A1 antinociception, tolerance, and withdrawal.

We examined the interactions among three classes of peripherally-acting antinociceptive agents (mu-opioid, alpha 2-adrenergic, and A1-adenosine) in the development of tolerance and dependence to their antinociceptive effects. Antinociception was determined by assessing the degree of inhibition of prostaglandin E2 (PGE2)-induced mechanical hyperalgesia, using the Randall-Selitto paw-withdrawal test. Tolerance developed within 4 hr to the antinociceptive effect of the alpha 2-adrenergic agonist clonidine; dependence also occurred at that time, demonstrated as a withdrawal hyperalgesia that was precipitated by the alpha 2-receptor antagonist yohimbine. These findings are similar to those reported previously for tolerance and dependence to mu and A1 peripheral antinociception (Aley et al., 1995). Furthermore, cross-tolerance and cross-withdrawal between mu, A1, and alpha 2 agonists occurred. The observations of cross-tolerance and cross-withdrawal suggest that all three receptors are located on the same primary afferent nociceptors. In addition, the observations suggest that the mechanisms of tolerance and dependence to the antinociceptive effects of mu, A1, and alpha 2 are mediated by a common mechanism. Although any of the agonists administered alone produce antinociception, we found that mu, A1, and alpha 2 receptors may not act independently to produce antinociception, but rather may require the physical presence of the other receptors to produce antinociception by any one agonist. This was suggested by the finding that clonidine (alpha 2-agonist) antinociception was blocked not only by yohimbine (alpha 2-antagonist) but also by PACPX (A1-antagonist) and by naloxone (mu-antagonist), and that DAMGO (mu-agonist) antinociception and CPA (A1-agonist) antinociception were blocked not only by naloxone (mu-antagonist) and PACPX (A1-antagonist), respectively, but also by yohimbine (alpha 2-antagonist). This cross-antagonism of antinociception occurred at the ID50 dose for each antagonist at its homologous receptor. To test the hypothesis that the physical presence of mu-opioid receptor is required not only for mu antinociception but also for alpha 2 antinociception, antisense oligodeoxynucleotides (ODNs) for the mu-opioid and alpha 2C-adrenergic receptors were administered intrathecally to reduce the expression of these receptors on primary afferent neurons. These studies demonstrated that mu-opioid ODN administration decreased not only mu-opioid but also alpha 2-adrenergic antinociception; A1 antinociception was unaffected. In contrast, alpha 2C-adrenergic ODN decreased antinociception induced by all three classes of antinociceptive agents. In conclusion, these data suggest that peripheral antinociception induced by mu, alpha 2, and A1 agonists requires the physical presence of multiple receptors. We propose that there is a mu, A1, alpha 2 receptor complex mediating antinociception in the periphery. In addition, there is cross-tolerance and cross-dependence between mu, A1, and alpha 2 antinociception, suggesting that their underlying mechanisms are related.

Different mechanisms mediate development and expression of tolerance and dependence for peripheral mu-opioid antinociception in rat.

The mu-opioid [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) exerts a peripheral antinociceptive effect against prostaglandin E2 (PGE2)-induced mechanical hyperalgesia in the hindpaw of the rat. Tolerance and dependence develop to this effect. We have shown previously that tolerance and dependence can be dissociated and are mediated by different second messenger systems. In the present study, we evaluated whether the same or different second messenger systems mediate the development of this peripheral opioid tolerance or dependence compared with the expression of the loss of antinociceptive effect or rebound opioid antagonist hyperalgesia (i. e., expression of tolerance and dependence). DAMGO-induced tolerance was prevented by pretreatment with the nitric oxide synthase inhibitor NG-methyl-L-arginine (NMLA) but not by the protein kinase C (PKC) inhibitor chelerythrine, the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (ddA), or the calcium chelators 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8) and 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (Quin-2). Once established, however, expression of DAMGO tolerance was acutely reversed by TMB-8 or Quin-2 but not by chelerythrine or NMLA. In contrast, naloxone-precipitated hyperalgesia in DAMGO-tolerant paws, a measure of dependence, was blocked by pretreatment with chelerythrine but not by NMLA, ddA, TMB-8, or Quin-2. Naloxone-precipitated hyperalgesia in DAMGO-tolerant paws was acutely reversed by chelerythrine, ddA, TMB-8, or Quin-2 but not by NMLA. Taken together, these results provide the first evidence that different mechanisms mediate the development and expression of both tolerance and dependence to the peripheral antinociceptive effect of DAMGO. However, although the development of tolerance and dependence are entirely separable, the expression of tolerance and dependence shares common calcium-dependent mechanisms.

Role of protein kinase A in the maintenance of inflammatory pain.

Although the initiation of inflammatory pain (hyperalgesia) has been demonstrated to require the cAMP second messenger signaling cascade, whether this mechanism and/or other mechanisms underlie the continued maintenance of the induced hyperalgesia is unknown. We report that injection of adenylyl cyclase inhibitors before but not after injection of direct-acting hyperalgesic agents (prostaglandin E2 and purine and serotonin receptor agonists) resulted in reduction in hyperalgesia, evaluated by the Randall-Selitto paw-withdrawal test. In contrast, injection of protein kinase A (PKA) inhibitors either before or after these hyperalgesic agents resulted in reduced hyperalgesia, suggesting that hyperalgesia after its activation was maintained by persistent PKA activity but not by adenylyl cyclase activity. To evaluate further the role of PKA activity in the maintenance of hyperalgesia, we injected the catalytic subunit of PKA (PKACS) that resulted in hyperalgesia similar in magnitude to that induced by the direct-acting hyperalgesic agents but much longer in duration (>48 vs 2 hr). Injection of WIPTIDE (a PKA inhibitor) at 24 hr after PKACS reduced hyperalgesia, suggesting that PKACS hyperalgesia is not independently maintained by steps downstream from PKA. In summary, our results indicate that, once established, inflammatory mediator-induced hyperalgesia is no longer maintained by adenylyl cyclase activity but rather is dependent on ongoing PKA activity. An understanding of the mechanism maintaining hyperalgesia may provide important insight into targets for the treatment of persistent pain.