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The purpose of this study was to evaluate potential mechanisms of ischemia-evoked amino acid transmitter release. Changes in extracellular levels of transmitter amino acids and lactic acid dehydrogenase (LDH) in rat cerebral cortex during and following four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked release of glutamate, aspartate and γ-amino-butyric acid (GABA) was compared in control vs. drug-treated animals. Tetrodotoxin and antagonists of glutamate receptors (DNQX, MK-801, and AP-3) depressed the initial rate of increase in extracellular glutamate and aspartate without altering the total amount of these amino acids collected in the conical superfusates. Cobalt, a calcium channel antagonist, failed to alter efflux. Acidic amino acid transport inhibitors (dihydrokainate, L-trans-PDC) depressed the rate of onset of glutamate and aspartate release and dihydrokainate depressed total release by 44%. PD 81723, an allosteric enhancer at the A, adenosine receptor, depressed glutamate efflux, as did L-NAME, an inhibitor of nitric oxide synthase. Extracellular increases in GABA levels were depressed by tetrodotoxin and L-trans-PDC. The GABA transport inhibitor, nipecotic acid, increased the initial rate of onset of GABA release. Increases in LDH levels in the extracellular fluid became apparent during the period of ischemia and continued to increase during the subsequent 90 min of reperfusion. These results suggest that ischemia evokes a release of neurotransmitter amino acids that is only partially dependent upon Ca2+ influx activation or the reversal of amino acid transporters. Nonselective mechanisms, resulting from the disruption of plasma membrane integrity, may contribute significantly to the total ischemia-evoked release of excitatory amino acids.

The high-affinity uptake of the acidic amino acid D-aspartate was inhibited in a dose- and time-dependent manner, when C6 cells were exposed to trypsin. The protease decreased the maximal velocity for uptake but not its Km, consistent with a reduction in the number of competent carriers at the plasma membrane. Cellular energy production and [K+]i were unaffected, indicating that the transporter itself was the site of trypsin action. Maximum inhibition of uptake was 50%, which suggests the presence of a heterogeneous population of transporters, only half of which is sensitive to trypsin. These results support our earlier postulate that in glial cells, the high-affinity transporter for acidic amino acids is a transmembrane protein, part of which extends into the external environment.

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.

Kinetic studies of uptake demonstrate the existence of an active transport system for d - and l -acidic amino acids in Neurospora crassa. This system, designated amino acid Transport System IV, has K m ' s for l -cysteic, l -aspartic and l -glutamic acids of 7, 13 and 16 μM, respectively. It has higher affinity for d -aspartic acid than for l -aspartic acid but lower affinity for d -glutamic than for l -glutamic acid. Transport System IV has low activity in growing mycelial pads but has higher activity in carbon-, nitrogen- or sulfur-starved pads. The characterization of Transport System IV appears to complete the outlining of the major amino acid transport systems in Neurospora. A comparison is made among the substrate affinities and regulation of several amino transport systems in Neurospora and in two other fungi.

By selection on a medium containing both ethionine and p-fluorophenylalanine a mutant strain of Neurospora has been isolated. This strain, nap, shows greatly decreased transport of neutral and acidic amino acids. The mutation also confers resistance to 4-methyltryptophan, aminoprotein and glycine. The physiological defect appears to be specific since the mutant showed a normal doubling time for protein, normal oxygen consumption and normal transport of glucose, sulfate and basic amino acids. This gene represents a third locus which governs neutral and acidic amino acid transport in Neurospora, the two previously described being un-t (55701) and mtr.

1. 1. l -Aspartate transport activity was optimally induced following 6 h growth on acetate minimal media. 2. 2. The observed aspartate (and glutamate) transport activity was attributed to the general amino acid transport system since l -phenylalanine, but not l -cysteic acid, inhibited transport activity at all pH values tested. 3. 3. Changes in K m values for l -aspartate and l -glutamate transport following growth on acetate minimal media indicated that apparent “affinities” of the “acidic amino acid transport systems” dramatically increased during the stages of active growth.