ABSTRACT
While the physiological role of GHB is unclear, it is known that a large variety of pharmacological responses are produced by the administration of high doses (100400mg/kg) of exogenous GHB (Maitre 1997; Table 3.2a). Oral, intraperitoneal or intravenous application of GHB or of its metabolic precursor γ-butyrolactone (GBL, Fig. 3.1) dose-dependently (1) depresses CNS function and induces sedation, sleep and coma (Maitre 1997), (2) induces EEG hypersynchronization sometimes associated with absence-like seizures (Snead 1991), (3) hyperpolarizes neuronal membranes (Olpe and Koella 1979; Harris et al. 1989; Xie and Smart 1992; Williams et al. 1995), and (4) alters synthesis and/or release of various neurotransmitters (GABA, glutamate or dopamine) (Waldmeier 1991; Engberg and Nissbrandt 1993; Nissbrandt et al. 1994; Banerjee and Snead 1995; Bernasconi et al. 1995; Emri et al. 1996; Feigenbaum and Howard 1997; Hu et al. 2000). These behavioral, EEG, cellular and biochemical responses occur only when GHB levels are increased 50-100-fold above their endogenous concentrations (Cash 1994). This discrepancy suggests that the effects of exogenous GHB are not only
Figure 3.1 Structures of some agonists and antagonists of GHB binding sites and GABAB receptors, respectively. GBL does not bind to GHB binding sites or to GABAB receptors and is inactive per se. However, as GBL is rapidly and irreversibly hydrolyzed to GHB by peripheral γ-lactonase and is more rapidly and reproducibly absorbed, it is often used as a GHB precursor in animal experiments. (GHB: γhydroxybutyric acid; HA-966: S-(−)-3-amino-lhydroxy-pyrrolidin-2one; GBL: γ-butyrolactone; TCHA: trans-γ-hydroxycrotonic acid; NCS-382:6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6ylideneacetic acid; R-(−)baclofen: 4-amino-3-(R)-3-(4-pchlorophenyl)-butyric acid; CGP 27492: (3-amino-propyl)- phosphinic acid; CGP 35348: (3-amino-propyl)-diethoxymethylphosphinic acid; CGP 36742: (3-amino-propyl)-n-butyl-phosphinic acid; CGP 64213:3-{l-(R)-[2-(S)-hydroxy-3(hydroxy-{5-[3-(4hydroxy-3-iodo-phenyl)-propionylamino]-pentyl}-phosphinoyl)- propylamino]-ethyl}-benzoic acid.)
On the occasion of his eightieth birthday, the authors would like to dedicate this work to Dr H. Keberle, who synthesized baclofen about forty years ago. mediated through stimulation of GHB binding sites, but also through other mechanisms. Indeed, many biological effects secondary to systemic administration of high doses (100400 mg/kg) can be blocked by GABAB receptor antagonists, but are not suppressed by NCS-382, an antagonist at [3H] GHB binding sites (Maitre et al. 1990). Moreover, some pharmacological properties of exogenous GHB are shared by GABAB receptor agonists such as R-(−)-baclofen (Xie and Smart 1992; Engberg and Nissbrandt 1993; Williams et
Table 3.1 Displacement of various radioligands by GHB
Radioligand Binding site GHB affinity Reference
[3H]GHB GHB K d 1=30-580 nMa
Maitre (1997)
K d 2=2.3-16 Ma
[3H]baclofen GABABK i=125 M bBernasconi et al. (1992)
[3H]baclofen GABABK i=398 M b,cIto et al. (1995)
[3H]baclofen GABABK i = 630 M b,cIshige et al (1993, 1996)
[3H]CGP 27492 GABABK i=80-120 Mb,d
Mathivet et al. (1997)
[3H]GABAe GABABK i=80-120 Mb,d
Mathivet et al. (1997)
[3H]CGP 54626 GABABK i = 3,300 M fMathivet et al. (1997)
[3H]muscimol GABAA-Bernasconi et al. (1992)
[35S]TBPSg Cl− channel — Serra et al. (1991)
[3H] flunitrazepamh Benzodiazepine — Serra et al. (1991)
[3H]naloxone -Opioid — Bernasconi et al. (1992)
[3H]DAGO -Opioid — Feigenbaum and Simantov (1996)
[3H] diprenorphine -Opioid — Feigenbaum and Simantov (1996)
[3H]etophirne -Opioid — Feigenbaum and Simantov (1996)
[3H]prazosin 1-Adrenoceptor — Bernasconi et al. (1992)
[3H]clonidine 2-Adrenoceptor — Bernasconi et al. (1992)
[3H]DHA -Adrenoceptor — Bernasconi et al. (1992)
[3H]5-HT 5-HT1-Bernasconi et al. (1992)
[3H]doxepine Histamine H1 — Bernasconi et al. (1992)
[3H]QNB Muscarinic — Bernasconi et al. (1992)
[3H]CHA Adenosine A1 — Bernasconi et al. (1992)
[3H] substance P Substance P — Bernasconi et al. (1992)
[3H]MK801 NMDA — Gessa et al. (1993)
al. 1995; Madden and Johnson 1998; Tables 3.2a and b). These findings, suggesting that exogenous GHB can act as an agonist at GABAB receptors, were confirmed by binding studies demonstrating that GHB is a weak, but selective, agonist of GABAB receptors (Bernasconi et al. 1992; Mathivet et al. 1997). More recently, Lingenhoehl et al. (1999) have demonstrated that GHB activates recombinant heteromeric GABABR1/R2 receptors expressed in Xenopus oocytes. However, these data do not imply that GABAB receptors mediate all the actions of endogenous or exogenous GHB (Feigenbaum and Howard 1996; Maitre 1997; Mathivet et al. 1997). It is unlikely that endogenous GHB activates The methods used for the [3H]GHB binding and for the GABAB receptor assay using agonist and antagonist radioligand are different. Crude membrane fractions prepared from total rat brain bind [3H]GHB in a saturable and reversible manner (Benavides et al. 1982a). The binding reaches equilibrium within 15-20 min at 0°C in a buffered medium. Maximum binding occurs at pH 5.5. Absence of Ca2+ in the incubation buffer does not change the binding characteristics. The GABAB receptor-binding assays are performed using frozen and thawed crude membranes prepared from rat cerebral cortex (Bittiger et al. 1996). The radioreceptor assay is performed at pH 7.4 and at 20 °C for 40 min. Ca2+ is necessary for the high affinity of GABA on native and recombinant GABAB receptors (Galvez et al. 2000). Notes a K d1=high-affinity and K d2=low-affinity GHB binding sites. b K i values were calculated from the IC50 values by means of the Cheng-Prusoff equation (Leff and Douglas 1993). c Ito et al. (1995) and Ishige et al. (1996) use cultured cerebellar granule cells and mouse cerebellar membrane, respectively, d Similar K i of GABAB receptors were measured in membranes prepared from rat hippocampus or cerebellum, e This binding assay is performed in the presence of the GABAA agonist isoguvacine hydrobromide. f The IC50 values of GABAB receptor agonists are much higher in the antagonist [
3H]CGP 54626 assay than in the [3H]baclofen and [3H]CGP 27492 agonist radioligand assays (Bittiger et al. 1992). g GHB does not modify in vitro muscimol-stimulated36 Cl-uptake. In vivo administration of sedative and hypnotic doses of GHB (300-700 mg/kg) fail to induce any significant changes in the [35S]TBPS binding. Moreover, GHB also does not antagonize the increase in [35S]TBPS binding induced by isoniazid. h GHB fails to modify the modulatory action of GABA on [3H]flunitrazepam binding. TBPS: t-butylbicyclophosphorothionate; DAGO: (D-Ala2, N-Me-Phe4, glycinol5)-enkephalin; DHA: dihydroalprenolol; QNB: quinuclidinyl benzylate; CHA: cyclohexyl-adenosine;—: no displacement of specific binding by GHB up to10−4M.
