Release Of Inhibitory Amino Acids Research Articles

The Effects of Angiotensin II and Angiotensin-(1–7) in the Rostral Ventrolateral Medulla of Rats on Stress-Induced Hypertension

We have shown that angiotensin II (Ang II) and angiotensin-(1–7) [Ang-(1–7)] increased arterial blood pressure (BP) via glutamate release when microinjected into the rostral ventrolateral medulla (RVLM) in normotensive rats (control). In the present study, we tested the hypothesis that Ang II and Ang-(1–7) in the RVLM are differentially activated in stress-induced hypertension (SIH) by comparing the effects of microinjection of Ang II, Ang-(1–7), and their receptor antagonists on BP and amino acid release in SIH and control rats. We found that Ang II had greater pressor effect, and more excitatory (glutamate) and less inhibitory (taurine and γ-aminobutyric acid) amino acid release in SIH than in control animals. Losartan, a selective AT1 receptor (AT1R) antagonist, decreased mean BP in SIH but not in control rats. PD123319, a selective AT2 receptor (AT2R) antagonist, increased mean BP in control but not in SIH rats. However, Ang-(1–7) and its selective Mas receptor antagonist Ang779 evoked similar effects on BP and amino acid release in both SIH and control rats. Furthermore, we found that in the RVLM, AT1R, ACE protein expression (western blot) and ACE mRNA (real-time PCR) were significantly higher, whereas AT2R protein, ACE2 mRNA and protein expression were significantly lower in SIH than in control rats. Mas receptor expression was similar in the two groups. The results support our hypothesis and demonstrate that upregulation of Ang II by AT1R, not Ang-(1–7), system in the RVLM causes hypertension in SIH rats by increasing excitatory and suppressing inhibitory amino acid release.

Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion

To evaluate changes in neurotransmission induced by a psychoactive beverage ayahuasca in the hippocampus and amygdala of naive rats. The level of monoamines, their main metabolites and amino acid neurotransmitters concentrations were quantified using high performance liquid chromatography (HPLC). Four groups of rats were employed: saline-treated and rats receiving 250, 500 and 800 mg/kg of ayahuasca infusion (gavage). Animals were killed 40 min after drug ingestion and the structures stored at -80 °C until HPLC assay. The data from all groups were compared using Analysis of variance and Scheffé as post test and P < 0.05 was accepted as significant. The results showed decreased concentrations of glycine (GLY) (0.13 ± 0.03 vs 0.29 ± 0.07, P < 0.001) and γ-aminobutyric acid (GABA) (1.07 ± 0.14 vs 1.73 ± 0.25, P < 0.001) in the amygdala of rats that received 500 of ayahuasca. Animals that ingested 800 mg/kg of ayahuasca also showed a reduction of GLY level (0.11 ± 0.01 vs 0.29 ± 0.07, P < 0.001) and GABA (0.98 ± 0.06 vs 1.73 ± 0.25, P < 0.001). In the hippocampus, increased GABA levels were found in rats that received all ayahuasca doses: 250 mg/kg (1.29 ± 0.19 vs 0.84 ± 0.21, P < 0.05); 500 mg/kg (2.23 ± 038 vs 084 ± 0.21, P < 0.05) and 800 mg/kg (1.98 ± 0.92 vs 0.84 ± 0.21, P < 0.05). In addition, an increased utilization rate of all monoamines was found in the amygdala after ayahuasca administration in doses: 250 mg/kg (noradrenaline: 0.16 ± 0.02 vs 0.36 ± 0.06, P < 0.01; dopamine: 0.39 ± 0.012 vs 2.39 ± 0.84, P < 0.001; serotonin: 1.02 ± 0.22 vs 4.04 ± 0.91, P < 0.001), 500 mg/kg (noradrenaline: 0.08 ± 0.02 vs 0.36 ± 0.06, P < 0.001; dopamine: 0.33 ± 0.19 vs 2.39 ± 0.84, P < 0.001; serotonin: 0.59 ± 0.08 vs 4.04 ± 0.91, P < 0.001) and 800 mg/kg (noradrenaline: 0.16 ± 0.04 vs 0.36 ± 0.06, P < 0.001; dopamine: 0.84 ± 0.65 vs 2.39 ± 0.84, P < 0.05; serotonin: 0.36 ± 0.02 vs 4.04 ± 0.91, P < 0.001). Our data suggest increased release of inhibitory amino acids by the hippocampus and an increased utilization rate of monoamines by the amygdala after different doses of ayahuasca ingestion.

Mechanisms of Inhibitory Amino Acid Release in the Brain Stem Under Normal and Ischemic Conditions

This article deals with the release of GABA, glycine and taurine from the brain stem under normal conditions and in ischemia. The release mechanisms, the effects of glutamate and adenosine receptors, and the roles of nitric oxide and second messengers are reviewed.

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Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn

Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca 2+, yet is not fully blocked in most neurons in the presence of Cd 2+, suggesting possible Ca 2+ entry through AMPA receptors. Postsynaptic Ca 2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.

Implications of intrathecal pertussis toxin animal model on the cellular mechanisms of neuropathic pain syndrome.

Like opioid tolerance, neuropathic pain syndrome manifested by hyperalgesia and allodynia responds poorly to opioids. Hitherto, its development is still not clear and its treatment and prevention are still disputable. Pertussis toxin (PTX) which ADP-ribosylates the alpha-subunit of inhibitory guanine nucleotide binding regulatory proteins (Gi/Go), is used to induce morphine tolerance through intrathecal (i.t.) injection. It decreases the antinociceptive effect of opioid receptor agonists, and produces a thermal hyperalgesia as well. With treatment of PTX the inhibitory Gi- and Go-proteins signal transduction is inactivated. Inhibition of the inhibitory system would likely lead to a predominance of the excitatory system. Intrathecal PTX administration has also been suggested as a model for study of the central mechanisms of neuropathic pain. In our previous studies, with intrathecal microdialysis and drug delivery techniques, we correlated the biochemical and pharmacological effects on the behavioral expressions of i.t. PTX-treated rats. Intrathecal PTX administration would induce thermal hyperalgesia in rats, with accompaniments of a prolonged increase in the concentrations of excitatory amino acids (EAAs), glutamate and aspartate, and a decrease in the concentration of the inhibitory amino acid (IAA) glycine in the spinal CSF dialysates. The PTX-induced thermal hyperalgesia peaked between day 2 and 4, but no cold allodynia is observed; i.t. administration of N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosponovaleric acid (D-AP5), glycine and protein kinase C (PKC) inhibitor chelerythrine attenuated the thermal hyperalgesia. The PKC gamma content of both synaptosomal and cytosolic fractions were significantly increased in PTX-treated rats. In contrast, the levels of PKC alpha, beta I, or beta II isozymes in these fractions were unaffected. Infusion of NMDA antagonist D-AP5 prevented both the thermal hyperalgesia and the increase in PKC gamma expression in PTX-treated rats. Similar to our previous report, i.t. PTX reduced morphine's analgesic effect. PKC inhibitor chelerythrine attenuated this reduction of morphine's analgesia, and an inhibition of the morphine-evoked EAAs release was observed in PTX-treated rats as well. Taken together, i.t. PTX-induced neuropathic pain syndrome is accompanied by increasing of EAAs, decreasing of IAA release, and a selective increasing of PKC gamma expression in the spinal cord. Inhibition of PKC not only blocked thermal hyperalgesia, but also reversed the reduction of morphine's analgesic effect in PTX-rats. These results suggest that PTX-induced neuropathic pain syndromes are involved in EAAs, IAAs and PKC alternations.

Effect of leptin on hypothalamic release of GnRH and neurotransmitter amino acids during sexual maturation in female rats.

The purpose of the present study was to analyse the effect of leptin treatment on the hypothalamic release of GnRH, GABA, and the excitatory amino acids (EAA), aspartate (ASP) and glutamate (GLU) involved in NMDA neurotransmission in prepubertal (15 day old) and peripubertal (30 day old) female rats. The animals were treated with a single dose of leptin (30 microg/kg i.p.) and sacrificed 60 min later. Hypothalamic samples were incubated in Earle's medium; GnRH was determined by RIA and GLU, ASP and GABA by HPLC by UV detection. The hypothalamic release of GnRH was increased by leptin at both ages, the release being significantly higher in peripubertal than in prepubertal rats. The levels of hypothalamic GABA release were different in the two groups; whereas in prepubertal rats the hypothalamic release of GABA increased with leptin administration, the neurotransmitter release decreased in the peripubertal group. On the other hand, the release of ASP was modified only in the peripubertal group, where leptin significantly increased its hypothalamic release. No modifications in leptin-induced hypothalamic release of GLU were observed at the two ages studied. In conclusion, the results showed that leptin increased GnRH release by the hypothalamus of prepubertal and peripubertal rats. In peripubertal rats this increase was accompanied by a significant decrease in the hypothalamic release of GABA as well as an enhanced release of ASP. These results and previous reports suggest that at this stage of sexual maturation, leptin exerts an stimulatory effect on GnRH by inducing release of excitatory amino acids (ASP) and reducing release of inhibitory amino acids (GABA) involved in GnRH control. In prepubertal rats the stimulating effect of the adipocyte hormone on GnRH appears to be related to its stimulative action on GABA which at this age increases GnRH release.

The SLC32 transporter, a key protein for the synaptic release of inhibitory amino acids.

The SLC32 family comprises a single member: the vesicular inhibitory amino acid transporter (VIAAT) or vesicular GABA transporter (VGAT). It belongs to a eukaryotic-specific superfamily of H(+)-coupled amino acid transporters, which also comprises the mammalian SLC36 and SLC38 transporters. VIAAT exchanges GABA or glycine for protons. It is present on synaptic vesicles of GABAergic and glycinergic neurons, and in some endocrine cells, where it ensures the H(+)-ATPase-driven uptake, and subsequent exocytotic release, of inhibitory amino acids. Despite a similar function in vesicular neurotransmitter loading, VIAAT is not related to the vesicular glutamate transporter (VGLUT, SLC17) or the vesicular monoamine transporter/vesicular acetylcholine transporter (VMAT/VACHT, SLC18) proteins.

Changes in Inhibitory Amino Acid Release Linked to Pontine-Induced Atonia: AnIn VivoMicrodialysis Study

We hypothesized that cessation of brainstem monoaminergic systems and an activation of brainstem inhibitory systems are both involved in pontine inhibitory area (PIA) stimulation-induced muscle atonia. In our previous study (Lai et al., 2001), we found a decrease in norepinephrine and serotonin release in motoneuron pools during PIA stimulation-induced muscle tone suppression. We now demonstrate an increase in inhibitory amino acid release in motor nuclei during PIA stimulation in the decerebrate cat using in vivo microdialysis and HPLC analysis techniques. Microinjection of acetylcholine into the PIA elicited muscle atonia and simultaneously produced a significant increase in both glycine and GABA release in both the hypoglossal nucleus and the lumbar ventral horn. Glycine release increased by 74% in the hypoglossal nucleus and 50% in the spinal cord. GABA release increased by 31% in the hypoglossal nucleus and 64% in the spinal cord during atonia induced by cholinergic stimulation of the PIA. As with cholinergic stimulation, 300 msec train electrical stimulation of the PIA elicited a significant increase in glycine release in the hypoglossal nucleus and ventral horn. GABA release was significantly increased in the hypoglossal nucleus but not in the spinal cord during electrical stimulation of the PIA. Glutamate release in the motor nuclei was not significantly altered during atonia induced by electrical or acetylcholine stimulation of the PIA. We suggest that both glycine and GABA play important roles in the regulation of upper airway and postural muscle tone. A combination of decreased monoamine and increased inhibitory amino acid release in motoneuron pools causes PIA-induced atonia and may be involved in atonia linked to rapid eye-movement sleep.

The release of inhibitory amino acids in the hypothalamus is tonically modified by impulses from aortic baroreceptors as a consequence of blood pressure fluctuations.

We investigated in conscious, freely moving rats whether the release of GABA, taurine and arginine in the hypothalamus is influenced by impulses originating from peripheral baroreceptors. The posterior hypothalamic nucleus was superfused with artificial cerebrospinal fluid through a push-push cannula and the release of amino acids was determined in the hypothalamic superfusate of control rats, as well as of rats after bilateral aortic denervation (AD). AD led to hypertension and increased the lability of arterial pressure. In sham-operated rats, intravenous infusion of phenylephrine increased blood pressure and the hypothalamic release of GABA and taurine. AD almost abolished the phenylephrine-induced release of the inhibitory amino acids. Similarly, the pressor response to hypervolaemia, elicited by blood injection, enhanced the release rates of GABA and taurine only in sham-operated rats. Baroreceptor unloading evoked either by intravenous infusion of nitroprusside, or by haemorrhage, decreased the release rates of GABA and taurine in sham-operated rats but not in AD rats. Electrical stimulation of the afferent aortic depressor nerve enhanced extracellular GABA and taurine in the posterior hypothalamic nucleus. The release rate of arginine was not influenced by alterations in baroreceptor activity either in sham-operated or in AD rats. The findings support the idea that, in the hypothalamus, GABA and taurine are involved in central blood pressure regulation. The release of these two amino acids seems to be driven tonically by baroreceptor impulses. Moreover, the findings indicate that the baroreceptors of the aortic arch play a crucial role in the mediation of changes in hypothalamic GABA and taurine outflow so as to counteract blood pressure fluctuations.

The GABAergic control of gonadotropin-releasing hormone secretion in male rats during sexual maturation involves effects on hypothalamic excitatory and inhibitory amino acid systems.

In order to evaluate the possible participation of the hypothalamic excitatory and inhibitory amino acid neurotransmitter systems in the GnRH release response to GABAergic drugs, hypothalami (preoptic and mediobasal area) of immature (26 days of age) and adult male rats were perifused with GABA-A and -B agonists and antagonists. GnRH and amino acid neurotransmitter concentrations (glutamate, taurine, GABA) were measured in perfusate samples collected every 15 min during 150 min. In immature rats, muscimol and baclofen (GABA-A and GABA-B agonists, respectively) increased GnRH, glutamate and GABA release and decreased taurine output, while in adults these agonists showed opposite effects on GnRH and glutamate release, and increased GABA and taurine output. On the other hand, in immature rats bicuculline and phaclofen (GABA-A and GABA-B antagonists, respectively) decreased GnRH, glutamate and GABA release, increasing taurine outflow. In adult animals, these antagonists enhanced GnRH and glutamate release, decreasing taurine and GABA outflow. These results indicate that GABA stimulates GnRH release in immature male rats and confirm the inhibitory role of this amino acid neurotransmitter in adult animals. This effect might be associated, at least partially, with the modifications observed in the excitatory and inhibitory amino acid release. On the other hand, in immature rats, stimulation of GABA-A and GABA-B receptors increased GABA release. Although ultrastructural studies have not produced any evidence of GABA-GABA neurointeractions, our results suggest the existence of a positive feedback mechanism of GABA autoregulation active during the prepubertal stage. Participation of this mechanism in the onset of puberty cannot be discarded.

The effect of central amino acid neurotransmitters on the antihypertensive response to angiotensin blockade in spontaneous hypertension

To investigate the effects of central amino acid neurons on the antihypertensive action of a newly developed angiotensin II type 1 receptor (AT1) antagonist, CV 11974. We measured the release of various amino acids in the rostral ventrolateral medulla using the brain microdialysis technique. A microdialysis probe was inserted into the exposed rostral ventrolateral medulla in male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats anaesthetized with urethane. Mean arterial pressure and the release of amino acids (glutamate, glycine, glutamine, taurine and gamma-aminobutyric acid) were monitored before and after intravenous administration of CV 11974 (5 mg/kg), nitroglycerin (5 mu g/kg per min) or vehicle. In SHR, CV 11974 decreased mean arterial pressure (-40 +/- 6 mmHg) accompanied by significant increases in the release of inhibitory amino acids, glycine (411 +/- 83%) and gamma-aminobutyric acid (363 +/- 71%) in the rostral ventrolateral medulla, whereas intravenous nitroglycerin produced a decrease in mean arterial pressure (-35 +/- 4 mmHg) without changes in amino acid release. In WKY rats, both intravenous CV 11974 and intravenous nitroglycerin produced smaller but significant decreases in mean arterial pressure (CV 11974, -18 +/- 5 mmHg; nitroglycerin, -20 +/- 7 mmHg) without change in the release of amino acids in the rostral ventrolateral medulla. Selective perfusion of glycine or gamma-aminobutyric acid into the rostral ventrolateral medulla caused a larger mean arterial pressure reduction in SHR than in WKY rats. Furthermore, the use of a specific antagonist of glycine or of the gamma-aminobutyric acid receptor in the rostral ventrolateral medulla attenuated the antihypertensive response induced by the intravenous AT1 antagonist in SHR. The present results suggest that the release of the inhibitory amino acids glycine and gamma-aminobutyric acid in the rostral ventrolateral medulla contributes to the depressor action of this AT1 receptor antagonist in the genetic hypertensive rat model.

Effects of excitatory amino acids, and of their agonists and antagonists on the release of neurotransmitters from the chick retina.

Effects of glutamic, aspartic, and cysteic acid, and of kainic acid and N-methyl aspartate on the release of labeled GABA, glycine, and taurine were examined in isolated, perfused chick retina. Glutamic acid (0.5-2 mM), increased the release of 3H-GABA by more than four times and that of 14C-glycine by about two times. The release of GABA decreased 50% and that of glycine 95% in the presence of the antagonist of glutamic acid receptors, glutamate diethyl ester (300 microM). N-methyl aspartate, used as an agonist of aspartic acid receptors, preferentially increased the release of GABA (seven times) over that of glycine (three times). The stimulatory effect of N-methyl aspartate was antagonized by D-alpha-aminoadipate and by Mg. Kainic acid (10 microM) induced the release of glycine but not that of GABA. Cysteic acid failed to modify the release of any of the amino acids examined. The efflux of labeled taurine was practically unaffected by all the compounds utilized. The release of GABA by the excitatory amino acids and agonists was Ca-independent but Na-dependent, whereas the release of glycine was markedly Ca-dependent. The evidence presented here suggests that experimental conditions activating receptors of excitatory amino acids differently affect the release of inhibitory amino acids.

The in vitro inhibition of GABA release by tetanus toxin

The in vitro effects of tetanus toxin on the potassium(K +)-evoked release of radiolabelled γ-aminobutyric acid (GABA) from slices prepared from substantia nigra of the rat have been investigated. The addition of tetanus toxin (750–15,000 mouse LD 50ml −1) to the slices produced a dose- and time-dependent reduction in the K + -evoked release of [ 3H]GABA. These effects of the toxin were diminished at low temperature and in the absence of calcium ions in the bathing medium. It is suggested that this relatively simple in vitro system may allow more quantitative studies to be made concerning the action of the toxin on inhibitory amino acid release from the mammalian central nervous system.