Effect of elevated extracellular potassium on the release of labelled noradrenaline,glutamate, glycine, β-alanine and other amino acids from rat brain cortex slices

The effect of an elevated concentration (46 mm) of extracellular potassium on the release, from superfused rat brain cortex slices, of labelled γ-aminobutyrate, glutamate, glycine, α- and β-alanine, aspartate, taurine and α-aminoisobutyrate has been studied; and a comparison made with the release of soluble (non-vesicular) noradrenaline (i.e. that transported into slices taken from the brains of animals pretreated with reserpine; the slices were incubated with nialamide). In all cases, with the possible exception of l-α-alanine, elevated potassium induced an increased efflux of these substances. Omission of calcium in the superfusing fluid markedly diminished the release of all the test substances, except, in part, that of β-alanine. It was also found that the time course of the induced efflux varied considerably for the different substances: substances such as noradrenaline and γ-aminobutyrate that are transported predominantly into axons and axon terminals, showed a ‘peak’ time course, with a maximal release within 2–4 min following potassium elevation, and the rate of release diminished rapidly in spite of the continued presence of high potassium. Such a decreased release was not due to exhaustion of the tissue stores of these substances. On the other hand, glutamate and glycine, substances that are thought to be transported predominantly into glial cells, attained their maximal release rates only 10 min after potassium was elevated, and such release was maintained without decrement, β-alanine showed a mixed type of release, with a small initial peak resembling that of the axonally located substances, and a delayed release similar to that of glycine and glutamate. The release of the rest of the amino acids also resembled that of glycine and glutamate. A correlation was found (r = 0.99, P < 0.01) between the area of the efflux curve that had a ‘peak’ shape, and the percentage of the substance that was transported into an axonal compartment.It is concluded that although elevated potassium concentrations can release substances from both axons and glia, and that a calcium dependency may exist in both cases, the time course of the induced efflux is markedly different when the substances are located in axons or in glial cells. Such a procedure may prove valuable for establishing grossly into which compartment a given substance is transported.     

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.     

The release of endogenous amino acids from the rat visual cortex.

The release of endogenous taurine, GABA, glycine, aspartate, glutamate, glutamine and alanine from the rat visual cortex was measured using a cortical cup technique. The electrocorticogram (ECoG) was monitored throughout most experiments. 2. Spreading depression, evoked by the dropwise placement of 10% KCl solution on to the brain outside the cup was associated with a significant increase in the release of GABA and glutamine but a marked fall in that of glutamate. The evoked release of GABA and glutamate but not of glutamine was Ca2+ dependent. 3. A solution containing 50 mM-K+ placed within the cup elicited a significant increase in the release of taurine and GABA, whereas 100 mM-K+ additionally released aspartate and glutamate. The K+-evoked release of these amino acids with the exceptions of taurine and glutamine was Ca2+-dependent. 4. Three series of experiments were carried out in which the preparations were stimulated electrically. Bipolar stimulation (100 Hz, 1 msec pulse width, 2-5 mA for 5 min) with the electrode within the cup was followed by significant increases in taurine, GABA and glutamate release; using a 5 mA current, there was an additional release of aspartate and alanine. Only the evoked release of GABA and glutamate was Ca2+ dependent. 5. In the second and third series of experiments, the electrode was sited adjacent to the cup or on the contralateral cortex respectively. Following stimulation (100 Hz, 1 msec pulse width, 2-5 mA for 5 min) there was a significant increase in taurine and GABA release and a significant fall in the release of aspartate and glutamate. With the exception of taurine, these changes in release were Ca2+ dependent. Reducing the stimulus current to 1-5 mA or the period of stimulation to 2-5 min initiated similar but statistically insignificant changes in release. A range (10-100 Hz) of stimulation frequencies was examined: the evoked release of GABA was linearly related to frequency whereas that of taurine was frequency-independent. The fall in aspartate and glutamate release was maximal at a frequency of about 50 Hz. 6. The results are discussed in relation to (a) the possible sites of release of the amino acids and (b) the proposed neurotransmitter roles of the physiologically active amino acids.     

Release of taurine from cultured cerebellar granule cells and astrocytes: co-release with glutamate

The properties of the release of preloaded [3H]taurine and endogenous taurine were studied with cultured cerebellar granule cells (7-8 days in vitro) and astrocytes (14-15 days in vitro) from the rat. The spontaneous release of taurine from both cell types was slow. The release from both neurons and astrocytes was significantly enhanced by 0.1 mM veratridine, the stimulatory effect being more pronounced in granule cells than in astrocytes. No homo or heteroexchange with extracellularly added taurine or its structural analogues could be detected, suggesting that the efflux is probably not mediated via the membrane transport sites. Kainate stimulated the release more from granule cells than from astrocytes, the effect apparently being mediated by kainate-sensitive receptors. Depolarization of cell membranes by 50 mM K+ induced co-release of endogenous taurine and glutamate from both cell types. Preloaded [3H]taurine was readily released from astrocytes by potassium stimulation. Stimulated release occurred from granule cells if they had been cultured for 4 days with the label but not from the cells preloaded for only 15 min.     

Preferential release of neuroactive amino acids from the ventrolateral medulla of the rat in vivo as measured by microdialysis

The basal overflow of extracellular endogenous amino acids was measured from the ventrolateral medulla of urethane anaesthetized rats in vivo by microdialysis. Inclusion of a mercury salt, p-chloromercuriphenylsulphonic acid, in the dialysate (Krebs' solution), results in a preferential increase in the overflow of aspartate, glutamate, glycine and GABA. A smaller increase in the overflow of the glutamate precursor and metabolite, glutamine, was also found. There was no significant change in the basal extracellular levels of taurine, asparagine, alanine, serine, ornithine or lysine. Inclusion of a specific GABA uptake inhibitor, nipecotic acid, in the dialysate results in an immediate, dose dependent increase in the overflow of GABA, and to a lesser extent, taurine. Since it is likely that mercury salts increase neurotransmitter release by increasing free intracellular calcium ion concentrations, it is suggested that these results provide further evidence for a physiologically relevant neurotransmitter role for aspartate, glutamate, glycine and GABA in the ventrolateral medulla.     

Met-enkephalin selectively increases the spontaneous release of amino acid neurotransmitters from rat striatal slices

Met-enkephalin (1 microM) increased the spontaneous release of endogenous glutamate (+155%), taurine (+80%) and glycine (+50%) from rat striatal slices, but was without effect in the cerebral cortex. This effect was calcium-dependent and significantly reduced in the presence of naloxone (1 microM). Naloxone alone had no effect on release of any substance. Release of aspartate, gamma-aminobutyric acid and [3H]acetylcholine was not significantly affected. Met-enkephalin did not affect potassium (35 mM)-evoked neurotransmitter release, nor did it affect the uptake of D-[3H]aspartate, [3H]taurine or [14C]glycine. The data indicate that opioid receptor activation selectively increases the spontaneous calcium-dependent release of putative amino acid neurotransmitters in the rat striatum.     

Continuous glutamate leakage from brain cells is balanced by compensatory high-affinity reuptake transport

The glutamate (and aspartate) uptake blocker threo-3-hydroxyaspartate (20 microM) was added to superfusion fluids employed for in vivo microdialysis of corpus striatum, and to incubation medium for striatal slices (5 microM). In vivo it caused an increase in glutamate and aspartate concentrations in the superfusion fluid. In vitro it caused increases in the levels of glutamate, aspartate, GABA, taurine and glutamine in the incubation fluid. Tetrodotoxin (1 microM) did not influence the rises in glutamate or aspartate. It is concluded from these results that there is a continuous outward leakage of glutamate, and aspartate, from neural cells which is normally balanced by an inward flux due to reuptake processes. This leakage is distinct from synaptic release of these substances due to spike activity, since tetrodotoxin added to striatal slices did not diminish the action of threo-3-hydroxyaspartate. The significance of the findings for mechanisms leading to ischaemic or hypoxic brain damage, and basic mechanisms in epilepsy is discussed.     

Involvement of nitric oxide,cyclic GMP and phosphodiesterase 5 in excitatory amino acid and GABA release in the nucleus accumbens evoked by activation of the hippocampal fimbria

It is known that the nucleus accumbens contains all elements of the nitric oxide (NO)-cyclic GMP (cGMP) system but the role of NO in this nucleus is not well understood. We investigated the contribution of the NO-cGMP system in the neurotransmission elicited by hippocampal nerve signals which are propagated to the nucleus accumbens via the fornix/fimbria. This glutamatergic hippocampus-accumbens projection was electrically stimulated for short periods in the urethane-anaesthetized rat. The nucleus accumbens was simultaneously superfused by the push-pull technique with compounds that influence the NO system and the released glutamate, aspartate and GABA were determined in the superfusate.Superfusion of the nucleus accumbens with the NO donor, PAPA/NO, enhanced basal release of the investigated amino acids with a complex concentration dependency. The release of glutamate and aspartate was also increased by the inhibitor of phosphodiesterase 5, UK-114,542. The PAPA/NO-elicited release of glutamate and aspartate was diminished by superfusion with the inhibitor of guanylyl cyclase, NS 2028. Basal release of amino acid transmitters was not influenced by NS 2028 and the NO synthase inhibitor, 7-NINA.Electrical stimulation of the fornix/fimbria increased the outflow of aspartate, glutamate and GABA in the nucleus accumbens. The stimulation-evoked release was abolished by superfusion of the nucleus with tetrodotoxin and strongly diminished by NS 2028, 7-NINA and N(G)-nitro-L-arginine methyl ester (L-name), while PAPA/NO facilitated stimulation-evoked release of these neurotransmitters. UK-114,542 also enhanced the evoked release of glutamate and aspartate while evoked GABA release was not influenced by the phosphodiesterase inhibitor.These findings indicate that NO plays the role of an excitatory transmitter in the nucleus accumbens and that nerve signals from the hippocampus propagated via fornix/fimbria induce NO synthesis in the nucleus accumbens. NO does not exert a tonic influence on basal release but facilitates release of aspartate, glutamate and GABA through increased cGMP synthesis. Phosphodiesterase 5 seems to be involved in the termination of the NO effect in glutamatergic but not in GABAergic neurons.     

Neurochemical and pharmacological correlates of inferior olive destruction in the rat: attenuation of the events mediated by an endogenous glutamate-like substance

The effects of a lesion of the inferior olive (the source of climbing fibres) has been investigated on a number of parameters of cerebellar excitatory amino acid function. Protoveratrine elicits a large increase in guanosine cyclic 3',5'-monophosphate levels in cerebellar slices, probably through the release of excitatory amino acids such as glutamate or aspartate, since the receptors involved are susceptible to specific antagonists. While the direct stimulatory (postsynaptic) effects of glutamate and related amino acids were not altered after loss of climbing fibres induced by 3-acetylpyridine, the guanosine cyclic 3',5'-monophosphate response to protoveratrine was markedly (40%) attenuated. Concomitant with this was a similar reduction in the high-affinity uptake of D-[3H]aspartate, a proposed marker for glutamate/aspartate terminals. Experiments to investigate the calcium-dependent, potassium-stimulated release of glutamate and aspartate, failed to reveal any reduction following acetylpyridine lesions. Although this latter finding does not exclude the possibility that these amino acids could be the transmitter of the climbing fibres, our data would rather suggest the involvement of some glutamate-like, but as yet unidentified, neuroexcitatory substance.     

Calcium-dependent increase in efflux of [1-3H] taurine from the superfused rat cerebellar cortex in vivo

Summary High (40 mM) potassium stimulation has been shown to increase the effluxin vivo of [1-³H] taurine from the superfused rat cerebellar cortex by 46%, P<0.001. During superfusion with calcium-free media this increase in efflux is abolished. The cellular location of [1-³H] taurine in the cerebellar cortex is not yet known, but this region of the brain contains exceptionally high levels of endogenous taurine. The calcium dependency of the release of labelled taurine raises the possibility that taurine has some neuron-humoral role in the cerebellar cortex.     

Aspartate release from rat hippocampal synaptosomes

Certain excitatory pathways in the rat hippocampus can release aspartate along with glutamate. This study utilized rat hippocampal synaptosomes to characterize the mechanism of aspartate release and to compare it with glutamate release. Releases of aspartate and glutamate from the same tissue samples were quantitated simultaneously. Both amino acids were released by 25 mM K(+), 300 microM 4-aminopyridine (4-AP) and 0.5 and 1 microM ionomycin in a predominantly Ca(2+)-dependent manner. For a roughly equivalent quantity of glutamate released, aspartate release was significantly greater during exposure to elevated [K(+)] than to 4-AP and during exposure to 0.5 than to 1 microM ionomycin. Aspartate release was inefficiently coupled to P/Q-type voltage-dependent Ca(2+) channels and was reduced by KB-R7943, an inhibitor of reversed Na(+)/Ca(2+) exchange. In contrast, glutamate release depended primarily on Ca(2+) influx through P/Q-type channels and was not significantly affected by KB-R7943. Pretreatment of the synaptosomes with tetanus toxin and botulinum neurotoxins C and F reduced glutamate release, but not aspartate release. Aspartate release was also resistant to bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase, whereas glutamate release was markedly reduced. (+/-) -Threo-3-methylglutamate, a non-transportable competitive inhibitor of excitatory amino acid transport, did not reduce aspartate release. Niflumic acid, a blocker of Ca(2+)-dependent anion channels, did not alter the release of either amino acid. Exogenous aspartate and aspartate recently synthesized from glutamate accessed the releasable pool of aspartate as readily as exogenous glutamate and glutamate recently synthesized from aspartate accessed the releasable glutamate pool. These results are compatible with release of aspartate from either a vesicular pool by a "non-classical" form of exocytosis or directly from the cytoplasm by an as-yet-undescribed Ca(2+)-dependent mechanism. In either case, they suggest aspartate is released mainly outside the presynaptic active zones and may therefore serve as the predominant agonist for extrasynaptic N-methyl-D-aspartate receptors.     

Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes

Cultures of differentiated, glial fibrillary acidic protein-positive astrocytes from early postnatal rat cerebral hemispheres respond with depolarization of 2-36 mV to glutamate, gamma-aminobutyric acid (GABA) and aspartate but not to glycine or taurine. While GABA resulted in a transient depolarization, the effect of glutamate and aspartate persisted during the application. Since neurons were not present in these cultures a contribution of transmitter-mediated K+ release from adjacent neurons could be excluded. The depolarization triggered by these neurotransmitters is therefore an intrinsic reaction of astrocytes.     

Characterization of spinal amino acid release and touch-evoked allodynia produced by spinal glycine or GABA(A) receptor antagonist

Intrathecal strychnine (glycine antagonist) or bicuculline (GABA(A) antagonist) yields a touch-evoked agitation that is blocked by N-methyl-D-aspartate receptor antagonism. We examined the effects of intrathecal strychnine and bicuculline on touch-evoked agitation and the spinal release of amino acids. Fifty-two Sprague-Dawley rats were prepared under halothane anesthesia with a lumbar intrathecal catheter and a loop dialysis catheter. Four days after implantation, rats were randomized to receive an intrathecal injection of N-methyl-D-aspartate (3 microg), strychnine (3 microg) or bicuculline (10 microg), or a combination of N-methyl-D-aspartate with bicuculline or strychnine. The agitation produced by brief light tactile stroking of the flank (tactile allodynia), and the spontaneous spinal release of glutamate, taurine and serine was measured. Intrathecal N-methyl-D-aspartate, strychnine and bicuculline produced similar touch-evoked allodynia. Intrathecal bicuculline and N-methyl-D-aspartate alone evoked a transient spinal release of glutamate and taurine, but not serine, in the 0- 10 min sample, while strychnine did not affect spinal transmitter release at any time. As GABA(A) but not glycine receptor inhibition at equi-allodynic doses increases glutamate release, while the allodynia of both is blocked by N-methyl-D-aspartate receptor antagonism, we hypothesize that GABA(A) sites regulate presynaptic glutamate release, while glycine regulates the excitability of neurons postsynaptic to glutamatergic terminals.     

Noradrenaline- and glutamate-induced taurine release from bulk isolated adult rat astrocytes.

The influence of noradrenaline and various agonists of glutamatergic receptors on preloaded [3H]taurine release from bulk isolated adult rat brain astrocytes was investigated by a superfusion technique. In the presence of 1 mM noradrenaline a stimulation of taurine release, resembling that observed in astroglial cultures, was preceded by an inhibition of the efflux, thus demonstrating different dynamics of noradrenaline-evoked taurine release from that observed with beta-agonists on cultured astroglia. Application of 1 mM glutamate and kainate produced stimulation of the release, while 1 mM N-methyl-D-aspartate (NMDA) and 1 mM NMDA together with 65 mM K+ had no effect on the [3H]taurine release. These data suggest the presence of kainate-sensitive and the absence of NMDA-sensitive glutamate receptors on bulk isolated astrocytes, which is consistent with previous observations on astrocytes in culture.     

Alterations in neurotransmitter amino acid content in the aging rat striatum are subregion dependent

The topographical distribution of putative neurotransmitter amino acids in both 6- and 20-month-old Fischer 344 rats was studied in eight striatal subregions. Tissue levels of glutamate, aspartate, GABA, and taurine in the 20-month-old rats were elevated in virtually all of the anterior striatal subregions examined. In addition, aspartate levels were higher in all dorsomedial subregions, while glutamate and taurine levels were elevated in all lateral and ventrolateral subregions, respectively. Other amino acids such as glutamate, serine, and alanine did not display any specific subregional changes. These findings demonstrate specific striatal subregion changes in neurotransmitter amino acid content as a function of aging.     

Release of taurine and GABA from cerebellar slices from developing and adult mice

The properties of the release of exogenous radioactive taurine and GABA from cerebellar slices from developing and adult mice were investigated using a superfusion system. Potassium stimulation (50 mM K+) caused, approximately, a 1.4-fold enhancement in the release of preloaded taurine from slices from adult mice, while the response to potassium in 7-day-old mice was about 6-fold. The potassium stimulation caused, approximately, a 3-fold increase in the release of preloaded GABA from cerebellar slices from 7-day-old mice, whereas the enhancement was about 10-fold in the adult. The actual molar amount of taurine released from the immature cerebellum was strikingly large, about 16 times larger than the amount of GABA released upon the same stimulus. Spontaneous taurine efflux was potentiated by taurine and GABA, the responses being more pronounced in the 7-day-old cerebellum, suggesting that the immature cerebellum is more prone to stimulation by homo- and heteroexchange than the mature cerebellum. Potassium-stimulated taurine release was inhibited by GABAergic substances in the adult but not in the developing cerebellum. Potassium-stimulated GABA release from the adult cerebellum was greatly increased by GABA and also moderately by muscimol and bicuculline, the effect of the latter being antagonized by taurine and hypotaurine. Taurine was thus able to modulate GABA release through bicuculline-sensitive receptors, but this modulation was not evident in cerebellar slices from 7-day-old mice. An exposure of the slices to sodium-free media greatly enhanced taurine and GABA release in both age groups. The stimulated release of GABA generally exhibited a similar calcium dependency in the adult and 7-day-old cerebella but in 7-day-old mice the stimulated release of taurine was not strictly calcium-dependent.     

Volume-activated taurine efflux from the in situ perfused lactating rat mammary gland

The effect of cell swelling on the efflux of amino acids from the in situ perfused lactating rat mammary gland has been examined. Cell swelling, induced by a hyposmotic shock, increased the fractional release of [³H]taurine. In contrast, a hyposmotic shock did not stimulate the efflux of D -[³H]aspartate, suggesting that the effect of a hyposmotic challenge on taurine release cannot be attributed to cell lysis. Volume-activated taurine efflux was reversible, dependent upon the extent of the osmotic challenge and inactivated with a prolonged hyposmotic shock. The release of taurine was also reversibly increased following isosmotic cell swelling (using urea). The results confirm the presence of a volume-sensitive taurine efflux transport system in lactating rat mammary tissue and suggest that the volume-activated amino acid efflux pathway is located at the blood-facing aspect of the mammary epithelium.     

Positive correlation between contamination by blood and amino acid levels in cerebrospinal fluid of the rat

The degree of contamination by blood in macroscopically clear cerebrospinal fluid (CSF) from the rat was assessed by the red blood cell count. Amino acid concentrations in the same samples were determined using high performance liquid chromatography. A significant positive correlation between the number of erythrocytes and amino acid concentration was found for alanine, asparagine, aspartate, citrulline, glutamate, glycine, phenylalanine and taurine but not for glutamine, histidine, isoleucine, leucine, lysine, methionine, ornithine, serine, threonine, tyrosine and valine. The difference in amino acid concentration between samples that were or were not contaminated by blood was as much as one order of magnitude for aspartate, glutamate, glycine and taurine; the concentrations of these amino acids were highly correlated with the erythrocyte counts (r = 0.87, P = 0.000002 for glutamate). The results suggest that macroscopical inspection is often not sufficient to judge contamination by blood in the CSF.     

The effect of cochlear nerve lesion on the release of glutamate,aspartate, and GABA from cat cochlear nucleus,in vitro

Summary Pool studies of glutamate and aspartate have suggested a transmitter role for these amino acids in cochlear nerve endings. As further evidence. the K⁺-evoked release of glutamate, aspartate and GABA was measured in cat cochlear nucleus slices in vitro and compared to the release following a cochlear nerve lesion. Using [³H]glutamine as precursor, the K⁺-evoked release of glutamate and -aminobutyric acid (GABA) was respectively 4.1 and 7.2 times the spontaneous release. Using [¹⁴C]glutamate as a marker, the K⁺-evoked release of glutamate and GABA was respectively 7.1 and 2.8 times the basal release. All K⁺-evoked releases were Ca⁺⁺-dependent. Nine days after unilateral lesion of the cochlear nerve in the cat, the glutamate release decreased by about 75% on the lesioned side compared to the intact one, whereas the GABA release was not decreased. The labelled tissue glutamate, either synthesized from [³H]glutamine or labelled with [¹⁴C]glutamate, was also markedly decreased on the lesioned side. In comparison, the evoked release of aspartate, newly synthesized from [¹⁴C]glutamate, remained low and was only decreased by about 45% after cochlear nerve lesions. Comparing cat with rat cochlear nucleus, the glutamate release was similar in both animals, whereas the GABA release was much higher in the rat.It is concluded that glutamate and to a lesser extent aspartate are likely to be released from cochlear nerve terminals, supporting a transmitter role in these nerve fibres for both amino acids.     

Effects of aniracetam on extracellular levels of transmitter amino acids in the hippocampus of the conscious gerbils: an intracranial microdialysis study

The effects of aniracetam on extracellular amino acid levels in the hippocampus of conscious gerbils, with or without transient cerebral ischemia/reperfusion, were measured by microdialysis and reverse phase-high performance liquid chromatography. Increased extracellular levels of aspartate and glutamate that were observed in the hippocampus of conscious gerbils during transient global forebrain ischemia were reversed by aniracetam. In contrast, the level of extracellular gamma-aminobutyric acid was increased, while taurine was maintained at a higher level than other amino acids by administration of aniracetam (100 mg/kg, p.o.) 60 min before ischemia. Further, in contrast to ischemic animals, administration of aniracetam (100 mg/kg, p.o.) enhanced the release of glutamate and aspartate in the normal gerbil hippocampus. The results suggest that these effects might be due to a partial calcium agonist activity of aniracetam, and that the effects of aniracetam on amino acid levels might be a mechanism of protection against delayed neuronal death in the ischemic hippocampus, thereby improving memory dysfunction induced by ischemia/reperfusion.