Mechanism of glutamate release from rat hippocampal slices during in vitro ischemia.

There was a large release of endogenous glutamate and of pre-accumulated [3H]-D-aspartate from rat hippocampal slices during deprivation of oxygen and glucose (in vitro ischemia). The role of Na(+)-dependent glutamate transporters in this process was investigated. The release of both glutamate and [3H]-D-aspartate was largely blocked by two competitive substrate analogues of the Na(+)-dependent glutamate transporters (L-trans-pyrrolidine-2,4-dicarboxylate and D,L-threo-B-hydroxyaspartate) if the substrate analogues were intracellularly loaded prior to the ischemia. The pre-loaded analogue, D,L-threo-B-hydroxyaspartate, did not block exocytotic release of glutamate, induced by high-potassium. Dihydrokainate, an inhibitor of a subset of the Na(+)-dependent transporters, did not inhibit ischemia-induced release of glutamate or [3H]-D-aspartate. However, it did block release induced by veratridine, which was also blocked by the pre-loaded substrate analogues. Dihydrokainate could still inhibit veratridine-induced release during ischemia, showing that conditions during ischemia did not reduce its efficacy. It is concluded that release of glutamate during ischemia is largely via reversal of the Na(+)-dependent glutamate transport system. The differential effects of dihydrokainate and the competitive substrate analogues on ischemia-induced release indicate that this release occurs via a subset of the glutamate transporters that are present in the hippocampus.

Evidence for amino acid concentration gradients between CSF and extracellular fluid.

A small volume in the extracellular space of the medulla in the anesthetized cat was perfused with cisternal cerebrospinal fluid (CSF) using a push-pull technique. The recovered perfusate was a mixture of pushed CSF and the extracellular fluid. HPLC-EC analysis showed that the concentration of some primary amino acids in recovered perfusate often differed from their concentrations in CSF. These results suggested that amino acid gradients existed between CSF and the extracellular space.

Multiple acyl-coenzyme A dehydrogenation disorder responsive to riboflavin: substrate oxidation, flavin metabolism, and flavoenzyme activities in fibroblasts.

Multiple acyl-CoA dehydrogenation disorders result from generalized defects in intramitochondrial acyl-CoA dehydrogenation. Fibroblasts from a riboflavin-responsive multiple acyl-CoA dehydrogenation disorder patient catabolized 14C-butyrate, -octanoate, and -leucine normally after culture in riboflavin-supplemented medium (2 mg/L). After culture in riboflavin-depleted medium (< or = 1.4 micrograms/L), his cells oxidized the same substrates poorly at 20 to 33% of control (p < 0.05). Patient cells incubated in a wide range of D-[2-14C]riboflavin concentrations (3, 31.4, and 100 micrograms/L) synthesized 14C-flavin mononucleotide and 14C-flavin adenine dinucleotide (FAD) normally and had normal cytosolic 14C-flavin mononucleotide and 14C-FAD contents, which argues against defects in cellular riboflavin uptake and conversion to flavin mononucleotide and FAD. After culture in 31.4 micrograms 14C-riboflavin/L for 2 wk, 14C-FAD specific radioactivities plateaued and were similar in patient and control cells. However, culturing these uniformly labeled cells in riboflavin-depleted medium for 2 wk lowered the patient's cellular 14C-FAD content to only 23% of control levels. Similarly, after incubation in low 14C-riboflavin concentrations (4.4 micrograms/L), the patient's mitochondrial 14C-FAD content was only 51% of control after 1 h and 29% of control at 4 h. After a 4-h incubation in a high physiologic concentration of 14C-riboflavin (31.4 micrograms/L), which raised the patient's cellular 14C-FAD levels 3- to 4-fold, his mitochondrial 14C-FAD content rose to normal; control values did not change. We also investigated possible defective FAD binding to flavoenzymes essential for acyl-CoA dehydrogenation.(ABSTRACT TRUNCATED AT 250 WORDS)

Theoretical studies of impulse propagation in serotonergic axons.

Impulse propagation in small-diameter (1-3 microns) axons with inhomogeneous geometry was simulated. The fibres were represented by a series of 3 microns-long compartments. The cable equation was solved for each compartment by a finite-difference approximation (Cooley and Dodge 1966). First-order differential equations governing temporal changes in membrane potential or Hodgkin-Huxley (1952) conductance parameters were solved by numerical integration. It was assumed that varicosity and intervaricosity segments had the same specific cable constants and excitability properties, and differed only in length and diameter. A single long varicosity or a 'clump' of 3 microns-long varicosities changed the point-to-point (axial) conduction velocity within as well as to either side of the geometrically inhomogeneous regions. When 2 microns-diameter, 3 microns-long varicosities were distributed over the 1 micron-diameter fiber length as observed in serotonergic axons, mean axial conduction velocity was between that of uniform-diameter 1 and 2 microns fibers, and changed predictably with different cable parameters. Fibers with inexcitable varicosity membranes also supported impulse propagation. These simulations provided a general theoretical basis for the slow (less than 1 M/s) conduction velocity attributed to small-diameter unmyelinated varicose axons.

HPLC-EC determination of free primary amino acid concentrations in cat cisternal cerebrospinal fluid.

This paper describes an HPLC-EC method for measuring the concentrations of 9 free primary amino acids in cerebrospinal fluid (CSF) withdrawn from the cisterna magna of Nembutal-anesthetized adult cats. Amino acid derivatives were formed with o-phthalaldehyde and beta-mercaptoethanol; subsequently, excess thiol reagent was removed with iodoacetamide. During elution through a C18 5-micron column, the electrochemical detector's sensitivity was switched to accommodate the wide ranges of CSF amino acid concentrations. The analysis was acceptably precise and linear at and above the CSF levels and did not require CSF deproteinization. During the 23 min elution, the concentrations of 8 CSF amino acids were determined: alanine, asparagine, glutamate, glutamine, glycine, serine, taurine, and tyrosine; measurable concentrations were between 1 and 800 microM. The concentration of GABA was below its detection limit (0.5 microM). To assess the ability to detect small concentration increases which might occur due to experimental manipulations, the minimum detectable increments in CSF amino acid concentrations above endogenous levels were determined.

Identification of gamma-aminobutyric acid-like immunoreactive neurons in the rat cuneate nucleus.

Neurons in the cuneate nucleus of the rat were examined for gamma-aminobutyric acid-like immunoreactivity (GABA-LI) using antiserum raised against GABA-glutaraldehyde-keyhole limpet hemocyanin. GABA-LI neurons were analyzed for size, shape, and distribution and compared to Nissl-stained neurons. GABA-LI cell bodies were located at all rostral-caudal levels and were distributed randomly throughout the nucleus except at the level of the obex, where they were limited to the peripheral region of the cuneate nucleus. GABA-LI cell bodies had a significantly smaller mean cross-sectional area than the total cuneate neuronal population and comprised 21.5% of the total neuronal population as assessed with Nissl-staining. These results are consistent with the hypothesis that GABA is involved in processing somatosensory information in the rat dorsal column nuclei.

14C-labeled substrate catabolism by human diploid fibroblasts derived from infants and adults.

Untransformed diploid skin fibroblasts from eight normal adults, aged 24 to 74 years, catabolized several 14C-labeled substrates less effectively than cells from ten normal male infants. 14C-labeled substrate metabolism was quantitated either by measuring the evolution of 14CO2 from the 14C-labeled compounds or the incorporation of 14C into cellular protein via transamination of tricarboxylic acid cycle intermediates derived from the 14C-labeled substrates. With these methods, adult cells catabolized [1-14C]butyrate, [1-14C]octanoate, and 1-[2-14C]leucine at rates 44 to 64% of those found in infant cells. The oxidation of [1,4-14C]succinate and [U-14C]malate was identical in both infant and adult cells, while [2,3-14C]succinate catabolism was mildly decreased in adult cells (65-80% of control). These observations parallel those made in rat tissues and confirm that the same phenomenon occurs in cultured human fibroblasts.