Fernando G. de Mello

Taurine prevents the neurotoxicity of β-amyloid and glutamate receptor agonists: activation of GABA receptors and possible implications for Alzheimer’s disease and other neurological disorders

Alzheimers disease (AD) and several other neurological disorders have been linked to the overactivation of glutamatergic transmission and exci‐totoxicity as a common pathway of neuronal injury. The β‐amyloid peptide (Aβ) is centrally related to the pathogenesis of AD, and previous reports have demon¬strated that the blockade of glutamate receptors pre¬vents Aβ‐induced neuronal death. We show that tau¬rine, a β‐amino acid found at high concentrations in the brain, protects chick retinal neurons in culture against the neurotoxicity of Aβ and glutamate receptor ago¬nists. The protective effect of taurine is not mediated by interaction with glutamate receptors, as demon¬strated by binding studies using radiolabeled glutamate receptor ligands. The neuroprotective action of taurine is blocked by picrotoxin, an antagonist of GABAA receptors. GABA and the GABAA receptor agonists phenobarbital and melatonin also protect neurons against Aβ ‐induced neurotoxicity. These results suggest that activation of GABA receptors decreases neuronal vulnerability to excitotoxic damage and that pharmaco¬logical manipulation of the excitatory and inhibitory neurotransmitter tonus may protect neurons against a variety of insults. GABAergic transmission may repre¬sent a promising target for the treatment of AD and other neurological disorders in which excitotoxicity plays a relevant role.—Louzada, P. R., Lima, A. C. P., Mendonca‐Silva, D. L., Noël, F., de Mello, F. G., Ferreira, S. T. Taurine prevents the neurotoxicity of β‐amyloid and glutamate receptor agonists: activation of GABA receptors and possible implications for Alz¬heimers disease and other neurological disorders.

Regulation of acetylcholine synthesis and storage

Acetylcholine is one of the major modulators of brain functions and it is the main neurotransmitter at the peripheral nervous system. Modulation of acetylcholine release is crucial for nervous system function. Moreover, dysfunction of cholinergic transmission has been linked to a number of pathological conditions. In this manuscript, we review the cellular mechanisms involved with regulation of acetylcholine synthesis and storage. We focus on how phosphorylation of key cholinergic proteins can participate in the physiological regulation of cholinergic nerve-endings.

Developmental immunoreactivity for GABA and GAD in the avian retina: possible alternative pathway for GABA synthesis.

Although the distribution of GABAergic neurons in chick retina has been previously described by several investigators, the early appearance of these neurons has not been reported. In the present study immunohistochemical methods were used to localize GABAergic neurons with antisera to both GABA and its synthesizing enzyme, glutamate decarboxylase (GAD), in embryonic chick retina at several stages of development and beyond hatching. GABA-positive neuroblast-like cells were clearly detected in retinas as early as embryonic day 6. In contrast, GAD-containing cells were not observed in retinas until embryonic day 10. These findings indicated that immunocytochemically detectable amounts of GAD were not present in young GABAergic cells. Our data on the developmental appearance of GABA and GAD immunoreactivities are consistent with previous biochemical data for the development of GABA concentration and GAD activity in the chick retina. Together, these data suggest that retina cells from the early stages of development may synthesize GABA from an alternative pathway in which the most likely precursor is putrescine.

Adenosine‐Elicited Accumulation of Adenosine 3′, 5′‐Cyclic Monophosphate in the Chick Embryo Retina

Abstract: The cyclic AMP level of 17‐day‐old chick embryo retina increased from 20 to 331 pmol/mg protein when the tissue was incubated for 20 min in the presence of 4‐(3‐butoxy‐4‐methoxybenzyl‐2‐imidozolinone) (RO 20‐1724). The addition of 0.5 mm‐3‐isobutyl‐1‐methylxanthine (IBMX) or 0.5 units/ml of adenosine deaminase (EC 3.5.4.4) to the medium reduced the increase of cyclic AMP content from 20 to 100 pmol/mg protein. Dipyridamole did not interfere with the rise of the retina cyclic AMP level observed with RO 20‐1724. The EC50 of 6‐amino‐2‐chloropurine riboside (2‐chloroadenosine)‐elicited accumulation of cylic AMP of retinas incubated in the presence of RO 20‐1724 plus adenosine deaminase was approximately 1 μm. When retina incubation was carried out in the presence of 0.5 mm‐IBMX, the 2‐chloroadenosine dose–response curve was shifted to the right two orders of magnitude. Maximal stimulation of the cyclic AMP level of 17‐day‐old chick embryo retina incubated in the presence of 0.5 mm‐IBMX was observed at 1 mm‐adenosine concentration. This effect was not blocked by dopamine antagonists. Guanosine and adenine did not affect the retinal cyclic AMP level. AMP and ATP had a slight stimulatory effect. Adenosine response of embryonic retina increased sharply from the 14th to the 17th embryonic day. A similar, but not identical, adenosine effect was observed in cultured retina cells.

Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393

Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G‐protein‐coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. Ionic currents induced by fast application of N‐methyl‐D‐aspartate (NMDA) were recorded under whole‐cell patch‐clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 μM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. The D1 receptor agonist (+)‐SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 μM on the NMDA response, while the enantiomer (−)‐SKF38393 was ineffective at 50 μM. However, the D1 antagonist R(+)‐SCH23390 did not prevent the inhibitory effect of (+)‐SKF38393. The degree of inhibition by dopamine and (+)‐SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage‐dependent block by dopamine was also observed in the presence of MgCl2 1 mM. Single‐channel recordings showed that the open times of NMDA‐gated channels were shortened by (+)‐SKF38393. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1‐type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)‐SKF38393 are NMDA channel ligands.

Induced Release of γ‐Aminobutyric Acid by a Carrier‐Mediated, High‐Affinity Uptake of L‐Glutamate in Cultured Chick Retina Cells

Abstract: [3H]γ‐aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse‐stimulated (2 min) with 0.1 mM L‐glutamate but not with D‐glutamate. The EC50 for L‐glutamate to evoke [3H]GABA release was approximately 15 μM. This value is close to the Km for high‐affinity uptake of L‐glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L‐glutamate‐induced release of GABA was abolished. Moreover. L‐[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L‐Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D‐Aspartate, which is taken up by the same high‐affinity uptake mechanism as L‐glutamate, induced an increase in [3H]GABA efflux comparable to L‐glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L‐glutamate in eliciting [3H]GABA release. The addition of both GABA and L‐glutamate to the incubation medium indicated that [3H]GABA efflux due to L‐glutamate and GABA was additive. L‐Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L‐aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA‐promoted GABA release‐homoexchange, (b) a Ca2+‐dependent L‐aspartate‐promoted release, and (c) a Ca2+‐independent, Na+‐dependent L‐glutamate‐evoked release. In addition, our data strongly suggest that the L‐glutamate‐promoted GABA release is due to a process of exchange of L‐glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.

Expression of A1 adenosine receptors modulating dopamine-dependent cyclic AMP accumulation in the chick embryo retina.

Abstract: Dopamine and 2‐chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16‐day‐old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine‐dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose‐dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12‐day‐old chick embryo retinas, with an IC50 of approximately 1 μM. This effect was not blocked by dipyridamole. N6‐ (l‐Phenylisopropyl)adenosine, (l‐PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 μM which was two orders of magnitude lower than its stereoisomer cf‐PIA (10 μM). The maximal inhibition of the dopamine‐elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3‐isobutyl‐l‐methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective, whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11‐ to 12‐day‐old embryos and 2‐chloroadenosine did not interfere with the apomorphine‐mediated shift in cyclic AMP levels. In the retinas from 5‐day‐old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP. In this case, 2‐chloroadenosine did not interfere with the response elicited by either one of the compounds. The data suggest the existence of two populations of D1 dopamine receptors in the chick embryo retina—one that is negatively modulated by adenosine and another that is not under purinergic control.

Differential ontogenesis of D1 and D2 dopaminergic receptors in the chick embryo retina

The differentiation of D1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our results reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10(-4) M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D1 receptors that does not desensitize significantly during retina development.

Expression of functional receptors and transmitter enzymes in cultured Muller cells

Glia represents the most numerous group of nervous system cells and CNS development and function depend on glial cells. We developed a purified Muller glia culture to investigate the expression of several neurotransmitter markers on these cells, such as dopaminergic, cholinergic, GABAergic and peptidergic receptors or enzymes, based on functional assays measuring second messenger levels or Western blot for specific proteins. Purified Muller cell culture was obtained from 8-day-old (E8) embryonic chick. Glial cells cultured for 15 days (E8C15) expressed D1A and D1B receptors mRNAs, but not D1D, as detected by RT-PCR. The binding of [3H]-SCH 23390 revealed an amount of expressed receptors around 40 fmol/mg protein. Dopamine (100 microM), PACAP (50 nM) and forskolin (10 microM) induced a 50-, 30- and 40-fold cAMP accumulation on glial cells, respectively, but not ip3 production. The dopamine-promoted cAMP accumulation was blocked by 2 microM SCH 23390. Carbachol stimulated a 3-fold ip3 accumulation. Western blot analysis also revealed the expression of tyrosine hydroxylase, L-dopa decarboxylase, PAC1 receptor, GAD67 and beta2-nicotinic receptor subunit by these cells. These results indicate that several components of neurotransmitter signaling and metabolism are found in cultured Muller cells.

GABAergic system in the developing mammalian retina: dual sources of GABA at early stages of postnatal development

In the present work, we have characterized the maturation of the GABAergic system in mammalian retina. Immunoreactivity for GABA, GAD (glutamic acid decarboxylase, EC 4.1.1.15) ‐65 and ‐67 in the adult rat retina was localized in cells in the inner nuclear and ganglion cell layers. This pattern was established around postnatal day 8 and included transient GABA and GAD‐67 expression in horizontal cells. GAD activity was very low at P1 and P4, increasing after P8, reaching maximal activity by P21 and decreasing to attain adult values by P30. GABA content was approximately constant from P1 to P13, increasing thereafter to reach adult levels. GAD protein content increased progressively with postnatal development and the two isoforms could be distinguished at P8.