Influence of pipecolic acid on the release and uptake of [3H]GABA from brain slices of mouse cerebral cortex

Recently, pipecolic acid (PA) has been involved in the functioning of the GABAergic system. In the present work we have studied the effect of PA on GABA uptake and release in cerebral cortex slices. PA (100 M) was able to increase the release of [³H]GABA (90%) stimulated by mild depolarization with 15 mM potassium. If during the labeling of the tissue with [³H]GABA, -alanine was present, PA also enhanced the release (42%). However, when nipecotic acid was present instead -alanine, no stimulation of [³H]GABA release by potassium was observed neither in the control nor in the presence of PA. Spontaneous release was not affected by PA in any of the experimental conditions tested. In uptake experiments, only when -alanine was present in the medium PA significantly diminished the uptake (36%) of [³H]GABA. These results suggest that the effect of PA is mostly at the presynaptic level, inhibiting the neuronal GABA uptake and/or enhancing its release.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.     

Chemical integrity of [3H]GABA used in binding studies

A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using [³H]GABA. The method was found to be unsuitable for³H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with [¹⁴C]GABA.     

Purification and characterization of a benzodiazepine-like substance from mammalian brain

An endogenous brain ligand which competes with [³H]-flunitrazepam for the binding to benzodiazepine receptor has been isolated and purified to homogeneity. The purification procedures involve the extraction of the endogenous ligand by homogenizing the brain tissue in water containing various protease inhibitors followed by filtration through a PM 10 membrane (exclusion limit: 10,000-dalton), column chromatographies on Sephadex G-50, Bio-Rad P2 and a series of C₁₈ reverse phase HPLC columns. The purified endogenous ligand was eluted as a single and symmetrical peak monitored at either 220 or 280 nm. Furthermore, the ligand activity coincided with the absorption peak. The purified endogenous ligand is thermostable, insensitive to various peptidases and proteolytic enzymes, resistant to DNAse, RNAse, and carbohydrate enzyme e.g. neuraminidase (EC 3.2.1.18) and acid treatment. It has a major absorption peak at 220 nm and a minor one at 313 nm. The endogenous ligand appears to be quite specific since it only inhibits the binding of ligand to the central type benzodiazepine receptor but not to other receptors, e.g. peripheral type benzodiazepine receptor, ₁-adrenoceptor, ₂-adrenoceptor, -adrenoceptor and muscarinic cholinergic receptor. Furthermore, the inhibition of the receptor binding by the endogenous ligand is enhanced by GABA suggesting that the endogenous ligand is a benzodiazepine receptor agonist. The structure of the endogenous ligand is unknown.Special issue dedicated to Dr. Elling Kvamme     

Substance P affects the GABAergic system in the hypothalamo-pituitary axis

In the present work we examined the effect of the neutralization of endogenous substance P by the administration of an anti-substance P serum (ASPS) on GABA concentration in the anterior pituitary in hyperprolactinemic conditions induced by 5-hydroxytryptophan or by grafting anterior pituitaries. ASPS reduced the increase in the anterior pituitary GABA concentration induced by hyperprolactinemia. In vitro experiments showed that substance P inhibited K⁺-evoked GABA efflux from hypothalamic fragments and decreased GABA concentration in the anterior pituitary but ASPS increased it. Our results demonstrate that substance P modifies hypothalamic GABA release and anterior pituitary GABA concentration and suggest that an interaction exists between substance P and GABA.     

Tetanus Toxin-Induced Effects on Extracellular Amino Acid Levels in Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: Tetanus toxin is a potent neurotoxin that is widely considered to produce its effect through impairment of inhibitory neurotransmission. We report the effect of a single unilateral intrahippocampal injection of tetanus toxin on extracellular levels of neuroactive amino acids in freely moving rats, at times ranging between 1 and 7 days posttreatment. Tetanus toxin treatment did not alter extracellular levels of aspartate, glutamate, and taurine at any time during the study. However, although extracellular GABA levels were unaffected by toxin injection 1, 2, and 3 days after treatment, they were reduced (45 ± 8% of contralateral vehicle-injected level) at day 7. Challenge with a high K⁺ concentration, 7 days after treatment, produced elevations in extracellular levels of taurine and GABA in both vehicle- and toxin-injected hippocampi, with evoked levels of GABA being lower in the toxin-treated side (39 ± 16% of contralateral vehicle-injected level). Aspartate and glutamate levels were not increased by high-K⁺ infusion. These findings are discussed in relation to the possible role that an imbalance in excitatory/inhibitory tone may play in the production of tetanus toxin-induced neurodegeneration.     

Responses of non-spiking giant interneurons to substrate tilt in the crayfish, with special reference to multisensory control in the compensatory eyestalk movement system

In the brain of the intact crayfish, three pairs of non-spiking giant interneurons (G1, G2, G3; NGIs) scarcely responded to substrate tilt about the longitudinal axis of the body either in the dark or in the presence of an overhead light. However, when the statolith was removed, these NGIs responded with depolarizing and hyperpolarizing potentials respectively to upward movements of the ipsilateral legs (2nd–5th pereiopods) and upward movements of the contralateral legs produced by substrate tilt. The relationships between the polarity of the potential and the direction of movement in the contralateral legs were opposite to those in the ipsilateral legs. The amplitude of the responses was proportional to the frequency (0.5-0.05 Hz) and amplitude of tilting. When the legs were moved unilaterally, the NGIs responded with depolarizing and hyperpolarizing potentials to upward movements of the ipsilateral legs and to upward movements of the contralateral legs, respectively. When the legs were moved bilaterally in the same direction by upward or downward movement of the substrate, the NGIs scarcely responded to the leg movements. A hypothetical model is presented to account for the pathways of sensory inputs to the NGIs and the role of NGIs in compensatory oculomotor system.     

A role for GABAergic inhibition in electrosensory processing and common mode rejection in the dorsal nucleus of the little skate, Raja erinacea

The electrosensory primary afferents in elasmobranchs are responsive to electric potentials created by the animal's own ventilation, while the second-order neurons (AENs) which receive this afferent input in the medulla suppress responses to ventilatory potentials but retain their extreme sensitivity to electric signals in the environment. Ventilatory potentials are common mode signals in elasmobranchs and a common mode rejection mechanism is one way the AENs suppress ventilatory noise. By pressure injecting the GABA-A receptor antagonist SR95531 while extracellularly recording from AENs, we tested the hypothesis that the subtractive circuitry that selectively reduces common mode signals in AENs utilizes GABA, and that a GAB-Aergic component of the dorsal nucleus commissural pathway mediates crossed inhibition of AENs. Local application of SR95531 increased the spontaneous activity and the responsiveness of AENs to electrosensory stimuli. AEN responses to a common mode stimulus were selectively increased compared to responses to a localized stimulus due to SR95531 application. Contralateral inhibition of AENs was blocked by SR95531, indicating that GABAergic commissural cells may inhibit AENs when the contralateral side of the body is stimulated, as with common mode stimulation. We conclude that GABAergic inhibition contributes significantly to the shaping of AEN responses including common mode rejection.Abbreviations AENs ascending efferent neurons - GABA gamma-aminobutyric acid