Introduction

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

The -aminobutyric acid_B (GABA_B) receptor (GBR) was identified nearly 20 years ago as a result of studies aimed at determining whether chloride-dependent GABA receptors are present on peripheral nerve terminals as they are on primary afferent terminals in spinal cord. Although the data indicated that GABA receptors are present on sympathetic nerve endings and that their activation reduced the evoked release of neurotransmitters, their pharmacological properties were quite different from those of the classic GABA receptor. For example, GABA-mediated inhibition of neurotransmitter release from rat sympathetic nerve terminals is not inhibited by bicuculline and is not mimicked by isoguvacine, an antagonist and agonist, respectively, for the chloride-dependent GABA receptor. On the other hand, this receptor was stereoselectively activated by -chlorophenyl GABA (baclofen), an antispastic agent that displays no affinity for the classic GABA receptor. Together, these and other findings suggested the existence of a GABA receptor subtype distinct from the chloride-dependent receptor system. This new receptor, termed GABA_B, like the classic GABA_A receptor, is widely distributed throughout the mammalian brain and spinal cord, suggesting it plays an important role in maintaining central nervous system function (Hill and Bowery, 1981).

	GBR Function

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

Ligand binding and second messenger assays revealed that the GBR is metabotropic, distinguishing it further from the ionotropic GABA_A receptor. Thus, GBRs are coupled to G proteins, with activation causing a decrease in Ca²⁺ and an increase in K⁺ membrane conductance (Fig. 1; also see Bowery, 1993). The GBR-mediated changes in Ca²⁺ conductance appear to be associated primarily with P/Q- and N-, and possibly L-, type Ca²⁺ channels and with several different types of K⁺ channels.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the GBR indicating the dimeric nature of the structure. R1a, the a isoform of GBR1; R1b, the b isoform of GBR1; , , and , G protein subunits; Kir, inwardly rectifying K+ channel; AC, adenylate cyclase. Adapted from Bettler et al., 1998.

In spinal cord, GBR activation decreases the duration of orthodromic action potentials and of excitatory neurotransmitter release from 1a afferent fibers (Curtis et al., 1997). Both of these effects are explained on the basis of GBR-mediated inhibition of Ca²⁺ influx into 1a afferent presynaptic terminals. In some circumstances, such as with rodent CA1 hippocampal fibers, regulation of GBR-mediated neurotransmitter release appears independent of effects on Ca²⁺ or K⁺ channel activity (Jarolimek and Misgeld, 1997). In this case, evidence suggests stimulation of GBRs enhances protein kinase C activity, which in turn regulates neurotransmitter release. Although there is strong evidence linking GBRs to protein kinase C in rat hippocampus, this coupling is most evident for only the first 14 days of life, diminishing thereafter.

Low threshold Ca²⁺ T currents, which are inactivated at normal resting membrane potentials, may also contribute to the GBR-mediated response, especially in the thalamus (Crunelli and Leresche, 1991). In this brain region, GBR activation produces a prolonged postsynaptic hyperpolarization, which in turn initiates Ca²⁺ spiking activity in thalamocortical cells. This effect is believed to be responsible for the spike-and-wave activity characteristic of absence seizures, suggesting a possible role for the GABAergic system and GBRs in this condition.

Early evidence of the metabotropic properties of GBRs was provided by the finding that their activation causes inhibition of basal and forskolin-stimulated adenylate cyclase activity in brain tissue (Bowery, 1993). Although this effect does not appear to be related to GABA_B-mediated changes in neuronal Ca²⁺ or K⁺ channel activity, like the channel effects it is a consequence of the dissociation of G_i or G_o proteins. Moreover, studies with brain slices revealed that simultaneous activation of GBRs and receptors coupled to G_s (e.g., -adrenergic, adenosine) results in an enhanced production of cAMP over that resulting from exposure to the -adrenergic or adenosine agonist alone. This augmentation of cAMP accumulation is due to stimulation, or enhancement, of adenylate cyclase activity by G protein subunits liberated by GBR activation, which by themselves are unable to stimulate the enzyme except in the presence of G_s (Enna, 1999). The in vitro relevance of these findings is illustrated by microdialysis experiments showing that both baclofen and GABA reduce forskolin-stimulated cAMP production and that baclofen enhances isoproterenol-stimulated cAMP accumulation in the cerebral cortex of freely moving rats (Hashimoto and Kuriyama, 1997).

Although the functional significance of the dual effect on cAMP production is unknown, these phenomena are useful in characterizing GABA_B responses for both wild-type and cloned receptors (Kaupmann et al., 1997). It has also been suggested that the two different effects on cAMP production may be characteristic of pharmacologically distinct GBRs (Cunningham and Enna, 1996).

	GBR Agonists and Antagonists

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

Although a host of phosphinic acid-based GBR agonists have been synthesized and tested, the number of potent and selective compounds is limited (Froestl et al., 1995a). Indeed, baclofen, which was introduced some 30 years ago, remains one of the most potent and selective agents for stimulation of the GBR, and its stereoselectivity makes it a particularly useful tool for characterization of this system (Fig. 2; also see Bowery et al., 1981). Among the agonists synthesized, 3-aminopropyl phosphinic acid and its methyl homolog AMPPA (SKF97541) are the most active, being 3- to 7-fold more potent as GBR agonists than the active isomer of baclofen (Fig. 2, Table 1). To date, no agonist has been found that unequivocally differentiates between putative GBR subtypes.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Chemical structures of various GABAB receptor agonists and antagonists.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities of various compounds for wild-type and expressed GABAB receptors Adapted from Kaupmann et al, 1998; White et al, 1998; and Jones et al, 1998.

The development of high-affinity, selective, and systemically active GBR antagonists played a crucial role in characterizing the physiological and structural properties of GBRs. The first members of this class were phaclofen and 2-hydroxysaclofen (Kerr and Ong, 1995). Although these compounds display rather low affinities for the receptor (100 and 12 µM, respectively), they were valuable for initial characterization of the GABA_B binding site in brain tissue. The next significant advance came with the development of high-affinity, selective, and systemically active GBR antagonists (Froestl et al., 1995b). The initial compounds in this series, CGP35348 and CGP36742 (Fig. 2), were the first GBR antagonists to display central nervous system activity after peripheral administration. These agents were subsequently supplanted by the synthesis of systemically active antagonists displaying affinities for the GBR up to 10,000 times greater than that of the earlier compounds. This increase in affinity resulted from the addition of a dichlorobenzene moiety to the structure of existing GABA_B antagonists (Fig. 2; also see Froestl et al., 1995b). One of these, CGP64213, was radiolabeled with ¹²⁵I and used as a tag for the initial expression cloning of the GBR (Kaupmann et al., 1997). The only systemically active compound outside of the dichlorobenzene series displaying significant central nervous system activity is ()-(R)-5,5-dimethylmorpholinyl-2-acetic acid ethyl ester HCl (SCH50911), which, unlike the other agents, is not a phosphinate (Fig. 2; also see Carruthers et al., 1998). A comparison of these chemical structures may yield important clues about the optimal configuration of GBR antagonists (Frydenvang et al., 1997).

	Molecular Characteristics of GBR

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

The GBR was first isolated by expression cloning in Cos-1 cells using a cDNA library of rat brain tissue and the high-affinity radioligand ¹²⁵I-CGP64213 (Kaupmann et al., 1997). This approach revealed two isoforms of the receptor, GBR1a and GBR1b, with molecular masses of 130 and 100 kDa, respectively. The structures of these two variants differ only marginally at the N terminus, with both possessing the same GABA binding domain on the extracellular chain. The isolated protein, like other G protein-coupled receptors, has seven membrane-spanning domains. The GBR1 has little or no structural homology with other metabotropic receptors, being most similar (35%) to metabotropic glutamate receptors (Kaupmann et al., 1997).

Although the GBR1 expressed in cell lines has antagonist binding and biochemical characteristics similar to those of wild-type GBRs, the affinity of agonists is some 100-fold less for expressed than wild-type receptors (Table 1). It was also perplexing that regardless of the cell type used, it was not possible to detect functional membrane-bound receptors after the expression of GBR1. Thus, the incorporation of GBR1 cDNA into Xenopus oocytes, human embryonic kidney 293, Chinese hamster ovary, or NG108 cells fails to produce a receptor that is stimulated by GBR agonists. Nevertheless, radiolabeled GBR antagonist binding is robust in cellular homogenates from tissues expressing GBR1 (Table 1).

The explanation for these findings was provided by recent discoveries showing that the GBR exists as a heterodimer, with a companion protein linked in a stoichiometric 1:1 ratio to the GBR1 through coiled domains at the C terminus (Fig. 1; also see Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). This newly identified protein, designated GBR2, has many of the structural features of GBR1, including a similar molecular mass, a seven transmembrane-spanning region, and a long extracellular chain at the N terminus (Fig. 1). The GBR2 has 35% homology and 54% similarity to GBR1. Although no GABA_B ligand binding has been detected using GBR2 protein alone, expression of GBR2 by itself yields a functional GBR, and like GBR1 alone, GBR2 couples to adenylate cyclase in Chinese hamster ovary cells (Kaupmann et al., 1998; Martin et al., 1999). Moreover, knockdown of GBR1 in melanotrope cells of the pituitary intermediate lobe eliminates baclofen-mediated inhibition of Ca²⁺ channels, indicating the need for the GBR1 subunit to regulate the activity of these high voltage-activated channels (Morris et al., 1998). Of greatest interest is the fact that coexpression and coupling of GBR2 and GBR1 result in the functional expression of GBRs in plasma membranes. These receptors, as with the wild type, possess high-affinity binding sites for both agonists and antagonists and display GABA_B-mediated changes in K⁺ channel conductances (Table 1; also see Kaupmann et al., 1998; White et al., 1998). Although the GBR1 expressed as part of the heterodimer has the same affinity for GABA_B agonists as wild-type receptors, no radiolabeled ligand binding has yet been demonstrated for the GBR2 component (White et al., 1998). Because there is no evidence for the formation of functional GBR1 or GBR2 homodimers, it appears that the GBR is the first functional heterodimer to be identified within the metabotropic receptor class.

Although it has been established that both GBR1 and GBR2 are capable of modulating adenylate cyclase, there is as yet no precise information about the type of G proteins that couple to the heterodimer (Kaupmann et al., 1998; Martin et al., 1999). Thus, it is possible that the receptor complex couples to two different types of G proteins, such as G_i and G_o, which act in concert on activation of this site. It is also possible that some GBRs have identical G proteins bound to the two subunits. In any event, the heterodimeric nature of the GBR suggests it may function, and be regulated, in ways that are quite distinct from those of more conventional metabotropic and ionotropic receptors.

	GBR Distribution and Localization

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

The GBR complex, as well as the individual GBR1 and GBR2 subunits, are widely distributed throughout the mammalian central nervous system. Although the distribution of GBR1 and GBR2 in rat brain closely parallels GBR binding, there are notable exceptions to this pattern (Clark et al., 1998; Jones et al., 1998). Thus, although GBR1 and GABA_B binding and function are present in the caudate-putamen, GBR2 is absent in this brain region. Likewise, the level of GBR2 mRNA is quite low relative to the amount of GBR1 mRNA in the hypothalamus. A differential up-regulation of GBR1 mRNA and GBR2 mRNA occurs in the rat dorsal spinal cord in response to pain, with the GBR2 message being elevated to a greater extent than GBR1 (McCarson and Enna, 1999). These findings related to the regulation of GABA_B expression suggest the existence of other, as yet unidentified, GBR subunits capable of dimerizing with GBR1 or GBR2 or indicate that homodimers may, in some circumstances, constitute functionally active GBRs.

Rat brain regions with the highest density of GABA_B binding sites include the thalamic nuclei, the molecular layer of the cerebellum, the cerebral cortex, interpeduncular nucleus, and laminae II and III of the spinal cord (Bowery et al., 1987). Because these data were obtained from ligand binding experiments, it is not possible to differentiate presynaptic and postsynaptic receptors. In situ hybridization analysis reveals that GBR1a may be more associated with presynaptic receptors than GBR1b, which tends to be more highly concentrated postsynaptically (S. Towers, A. Billinton, B. Bettler, L. Urban, J.-M. Castro-Lopes and N.G.B., submitted). For example, the density of GBR1a mRNA in rat dorsal root ganglia represents more than 90% of the total GBR mRNA, with GBR1b comprising 10% or less in this tissue. Likewise, GBR1a mRNA is present in rat and human cerebellar granule cells, whereas GBR1b mRNA is primarily associated with Purkinje cells in this brain region, a localization suggesting presynaptic expression for the former and postsynaptic for the latter.

Outside the central nervous system GBRs are found on enteric neurons innervating the intestine (Ong and Kerr, 1990). Indeed, GABA neurons, as well as an abundance of GBRs, are present in intestinal tissue, and this system responds to GABA_B agonists. In contrast, most cardiovascular, respiratory, and endocrine responses to GBR agonists and antagonists appear to be centrally mediated (see Bowery, 1993). However, reverse transcription-polymerase chain reaction analysis has revealed GBR1 mRNA in a number of peripheral tissues, including heart, lung, intestine, kidney, and urinary bladder, suggesting that the GABA_B system may exert direct control over a number of organs (Isomoto et al., 1998). However, the physiological and pharmacological relevance of this finding will not be fully known until GABA neurons are identified in these tissues or it is demonstrated that the concentration of circulating GABA is sufficient to exert a hormone-like action on these organs.

	GBR Subtypes

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

Inasmuch as subtypes have been identified for virtually all G protein-coupled receptors, it seems likely that there are pharmacologically and molecularly distinct GBRs as well. Indeed, heterodimers composed of different forms of GBR1 and GBR2, or of other GBR proteins, would provide sufficient structural differentiation to constitute subclasses of this receptor. To date, electrophysiological and neurochemical studies suggest only subtle distinctions between presynaptic and postsynaptic GBRs (Gemignani et al., 1994; Cunningham and Enna, 1996). A major problem in unequivocally establishing pharmacologically distinct GBRs is the paucity of highly selective ligands for these putative sites. This has led to conflicting interpretations of results comparing the relative affinities of GABA_B agonists and antagonists in different systems. A definitive conclusion regarding GBR subtypes awaits further information on the heterogeneity of the receptor complexes and the development of subtype-specific agonists and antagonists.

	Therapeutic Application of GABA Receptor Agonists and Antagonists

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

Baclofen has been used for years for the treatment of spasticity, with its effectiveness believed to be related to GBR-mediated inhibition of excitatory amino acid release onto motoneurons in the ventral horn of the spinal cord. Side effects (in particular, sedation) limit its clinical usefulness. This is avoided by intrathecal administration, which is used for the treatment of spasticity associated with brain and spinal cord injuries, cerebral palsy, tetanus, multiple sclerosis, stiff-man syndrome, and dystonia (Penn and Mangieri, 1993).

Baclofen is an effective adjunct in reducing pain associated with trigeminal neuralgia, stroke, spinal cord injury, or musculoskeletal disorders (Loubser and Akman, 1996). Baclofen may also be of some benefit in the treatment of migraine headache, where in some cases, it appears to reduce the severity and duration, if not the frequency, of attacks. Other clinical uses of this prototype GABA_B agonist include intractable hiccups and cough.

Although baclofen displays significant antinociceptive properties in a variety of animal models of pain, its clinical usefulness is limited in this regard. This may be because tolerance develops rapidly to the analgesic effects of baclofen or the dose necessary to attenuate pain exceeds the sedative dose. Data suggest the antinociceptive effects of baclofen are mediated, at least in part, at the level of the spinal cord, where GBRs are localized in the dorsal horn on small diameter afferent fiber terminals (Hammond and Washington, 1993). Activation of these sites decreases the evoked release of sensory transmitters, such as substance P and glutamate, attenuating the transmission of a painful impulse (Kangra et al., 1991; Malcangio and Bowery, 1996).

There are suggestions the antinociceptive response to GABA_B agonists may be mediated by effects in the brain. For example, studies with tiagabine, a GABA uptake inhibitor, and therefore a nonselective activator of GABA receptors, have revealed it displays antinociceptive properties in a variety of pain models (Ipponi et al., 1999). This antinociceptive response is most associated with an increase in the extracellular concentration of thalamic GABA and is blocked by a GBR antagonist.

Baclofen has been shown to attenuate allodynia at subsedative doses in a rat model of trigeminal neuropathic pain (Idanpaan-Heikkila and Guilbaud, 1999). Although the authors suggest these data point to deficits in GABAergic transmission in the pathogenesis of pain in this animal model, this conclusion is at variance with results indicating an increase in GABA levels associated with inflammatory pain (Malcangio and Bowery, 1996). It is conceivable the increase in GABA content may represent a compensatory response in the spinal cord for modulating an increased release of sensory transmitters.

It has also been reported that baclofen reduces craving for cocaine (Roberts and Andrews, 1997). Thus, at modest doses, baclofen suppresses cocaine self-administration in rat without affecting responses for food reinforcement, and baclofen treatment prevents the reinstatement of cocaine-seeking behavior. These data hint at a possible role for GBRs in drug addiction and suggest a new therapeutic application for GBR agonists.

As yet, GBR antagonists have not been examined clinically; however, preclinical data indicate such agents improve cognitive performance (Mondadori et al., 1993). Moreover, the administration of GBR antagonists prevents the thalamic spike-and-wave discharges characteristic of absence epilepsy (see Bowery, 1993). Although it has been suggested that GBR antagonists are themselves convulsant, the doses necessary for this response are far in excess of those that are anticonvulsant or enhance cognition, making it questionable whether this toxic central nervous system effect reflects a specific interaction with GBRs.

Data suggest that GBR antagonists are efficacious in models of depression (see Bowery, 1993). Moreover, like conventional antidepressants, GBR antagonists increase rat brain levels of nerve growth factor and brain-derived neurotrophic factor (Heese et al., 1999), which may be associated with an antidepressant effect. It is possible these changes in brain levels of growth factors predict a neuroprotectant role for GBR antagonists as well.

	Conclusions

Top Abstract Introduction GBR Function GBR Agonists and Antagonists Molecular Characteristics of... GBR Distribution and... GBR Subtypes Therapeutic Application of GABA... Conclusions References

Characterization of GBRs has provided new insights into the structural composition and assembly of G protein-coupled receptors. Because GBRs are one of many coupled to G_i and G_o, which regulate K⁺ and Ca²⁺ entry into neurons and inhibit transmitter release, information on its structure, function and pharmacology are of fundamental neurobiological and clinical importance. From a therapeutic standpoint, the next advances in GABA_B pharmacology will be the delineation of the clinical potential of GABA_B antagonists and the identification of GBR subtypes that can be selectively manipulated for therapeutic gain.