Metabotropic Glutamate Receptors

16 May

mGluRs belong to family 3 of G protein-coupled receptors, which can trigger long-lasting intracellular processes and plastic changes. They are characterized by a seven-transmembrane domain topology and a large N-terminal extracel- lular domain, which contains important residues for ligand binding and forms two lobes that close like a Venus’ flytrap upon ligand binding (Bockaert and Pin 1999). The second intracellular loop determines G protein specificity and the intracellular C-terminal interacts directly with intracellular proteins such as Homer, which are involved in receptor trafficking, synaptic anchoring, cell signaling, and constitutive (basal) receptor activity (Bockaert and Pin 1999; De Blasi et al. 2001; Gasparini et al. 2002; Bhave et al. 2003). Eight mGluR subtypes have been cloned and are classified into groups I (mGluRs 1 and 5), II (mGluRs

2 and 3), and III (mGluRs 4, 6, 7, and 8) based on their sequence homology, signal transduction mechanisms, and pharmacological profile (Schoepp et al.

1999; De Blasi et al. 2001; Neugebauer 2001; Gasparini et al. 2002; Varney and Gereau 2002; Lesage 2004; Swanson et al. 2005; see Table 1). Several splice vari- ants have been identified that may differ with regard to their pharmacology and G protein coupling.

Group I mGluRs couple through Gq proteins to the activation of phospholi- pase C (PLC), which leads to the formation of inositol-1,4,5-trisphosphate (IP3)

and diacylglycerol (DAG), resulting in calcium release from intracellular stores and activation of protein kinase C (PKC), respectively (Anwyl 1999; Schoepp et al. 1999; Neugebauer 2001; Gasparini et al. 2002). Tyrosine kinase activa- tion is another signaling pathway of group I mGluRs. Both PKC- and tyrosine kinase-dependent pathways can involve mitogen-activated protein (MAP) ki- nases such as the extracellular signal-regulated kinases 1/2 (ERK1/2) (Varney and Gereau 2002). Stimulation of adenylyl cyclase (AC) by group I mGluRs has been reported but the underlying mechanism is not yet known. Group II and group III mGluRs are negatively coupled to AC through Gi/Go proteins, thereby inhibiting cyclic AMP (cAMP) formation and cAMP-dependent pro- tein kinase (PKA) activation (Anwyl 1999; Schoepp et al. 1999; Neugebauer

2001; Gasparini et al. 2002).

Individual mGluR subtypes show distinct synaptic localization and function

(Schoepp et al. 1999; Neugebauer 2001; Gasparini et al. 2002; Varney and Gereau

2002; Lesage 2004; Swanson et al. 2005). Group I mGluR1 and 5 are most often localized postsynaptically whereas group II mGluR2 and group III mGluR8 are largely presynaptic. Group II mGluR3 and group III mGluR4 and 7 can be found both pre- and postsynaptically. In general, the predominant effect of group I mGluR activation is enhanced neuronal excitability and synaptic transmission whereas groups II and III typically mediate inhibitory effects, although excep- tions exist. The mGluRs can regulate neuronal  excitability through  direct or indirect effects on a variety of voltage-sensitive ion channels, including high

voltage-activated Ca2+ channels, K+ channels, and nonselective cationic chan- nels (Anwyl 1999; Schoepp et al. 1999; Neugebauer 2001). The modulation  of

ligand-gated ion channels by mGluRs includes the group I mGluR-mediated enhancement of ionotropic glutamate receptor function, which likely involves

receptor  phosphorylation (Anwyl 1999; Fundytus  2001; Neugebauer  2001). Group I mGluRs also potentiate  the function  of the capsaicin/vanilloid  re- ceptor, TRPV1 (Hu et al. 2002). Convincing evidence suggests that mGluRs interact with the opioid system and play a role in the development of opioid

tolerance and dependence (Fundytus 2001). mGluRs can also modulate the re- lease of transmitters by acting as autoreceptors (glutamate) or heteroreceptors (GABA, substance P, serotonin,  dopamine,  and acetylcholine) (Cartmell and

Schoepp 2000).

Several potent  and  mGluR subgroup/subtype-selective compounds  have been developed (Schoepp et al. 1999; Neugebauer 2001; Gasparini et al. 2002; Varney and  Gereau 2002; Lesage 2004; Swanson et al. 2005; see Table 1). Currently  available agonists  are selective for subgroups  I (DHPG, (S)-3,5- dihydroxyphenylglycine),  II (LY354740, [1S, 2S,5R,6S]-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic  acid; LY379268, (−)-2-oxa-4-aminobicylco  hexane-

4,6-dicarboxylic acid), and  III (LAP4, l-(+)-2-amino-4-phosphonobutyric- acid). LY354740 has been tested in phase II clinical trials. Several subtype- selective agonists have become available. (RS)-2-chloro-5-hydroxyphenylgly- ine (CHPG) is selective for mGluR5. (S)-3,4-dicarboxyphenylglycine [(S)-3,4-

DCPG] activates mGluR8 with nanomolar  potency and has greater than 280- fold selectivity over other mGluR subtypes (Thomas et al. 2001). Positive al- losteric activators have been identified for mGluR4 (N -phenyl-7-(hydoxylimi- no)cyclopropa[b]chromen-1a-carboxamide [PHCCC]; Maj et al. 2003; Shipe et al. 2005) and mGluR7 (N,N -dibenzhydrylethane-1,2-diamine dihydrochlo- ride [AMN082]; Conn and Niswender 2006; Mitsukawa et al. 2005). Antagonists for group I mGluRs include the competitive mGluR1 subtype-selective antag-

onist LY367385 [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid] and noncompetitive (allosteric) antagonists for mGluR1 [CPCCOEt, 7-(hydroxyimi- breakno)  cyclopropa[b]chromen-1a-carboxylate  ethyl  ester]  and  mGluR5-

(MPEP,  2-methyl-6-(phenylethynyl)pyridine).  Competitive  group  II  and- group III mGluR antagonists are available; they are subgroup- but not subtype- selective (Table 1). The activity of mGluRs can be regulated not only by receptor

agonists and antagonists but also through receptor phosphorylation, including PKC-mediated desensitization of group I mGluRs and uncoupling of groups II and III mGluRs from G proteins by PKC and PKA (Karim et al. 2001; Neuge-

bauer 2001; Varney and Gereau 2002).

Glutamatergic Nociceptive Transmission in the Peripheral and Central Nervous System

The role of iGluRs and mGluRs in nociceptive transmission has been reviewed in detail in a number of recent articles (Fisher et al. 2000; Carlton 2001; Willis

2001; Neugebauer and Carlton 2002; Neugebauer 2002; Varney and Gereau 2002; Ruscheweyh and Sandkuhler 2002; Hewitt 2003; Garry et al. 2004; Lesage 2004). The following is a summary  of glutamate  receptor  function  in nociceptive processing in naïve animals; the involvement of iGluRs and mGluRs in pain- related plasticity will be discussed in the context of preclinical pain models (Sect. 3).

Ionotropic Glutamate Receptors

Functional NMDA, AMPA, and kainate receptors are present along the pain neuraxis from the peripheral nervous system to the brain.

Periphery

It is firmly established now that iGluRs (particularly NR2B, GluR1, and GluR5) are localized in the periphery on nociceptive primary afferent terminals and on their cell bodies (Carlton 2001). Injections of NMDA, AMPA, or kainate into peripheral  tissues excite and sensitize nociceptors in electrophysiologi-

cal studies and produce pronociceptive  behavioral effects including thermal and mechanical hyperalgesia as well as spontaneous lifting and licking behav- iors (Lawand et al. 1997; Carlton 2001). These effects can be blocked by the appropriate antagonists. Although the details of iGluR-mediated signal trans- duction at the peripheral nerve terminals need to be determined, it is clear that neuronal as well as nonneuronal elements serve as the sources for glutamate (Carlton 2001). Importantly, glutamate levels are increased in the joints of pa- tients with arthritis, which may suggest a role of peripheral iGluRs in clinical pain associated with tissue damage (McNearney et al. 2000).

2.1.2

Spinal Cord

In the spinal dorsal horn, NMDA, AMPA, and kainate receptors have now been shown to be localized pre- and postsynaptically, particularly in the superficial laminae I and II (Tolle et al. 1993; Willis and Coggeshall 2004; Lu et al. 2005). NMDA receptors (NR1) are present on the terminals of primary afferents and of GABAergic interneurons as well as on nearly all dorsal horn neurons. The distribution of the different NR2 subunits  appears to be more complex, but NR2B is found predominantly in small-diameter primary afferents and in the superficial dorsal horn whereas NR2A is highly concentrated  in the deep dor- sal horn (Nagy et al. 2004). AMPA receptors are expressed on the terminals of unmyelinated (GluR1) and myelinated (GluR2) afferents and of GABAergic interneurons (GluR4) as well as on neurons  in the superficial (GluR1 and 2) and deep (GluR3 and 4) dorsal horn. Although the AMPA receptor  subunit distribution is controversial, it has been suggested that unmyelinated C-fibers are positioned to activate calcium-permeable AMPA receptors (lacking GluR2) on inhibitory (and some excitatory) interneurons in the superficial dorsal horn whereas (small) myelinated fibers make contact with GluR2-containing AMPA receptors  on excitatory interneurons (Willis and Coggeshall 2004). Such an arrangement  would limit the nociceptive transmission  from C-fibers under normal conditions. Kainate receptor subunits KA1 and KA2 are found presy- naptically on primary afferents and GABAergic interneurons as well as postsy- naptically on dorsal horn neurons (Willis and Coggeshall 2004; Lu et al. 2005). Importantly,  essentially all of the kainate receptor-expressing  primary affer- ents appear to be nociceptors, and GluR5 is the predominant kainate receptor subunit expressed in primary afferents (Carlton 2001).

Presynaptic NMDA, AMPA, and kainate receptors have been shown to act as autoreceptors on primary afferents to decrease the release of glutamate through

a mechanism that involves primary afferent depolarization,  thus preventing the propagation  of action potentials into the central terminals or, more likely, reducing their size (Lee et al. 2002; Bardoni et al. 2004). The consequence of

such inhibitory action for spinal nociceptive processing remains to be deter- mined, but inhibitory effects of kainate receptor activation on spinal excitatory

neurotransmission have been described (Ruscheweyh and Sandkuhler 2002; Youn and Randic 2004). The predominant effect, however, of spinal NMDA, AMPA, or kainate receptor  activation is the excitation of spinal dorsal horn neurons and the increases of their responses to innocuous and noxious stimuli (Fundytus 2001; Ruscheweyh and Sandkuhler 2002; Youn and Randic 2004). Accordingly, intrathecal administration of NMDA, AMPA, and kainate recep- tor agonists produces spontaneous nociceptive behavior and thermal and me- chanical hyperalgesia (Fundytus 2001). Conversely, there is good evidence that spinal administration of NMDA, AMPA, and kainate receptor antagonists can inhibit nociceptive transmission  in dorsal horn  neurons  and pain behavior (Stanfa and Dickenson 1999; Moore et al. 2000; Fundytus 2001; Willis 2001; Schaible et al. 2002; Ruscheweyh and Sandkuhler 2002; Garry et al. 2004). It should be noted, however, that AMPA receptor activation is also involved in the transmission of non-nociceptive information whereas blockade of NMDA and kainate receptors does not affect all forms of nociceptive transmission. NMDA receptors may be involved predominantly in tonic pain responses and persis- tent pain states (see Sect. 3). The role of kainate receptors is only beginning to emerge.

Brain

Activation of NMDA and AMPA receptors  in the brainstem,  including peri- aqueductal  gray (PAG) and rostral  ventromedial  medulla (RVM), produces antinociceptive  effects, which is consistent  with the key role of these brain- stem areas in the descending  inhibition  of nociception  and  pain  (Berrino et al. 2001; Ren and Dubner 2002; Gebhart 2004; Vanegas and Schaible 2004). More recently, however, it has been shown that glutamate or NMDA, but not AMPA, micro-injected into the RVM facilitates spinal nociceptive processing (Ren and Dubner 2002; Gebhart 2004). The dual facilitatory and inhibitory role of iGluRs in the brainstem reflects the bidirectional modulation of pain by the descending control system. These differential effects appear to be activity and time-dependent as is evident in pain models (see Sect. 3). Antagonists for non-NMDA, but not NMDA, receptors in the RVM produce facilitation of nociceptive responses in naïve animals, suggesting that a glutamatergic  de- scending inhibitory system is tonically active (Urban et al. 1999). The role of kainate receptors in descending pain modulation is not yet known.

The contribution of iGluRs to nociceptive processing in higher brain areas has been determined  in the thalamus (Salt 2002) and amygdala (Neugebauer

et al. 2004). Sensory thalamic relay nuclei such as the ventrobasal thalamus (VB) play an important role in gating and processing sensory information transmitted to the cerebral cortex. Sensory transmission  to the VB complex

involves both NMDA and non-NMDA receptors. NMDA receptors contribute strongly to nociceptive responses of VB neurons and prolonged or repetitive

synaptic input. Non-NMDA receptors mediate non-nociceptive responses and the fast component of sensory-evoked synaptic responses but contribute little to nociceptive transmission.  Kainate receptors  do not appear  to contribute to the responses of thalamic relay cells to sensory inputs, but studies using LY382884 (kainate  GluR5 antagonist)  suggest there  is activation  of kainate receptors  on GABAergic axons from the thalamic  reticular  nucleus, which results in disinhibition through decreased recurrent inhibition. This has been proposed  to play an important  novel role in extracting sensory information from background noise (Salt 2002).

The latero-capsular  part  of the central  nucleus of the amygdala (CeLC) represents the nociceptive amygdala, which is believed to be concerned with the emotional-affective component and modulation of pain (Neugebauer et al.

2004). Nociceptive responses of CeLC neurons in naïve animals are mediated by a combination  of NMDA and non-NMDA receptors whereas non-nociceptive

inputs and basal synaptic transmission activate only non-NMDA receptors (Li and Neugebauer 2004b; Bird et al. 2005). The relative contribution of AMPA

and kainate receptors is not yet known. Enhanced NMDA receptor function appears to be an important  mechanism in pain-related plasticity in the CeLC (see Sect. 3).

Metabotropic Glutamate Receptors

The important role of group I mGluRs in peripheral and central nociceptive processing and pain behavior is now well established whereas the functions of groups II and III mGluRs are less well known (Fundytus 2001; Neugebauer

2001; Neugebauer 2002; Varney and Gereau 2002; Lesage 2004).

Periphery

Group I mGluR1 and mGluR5 have been demonstrated on a subset (∼20%–

30%) of unmyelinated and myelinated peripheral axons (Neugebauer and Carl- ton 2002; Lesage 2004). There is evidence for a colocalization of mGluR5 with the capsaicin/vanilloid receptor (TRPV1), while mGluR1 may also be present on sympathetic efferent fibers. Group II mGluR2 and mGluR3 and group III mGluR7 and mGluR8 are also present in a subpopulation of small-diameter primary  afferent neurons,  and there is some colocalization of group I and group II mGluRs (Neugebauer and Carlton 2002; Varney and Gereau 2002). Exogenous activation of peripheral group I mGluRs (Neugebauer and Carlton

2002; Varney and Gereau 2002; Lesage 2004) produced long-lasting thermal hy- peralgesia in mice (but not rats), which was significantly reduced by peripheral

injections of mGluR1 antagonists  (CPCCOEt and LY367385) and completely blocked by an mGluR5 antagonist  (MPEP). Peripheral  injections of a selec-

tive mGluR5 agonist (CHPG) and the mGluR1/5 agonist (DHPG) were equally potent in producing  mechanical allodynia in rats. The mechanical allodynia was blocked by a selective mGluR5 antagonist  (MPEP) but not by a selec- tive mGluR1 antagonist (4CPG, (S)-4-carboxy-phenylglycine). These data may suggest a role of peripheral  mGluR5 in mechanical allodynia whereas both mGluR1 and mGluR5 are involved in thermal hyperalgesia.

There is good evidence to suggest the involvement and endogenous acti- vation of peripheral  group I mGluRs in persistent  pain states (see Sect. 3) but not in normal nociception. Subcutaneous injection of a group II mGluR agonist (APDC; 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate) into the mouse hindpaw did not alter mechanical and thermal sensitivity in behavioral studies (Yang and Gereau 2002; Yang and Gereau 2003), but very low concentrations of APDC inhibited the extracellularly recorded responses of nociceptive fibers to heat stimuli in an in vitro skin-nerve preparation (Neugebauer and Carlton

2002). Peripheral group II mGluRs may be particularly useful targets in pain states (see Sect. 3). The role of peripheral  group III mGluRs remains  to be

determined.

Spinal Cord

Group I mGluR1 and mGluR5 are functionally expressed in the spinal dor- sal horn: mGluR5 expression is particularly strong in the superficial laminae whereas high levels of mGluR1 are found in the deep dorsal horn. Anatom- ical and electrophysiological data further suggest that mGluR1 and mGluR5 are mainly postsynaptic  but can also act presynaptically to facilitate spinal neurotransmission (Neugebauer 2001, 2002; Varney and Gereau 2002; Lesage

2004). Importantly, mGluR5 was identified on GABA and non-GABAergic dor- sal horn  neurons.  Activation of spinal group  I mGluRs generally produces

pronociceptive effects in behavioral and electrophysiological assays, although mixed excitatory and inhibitory  effects have been reported  (Fundytus 2001; Neugebauer 2001; Varney and Gereau 2002; Lesage 2004). Intrathecal admin-

istration  of group I agonists such as DHPG evokes spontaneous  nociceptive behavior, thermal  and mechanical hyperalgesia, and mechanical allodynia, which can be blocked with antagonists or antibodies for mGluR1 or mGluR5,

where tested  (Fundytus  2001; Neugebauer  2001; Varney and  Gereau 2002; Lesage 2004). Activation of spinal group I mGluRs can produce facilitation or inhibition  of synaptic transmission  and of dorsal horn neuron  responses to

innocuous and noxious stimuli (Gerber et al. 2000a; Neugebauer 2001; Lesage

2004). Antagonists selective for mGluR1 (CPCCOEt) or mGluR5 (MPEP) can block these facilitatory effects but do not appear to affect nociceptive behav- ior under normal conditions (Fundytus 2001; Neugebauer 2001; Varney and Gereau 2002; Lesage 2004; Soliman et al. 2005). Spinal mGluR5 may mediate the inhibitory effects of groupI mGluR activation.

The roles of group II and group III mGluRs in spinal nociceptive processing are less clear (Neugebauer 2001; Neugebauer 2002; Varney and Gereau 2002). Both group II mGluR2/3 and group III mGluR4 and 7, but not mGluR6 and 8, are present in the spinal cord. Group III mGluRs are localized predominantly on presynaptic  terminals  in the dorsal and ventral horns  whereas group II mGluRs have been detected on presynaptic terminals in the superficial dor- sal horn as well as on postsynaptic  elements in deeper laminae. Agonists of group II (LCCG, (2S,1 S,2 S)-2-(carboxycyclopropyl)glycine)  and III (LAP4) mGluRs can inhibit synaptic transmission  in the dorsal horn in spinal cord slices in vitro (Gerber et al. 2000b), but behavioral studies found little evi- dence for antinociceptive  effects of group II or group III mGluR activation (Neugebauer  2001; Neugebauer 2002; Varney and Gereau 2002). Intrathecal administration of selective group II (APDC) and group III (LAP4) agonists had no effect on mechanical withdrawal response thresholds in the absence of tissue damage (Soliman et al. 2005). Similarly, systemic administration of a se- lective group II agonist (LY379268) had no significant effects on the tail flick or paw withdrawal tests of acute thermal nociceptive function (Simmons et al.

2002). Intraspinal  administration of LY379268 had no effect on the responses of nociceptive dorsal horn neurons (spinothalamic tract cells) recorded under normal conditions whereas LAP4 inhibited the responses to brief noxious and

innocuous mechanical cutaneous stimuli (Neugebauer et al. 2000).

Brain

In the brainstem, activation of group I mGluR1/5 (DHPG) or mGluR5 (CHPG) in the PAG had antinociceptive  effects in the hotplate  test in naïve animals (Maione et al. 1998; Lesage 2004). Intra-PAG administration of an mGluR1 antagonist  (CPCCOEt) attenuated  the effect of DHPG whereas an mGluR5 antagonist  (MPEP) blocked the effect of CHPG, indicating  the involvement of both mGluR1 and mGluR5 subtypes. When administered  without exoge- nous agonists, MPEP, but not CPCCOEt, had pronociceptive effects, suggesting the presence of tonic descending inhibition mediated by mGluR5 in the PAG. Activation of mGluR5 (CHPG) in the RVM, however, produced cold hypersen- sitivity that was prevented by MPEP (Lesage 2004). The possibly differential roles of mGluR1 and mGluR5 in descending inhibitory  and facilitatory sys- tems await a detailed analysis. Different from group I mGluRs, activation of groups II and III mGluRs in the PAG appears to decrease the descending in- hibition of pain behavior (Maione et al. 1998). Agonists of group II (LCCG) and III (LSOP, l-serine-O-phosphate)  receptors  administered  into the PAG decreased the latency of the nociceptive reaction in the hot plate test. Antag-

onists of group II [EGLU, (2S)-α-ethylglutamic acid] and III [MSOP, (RS)-α- methylserine-O-phosphate] receptors antagonized these pronociceptive effects

of LCCG and LSOP. Interestingly, MSOP, but not EGLU, alone increased the la-

tency in the hot plate test, perhaps suggesting the tonic activation of group III

mGluRs in the PAG.

In the VB, mGluR1 and mGluR5 are involved in the processing of nocicep- tive, but not non-nociceptive, information (Salt and Binns 2000; Lesage 2004). Intrathalamic  administration of antagonists selective for mGluR1 (LY367385) and mGluR5 (MPEP) inhibited the responses of VB neurons to brief noxious heat but not innocuous stimuli. Intrathalamic MPEP had no effect on noxious mechanical stimuli; the role of thalamic mGluR1 in mechanonociception re- mains to be determined.  Group II and III mGluRs mediate the presynaptic reduction of GABAergic inhibition in the VB, producing disinhibition of sen- sory (including nociceptive) processing (Salt et al. 1996; Salt 2002). Agonists of group II (LY354740 and APDC) and III (LAP4) decreased the inhibitory trans- mission from the thalamic reticular  nucleus and the respective antagonists

[MCCG (α-methyl-CCG-I) and MAP4 (α-methyl-l-AP4)] blocked these disin- hibitory effects. Accordingly, administration of a group II antagonist (EGLU) in the thalamic reticular nucleus produced antinociceptive effects presumably

by blocking the activation of group II mGluRs by the noxious input, thus dis- inhibiting the GABAergic neurons  that inhibit thalamic relay neurons  (Neto and Castro-Lopes 2000). When evaluating the therapeutic  potential  of these drug targets, it is important to consider possible facilitatory effects of other-

wise inhibitory groups II and III mGluRs in brain areas such as the PAG and thalamus.

In the amygdala (Neugebauer et al. 2004), activation of group I mGluR1/5

(DHPG) or mGluR5 (CHPG) enhanced the responses of CeLC neurons to in- nocuous  and noxious stimuli in naïve animals (Li and Neugebauer 2004a). Likewise, DHPG and CHPG potentiated normal synaptic transmission of pre- sumed nociceptive input from the pontine  parabrachial  area to the CeLC in brain slices (Neugebauer et al. 2003). An mGluR5 antagonist (MPEP) inhibited brief nociceptive responses of CeLC neurons under normal conditions whereas an mGluR1 antagonist (CPCCOEt) had no effect. Similarly, MPEP, but not CPC- COEt, inhibited basal synaptic transmission  in CeLC neurons  in slices from naïve animals. The role of group II and III mGluRs in nociceptive processing in the amygdala is not yet known.

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