Norman G. Bowery

Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals.

The action of gamma-aminobutyric acid (GABA) and related compounds on rat isolated atria and mouse and guinea pig isolated vas deferens has been studied. GABA depressed the evoked but not basal release of [3H]noradrenaline from atria (IC50 4 micro M) and reduced the twitch responses of the vas deferens (IC50 3 micro M) in a dose-dependent manner. These depressant effects were not prevented by recognized GABA antagonists such as bicuculline and picrotoxin. Numerous GABA analogues, in particular 3-aminopropanesulphonic acid, failed to mimic the action of GABA. However, beta-p-chlorophenyl GABA (baclofen) was stereospecifically active. Other related beta-substituted derivatives were also active but to a lesser degree than GABA. Pretreatment of the vas deferens with the neuronal GABA uptake inhibitors 2,4-diaminobutyric acid or cis-3-aminocyclohexanecarboxylic acid potentiated the action of GABA. These data suggest the presence of a bicuculline-insensitive GABA receptor on autonomic nerve terminals. Preliminary observations indicate a lack of chloride ion dependence in the action of GABA at this site.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes

1 Saturable binding of (±)‐[3H]‐baclofen and [3H]‐γ‐aminobutyric acid ([3H]‐GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]‐baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+‐dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]‐baclofen formed 20.3 ± 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 μm), (–)‐baclofen (0.04 μm) and to a much lesser extent by (+)‐baclofen (33 μm). Isoguvacine, piperidine‐4‐sulphonic acid and bicuculline methobromide were inactive (up to 100 μm) and muscimol was only weakly active (IC50 = 12.3 μm). 4 Saturable binding of [3H]‐GABA increased on adding CaCl2 or MgSO4 (up to 2.5 Mm and 5.0 Mm respectively) to the Tris‐HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline‐sensitive binding of GABA (GABAA site binding) and could be completely displaced by (–)‐baclofen (IC50 = 0.13 μm). 5 Increasing the Ca2+ concentration (0 to 2.5 Mm) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]‐GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non‐ionic detergent Triton‐X‐100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]‐GABA from GABAB sites correlated well with that for [3H]‐baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]‐baclofen or [3H]‐GABA are both ligands for the same bicuculline‐insensitive, divalent cation‐dependent binding sites in the rat brain.

Light microscopic autoradiographic localisation of [3H] glycine and [3H] strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.

Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.

1 An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK‐801, in rat and gerbil brain sections. 2 Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nm [3H]‐MK‐801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 μm unlabelled MK‐801 amounted to 55–60% of total. 3 Phencyclidine, (±)‐SKF 10047, ketamine and 2‐aminophosphonovaleric acid (APV) (all 100 μm) prevented the specfic binding of [3H]‐MK‐801. l‐Glutamate and N‐methyl d‐aspartate (NMDA) (100 μm) had no effect. However, l‐glutamate prevented the inhibition by APV. 4 The highest concentrations of [3H]‐MK‐801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5 The distribution of [3H]‐MK‐801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]‐TCP) but not with high‐affinity σ sites labelled with [3H]‐3‐PPP. 6 The density of [3H]‐MK‐801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7 These results indicate that binding sites for [3H]‐MK‐801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.

Effect of interleukin-1β on the release of substance P from rat isolated spinal cord

Superfusion of rat spinal cord slices with rat interleukin-1 beta resulted in a significant enhancement of electrically evoked substance P-like immunoreactivity with a maximal effect (> 2-fold increase) at 0.1 ng/ml, whereas higher concentration (10-50 ng/ml) of the cytokine inhibited (approximately 50%) the release of the neuropeptide. Interleukin-1 beta (0.1 ng/ml) potentiation of substance P-like immunoreactivity release was abrogated by co-perfusion with interleukin-1 receptor antagonist (10-100 ng/ml) or with indomethacin (1 microM). Superfusion of spinal cord with interleukin-1 beta inhibited electrically evoked calcitonin gene-related peptide-like immunoreactivity release. Modulation of substance P-like immunoreactivity release from the spinal cord by interleukin-1 beta may represent a mechanism responsible for the hyperalgesic action of the cytokine characteristic of the inflammatory response.

GABA, glutamate and substance P‐like immunoreactivity release: effects of novel GABAB antagonists

1 The effects of various GABA receptor ligands on the electrically‐evoked release of endogenous GABA, glutamate and substance P‐like immunoreactivity from the dorsal horn of rat isolated spinal cord were examined. 2 Exogenous GABA (10–300 μm) significantly decreased the evoked, but not basal, release of endogenous glutamate in a concentration‐dependent manner. The GABAA agonist, isoguvacine (1–100 μm), failed to decrease the release of glutamate although it did reduce the release of GABA. Baclofen (0.1–1000 μm), the GABAB agonist, reduced the release of GABA and glutamate in a stereospecific and concentration‐dependent manner. 3 The actions of five GABAB antagonists on these release systems were compared. CGP36742, CGP52432, CGP55845A and CGP57250A significantly increased the evoked release of GABA and glutamate. They also reversed the effects of (−)−baclofen in a concentration‐dependent manner. On the other hand, while CGP56999A had no effect on glutamate release, it was an effective antagonist of the baclofen‐induced inhibition of GABA and substance P release. 4 These results suggest that GABAB receptors on nerve terminals within the dorsal horn spinal cord may be heterogeneous. However, this is based solely on the data obtained with CGP56999A which affected only GABA and substance P, but not glutamate, release.

Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of the GABAB receptor system

1 Monoarthritis was induced in Lewis rats by interdermal injection in the left hind paw of a suspension of Mycobacterium tubercolusis in mineral oil (500 μg 100 μ1−1). Controls were injected with 100 μ1 mineral oil. 2 Withdrawal latencies to thermal stimuli of the inflamed paw, the contralateral and both paws of control rats were measured at daily intervals after injection by the plantar test. 3 After detection of the pain threshold, rat spinal cords were removed and horizontal dorsal slices were mounted in a 3‐compartment bath to measure electrically‐evoked release of substance P‐like immunoreactivity (SP‐LI). 4 The inflamed paw of monoarthritic rats exhibited a lower pain threshold to thermal stimuli than the contralateral paw of the same animals and both paws of control rats. Inflamed paw hyperalgesia was maximal two days after injection, and declined gradually between 7 to 21 days with no evidence of excitability of withdrawal reflexes after 28 days. 5 During the 28 days study, monoarthritic rats gained less weight than control rats. 6 Electrical stimulation of the dorsal roots attached to rat isolated spinal cord slices induced a significant increase (174 ± 18% of basal outflow which was 30.3 fmol 8 ml−1 n = 5) in SP‐LI release. 7 One‐week after induction of inflammation no differences in the amount of SP‐LI released from the spinal cord of incomplete Freunds adjuvant‐treated rats (IFA) and Freunds adjuvant‐treated rats (CFA) were detected. Two weeks after, CFA spinal cord tended to release more SP‐LI than IFA cords and, 21 days after injection, the spinal cord of CFA rats released significantly more peptide than IFA rats (17.8 ± 2.8 fmol ml−1, n = 12 and 6.9 ± 3.2 fmol ml−1, n = 9, respectively). 8 Twenty‐one days after treatment, the evoked release from monoarthritic rat spinal cords was increased by 263 ± 42% (n = 3) in the presence of the GABAB receptor antagonist, CGP 36742 (100 μm) which also significantly potentiated monoarthritis‐induced hyperalgesia up to 45min after injection (100mg kg−1, i.p.). 9 These findings may provide a basis for a novel approach to chronic pain therapy but also an explanation for the lack of analgesia produced by the GABAB agonist, baclofen, in chronic as compared to acute pain.

Plasticity of GABAB receptor in rat spinal cord detected by autoradiography

Prolonged administration of (+/-)-baclofen induces tolerance to its antinociceptive effect in mice. In the present study we compare the effects of 21 days administration of (-)-baclofen or GABAB (gamma-aminobutyric acidB) receptor antagonists, with saline on the density of GABAB receptor binding sites in rat spinal cord using receptor autoradiography. Treatment with (-)-baclofen significantly reduced the number of silver grains (71%) whilst GABAB receptor antagonists, CGP 36742 (3-aminopropyl-n-butyl-phosphinic acid) and CGP 46381 (3-aminopropyl-cyclohexylmethylphosphinic acid), increased the grain count by 114% and 89%, respectively. These data indicate that GABAB receptors in the spinal cord can be influenced by prolonged administration of receptor ligands.

Possible therapeutic application of GABAB receptor agonists and antagonists

After their discovery within the mammalian periphery in 1981, gamma-aminobutyric acid-B (GABAB) receptors have been characterized also in the central nervous system (CNS). The highest concentrations of GABAB binding sites appear to be in the cerebellum, frontal cortex, and thalamic nuclei, where they are located on pre- and postsynaptic neurons. On activation, the primary effects appear to be membrane hyperpolarization, suppression of transmitter release, and changes in the levels of cyclic nucleotides. GABAB receptors have been implicated in a variety of neurological phenomena and, as a consequence, receptor agonists and antagonists may well have therapeutic potential. This article is an introduction to GABAB receptor pharmacology and reviews the future of the receptor ligands. Particular attention is given to the role of spinal cord GABAB receptors in chronic pain.

Chronic (-)baclofen or CGP 36742 alters GABAB receptor sensitivity in rat brain and spinal cord.

Administration of the GABAB receptor agonist, (-)-baclofen 10 mg kg-1, i.p. daily for 21 days to rats prevented (-)-baclofen-induced hyperpolarizing responses and synaptically-evoked late inhibitory potentials (IPSPs) in olfactory cortical neurones recorded intracellularly from 450 microns brain slices. In contrast, pre-treatment with CGP 36742 induced a significant increase in (-)-baclofen-mediated post-synaptic responses and late IPSP amplitude. In the spinal cord, the potency of (-)-baclofen in inhibiting electrically-evoked substance P-like immunoreactivity or amino acid release was significantly reduced or increased in slices from rats pre-treated with the GABAB agonist or antagonist, respectively. These data suggest that functional responses to GABAB receptor activation in the mammalian central nervous system can be up- or down-regulated.