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.     

Effect of low concentration benzene vapor on metabolism of gamma-aminobutyric acid in brain mitochondrial fractions

The impact of benzene vapor (0.35 mg/l) of low concentration on the contents of free GABA, Glu and Asp, as well as on the enzyme activities of GDC and GABA-T in mitochondrial fractions of different brain regions of adult male rats was investigated. The conclusion was put forward that GABA increase by means of "keeping" inhibition provided for nerve cells defense from extremal excitation, taking place under the chronic exposure to the benzene low concentration.     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of repeated administration of insulin on pain threshold and gamma-aminobutyric acid level in mouse brain

In experiments on male Albino-Swiss mice weighing 18-22 g insulin given in doses of 2 i.u./kg caused no change in the time of reaction to pain, while the same dose administered daily for 7 days potentiated the analgesic action of morphine (3 mg/kg s.c.). Glucose caused no change in this effect of insulin. After 14 days of insulin treatment the time of reaction to pain in the animals subjected to the action of morphine returned to its initial value. Twenty-four hours after the last administration of morphine the level of gamma-aminobutyric acid (GABA) was found to be decreased in the animals receiving insulin with glucose. These results suggest that the central action of insulin is dependent not only on hypoglycaemia produced by it, but may be due also to its direct action on the central structures and an indirect action mediated by its effect on other neurotransmitter systems.     

Uptake of gamma-aminobutyric acid by catfish brain

Using homogenates of catfish whole-brain in an isotonic medium, we observed an accumulation of [3H]GABA that was temperature-sensitive and was dependent on the presence of sodium ions, the optimum concentration of which was 75 mM. A kinetic analysis showed that the [3H]GABA uptake mechanism became saturated with increasing GABA concentrations. A high-affinity system, only, was evident whose Km was calculated as 12 microM. Four structural analogues of GABA were found to be competitive inhibitors of uptake, and Ki values were determined. Nipecotic acid (Ki = 1.8 microM) and guvacine (Ki = 3.9 microM) were the most potent compounds, however 2,4-diaminobutyric acid (Ki = 8.9 microM) and beta-alanine (Ki = 55 microM) also had an effect. The characteristics of the uptake mechanism in catfish brain that we have studied are similar to those reported for uptake by mammalian brain except that in the latter, both a high- and a low-affinity transport processes are present. Our data, taken together with what is already known, strongly suggest that the biochemistry of the GABA system in lower vertebrates does not differ significantly from that in mammals.     

Antibiotic inhibitors of mitochondrial ATP synthesis.

Fourteen antibiotics have been found to inhibit oxidative phosphorylation and uncoupler-stimulated adenosinetriphosphatase in mitochondria. Four different types of binding sites for these inhibitors have been found. The first (1) binds aurovertin to purified MF1 ATPase in the stoichiometric ratio of two aurovertin molecules per molecule of ATPase. Site II is the locus for efrapeptin (A23871) and may be a catalytic site on purified ATPase. The remaining two sites have been demonstrated only in mitochondria or submitochondrial particles when the APTase is bound to other membrane components. Oligomycin, venturiciden, venturicidin X and ossamycin probably all bind at site III. Leucinostatin (A20668) binds at site IV. At low concentrations, this antibiotic acts like oligomycin; at higher concentrations it uncouples oxidative phosphorylation. Venturicidin appears to prevent leucinostation from binding at site IV for it allows uncoupling to occur at very low concentrations of the latter antibiotic. Venturicidin aglycone, which is a more effective inhibitor than its parent compound, does not exert this effect. It is concluded that sites III and IV are in juxtaposition and that when venturicidin binds at site III its sugar moiety projects into the area of site IV to prevent leucinostation from binding at its inhibitory site.     

Low-affinity gamma-aminobutyric acid transport in rat brain

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F₁ supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP.The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k ₊₁) and the reverse dissociation step (k _–1).k _–1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement ofk _–1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP.k ₊₁ was affected little by NADH oxidation. The dissociation constant (K _d ATP) increased many orders of magnitude during the transition from nonenergized to energized states.