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Rodrigo A Cunha

Publications and source records attributed to Rodrigo A Cunha.

34 records · Page 2Linked to original sources
PubMed2004

Presynaptic kainate receptors modulating glutamatergic transmission in the rat hippocampus are inhibited by arachidonic acid.

Kainate receptors are ionotropic glutamate receptors located postsynaptically, mediating frequency-dependent transmission, and presynaptically, modulating transmitter release. In contrast to the excitatory postsynaptic kainate receptors, presynaptic kainate receptor can also be inhibitory and their effects may involve a metabotropic action. Arachidonic acid (AA) modulates most ionotropic receptors, in particular postsynaptic kainate receptor-mediated currents. To further explore differences between pre- and postsynaptic kainate receptors, we tested if presynaptic kainate receptors are affected by AA. Kainate (0.3-3 microM) and the kainate receptor agonist, domoate (60-300 nM), inhibited by 19-54% the field excitatory postsynaptic potential (fEPSP) slope in rat CA1 hippocampus, and increased by 12-32% paired-pulse facilitation (PPF). AA (10 microM) attenuated by 37-72% and by 62-66% the domoate (60-300 nM)-induced fEPSP inhibition and paired-pulse facilitation increase, respectively. This inhibition by AA was unaffected by cyclo- and lipo-oxygenase inhibitors, indomethacin (20 microM) and nordihydroguaiaretic acid (NDGA, 50 microM) or by the free radical scavenger, N-acetyl-L-cysteine (0.5 mM). The K+ (20 mM)-evoked release of [3H]glutamate from superfused hippocampal synaptosomes was inhibited by 18-39% by domoate (1-10 microM), an effect attenuated by 35-63% by AA (10 microM). Finally, the KD (40-55 nM) of the kainate receptor agonist [3H]-(2S,4R)-4-methylglutamate ([3H]MGA) (0.3-120 nM) binding to hippocampal synaptosomal membranes was increased by 151-329% by AA (1-10 microM). These results indicate that AA directly inhibits presynaptic kainate receptor controlling glutamate release in the CA1 area of the rat hippocampus.

Animals↗
PubMed2003

Subcellular localization of adenosine A(1) receptors in nerve terminals and synapses of the rat hippocampus.

Adenosine is a neuromodulator in the CNS that mainly acts through pre- and postsynaptic A(1) receptors to inhibit the release of excitatory neurotransmitters and NMDA receptor function. This might result from a highly localized distribution of A(1) receptors in the active zone and postsynaptic density of CNS synapses that we now investigated in the rat hippocampus. The binding density of the selective A(1) receptor antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX), was enriched in membranes from Percoll-purified nerve terminals (B(max)=1839+/-52 fM/mg protein) compared to total membranes from the hippocampus (B(max)=984+/-31 fM/mg protein), the same occurring with A(1) receptor immunoreactivity. [3H]DPCPX binding occurred mainly to the plasma membrane rather than to intracellular sites, since the binding of the membrane permeable A(1) receptor ligand [3H]DPCPX to intact hippocampal nerve terminals (B(max)=1901+/-192 fM/mg protein) was markedly reduced (B(max)=321+/-30 fM/mg protein) by the membrane impermeable adenosine receptor antagonist, 8-sulfophenyltheophilline (25 microM). Further subcellular fractionation of hippocampal nerve terminals revealed that A(1) receptor immunoreactivity was strategically located in the active zone of presynaptic nerve terminals, as expected to understand the efficiency of A(1) receptors to depress neurotransmitter release. A(1) Receptors were also present in nerve terminals outside the active zone in accordance with the existence of a presynaptic A(1) receptor reserve. Finally, A(1) receptor immunoreactivity was evident in the postsynaptic density together with NMDA receptor subunits 1, 2A and 2B and with N-and P/Q-type calcium channel immunoreactivity, emphasizing the importance of A(1) receptors in the control of dendritic integration.

Adenosine A1 Receptor Antagonists↗
PubMed2003

Adenosine A3 receptors are located in neurons of the rat hippocampus.

Adenosine is a neuromodulator acting mainly via inhibitory A1 and facilitatory A2A receptors. Whole tissue PCR also identified adenosine A3 receptors in the brain and A3 receptor agonists affect CNS neuronal responses and viability. However, recent reports failed to detect A3 receptor expression in CNS neurons and showed that A3 receptor agonists can bind and activate A1 receptors. We now present evidence for the presence of A3 receptor mRNA in CNS neurons using single cell PCR analysis of laser dissected hippocampal neurons. Western blot analysis showed that A3 receptors are present in rat hippocampal nerve terminal membranes. This indicates that A3 receptors are present in CNS neurons in the hippocampus.

Animals↗
PubMed2003

Ecto-AMP deaminase blunts the ATP-derived adenosine A2A receptor facilitation of acetylcholine release at rat motor nerve endings.

At synapses, ATP is released and metabolised through ecto-nucleotidases forming adenosine, which modulates neurotransmitter release through inhibitory A1 or facilitatory A2A receptors, according to the amounts of extracellular adenosine. Neuromuscular junctions possess an ecto-AMP deaminase that can dissociate extracellular ATP catabolism from adenosine formation. In this study we have investigated the pattern of ATP release and its conversion into adenosine, to probe the role of ecto-AMP deaminase in controlling acetylcholine release from rat phrenic nerve terminals. Nerve-evoked ATP release was 28 +/- 12 pmol (mg tissue)-1 at 1 Hz, 54 +/- 3 pmol (mg tissue)-1 at 5 Hz and disproportionally higher at 50 Hz (324 +/- 23 pmol (mg tissue)-1). Extracellular ATP (30 microM) was metabolised with a half time of 8 +/- 2 min, being converted into ADP then into AMP. AMP was either dephosphorylated into adenosine by ecto-5'-nucleotidase (inhibited by ATP and blocked by 200 microM alpha,beta-methylene ADP) or deaminated into IMP by ecto-AMP deaminase (inhibited by 200 microM deoxycoformycin, which increased adenosine formation). Dephosphorylation and deamination pathways also catabolised endogenously released adenine nucleotides, since the nerve-evoked extracellular AMP accumulation was increased by either alpha,beta-methylene ADP (200 microM) or deoxycoformycin (200 microM). In the presence of nitrobenzylthioinosine (30 microM) to inhibit adenosine transport, deoxycoformycin (200 microM) facilitated nerve-evoked [3H]acetylcholine release by 77 +/- 9 %, an effect prevented by the A2A receptor antagonist, ZM 241385 (10 nM). It is concluded that, while ecto-5'-nucleotidase is inhibited by released ATP, ecto-AMP deaminase activity transiently blunts adenosine formation, which would otherwise reach levels high enough to activate facilitatory A2A receptors on motor nerve terminals.

AMP Deaminase↗
PubMed2003

Adenosine promotes neuronal recovery from reactive oxygen species induced lesion in rat hippocampal slices.

Reactive oxygen species (ROS) are believed to be involved in the pathogenesis of several neurological disorders. We now tested whether the endogenous neuroprotective substance, adenosine, attenuates the cell damage induced by ROS. In rat hippocampal slices, the xanthine oxidase (40 mU/ml) plus xanthine (1 mM) (X/XO) system produced a 27.8+/-7.3% (n=3) increase in ROS, measured by fluorimetry with 2',7'-dichlorodihydrofluorescein, a 246.9+/-18.4% (n=6) increase in the release of tritiated adenosine, and a decrease in synaptic transmission that fully recovered after washout. In the presence of the adenosine A(1) receptor selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (100 nM), X/XO induced a similar inhibition, however synaptic transmission only recovered to 70.7+/-5.8% of control (n=5). The blockade of A(2A) receptors was devoid of effect (n=4). Adenosine is released by ROS-generating systems, and attenuates the deleterious cellular consequences of ROS through A(1) receptor activation.

Adenosine↗
PubMed2003

ATP is released from nerve terminals and from activated muscle fibres on stimulation of the rat phrenic nerve.

Nerve stimulation increases the concentration of ATP in the synaptic cleft, which can act as a neurotransmitter or as a presynaptic neuromodulator. Using the luciferin-luciferase assay, we observed that the extracellular concentration of ATP increased by 11-26 nM over a basal concentration of 6 nM, in a frequency dependent manner (1-5 Hz), in the adult rat phrenic nerve-hemidiaphragm preparation. This ATP release depends on nerve activity since it was abolished by tetrodotoxin (1 microM) and is strictly dependent on the presence of extracellular calcium. However, more than half of this nerve-evoked release of ATP is derived from activated muscle fibres since the selective post-synaptic nicotinic receptor antagonist, alpha-bungarotoxin (1 microM), inhibited by over 60% the evoked release of ATP. The presently observed post-synaptic release of ATP together with the previously reported lack of post-synaptic effects of ATP and to the ability of ATP to act as a presynaptic modulator open the possibility that ATP may behave as a retrograde messenger at this neuromuscular junction.

Adenosine Triphosphate↗
PubMed2003

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.

Animals↗
PubMed2003

Presynaptic modulation controlling neuronal excitability and epileptogenesis: role of kainate, adenosine and neuropeptide Y receptors.

Based on the idea that seizures may arise from an overshoot of excitation over inhibition, all substances that may decrease glutamatergic function while having no effect or even increasing GABAergic neurotransmission are likely to be effective anticonvulsants. We now review the possible role of three such neuromodulators, kainate, adenosine, and neuropeptide Y receptors in controlling hyperexcitability and epileptogenesis. Particular emphasis is given on the robust neuromodulatory role of these three groups of receptors on the release of glutamate in the hippocampus, a main focus of epilepsy. Moreover, we also give special attention to the mechanisms of receptor activation and coupled signaling events that can be explored as attractive targets for the treatment of epilepsy and excitotoxicity. The present paper is a tribute to Arsélio Pato de Carvalho who has been the main driving force for the development of Neuroscience in Portugal, notably with a particular emphasis on the presynaptic mechanisms of modulation of neurotransmitter release.

Animals↗
PubMed2003

Neuroprotection by caffeine and adenosine A2A receptor blockade of beta-amyloid neurotoxicity.

Adenosine is a neuromodulator in the nervous system and it has recently been observed that pharmacological blockade or gene disruption of adenosine A(2A) receptors confers neuroprotection under different neurotoxic situations in the brain. We now observed that coapplication of either caffeine (1-25 micro M) or the selective A(2A) receptor antagonist, 4-(2-[7-amino-2(2-furyl)(1,2,4)triazolo (2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol (ZM 241385, 50 nM), but not the A receptor antagonist, 8-cyclopentyltheophylline (200 nM), prevented the neuronal cell death caused by exposure of rat cultured cerebellar granule neurons to fragment 25-35 of beta-amyloid protein (25 micro M for 48 h), that by itself caused a near three-fold increase of propidium iodide-labeled cells. This constitutes the first in vitro evidence to suggest that adenosine A(2A) receptors may be the molecular target responsible for the observed beneficial effects of caffeine consumption in the development of Alzheimer's disease.

Adenosine A2 Receptor Antagonists↗
PubMed2003

Decrease of adenosine A1 receptor density and of adenosine neuromodulation in the hippocampus of kindled rats.

Adenosine is a neuromodulator that has been proposed to be a major endogenous anticonvulsant acting via A1 receptors. We tested if implementation of kindling through stimulation of the amygdala affected A1 receptor-mediated neuromodulation in hippocampal slices taken from rats 4 weeks after the last stage 5 seizure. The A1 receptor agonist, N6-cyclopentyladenosine (CPA) (6-100 nm), inhibited field excitatory postsynaptic potential (fEPSP) slope with an EC50 of 19.1-19.5 nm in control and sham-operated rats, but was less potent in kindled rats (EC50 = 42.7 nm). This might result from a decreased number of A1 receptors in hippocampal nerve terminal membranes, because A1 receptor immunoreactivity decreased by 28 +/- 3% and the binding density of the A1 receptor agonist [3H]R-PIA decreased from 1702 +/- 64 to 962 +/- 78 fmol/mg protein in kindled compared with control rats. The tonic inhibition of hippocampal synaptic transmission by endogenous adenosine was also lower in kindled rats, because A1 receptor blockade with 50 nm 1,3-dipropyl-8-cyclopentyladenosine (DPCPX) enhanced fEPSP slope by 23 +/- 3% and theta-burst-induced long-term potentiation by 94 +/- 4% in control rats but was virtually devoid of effects in kindled rats. The evoked release of adenosine from hippocampal slices or nerve terminals was 56-71% lower in kindled rats probably due to the combined decrease in the capacity of adenosine transporters and decreased release of adenosine 5'-triphosphate (ATP), which was partially compensated by a higher extracellular catabolism of ATP into adenosine in kindled rats. These results indicate that, although adenosine might inhibit the onset of epileptogenesis, once kindling is installed, the efficiency of the adenosine inhibitory system is impaired.

Adenosine↗
PubMed2003

Enhanced adenosine A2A receptor facilitation of synaptic transmission in the hippocampus of aged rats.

Adenosine either inhibits or facilitates synaptic transmission through A1 or A2A receptors, respectively. Since A2A receptor density increases in the limbic cortex of aged (24 mo) compared with young adult rats (2 mo), we tested if A2A receptor modulation of synaptic transmission was also increased in aged rats. The A2A receptor agonist, CGS21680 (10 nM), caused a larger facilitation of the field excitatory postsynaptic potential (fEPSP) slope in hippocampal slices of aged (38%) than in young rats (19%), an effect prevented by the A2A receptor antagonist, ZM241385 (20 nM). In contrast to young rats, where CGS21680 facilitation of fEPSPs is prevented by the protein kinase C inhibitor, chelerythrine (6 microM), but not by the protein kinase A inhibitor, H-89 (1 microM), the CGS21680-induced facilitation of fEPSP slope in aged rats was prevented by H-89 (1 microM) but not by chelerythrine (6 microM). Also, in contrast to the beta-receptor agonist, isoproterenol (30 microM), CGS21680 (100-1,000 nM) enhanced cAMP levels in hippocampal nerve terminals of aged but not young rats. Finally, we observed a significant increase of both the binding density of [3H]CGS 21680 and the [3H]ZM241385 as well as of the anti-A2A receptor immunoreactivity in hippocampal nerve terminal membranes from aged compared with young rats. This shows that A2A receptor-mediated facilitation of hippocampal synaptic transmission is larger in aged than young rats due to increased A2A receptor density in nerve terminals and to the modified transducing system operated by A2A receptors, from a protein kinase C mediated control of A1 receptors into a direct protein kinase A dependent facilitation of synaptic transmission.

Adenosine↗
PubMed2003

Pharmacology of adenosine A2A receptors and therapeutic applications.

Adenosine A(2A) receptors were cloned about ten years ago and are known to be well conserved among mammals. Rather selective agonists and antagonists are available. In addition, two different knock-out mice have been prepared and extensively characterized. A(2A) receptors are highly enriched in the basal ganglia and on cells involved in inflammatory reactions. At these sites they are likely to play physiologically important roles. Efforts to develop new therapies based on A(2A) receptors have focused on these topics. However, A(2A) receptors are found on many other cell types and on them as well agonists can exert effect.

Animals↗
PubMed2002

Transducing system operated by adenosine A(2A) receptors to facilitate acetylcholine release in the rat hippocampus.

Although molecular biology studies indicate the presence of adenosine A(2A) receptors in the rat hippocampus, the pharmacological characterization of adenosine A(2A) receptor binding and of its putative facilitatory effects has revealed features essentially different from these found for adenosine A(2A) receptors in most preparations. We now confirmed that activation of adenosine A(2A) receptors with 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) or 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HENECA, 3-100 nM) facilitated the veratridine-evoked [3H]acetylcholine release from hippocampal synaptosomes with a maximal effect of 14+/-2% and 16+/-2%, respectively. These effects were prevented by the adenosine A(2A) receptor antagonists, 4-(2-[7-amino-2-[2-furyl][1,2,4]-triazolo[2,3a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM 241385, 20 nM) and 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261, 20 nM), but not by the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 20 nM). Adenosine A(2A) receptors may activate adenylate cyclase and protein kinase A since CGS 21680 (10 nM) facilitation of [3H]acetylcholine release was occluded by 8-bromo-cAMP (0.5 mM) and forskolin (10 microM) and prevented by H-89 (1 microM), but unaffected by phorbol-12,13-didecanoate (250 nM) or bisindolylmaleimide I (1 microM). The existence of adenosine A(2A) receptors in hippocampal nerve terminals was further confirmed by a Western blot immunoreactivity qualitatively identical to that found in the striatum. This constitutes the first pharmacological identification of canonical adenosine A(2A) receptors coupling to the expected cAMP/protein kinase A pathway in the hippocampus with the expected immunoreactivity.

Acetylcholine↗
PubMed2002

Effects of carbamazepine and novel 10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide derivatives on synaptic transmission in rat hippocampal slices.

The effects of carbamazepine on synaptic transmission in rat hippocampal slices were compared with those of two novel analogues (BIA2-093 and BIA2-024) with equivalent anticonvulsant efficacy but with fewer side effects. Carbamazepine (10-1000 microM) inhibited in a concentration-dependent manner the field excitatory postsynaptic potential (fPSP) response, with an EC50 of 263 microM, and also attenuated the presynaptic volley with a similar EC50 value. Carbamazepine was more potent to inhibit the NMDA receptor component of the fPSP (fPSPNMDA), with an EC50 of 160 microM. BIA2-093 and BIA2-024 were nearly equipotent with carbamazepine to inhibit synaptic transmission, and displayed similar potency to inhibit the fPSP (EC50 of 145 microM and 205 microM) and fPSPNMDA responses (EC50 of 198 microM and 206 microM). As with carbamazepine, BIA2-093 and BIA2-024 also attenuated the presynaptic volley with EC50 values ranging from 142 to 322 microM. These results indicate that carbamazepine and its analogues mostly inhibit synaptic transmission through inhibition of conduction, although carbamazepine, but not BIA2-093 and BIA2-024, may also depress NMDA receptor-mediated responses.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2002

Modulation of the rat hippocampal dinucleotide receptor by adenosine receptor activation.

Diadenosine pentaphosphate (Ap(5)A) and ATP stimulate an intracellular free calcium concentration ([Ca(2+)](I)) increase in rat hippocampal synaptosomes via different receptors as demonstrated by the lack of cross-desensitization between Ap(5)A and ATP responses. The ATP response was inhibited by P2 receptor antagonists and not by the dinucleotide receptor antagonist, diinosine pentaphosphate (Ip(5)I). In contrast, the Ap(5)A response was inhibited by Ip(5)I but not by P2 receptor antagonists. Studies in single hippocampal synaptic terminals showed that 31% of them responded to Ap(5)A by a [Ca(2+)](i) increase. Adenosine receptors (A(1), A(2A), and A(3)) were also present in isolated terminals as demonstrated by immunohistochemistry. The activation of A(1) or A(2A) receptors by specific agonists changed the sigmoid concentration-response curve for Ap(5)A (EC(50) = 33.5 +/- 4.5 microM) into biphasic curves. When the high-affinity adenosine receptors A(1) or A(2A) were activated, the Ap(5)A dose-response curves showed a high-affinity component with EC(50) values of 41.1 +/- 1.9 pM and 99.9 +/- 10.2 nM, respectively. The low-affinity component showed EC(50) values of 17.1 +/- 0.8 and 21.6 +/- 1.4 microM for A(1) and A(2A) receptor activation, respectively. However, the adenosine A(3) receptor activation induced a right shift of the dinucleotide concentration-response curve, showing an EC(50) value of 331.4 +/- 54.6 microM. In addition, in the presence of the A(2A) agonist, the Ap(5)A calcium influx responses were increased up to 300% of the control values. These results clearly demonstrate that the activation of presynaptic adenosine receptors is able to modulate the dinucleotide response in hippocampal nerve terminals.

Adenosine Triphosphate↗