GABA Pharmacology: The Search for Analgesics

Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABA_A and GABA_B receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.     

Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs

Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation–π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl^? channel.     

The GABA Synapse as a Target for Antiepileptic Drugs: A Historical Overview Focused on GABA Transporters

It is clear that normal neuronal function relies on a tight balance between excitatory and inhibitory neurotransmission. Inhibitory signaling through the GABAergic system can be tightly regulated at the level of GABA uptake via GABA transporters (GAT). As such, selectively modulating the GABA uptake process through pharmacological agents has been an area of active investigation over several decades. These studies have demonstrated that inhibition of astroglial, but not neuronal, GATs may be preferred for anticonvulsant action. To date, four distinct GAT subtypes have been identified and efforts to selectively target these transporters have led to the proliferation of pharmacological agents aimed at augmenting extrasynaptic GABA levels. These pharmacological tools have provided novel and informative insight into the role of GABA and GABAergic signaling in the brain, but have also provided critical information concerning the regulation of CNS disorders associated with an imbalance in inhibitory tone, such as epilepsy. One such compound with notable inhibitory effects at GATs, tiagabine, has demonstrated clinical anticonvulsant efficacy, and is, to date, the only approved GAT inhibitor for clinical use. Thus, efforts to identify and develop GAT subtype-specific compounds continue to be an area of active investigation for the management of epilepsy and other CNS disorders. Herein, the historical efforts to elucidate the role of GABA in the synapse, as well as the role of GAT inhibitors as anticonvulsants, are described.     

Muscimol as an Ionotropic GABA Receptor Agonist

Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol^®) and 4-PIOL.     

The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

GABA_A receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABA_A receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABA_A receptors. The two inhibitory current types are mediated by different subpopulations of GABA_A receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABA_A receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC₅₀ values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC₅₀ values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABA_A receptors located extrasynaptically using the antagonist, DPP-4-PIOL.     

Adsorption of γ-Aminobutyric acid to phosphatidylserine membranes

The interaction of the negatively-charged phosphatidylserine (PS) and γ-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10^?5-10^?4 M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of¹⁴C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

Activation of GABAB Receptors Ameliorates Cognitive Impairment via Restoring the Balance of HCN1/HCN2 Surface Expression in the Hippocampal CA1 Area in Rats With Chronic Cerebral Hypoperfusion

Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABA_B receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 μ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABA_B receptors were pathologically altered and investigated neuroprotective effects of GABA_B receptors activation and cross-talk networks between GABA_B receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABA_B R1, GABA_B R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABA_B receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABA_B R1, GABA_B R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 μ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABA_B R1 and GABA_B R2, and activation of GABA_B receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 μ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.     

Analysis of γ-Aminobutyric Acid (GABA) Type A Receptor Subtypes Using Isosteric and Allosteric Ligands

The GABA_A receptors (GABA_ARs) play an important role in inhibitory transmission in the brain. The GABA_ARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the ‘receptor-specific’ GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA_ARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABA_ARs. These binding sites depend on the GABA_AR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.     

Effect of glycine and gamma-aminobutyric acid on excitability of central terminals of primary afferent fibers

The effect of microelectrophoretically injected glycine and GABA on the excitability of intraspinal terminals of group Ia muscle afferents was studied in experiments on cats anesthetized with pentobarbital. The excitability of the terminals, studied by Wall's method, was reduced by the action of glycine but increased by that of GABA. The possible role of the two amino acids in presynaptic inhibition of spinal reflexes is discussed.     

Diethylpyrocarbonate modification of benzodiazepine receptors from calf cerebral cortex

Diethylpyrocarbonate (DEP), an amino acid modifying reagent, causes complete inactivation of particulate and deoxycholate-solubilized benzodiazepine-receptors from calf cerebral cortex. No heterogeneity was observed in DEP-sensitivity of the receptors. Protection from DEP-induced inactivation was provided by the centrally active benzodiazepines, diazepam and nitrazepam, but not by the peripherally active Ro5-4864, suggesting that DEP modifies a residue which is essential for the central actions of benzodiazepines. GABA did not protect against inactivation or influence the protection afforded by diazepam, indicating that the DEP-modifiable residue is independent of GABA binding sites, or that GABA binding sites are also sensitive to DEP. DEP-induced inactivation of benzodiazepine-receptors proceeds much faster at pH 10.1 than at pH 8.1 or 6.0, indicating the modification of a high pK_a side group, possibly the phenol of a tyrosyl residue. This postulation is in accord with our previous findings with the modifying reagents tetranitromethane and N-acetylimidazole.     

In vitro release of endogenous monoamines and amino acids from several CNS regions of the rat

The in vitro release of endogenous norepinephrine (NE), dopamine (DA), serotonin (5-HT), GABA, glutamate (GLU), aspartate (ASP), glycine (GLY), taurine (TAU) and alanine (ALA) from superfused slices of cerebral cortex (CTX), striatum (STR), hippocampus (HIP), hypothalamus (HYPO), midbrain (MB), thalamus (THAL), nucleus accumbens (ACC), pons-medulla (PM) and spinal cord (SC) was studied. Under resting conditions or with 60 mM K⁺ in the absence of Ca²⁺, there was little or no release of NE, DA, 5-HT, GABA, GLU or ASP from any region. In most regions, there was a measurable resting release of ALA, GLY and TAU; of these three amino acids, only GLY in the PM and SC showed an increased release in the 60 mM K⁺ plus 2.5 mM Ca²⁺ medium. In 8 of the regions studied, the release of both GABA and GLU were stimulated by 60 mM K⁺ in the presence of 2.5 mM Ca²⁺. For the amino acids, no reliable data were obtained for release from the ACC because of its small size. The highest amount of K⁺-stimulated, Ca²⁺-dependent release of GABA was found with slices from the HYPO, THAL and MB while the highest amount of GLU was released from slices of STR, HIP and CTX. In those regions where reliable levels of K⁺-stimulated, Ca²⁺-dependent release of ASP were observed (STR, CTX, THAL), the amount of ASP was at least 5-fold lower than the values for GLU. A K⁺-stimulated, Ca²⁺-dependent release of NE, DA and 5-HT was observed for all 9 CNS regions studied. The highest release of (a) DA occurred from slices of CTX, STR and ACC; (b) NE was found in the HYPO and ACC; and (c) 5-HT occurred in the HYPO. The data (a) do not support a transmitter role for ALA and TAU in the CNS; (b) support a major transmitter function for GLY only in the PM and SC; and (c) support a transmitter role for GABA, GLU, NE, DA and 5-HT in the CNS regions examined (with the exception of GABA and GLU in the ACC where no data were obtained).     

Blocking action of furosemide on chloride conductance induced by acetylcholine and gaba in molluscan neuron membrane

Experiments on isolated neurons of the molluskPlanorbarius corneus under membrane voltage clamp conditions showed that furosemide (2×10^?4 to 1×10^?3 g/mg) inhibits the increase in chloride conductance evoked by iontophoretic application of acetylcholine, suberyldicholine, and gamma-aminobutyric acid (GABA). If microelectrodes filled with potassium sulfate were used in the experiments furosemide did not shift the reversal potential, but when microelectrodes filled with potassium chloride were used the reversal potential of the chloride-dependent responses became less negative. In the last case, the action of furosemide evidently was exhibited not only on passive chloride conductance of the chemoreceptive membrane, but also on active chloride transport. Furosemide had no effect on sodium- and potassium-dependent responses evoked by activation of choline receptors. Unlike D-tubocurarine, which selectively blocks acetylcholine effects, furosemide also depressed conductance evoked by GABA. In the presence of furosemide chloride-dependent responses not only were reduced in amplitude, but also developed more slowly. It is postulated that the action of furosemide is aimed not at receptors, but at chloride channels of the chemoreceptive membrane common to both acetylcholine and GABA.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Pharmacological Identification of a Guanidine-Containing β-Alanine Analogue with Low Micromolar Potency and Selectivity for the Betaine/GABA Transporter 1 (BGT1)

The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [³H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC₅₀ value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.     

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Inhibitory systems sensitive to catecholamines and GABA in the inner plexiform layer of the frog retina

Local electroretinography was used to study the effect of pharmacological agents injected into the eye on lateral inhibitory interaction in the inner plexiform layer of the frog retina. Adrenoblocking agents, unlike cholinoblocking agents, were found to abolish dynamic lateral inhibition. It is postulated on the basis of the results that the inhibitory mediator in the system of dynamic lateral inhibition is a catecholamine. Yet another type of inhibition at the level of the amacrine cells, differing from dynamic inhibition (steady inhibition of adaptive type), sensitive to picrotoxin, was revealed with the aid of GABA and analeptics (strychnine and picrotoxin). It is postulated that GABA acts in this inhibitory system as an inhibitory mediator. On the basis of the results and analysis of the morphological picture of the plexiform layer, a model is proposed for the synaptic organization of lateral inhibition at the level of the amacrine cells.