Thermal and Mechanical Hyperalgesia

Transcription

1 Focus Thermal and Mechanical Hyperalgesia A Distinct Role for Different Excitatory Amino Acid Receptors and Signal Transduction Pathways? s. T. Metier There are three steps in the revelation of any truth: in the first, it is ridiculed; in the second, it is resisted; in the third, it is considered self-evident. Schopenhauer ( ) The mechanisms underlying hyperalgesia have been intensively studied over the last decade using a variety of molecular, electrophysiological, and behavioral approaches. These experiments have led to the generalized scheme that persistent nociceptor input, as a result of tissue or nerve injury, removes a magnesium block on spinal NMDA receptors, allowing for calcium influx, translocation of protein kinase C, and activation of nitric oxide synthase and soluble guanylate cyclase as important central components of hyperalgesia. However, the hypothesis proposed here suggests a need for a critical reevaluation of the generalized applicability of these events to our understanding of the mechanisms of hyperalgesia. It is proposed that thermal and mechanical hyperalgesia rely on activation of two different intracellular cascades in the spinal cord. Thermal hyperalgesia relies principally on activation of spinal NMDA receptors, translocation of protein kinase C, and #'roductio, n of nitric oxide, and cgmp. In contrast, mechanical hyperalge~ia relies principal~ly on c/o~ctivation of spinal AI~PA and~ metabotropic glutamate receptors, activation of phospholipase A2 (PLA2), and production of cyclooxygenase products. Key words: persistent pain, hyperalgesia, NMDA, AMPA, metabotropic glutamate receptor From the Department of Pharmacology, University of Iowa, Iowa City, IA. Reprint requests: Stephen T. Meller, PhD, Department of Pharmacology, Bowen Science Building, College of Medicine, University of Iowa, Iowa City, IA p ersistent pain resulting from tissue injury is often associated with an altered sensitivity to cutaneous stimuli and can be manifest as hyperalgesia (increased sensitivity to noxious stimuli) and allodynia (nonnoxious stimuli perceived as noxious). 112 As a consequence of tissue injury, inflammatory mediators are released at the site of injury, resulting in sensitization of nociceptors. 38,~5 The increase in neuronal activity leads to a neuronal plasticity in the dorsal horn of the spinal cord, and these functional alterations contribute to the development of hyperalgesia and allodynia. 24 Although recent efforts to describe and define hyperalgesia indicate that excitatory amino acids are intimately involved in mechanisms that result in long term, use dependent changes in neuronal excitability in the spinal cord, 13'15,24,79,11 the receptor subtypes and intracellular cascade of events subsequent to receptor activation that result in hyperalgesia are still poorly defined. Even less well-defined are the similarities or differences, in the neurotransmitters and intracellular events involved in thermal and mechanical hyperalgesia. It is crucial to the effective treatment of chronic and persistent pain to have a better understanding of the mechanisms that underly the processing of different types of hyperalgesia. Therefore, this focus article will emphasize the role of excitatory amino acid receptor subtypes and different signal transduction pathways in thermal and mechanical hypera!gesia. Although they likely play a modulatory role, no attention will be given here to the involvement of peptides in mechanisms of hyperalgesia. APS Journal 3(4): ,

2 21 6 FOCUS/Meller HYPERALGESIA: THE CURRENT GENERAL VIEW It has been proposed that after tissue or nerve injury, a barrage of C-fiber discharges releases glutamate and neurokinins (and other substances) from primary afferent terminals, leading to fast synaptic potentials produced by actions in non-nmda receptors (i.e., Na+-permeable AMPA/KA receptors; el-amino- 3 - hydroxy methyl - isoxazole proprionate/ kainate) and slow synaptic potentials produced by continuous depolarization and the release of peptides (e.g., substance P; calcitonin gene-related peptide, CGRP). As a consequence, a voltage-dependent magnesium block on NMDA receptors is removed allowing for an influx of calcium that triggers a cascade of intracellular events and activation of immediate-early genes leading to the development and maintenance of hyperalgesia. There have been many electrophysiological, molecular, and complementary behavioral studies over the last decade or so that have contributed to this current view on the mechanisms of hyperalgesia, 79,11,112-1~ While the electrophysiological and molecular studies have provided valuable information concerning cellular mechanisms, the functional interpretation of these results has been aided by behavioral studies where the expression of hyperalgesia in intact experimental animals can be examined. We have chosen to study hyperalgesia in awake animals by relying on the collective information gathered from two different approaches which, in general, has not been done. First, excitatory amino acid receptor agonists have been administered to the intrathecal space to determine which agonists, or combination of agonists, are able to produce an acute thermal or mechanical hyperalgesia, or both. The second approach has involved administration of antagonists at these excitatory amino acid receptors to experimental animals with persistent pain. This aspect of the approach has allowed for a determination of which receptors are involved in persistent thermal or mechanical hyperalgesia, or both. Using a combination of those two approaches, we will likely come closer to a meaningful functional interpretation of the electrophysiological and molecular findings. Further, those two approaches will allow a determination of which excitatory amino acid receptors are likely necessary and sufficient to produce a thermal or mechanical hyperalgesia, or both; and what other excitatory amino acids (or peptides) might be able to potentiate or modulate the hyperalgesia. This article will focus on mechanisms and mediators of thermal and mechanical hyperalgesia in the spinal cord. However, there has been a greater consideration of mechanisms of thermal hyperalgesia compared to mechanical hyperalgesia. Consequently, we know very little about the spinal mechanisms and mediators of mechanical hyperalgesia. This is likely due to two principal reasons. First, there is no agreement on what constitutes a valid and reliable stimulus to examine and test mechanical withdrawal thresholds. Second, is the underlying assumption that the mechanisms of both thermal and mechanical hyperalgesia might be similar. This unsubstantiated assumption, and the lack of information about mechanisms of mechanical hyperalgesia prompted us to initiate a series of experiments leading to evidence that the receptor subtypes and signal transduction systems for thermal and mechanical hyperalgesia in the spinal cord are not the same. HYPOTHESIS The working hypothesis, simply stated, is that the mechanisms underlying thermal and mechanical hyperalgesia rely on two divergent and different systems. While differences may exist at all levels, this article will focus on the differences in the spinal cord. In general, thermal hyperalgesia relies principally on activation of spinal NMDA receptors, activation of protein kinase C (PKC), and production of nitric oxide (NO) and cgmp; mechanical hyperalgesia relies principally on coactivation of spinal AMPA and metabotropic glutamate receptors, activation of phospholipase A2 (PLA2), and production of cyclooxygenase (COX) products. This hypothesis necessitates viewing the nervous system as a dynamic entity that is able to summate and differentiate information at many different sites from the peripheral receptor to the gene. In general support of this hypothesis: there are different receptors 2 and transduction mechanisms 9,4 for thermal and mechanical stimulation;somatic afferents do not respond or encode thermal and mechanical stimuli in the same manner; 9,4 all neurons in the spinal cord do not respond to both thermal and mechanical stimuli; 22,23 only 35% of spinal cord neurons respond to glutamate but 75% respond to NMDA, 87 suggesting there are glutamate insensitive neurons that are activated by an endogenous substrate that is not glutamate; selective endogenous NMDA and metabotropic glutamate receptor agonists are released from neural tissue; 18,89 different neurons have different glutamate receptors; 3,4 NMDA, AMPA, and metabotropic glutamate receptors are not ubiquitously distributed, but are predominantly localized to the dorsal horn; 3,31,43,65,

3 FOCUS/Meller 217 8s.97 Nitric oxide synthase (NOS) positive cells are not evenly distributed, but are predominantly distributed in the dorsal horn and around the central canal; 27,1 8 NMDA and non-nmda receptors occur together or individually as a homogeneous population at synaptic sites; 3,6,~9 a single neuron can have concentrated Iocalizations of a single receptor subtype at different places along the dendrite and soma; 3,4 intracellular calcium concentration at a synapse is greater than in other regions of the same neuron, 1 9 suggesting that microdomains in calcium concentration changes may occur; different glutamate receptor agonists result in activation of different intracellular enzymes, ~7,96 possibly as a result of these calcium microdomains; intracellular enzyme activation can selectively modulate different glutamate receptors; ~,6~ different glutamate receptor agonists induce gene expression by different routes of calcium entry; 53 high-frequency activation of C-fibers, a basic requisite for hyperalgesia, likely leads to activation of metabotropic glutamate receptors in the central nervous system.~ 2,35,117 Collectively, these data support the hypothesis that different coded afferent impulses (from thermal or mechanical stimuli) might activate different receptors in a spatial and temporal manner that cause the differential release of calcium and activation of different signal transduction mechanisms, second messenger systems, and immediate-early genes. For example, afferents might terminate on neurons with only NMDA receptors or on neurons with only AMPA and metabotropic receptors. Conversely, they might terminate on neurons that contain all of the different receptor subtypes, but on regions where there is a dense accumulation of a single receptor subtype. "By controlling the types of channels, their density and distribution on the surface, neurons generate a varied repertoire of electrical signals in different cells and in different parts of the same cell. These signals give neurons the complexity and flexibility that they need as the cellular components of the large signaling system that is our brain. ''37 EVIDENCE IN SUPPORT OF THE HYPOTHESIS Thermal Hyperalgesia Which Receptor Subtypes Are Involved? There is a large literature that suggests that nociceptive reflexes and responses of spinal dorsal horn neurons to noxious thermal stimuli are largely unaffected by NMDA receptor antagonists, but NMDA receptor antagonists block facilitation of these re- sponses to noxious thermal stimuli. 24,79 While many of these studies have determined that the maintenance of thermal hyperalgesia is dependent on NMDA receptor activation, fewer studies have addressed the role of excitatory amino acids other than NMDA in development of thermal hyperalgesia. Of these, Mao et al. 63 have reported that the development of thermal hyperalgesia in a model of neuropathic pain is dependent on activation of both non- NMDA and NMDA receptors while maintenance was dependent only on activation of NMDA receptors. However, the extent of the involvement of non-nmda receptors is still not clear as the doses of the non- NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) that were used in that study were sufficiently great enough to act at the glycine site on NMDA receptors. 46,54,91 While it is clear that activation of NMDA receptors are intimately involved in development and maintenance of thermal hyperalgesia, other than the report by Mao et al., 63 there have been relatively few studies that have addressed the possible contribution of other excitatory amino acid receptors in thermal hyperalgesia. Ren et al. 93 reported that the thermal hyperalgesia produced by intraplantar carrageenan was blocked by NMDA receptor antagonists, but was not affected by CNQX until very high doses were used. This supports a role for only NMDA receptor activation in thermal hyperalgesia. Complementary data suggests that only NMDA receptors and not AMPA receptors are involved in the heat hyperalgesia found after thermal injuryj 4 These data are consistent with recent findings that activation of AMPA (with AMPA), metabotropic (with trans-l-amino-l,3- cyclopentane-dicarboxylate, trans-acpd), or coactivation of AMPA and metabotropic (AMPA + trans- ACPD) receptors were not sufficient to produce an acute thermal hyperalgesiaz 8 Only activation of NMDA receptors produces a dose-dependent acute thermal hyperalgesia 28,48,49,73,78 although the affects of NMDA could be enhanced by trans-acpdz 8 These data are supported by the observations of Cerne and Randic 1~ where the cellular responses to NMDA in spinal cord slices can be potentiated by trans-acpd. Which Intracellular Events Are Involved? Nitric Oxide. Several recent studies have demonstrated that NO, like NMDA receptors, plays a pivotal role in mechanisms of thermal hyperalgesia produced by acute administration of NMDA 47,~,73,8,81 or the persistent NMDA-mediated thermal hyperalgesia produced in models of peripheral inflammationz 2,82

4 218 FOCUS/Meller Soluble Guanylate Cyclase (GC-S). One of the major targets of neuronal NO is activation of a soluble form of guanylate cyclase. 99 As such, it has been reported that the GC-S inhibitor methylene blue blocks the acute thermal hyperalgesia produced by intrathecal NMDA 73 as well as the persistent thermal hyperalgesia produced in a model of neuropathic pain. 82 It has been suggested that NO must leave the neuron where it is produced and travel extracellularly to activate GC-S in an adjacent structure, as the increase in intracellular content of calcium following NMDA receptor activation results in activation of a cgmp phosphodiesterase that inhibits cgmp accumulation. 7 The effect of hemoglobin on blocking thermal hyperalgesia 28,47,7a,81 is consistent with this hypothesis as hemoglobin is unable to cross cell membranes. Recently, however, is has been suggested that methylene blue is also able to inactivate the neuronal form of NOS, 69 clouding interpretation of results obtained with methylene blue. Nevertheless, a recent report has shown that cgmp is produced in rat spinal cord following acute administration of NMDA, 32 which supports observations that GC-S and cgmp are involved in thermal hyperalgesia. Protein Kinase C. Because NMDA receptor activation has been shown to result in activation and translocation of PKC from the cytosol to the membrane, 1 7 recent investigations have examined the role of PKC in NMDA-mediated nociceptive responses. For example, Mao and colleagues 39,8~-64 have reported that treatment with GM1 gangliosides attenuates the thermal hyperalgesia produced in a model of a peripheral nerve injury. One of the actions of GM1 gangliosides is to prevent membrane translocation of PKC. 1 7 In addition, Meller et al. have reported that a selective PKC inhibitor blocks the acute 78 and persistent 76 thermal hyperalgesia produced by NMDA administration or intraplantar zymosan, respectively. Phospholipase A2. While it is clear that activation of NOS, PKC, and GC-S are involved in acute NMDAproduced thermal hyperalgesia and persistent NMDA-mediated thermal hyperalgesia, the evidence reported for other signal transduction systems in thermal hyperalgesia is less clear. While activation of PLA2 has been linked to NMDA receptor activation, 1 4 mepacrine had no effect on thermal withdrawal latencies, 78 acute thermal hyperalgesia produced by NMDA, 78 or the persistent thermal hyperalgesia produced by intraplantar zymosan. 76 Collectively, these data suggest that activation of PLA2 is not involved in thermal hyperalgesia. Ecoisanoids. One of the major actions of PLA2 is to produce arachidonic acid (AA) 2s and presumably prostaglandins and leukotrienes as enzymatic products of AA. However, PLA2 does not appear to be involved in thermal hyperalgesia. 76,78 In support of this, the acute thermal hyperalgesia found after intrathecal NMDA is not affected by the selective lipoxygenase (LOX) inhibitor baicalein or the selective COX inhibitor indomethacin. 78 In addition, the persistent thermal hyperalgesia produced by intraplantar zymosan is unaffected by baicalein or indomethacin. 76 In contrast to these results, Malmberg and Yaksh 59 demonstrated that intrathecal administration of high doses of either AMPA, NMDA, or substance P produced a delayed (30 minute) and prolonged thermal hyperalgesia after an intense caudally directed biting and scratching behavior had resolved. This prolonged thermal hyperalgesia was attenuated by COX inhibitors. As PLA2 is not involved in thermal hyperalgesia, the attenuation of the NMDA-produced thermal hyperalgesia observed by Malmberg and Yaksh s9 following COX inhibitors suggest that prostaglandins might be liberated through an as yet to be described non-pla2 mechanism. Summary In short, the current literature indicates that activation of NMDA receptors are intimately involved in thermal hyperalgesia. There is little or no evidence in support of an obligatory role for other excitatory amino acid receptors in thermal hyperalgesia, although activation of metabotropic glutamate receptors may potentiate an NMDA-mediated thermal hyperalgesia (Figs. 1,2). There has only been a limited amount of information published to date with respect to the intracellular events associated with thermal hyperalgesia. However, as with NMDA receptors, the evidence is generally internally consistent except for the evidence on a role for ecosanoids. That is, production of NO and cgmp and activation and translocation of PKC are involved in acute and persistent thermal hyperalgesia. Mechanical Hyperalgesia Which Receptor Subtypes Are Involved? Very little is known about the excitatory amino acid receptor subtypes involved in mechanical hyperalgesia. Recent studies suggest that coactivation of AMPA and metabotropic receptors are involved in mechanical hyperalgesia. 74,7s,8 Although these initial results related directly to an acute, short-term mechanical hyperalgesia, the same mechanisms may

5 FOCUS/Meller 219 NO Ca 2+ D, j ; -... GC-S m 9 cgmp o L-arginine Figure 1. The proposed mechanisms for thermal hyperalgesia. Persistent nociceptor input as a result of tissue injury allows for a magnesium block on NMDA receptors to be removed, an influx of calcium, activation and translocation of protein kinase C (PKC) and/or activation of nitric oxide synthase (NOS), allowing for a production of nitric oxide [NO) from L-arginine, activation of a soluble form of guanylate cyclase [GC-S), and production of cgmp. However, it is not known whether PKC activation allows for phosphorylation of NOS [there are phosphorylation sites}. As NO is able to act on other systems, such as ADP-ribosylation, these also are possibilities in mechanisms of thermal hyperalgesia. In addition, NO must likely leave the neuron in which it is produced as hemoglobin abolished thermal hyperalgesia. also be important for mechanisms of persistent mechanical hyperalgesia. For example, Dougherty et al. 23 demonstrated that the sensitization of spinothalamic tract neurons to mechanical stimulation after intraarticular injection of carrageenan was enhanced by non-nmda receptor agonists such as quisqualate (QA) (which acts at both AMPA and metabotropic glutamate receptors); NMDA was without affect. In addition, Tal and Bennett ~ 3 have reported that the NMDA receptor antagonist dextrorphan abolished thermal but not mechanical hyperalgesia in a model of neuropathic pain. Consistent with this, Rueff et a124 have reported that the thermal hyperalgesia but not the mechanical hyperalgesia found following nerve growth factor treatment is blocked by MK-801. Further consistent with a role for AMPA and metabotropic receptors, and a lack of effect of NMDA receptors in mechanical hyperalgesia, is that the mechanical hypersensitivity found following spinal cord ischemia is abolished by AMPA but not NMDA receptor antagonists. 116 Activation of AMPA and metabotropic glutamate receptors also appear to be necessary for the persistent mechanical hyperalgesia produced by intraplantar zymosan. 76 A modulatory role for NMDA receptors is also suggested as the persistent mechanical hyperalgesia found after intraplantar zymosan was attenuated by the NMDA antagonist MK-801. TM Similar results have recently been described where the mechanical hyperalgesia produced by carrageenan was attenuated but not blocked by either MK-801 or AP5. 92 Therefore, while it has been shown that NMDA receptor activation alone is not sufficient to produce an acute mechanical hyperalgesia, 74,7S,8 it is possible under some circumstances, and not others, that NMDA receptor activation may enhance the effects of coactivation of AMPA and metabotropic glutamate receptors in producing mechanical hyperalgesia. That is, NMDA receptor activation may allow for an AMPA and metabotropic glutamate receptor-mediated mechanical hyperalgesia. The data suggesting an interaction between AMPA and metabotropic glutamate receptors are supported by several electrophysiological studies that have revealed a cooperativity between these two receptor subtypes on spinal cord neurons. TM In a study by Bleakman et al., 7 the metabotropic glutamate receptor agonist 1S,3R-ACPD enhanced the responses to AMPA, KA, and NMDA. In a study by Cerne and Randic, 1~ trans-acpd potentiated the responses of both NMDA and AMPA suggesting that metabotropic receptors may contribute to the strength of primary afferent nociceptive transmission. This supports our observations that AMPA + trans-acpd produce an acute mechanical hyperalgesia (NMDA + trans- ACPD did not 7S) and that trans-acpd potentiates an NMDA-produced acute thermal hyperalgesia. Therefore, the effects of metabotropic receptor activation on different excitatory amino acid receptors may be expressed in a functionally different manner. There also is anatomical and immunocytochemical evidence to support a role for AMPA and metabotropic receptor subtypes in spinal nociceptive mechanisms. While one recent immunocytochemical report showed that metabotropic glutamate receptors were relatively evenly spread throughout the gray matter of the spinal cord, 98 there are several reports that show differential distribution in the

6 220 FOCUS/Meller DAG I Ca 2+ I thermal hyperalgesia C Ca2+ camp kina~ / [ thermal hyperalgesia Figure 2. Two of the possible mechanisms where activation of metabotropic receptor activation may potentiate NMDAmediated thermal hyperalgesia. The upper panel suggests that activation of a metabotropic glutamate receptor might result in a phospholipase C (PLC)-mediated production of diacylglycerol (DAG) that in combination with a rise in intracellular calcium activates protein kinase C resulting in thermal hyperalgesia. In the lower panel, activation of a metabotropic glutamate receptor might result in an adenylate cyclase-mediated production of camp leading to activation of protein kinase A (PKA) and phosphorylation of a calcium-permeable NMDA receptor allowing for an influx of calcium and thermal hyperalgesia. spinal cord. 3,43,88 For example, QA-sensitive binding (to mglur) was predominantly localized in lamina II of the spinal cord 43 and mglur1 receptors have been localized predominantly to the dorsal horn of the spinal cord; 3 there appear to be no mglur2 receptors in the spinal cord. 88 In addition to a differential localization of metabotropic glutamate receptors, AMPA receptor subunits have also been shown to be differentially distributed. 3i'65'1 s For example, a recent immunocytochemical study showed that the distribution of several AMPA subunits (GluR1, GluR2/ 3/4c, and GluR4) are very different in the dorsal horn of the spinal cord. 65 An additional study has suggested that GluR2 showed the strongest expression in lamina II and III where GluR4 levels are low. 31 Further, a study by Tolle et al. 1 5 has reported that the distribution of GluR2 is highest in lamina II and III, while GluR3 and GluR4 is greatest on large neurons in lamina IV where GluR2 expression is low. Whether these differences in receptor localization implies any functional difference or if there are any differences in their pharmacological profiles remains to be seen. However, it is clear that homomeric or heteromeric combinations that include the GluR2 subunit allow free permeability to monovalent ions such as sodium (as has been reported for receptors involved in fast synaptic transmission in the spinal cord), but homomeric or heteromeric combinations that include GluR1, GluR3, and/or GluR4 (but not GluR2) all allow free permeability to divalent cations such as calcium. 1 We have previously suggested that the fast excitatory AMPA/KA receptors that are responsible for spinal cord transmission are different from those AMPA receptors that are involved in mechanical hy-

7 FOCUS/Meller 221 peralgesia (sodium versus calcium permeability?). 74 This hypothesis has not yet been examined in electrophysiological studies in the spinal cord. However, the immunocytochemical experiments reported by Tolle et al. 1 ~ suggest that Na+-gated AMPA receptors might be more numerous in lamina II while calcium-gated AMPA receptors might be more predominant in lamina IV. Coactivation of two receptor subtypes to produce a physiological or pathological event is an unusual, but not unprecedented occurrence in the central nervous system. However, the mechanism(s) by which an iontropic glutamate receptor and a metabotropic G-protein-linked glutamate receptor interact is not clear, although there are a number of possibilities. As recent evidence suggests that some AMPA-sensitive receptors are able to gate calcium, 1 and as an increase in intracellular calcium is important to sustained effects (i.e., hyperalgesia), 7~'83 it is possible that metabotropic receptor activation allows for a direct membrane-delimited action where activation of a G-protein directly controls how an ion channel (i.e., AMPA ) gates. This has only recently been suggested as a mechanism for signal transduction 68 and has not been directly examined in the spinal cord. Alternatively, there may be indirect cytoplasmic-mediated effects where activation of a G-protein results in production of a second messenger that phosphorylates an ion channel (i.e., AMPA), resulting in activation. Through either of these two mechanisms, activation of a metabotropic glutamate receptor leading to activation of a calcium-permeable AMPA receptor would allow for an influx of calcium and a cascade of intracellular events giving rise to mechanical hyperalgesia. In support, it has been shown that there are multiple phosphorylation sites on AMPA receptors 4~ but whether they exist on calcium-permeable AMPA receptors remains to be determined. However, it has been reported that activation of the mglur1 receptor (localized to the entire dorsal horn of the spinal cord) results in production of camp 9s which, in turn is able to activate camp-dependent kinases. The camp-dependent kinases have been shown to potentiate the effects of non-nmda receptors, likely through phosphorylation. 4S This may, therefore, be a mechanism whereby metabotropic glutamate receptor activation results in activation of a calcium-permeable AMPA receptor. However, a recent study has shown that the effects of metabotropic glutamate receptors on potentiating the effects of AMPA in the spinal cord where short-lived and were not dependent on an increase in intracellular calcium, z Therefore, it is possible that the influx in calcium also results in a sus- tained release of calcium from ryanodine-sensitive calcium stores as has been shown for other regions of the central nervous system. 83 For example, activation of the metabotropic receptor has been shown to result in release of calcium from inositol 1,4,5-trisphosphate (IP3)-sensitive calcium stores that may also cause further release from ryanodine-sensitive stores. 83 Which of these two potential mechanisms (direct membrane-delimited or indirect cytoplasmic-delimited), if either, exist for neurons in the spinal cord remains to be determined. The available behavioral data suggests that activation of both AMPA and metabotropic receptors may result in a sustained elevation of intracellular calcium and the potential for sustained physiological or pathophysiological effects such as hyperalgesia. What Intracellular Events Are Involved? NOS, GC-S, and PKC. Production of NO, cgmp, and activation of PKC have all been suggested and shown to be involved in thermal hyperalgesia. In contrast, these pathways have, in general, been shown not to be involved in the behavioral expression of mechanical hyperalgesia. 72,76,77,8,81 For example, acute mechanical hyperalgesia produced by coactivation of AMPA and metabotropic glutamate receptors is unaffected by intrathecal doses of L-NAME or methylene blue that completely block thermal hyperalgesia. 73,77 In addition, the persistent mechanical hyperalgesia produced by the intraplantar injection of zymosan 76 or carrageenan 72 is also unaffected by intrathecal L- NAME, hemoglobin, or methylene blue (these drugs do however, attenuate thermal hyperalgesia ). While activation and translocation of PKC is involved in thermal hyperalgesia, 28,62,7~ administration of selective PKC inhibitors do not alter the acute mechanical hyperalgesia produced by coactivation of AMPA and metabotropic glutamate receptors 78 or the persistent mechanical hyperalgesia produced by the intraplantar injection of zymosan. 76 These data indicate that production of NO, cgmp, and activation and translocation of PKC are not necessary for the behavioral expression of mechanical hyperalgesia. Phospholipase C (PLC). While activation of NOS, GC-S, and PKC do not appear to be involved in mechanical hyperalgesia, other signal transduction systems linked to activation of the metabotropic receptor such as PLC 29'96 have been studied. However, the PLC inhibitor neomycin did not affect the acute mechanical hyperalgesia produced by coactivation of AMPA and metabotropic glutamate receptors. 7z Neomycin was also found to be without affect on the

8 222 FOCUS/Meller persistent mechanical hyperalgesia produced by the intraplantar administration of zymosan. 76 Phospholipase A2. It has recently been shown that coactivation of AMPA and metabotropic glutamate receptors are able to produce AA through activation of PLA2. 2S Meller et al. 77 have shown that the PLA2 inhibitor mepacrine blocked the acute mechanical hyperalgesia produced by coactivation of AMPA and metabotropic glutamate receptors. Further, the persistent mechanical hyperalgesia produced by intraplantar zymosan was completely blocked by mepacrine. 76 The reports that both AMPA 1 '66'67 and metabotropic glutamate receptor binding ~ are selectively increased by PLA2 activation further support the hypothesis that there is a cooperativity between metabotropic glutamate receptors and AMPA receptors. In addition, PLA2 activation has been shown to enhance excitatory amino acid release and AMPAstimulated calcium influx. 5 These feedforward mechanisms are important to synaptic plasticity as an increased sensitivity to AMPA contributes to long-term potentiation (LTP). 2,44 This is supported by evidence in the spinal cord that there is a preferential increase in sensitivity of spinothalamic tract neurons to non- NMDA agonists such as AMPA that parallels the increase in mechanical sensitivity following kaolin and carrageenan-produced inflammation. 23 While NMDA receptor activation is able to activate PEA226'1 4 and produce AA, 5 -s2 it is unlikely that this pathway is involved in mechanical hyperalgesia as NMDA receptor activation is not a necessary requirement for mechanical hyperalgesia. Activation of the NMDA receptor may, however, play a supportive role and contribute to the potentiation of AMPA and metabotropic glutamate receptor-mediated mechanical hyperalgesia as NMDA receptor antagonists reduce the mechanical hyperalgesia produced by the intraplantar injection of zymosan. 76 Ecoisanoids. One of the major actions of PLA2 is to produce AA 34 and it has been recently suggested that AA may be involved in mechanisms of synaptic plasticity. $7 For example, it has been found that administration of exogenous AA induces a delayed LTP ~ and inhibitors of AA synthesis (and of PLA2) block the induction of LTP. s8 In addition, induction of LTP has been reported to be accompanied by an increase in the extra- and intracellular content of AA 8'57 and AA has been found to potentiate the release of glutamate. 21 Previous evidence has suggested that both prostaglandins and leukotrienes may play a role in hyperalgesia. 28,59,75-77,84,1 2,1 6 For example, the intrathecal administration of PGE2 produces a dose-dependent hyperalgesia in both thermal (tail-flick) and mechanical (RandalI-Selitto) nociceptive tests, 1 2 while PGD2 produces thermal hyperalgesia (mechanical was not tested). 1 6 In addition, a recent report suggests that intrathecal administration of PGF2, produces spontaneous agitation and touch-evoked agitation indicative of mechanical allodynia and hyperalgesia. 84 However, it is not at all clear whether there is modality-specificity for different metabolites of AA. Recent reports have suggested that prostaglandins are particularly important for the mechanisms that underlie both acute and persistent mechanical hyperalgesia 75,77 while they may not play any significant role in acute or persistent thermal hyperalgesia. 28,7e These results are in contrast to the effect of indomethacin on the prolonged thermal hyperalgesia produced by intrathecal administration of either AMPA, NMDA, or substance P that was reported by Malmberg and Yaksh, 59 although in that study they did not examine the acute thermal hyperalgesia produced by NMDA. They studied the effect of indomethacin on a late onset hyperalgesia produced following resolution of an intense biting and scratching behavior produced by intrathecal administration either NMDA, AMPA, or substance P; the behavior and hyperalgesia observed were the same with all three agonists. Summary There is little evidence on the mechanisms that results in behavioral expression of mechanical hyperalgesia. However, recent evidence suggests that coactivation of AMPA and metabotropic glutamate receptors are both necessary and sufficient for mechanical hyperalgesia (Figs. 3, 4). There appears to be little evidence in support of an obligatory role for NMDA receptors although they may potentiate AMPA and metabotropic glutamate receptor-mediated mechanical hyperalgesia (Figs. 3, 4). In addition, activation of PLA2 appears to be the major signal transduction pathway leading to production of ecoidanoids. There is no apparent behavioral evidence of a role for PKC, NOS, or GC-S in mechanical hyperalgesia. SPECULATION ON THE HYPOTHESIS The previous sections have detailed the direct and indirect experimental evidence in support of the hypothesis that there are two different pathways for thermal and mechanical hyperalgesia. If one accepts this evidence, then the functional implications of this hypothesis are far-reaching.

9 FOCUS/Meller AA m Ca2+ I lipo / ; \ PGs? LTs PI Figure 3. The proposed mechanisms that are both necessary and sufficient to produce mechanical hyperalgesia. High-frequency stimulation as a result of tissue injury allows for activation of a metabotropic receptor leading to activation of a calcium-permeable AMPA receptor, an infux of calcium and activation of a soluble form of phospholipase A2 (PLA2), generation of arachidonic acid (AA), and activation of cyclooxygenases (COX) and lipoxygenases (LOX) to produce prostaglandins (PGs) and leukotrienes (LTs), respectively. Convergence or Divergence in Mechanisms of Thermal and Mechanical Hyperalgesia It is interesting to contrast the receptor subtypes and intracellular events involved in both thermal and mechanical hyperalgesia as there appear to be different signaling systems in the spinal cord responsible for each (Fig. 5). For example, both require activation of a glutamate receptor subtype that is likely inactive under normal conditions (NMDA versus metabotropic or AMPA, or both), an increase in intracellular calcium (through different channels), activation of a primary effector enzyme (PKC versus PLA2), activation of a secondary effector enzyme (NOS versus COX), and generation of a labile intra- and intercellular messenger (NO versus AA). If the receptor subtypes and intracellular signaling mechanisms for thermal and mechanical hyperalgesia are different, as suggested by the present data, then how do neurons in the spinal cord differentiate and transduce the information? The answer may lie in viewing the nervous system as a dynamic entity that is able to adjust to different inputs that involve different regulatory mechanisms depending on the information to be processed. Certainly, different afferents may be involved. In addition, for hyperalgesia to develop there must be a critical threshold of stimulus strength and synapses must be activated at a relatively high frequency. As such, it has been shown that the voltage-gated NMDA receptor is inactive at resting membrane potential 16 but becomes active after high-frequency stimulation. 16,113,~4 This has been proposed to be a mechanism that is responsible for NMDA-mediated thermal hyperalgesia. However, if activation of the NMDA receptor alone cannot produce mechanical hyperalgesia, is there an analagous mechanism for mechanical hyperalgesia, or do different neurons in the spinal cord transduce the different messages? There are two general possibilities (Fig. 6). However, it is not clear which is the most likely. First, there may be different subsets of neurons involved in hyperalgesia that are distinct from those involved in normal sensation (Fig. 6). The subset of neurons involved in hyperalgesia would be able to show changes (i.e., plasticity) that would be reflected in an increased sensitivity to thermal stimuli, mechanical stimuli, or both. Alternatively, there may be different subsets of one overall class of neurons that can show an increased sensitivity to thermal stimuli, mechanical stimuli, or both (Fig. 6). That is, under normal conditions, the same neuron would be able to respond to thermal or mechanical stimuli. After injury and development of hyperalgesia, the neuron would still be able to respond to both stimuli but the increased sensitivity that the neuron would show would depend on receptor density, receptor location, and intracellular activity of that neuron. Which of these alternatives, if either, are correct is not known. It might be interesting to speculate that if a neuron had a greater proportion of receptors important for production of mechanical hyperalgesia (calciumpermeable AMPA and metabotropic receptors) than those associated with thermal hyperalgesia (NMDA receptors), that neuron may show an increase in mechanical sensitivity (and vice versa for NMDA and thermal stimuli). Alternatively, if a neuron had similar numbers of each receptor subtype but they were activated by different afferents to a differing degree, then

10 224 FOCUS/Meller f Ca2+ IP3 CaM kinase~ ~r IP4 campp!a~kina~// Ca2+ mechanical hyperalgesia Figure 4. One of the possible mechanisms whereby coactivation of metabotropic receptors and AMPA receptors could produce mechanical hyperalgesia. It has been shown that protein kinase A (PKA), produced by actions of a camp-dependent kinase, following activation of metabotropic glutamate receptors, is able to increase the opening time of non-nmda receptors (AMPA?). 36 Therefore, camp produced by metabotropic glutamate receptors (mglur1) might be able to activate AMPA receptors through a cytoplasmic affect to produce mechanical hyperalgesia; a membrane-delimited effect has not been ruled out. In addition, activation of AMPA receptors is able to inhibit IPa accumulation by increasing its conversion to IP4, by allowing influx through L-type voltage-sensitive calcium channels that activates a calmodulin kinase; this does not require protein kinase C. ~6 Activation of AMPA receptors may be able to inhibit the effects of metabotropic receptor activation that are not involved directly in mechanical hyperalgesia. Further, it has been shown that activation of trans- ACPD-sensitive metabotropic glutamate receptors are able to inhibit N-type calcium channels by a membrane-delimited effect; 1 1 this may be an additional mechanism whereby the calcium-mediated intracellular signal produced by actions at calcium-permeable AMPA receptors can be focused. thermal mechanical + r~/ I ca~- ~r, ~KC / _ ~... Soo ~a" "? guanine nucleotides NO L-arginine PI AA? cgmp? PGs LTs O Figure 5. Proposed general scheme of receptor-mediated and intracellular cascade of events resulting in thermal and mechanical hyperalgesia, and the potential interactions between the systems responsible for thermal and mechanical hyperalgesia that might possibly exist in a single neuron. The basic mechanisms of thermal and mechanical hyperalgesia are shown in Figures 1 and 3, respectively. In addition, the potential interactions between receptor subtypes for thermal and mechanical hyperalgesia are illustrated in Figures 2 and 4, respectively.

11 FOCUS/Meller 225 Figure 6. The two general possibilities that may exist in the spinal cord. The upper panel suggests that there may be two general and distinct groups of neurons. The left-hand neuron is a hard-wired neuron that responds to stimuli with a reflex output. This neuron is not able to be sensitized. The right-hand neuron represents a general group of neurons that are able to demonstrate synaptic plasticity and able to be sensitized but do not respond well to normal inputs. In this group, neurons may be able to be sensitized to thermal stimuli, mechanical stimuli, or possibly both. The lower panel represents the situation where all neurons are capable of synaptic plasticity and therefore hyperalgesia. As such, neurons may be sensitized to thermal stimuli, mechanical stimuli, or possibly both as well as reflex responses. There is no requirement that these neurons be the same or in the same location but the transduction system must be different for each. the increase in sensitivity of a neuron might depend on afferent strength and synaptic location. Recent support for this aspect of the hypothesis comes from studies that shows that different glutamate receptor subtypes have differential Iocalizations with differential densities on the same neuron. 3,4 In addition, C-thermoreceptors and A~mechanoreceptors may signal and synapse on different neurons or in different regions of the spinal dorsal horn. This suggests that all neurons are not the same in terms of receptor subtypes, density, and location of receptors. An additional requisite for the hypothesis is that AMPA or metabotropic glutamate receptors, or both are inactive (or in a different state) at resting membrane potential but are able to become activated with high-frequency stimulation. It is not clear whether the AMPA receptor is activated and allows the metabotropic receptor to become active or vice versa. However, one plausible suggestion is that metabotropic receptors becomes active after high-frequency stimulation, activates the AMPA receptor, and allows the AMPA receptor to gate calcium, resulting in mechanical hyperalgesia either by a membrane-delimited effect or a cytoplasmic effect. In support, it has been shown that metabotropic receptors in the hippocampus, 12 dorsolateral septal

12 226 FOCUS/Meller nucleus,~ 17 and the nucleus of the solitary tract 35 are activated by high-frequency stimulation. As high-frequency stimulation is a requisite for mechanisms of synaptic plasticity such as LTP 16 and hyperalgesia, ~2 activation of the metabotropic receptor, in an analogous manner to activation of NMDA receptors and production of thermal hyperalgesia, might initiate a different cascade of events that results in mechanical hyperalgesia by allowing either direct or indirect effects on the AMPA receptor. Another alternative would be that all neurons contain similar numbers of receptors that are activated equally and the intracellular cascade for thermal and mechanical hyperalgesia take place inside a single neuron. However, under these circumstances there would be no specificity. Therefore, to focus the increased sensitivity of a neuron on one modality, it would be necessary for the cascade of intracellular events involved with mechanical hyperalgesia to inhibit the events involved with thermal hyperalgesia and vice versa. In this case, the final output of a sensitized neuron would be focused on one stimulus modality, although the neuron would still be able to respond to both stimuli and also to iontophoretic application of different agonists. This hypothesis would fit with much of the available electrophysiological evidence. Therefore, if a single neuron were to be activated under high-frequency stimulation, NMDA receptors might initiate one cascade of intracellular events while activation of metabotropic receptors would initiate a different cascade of intracellular events in the same neuron. It appears that the intracellular mechanisms associated with thermal hyperalgesia may be able to inhibit those involved with mechanical hyperalgesia, and vice versa. For example, activation of PKC, which is important for production of thermal hyperalgesia, has been shown to inhibit metabotropic glutamate receptors. 6 Conversely, activation of PKC enhances actions at NMDA receptors in the spinal cord in a feed-forward mechanism. 33 Further, it also has been shown that NMDA receptor activation can inhibit metabotropic receptor activation directly, 9 although the mechanism remains unclear. Therefore, if NMDA receptors are more numerous on a neuron or activated to a greater extent, then the intracellular cascade of events subsequent to receptor activation might not only produce thermal hyperalgesia, but also inhibit the mechanisms that would produce mechanical hyperalgesia. What happens if AMPA and metabotropic glutamate receptors were more numerous or more predominantly activated? It has been shown that guanine nucleotides, such as GTP and GDP, are able to inhibit affinity 86 and binding 42,11S of agonists to the NMDA receptor through an action at the NMDA recognition site. Therefore, it is possible that activation of the metabotropic receptor might not only be able to initiate a cascade of events that result in mechanical hyperalgesia but also leads to an increase in guanine nucleotides that may be able to inhibit the events leading to thermal hyperalgesia. In addition to inhibiting thermal hyperalgesia, activation of the metabotropic receptor and the cascade of events produced is likely able to potentiate the mechanisms underlying mechanical hyperalgesia in a feed-forward mechanism. For example, PLA2 activation is able to enhance the binding of both AMPA 5'1 '66'67 and metabotropic glutamate receptor agonists. 1 In addition, it has been shown that protein kinase A (PKA), produced by actions of a camp-dependent kinase, is able to increase the opening time of non- NMDA receptors. 36 Therefore, camp produced by metabotropic glutamate receptors (mglur1) might be able to potentiate AMPA receptors through a cytoplasmic effect; a membrane-delimited effect cannot be ruled out. In addition to the enhancing effects described, activation of the metabotropic glutamate receptor is able to activate PLC 95 to produce IP3 and diacyglycerol. ~ However, PLC inhibitors do not affect acute mechanical hyperalgesia, z7 Therefore, a neuron would require a mechanism to inhibit the actions of IP3. In support, it has been shown that activation of AMPA receptors is able to inhibit IP3 accumulation by increasing its conversion to IP4 by allowing influx through L-type voltage-sensitive calcium channels that activates a calmodulin kinase that does not require PKC. 56 Activation of AMPA receptors may be able to inhibit the effects of metabotropic receptor activation that are not involved directly in mechanical hyperalgesia. In addition it also has been shown that activation of trans-acpd-sensitive metabotropic glutamate receptors are able to inhibit N-type calcium channels by a membrane-delimited effect. 1 ~ This may be an additional mechanism whereby the calcium-mediated intracellular signal produced by actions at calcium-permeable AMPA receptors can be focused. SUMMARY The majority of the evidence presented is consistent with a role for spinal NMDA receptors, NO, and cgmp in thermal hyperalgesia and for coactivation of spinal AMPA and metabotropic glutamate receptors, activation of PLA2, and production of COX products in mechanical hyperalgesia. These data suggest that, in

13 FOCUS/Meller 227 general, thermal and mechanical hyperalgesia rely on activation of two different intracellular cascades of events in the spinal cord. The hypothesis proposed would allow for minuteto-minute plasticity to be able to occur in the spinal cord. During the course of an inflammation, tissue or nerve injury, the spatial and temporal release of mediators in the periphery would change following an initial injury. Therefore, it is likely that different afferents are sensitized by different mediators and that different classes of afferents are activated at different times to differing extents after the initial injury. This would allow for diverse inputs to central neurons to become active or change over time allowing for an extraordinary degree of plasticity in the spinal cord. For example, an afferent, sensitized by mediator X, that contacts sites on central neurons that contain predominantly NMDA receptors (allowing for the neuron to show an increase sensitivity to thermal stimulation) might decrease firing because the mediators involved in activating that neuron are now not produced in the periphery. Now, at that same time, if mediatory is produced, this might allow for sensitization and activation of different afferents that might contact the same neuron, but on sites that contain predominantly AMPA and metabotropic glutamate receptors allowing for the same neuron to now show an increased sensitivity to mechanical stimulation. That is, the neuron may show a sensitivity to either thermal or mechanical stimuli during the time-course of an injury depending on the temporal and spatial production and release of mediators, the afferent input, and the density and location of excitatory amino acid receptors on central neurons. Further, the expression of the sensitivity may change from one modality to the other. Although experimental animals might show a continuous thermal or mechanical hyperalgesia, or both, it may not be produced by the same events in the same neurons throughout the complete time-course of the hyperalgesia. Acknowledgments The author wishes to thank Drs. G. F. Gebhart and A. Randich for reading drafts of the article. The work of the author described here has been supported by NS 29844, DA 02879, and an unrestricted pain research award from Bristol-Myers Squibb. References 1. AbdeI-Latif AA: Calcium-mobilizing receptors, phosphoinositides, and the generation of second messengers. Pharmacol Rev 38: , Andres KH, von During M: Morphology of cutaneous receptors, pp In Iggo A (ed): Handbook of sensory physiology. VoI. 2. Springer-Verlag, Heidelberg, Arancio O, MacDermott AB: Differential distribution of excitatory amino acid receptors on embryonic rat spinal cord neurons in culture. J Neurophysiol 65: , Arancio O, Yashimura M, Murase K, MacDermott AB: The distribution of excitatory amino acid receptors on acutely dissociated dorsal horn neurons from postnatal rats. Neuroscience 52: , Aronica E, Casabona G, Genazzani AA, et al: Mellitin enhances excitatory amino acid release and AMPAstimulated 45Ca2+ influx in cultured neurons. Brain Res 586:72-77, Bekkers JM, Stevens CF: NMDA and non-nmda receptors are co-localized at individual excitatory synapses in cultured rat hippocampus. Nature 341: , Bleakman D, Rusin KI, Chard PS, Glaum SR, Miller R J: Metabotropic glutamate receptors potentiate ionotropic glutamate responses in the rat dorsal horn. Mol Pharmacol 42: , BlissTV, Errington ML, Lynch MA, WilliamsJH: Presynaptic mechanisms in hippocampal long-term potentiation. Cold Spring Harb Symp Quant Biol 55: , Burgess PR, Perl ER: Cutaneous mechanoreceptors and nocicpetion, pp In Iggo A (ed): Handbook of sensory physiology. Vol. 2. Springer-Verlag, Heidelberg, Catania MV, Hollingsworth Z, Penney JB, Young AB: Phospholipase A2 modulates different subtypes of excitatory amino acid receptors: autoradiographic evidence. J Neurochem 60: , Cerne R, Randic M: Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans- 1-aminocyclopentane-l,3-dicarboxylic acid. Neurosci Lett 144: , Charpak S, Gahkwiler BH: Glutamate mediates a slow synaptic response in hippocampal slice cultures. Proc R Soc Lond 243: , Coderre T J, Katz J, Vaccarino AL, Melzack R: Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 53: 1-27, Coderre T J, Melzack R: Central neural mediators of secondary hyperalgesia following heat injury in rats: neuropeptides and excitatory amino acids. Neurosci Lett 131:71-74, Coderre T J: The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats. Mol Neurobiol 7: , Collingridge GL: The mechanisms of induction of NMDA receptor-dependent long-term potentiation in the hippocampus. Exp Physiol 77: , 1992

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