Comparison of the effect of gamma-aminobutyric acid and its derivative baclofen on the activity of Purkinje cells of frog cerebellum in vitro

The inhibitory effect of gamma-aminobutyric acid (GABA) and its synthetic derivative baclofen were compared in frog cerebellum in vitro. Baclofen inhibited synaptic transmission from parallel fibres to the Purkinje cells in EC50 concentrations approximately 200-fold lower than for GABA. In addition to its inhibitory effect, GABA induced temporary facilitation of responses in the dendrite zone by a mechanism dependent on the presence of a normal Cl- concentration; the inhibitory phase was only partly sensitive to reduction of the Cl- concentration in the medium and to the administration of picrotoxin. The action of baclofen, which was unaffected by these treatments, requires an intact catecholamine and serotonin pool, since it is ineffective in reserpine-treated animals. Both substances also influence the excitability of parallel fibres. In solutions with a high Mg2+ and a low Ca2+ concentrations GABA inhibits the spontaneous activity of Purkinje cells by acting on the postsynaptic membrane of the soma and the primary dendrites. The effect of baclofen is evidently the outcome of inhibition of transmitter release from presynaptic endings.     

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

The effect of phospholipases and proteases on the binding of gamma-aminobutyric acid to junctional complexes of rat cerebellum.

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with gamma-amino [14C]butyric acid at 25degreesC for 10 min, using [3H]sucrose as a marker for entrapped space, Total binding was determined in the absence of, and non-specific binding in the presence of, and excess of unlabelled gamma-aminobutyric acid. The difference bewteen the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or L-2,4-diaminobutyric acid. The above compounds had little effect on the non-specipic binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A2 (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with gamma-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with gamma-aminobutyric acid for binding to the receptor protein.     

Stimulating effect of gamma-aminobutyric acid on rat hippocampal neurons in vitro

Effects of GABA on the background and electrically stimulated activity of single neurons and population spikes were investigated in isolated hippocampal slices. Application of relatively large GABA concentrations (10(-3) mol/l and more) depressed an antidromic population spike, field EPSP and neuronal background activity. Low concentrations of GABA (less than 10(-3) mol/l) added to the bath increased the population spikes amplitude and the late component of field EPSP, facilitated single neurone responses, their background activity and epileptiform discharges. GABA-evoked depolarization was observed in the majority of the studied neurons. The duality of the GABA action on central neurons are discussed.     

In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation

Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract. Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena. Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function. The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved. GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml). Scatchard analysis of [(3)H]-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.). [(3)H]-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay). We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin. It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R.     

Enhancement of diazepam and gamma-aminobutyric acid binding by (+)etomidate and pentobarbital

Abstract: (+) Etomidate and pentobarbital enhance [³H]diazepam and [³H]γ-aminobutyric acid ([³H]GABA) binding to cerebral cortex membranes. Both (+)etomidate and pentobarbital increase the affinity of [³H]diazepam for its binding sites. In contrast, they increase the B _max of both the high- and low-affinity GABA receptor sites. The enhancement of [³H]diazepam and [³H]GABA by (+)etomidate and pentobarbital is blocked by GABA antagonists. These results indicate that hypnotic drugs such as (+)etomidate and pentobarbital, which are not structurally related, modulate diazepam and GABA binding sites via similar mechanisms.     

The effect of hypoxia on brain gamma-aminobutyric acid levels

(1) Animals were exposed to hypoxic environments either by supplying them with breathing mixtures low in oxygen or by exposing them in a decompression chamber to simulated altitude. Both methods of producing hypoxia brought about significant increases in brain GABA levels. (2) Elevated GABA levels occurred in all species tested (mouse, hamster, rat, guinea pig, and rabbit) and reached maximal concentration 60 min after the initiation of breathing the hypoxic mixtures. Extension of the exposure beyond 60 min brought about a gradual decline in GABA level from the maximal value reached. (3) A linear relation was found between the oxygen content of the gas mixture and the elevation of GABA level. For guinea pigs, at least, the critical oxygen content required to prevent elevation of GABA level was 8.1 per cent.     

The effect of gamma-aminobutyric acid on brain mitochondrial ATP synthesis

—Rat brain mitochondrial ATP synthesis was studied by measuring labeled orthophosphate incorporation into ADP to form ATP. GABA stimulates ATP synthesis, and this effect requires glutamate since with GABA alone little ATP is formed. The significance of this effect by GABA is unclear, but the mechanism may relate to induction of conformational change or chelation of cation.     

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.     

The effect of gibberellic acid on the response of leaf extension to low temperature

The effect of cooling on leaf extension rate (LER) and on relative elemental growth rate (REGR) was measured in both gibberellic acid (GA)-responsive dwarf barley and in the same barley variety treated with GA. Seedlings were maintained at 20 degrees C while their leaf extension zone (LEZ) temperature was reduced either in steps to -6 degrees C in short-term cooling experiments, or to 10 degrees C for 48 h in long-term cooling experiments. Short-term cooling resulted in a biphasic response in LER, with a clear inflection point identified. Below this point, the activation energy for leaf extension becomes higher. The short-term response of LER to cooling was altered by the application of GA, which resulted in a lower base temperature (Tb), inflection point temperature and activation energy for leaf extension. Both GA-treated and untreated seedlings were less sensitive to cooling maintained for a prolonged period, with LER making a partial recover over the initial 5 h. Although long-term cooling reduced maximum REGR, it resulted in a longer LEZ and an increase in the length of mature interstomatal cells in GA-treated and untreated seedlings. These changes in overall physiology appear to enhance the ability of the leaves to continue expansion at suboptimal temperatures. In both GA-treated and cold-acclimated tissue, the occurrence of a longer LEZ was associated with a lower temperature sensitivity in LER.     

The influence of rabies immunization of gamma-aminobutyric acid metabolism in the brains of animals]

Subcutaneous injection to albino rats (100-120 g) of lived fixed rabies virus was accompanied by a brief marked decrease in the content of gamma-aminobutryic acid in the animals' brain. There was also an increase in the activity of gamma-aminobutyric acid alpha-ketoglutaric acid transaminase in the brain tissue of animals vaccinated with live fixed rabies virus.     

The in vitro effect of temperature on motility and antioxidant response of common carp Cyprinus carpio spermatozoa

The effect of temperature on Cyprinus carpio spermatozoa in vitro was investigated with spermatozoa activated at 4, 14, and 24 °C. At 30 s post-activation, motility rate was significantly higher at 4 °C compared to 14 and 24 °C, whereas highest swimming velocity was observed at 14 °C. The thiobarbituric acid-reactive substance (TBARS) content was significantly higher at 14 °C and 24 °C than at 4 °C in motile spermatozoa. No significant differences in catalase and superoxide dismutase activity relative to temperature were observed. This study provides new information regarding effect of temperature on lipid peroxidation intensity and spermatozoon motility parameters in carp. The elevation of TBARS seen at higher temperatures could be due to inadequate capacity of antioxidant enzymes to protect the cell against the detrimental effects of oxidative stress induced by higher temperatures.