Effect of the L- or D-aspartate on ecto-5'nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A(2A) receptors.

Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5'-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5'-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5'-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A(2A) receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A(1) receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5'-nucleotidase and on cellular damage. The adenosine formed from ecto-5'-nucleotidase stimulation preferentially acted on adenosine A(2A) receptor which is probably co-operating with the neurotoxicity induced by amino acids.

Catalepsy and hypolocomotion induced by a nitric oxide donor: attenuation by theophylline.

Nitric oxide (NO) promotes adenosine release in the striatum and hippocampus. Behavioral effects of the nitric oxide donor sodium nitroprusside were studied in mice and included an examination of spontaneous locomotion and catalepsy, which are behaviors modulated by adenosine. Sodium nitroprusside caused a dose-dependent (2, 4 and 6 mg/kg) decrease in locomotor activity and catalepsy at the dose of 6 mg/kg. These effects were substantially attenuated by pretreatment with the non-selective adenosine receptors antagonist theophylline (10 and 30 mg/kg). Moreover, combined treatment with theophylline (30 mg/kg) and sodium nitroprusside (6 mg/kg) induced limbic seizures in 23% of animals. The pretreatment with the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine (CPT) (1.2 mg/kg) caused no effect on the spontaneous or sodium nitroprusside-induced behavior. These data suggest that these behavioral effects of sodium nitroprusside are at least partially mediated by adenosine in the striatum and hippocampus, probably via adenosine A(2A) receptors.

The modulation of ecto-nucleotidase activities by glutamate in cultured cerebellar granule cells.

Several enzymes hydrolyze ATP, producing ADP which is hydrolyzed to AMP. Ecto-5'-nucleotidase produces adenosine from AMP. Glutamate (Glu) is an excitatory neurotransmitter and increases extracellular adenosine levels, which is considered an important inhibitory neuromodulator. Here we show that Glu activates ADP and AMP hydrolysis. NMDA and kainic acid (KA) also increased these enzymatic activities, but 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) had no effect. Dihydrokainate (DHK), an inhibitor of glutamate uptake, also blocked glutamate-evoked activation of ecto-nucleotidases, suggesting that this activation was also Glu transporters dependent. Therefore, we suggest that the Glu-evoked stimulation of ecto-nucleotidases might contribute to the increase of adenosine in extracellular space induced by Glu.

Study of adenosine A2 receptors in membrane preparations from optic tectum of chicks.

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [3H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosin e (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [3H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K(D) value was 400.90 +/- 80.50 nM and the Bmax was estimated to be 2.51 +/- 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [3H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.

Desensitization of adenosine A1 receptor-mediated inhibition of adenylyl cyclase in cerebellar granule cells.

Agonist-induced desensitization of adenosine A1 receptor-mediated inhibition of adenylyl cyclase has been studied in cerebellar granule cells. Exposure of cells to the adenosine A1 receptor agonist R-phenylisopropyl adenosine (R-PIA) from 2 to 48 h brings about desensitization of this signal transduction pathway. Associated with the desensitization process, a decrease in radioligand binding performed in intact cells with the adenosine A1 receptor agonist [3H]cyclohexyladenosine (CHA) has been detected. Simultaneously, an increase of adenosine A1 radioligand binding has also been detected in microsomes. A decrease in the steady-state level of alpha-Gi in both, plasma membrane and microsomes also has been detected during the desensitization process. These data may account for the desensitization of the inhibitory pathway of the adenylyl cyclase in cerebellar granule cells described herein. After a transient increase in adenosine A1 receptor mRNA, no changes were observed in this parameter after 12 hr of treatment with the adenosine A1 agonist R-PIA, suggesting a post-transcriptional regulation of this receptor during long-term desensitization.

Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine.

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.

Effects of adenosine on cAMP production during early development in the optic tectum of chicks.

Accumulation of cyclic adenosine monophosphate (cAMP) elicited by adenosine was studied in slices and membrane preparations of optic tectum from chicks aged 1-13 days post-hatch. Accumulation of cAMP promoted by adenosine declined with age, the highest value being observed in three-day-old chicks and the lowest in 11-day-old chicks. However, when the slices were incubated with adenosine and the phosphodiesterase inhibitor-Ro 20-1724 the differences between the two ages were abolished, suggesting a higher phosphodiesterase activity in 11-day-old chicks. In membrane preparations, although basal adenylate cyclase activity was lower in three-day-old chicks, the guanylyl-imidodiphosphate (Gpp(NH)p) concentration curves for stimulation of adenylate cyclase activity indicated a higher sensitivity of G protein to Gpp(NH)p at this age. This hypothesis was reinforced by the observation that the binding of [3H]Gpp(NH)p to the membrane preparation was greater in three-day-old animals. In spite of these differences, the percentage of adenylate cyclase activity stimulation by 2-chloroadenosine (2CADO)+Gpp(NH)p was the same at both ages. These findings suggest that the decreased response evoked by adenosine during development is probably due to increased phosphodiesterase activity and a lower sensitivity of adenylate cyclase activity to Gpp(NH)p.

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum.

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 microM) was abolished by 8-phenyltheophylline (15 microM). Glutamate and the glutamatergic agonists kainate or trans-D, L-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 microM, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 microM 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)M-CPG], a metabotropic antagonist, while 1 mM L-2-amino-3-phosphonopropionate (L-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 microM and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.

Effects of undernutrition during suckling on foot-shock escape behavior and of post-traini9ng beta-endorphin administration on inhibitory avoidance task test behavior of young rats

The hypothalamic beta-endorphin system of young Wistar rats of both sexes (21-day-old) responds in a distinct way to behavioral situations when compared to adult rats (90 to 120-day-old). In the present study we investigated whether the post-training amnestic effect of beta-endorphin previously demonstrated in Wistar adult rats is also observed young (21-day-old) well-nourished and undernourished rats. rats were undernourished since birth by feeding their dams an 8% casein diet, while well-nourished off spring were fed by dams maintained on a 20% caseindiet beta-endorphin was administered after training in a step-down inhibitory avoidance task using a 0.2 or 0.8 m-A footshock. Retention was tested 24 h later. We observed that the dose of beta-endorphin (1 ug/kg, ip) previously reported to have an amnestic effect on adult rats was ineffective in weanling rats of both nutritional groups. At a higher dose (2 ug/kg, ip) and using a 0.2-mA shock, beta-endorphin impaired the retention only of well-nourished rats. Test-to training difference (in s) in step-down latency for well-nourished beta-endorphin-treated rats was 7 vs 25 s for well-nourished rats treated with saline (P<0.05). Undernourished rats were hyperreactive to this shock intensity. Footshock escape latency (in s) for undernourished rats was 3.56 vs 5.80 for well-nourished rats (P<0.05, experiment 1) and 5.01 vs 10.89 (P<0.05 in experiment 2) and showed better retenmtion than did well-nourished rats. Test-totraining step down latency (in s) for saline-treated undernourished rats was abouth 108 vs 28 saline-treated well-nourished rats (P<0.05). At 0.8-m-A, neither beta-endorphin nor undernutrition affected performance. These resultssuggest that well-nourished rats respons in a distinct way to post-training beta-enmdorphin when compared to adult rats of both sexes. The absence of amnesia in weanling undernourished rats may be related to the enhacenced sensitivity of both sexes. The absence of amnesia in weanling undernourished rats may be related to the enhanced sensitivity of these animals to a 0.2-mA footshock

Undernutrition during suckling does not change the specific or total activity of hypothalamic proline endopeptidase in adult rats

Undernutrition during suckling causes a decrease in hypothalamic beta-endorphin-like immunoreactivity in rats. Since proline endopeptidase (E.C. 3.4.21.26) has been proposed to play a role in the processing of Beta-endorphin, we examined the effects of undernutrition during suckling on the enzyme activity. Rats were undernourished by feeding their dams an 8% casein diet from the day of birth until weaning (21 days). Dams of well-nourished rats were fed a 25% casein diet during the same period. After weaning, all rats received a 20% protein diet until 90 to 120 days of age when they were killed for the enzyme assay. The specific and total activity of hypothalamic proline endopeptidase was not altered by undernutrition followed by nutritional rehabilitation(2.37 + or - 0.24 nmol sulphamethoxazole min-1 mg-1 for well-nourished rats vs 2.68 + or - 0.24 nmol sulphamethoxazole min-1 mg-1 for undernourished rats). This lack of correlation suggests that proline endopeptidase is probably not responsible for the low levels of hypothalamic Beta-endorphin found in adult rats submitted to undernutrition during suckling

Effects of undernutrition during suckling and of post-training ß-endorphin administration on avoidance performances of adult rats

1. The effects of undernutrition during suckling and of post-training ß-endorphin administration on avoidance task were invstigated in adult rats. 2. young rats were undernourished from delivery until weaning (21 days) by feeding their mothers a diet conatining 8% protein (w/w). Mothers of well-nourished rats were fed a 20% protein diet. After weaning, both groups of rats were fed a 20% protein diet until 90-120 days if age, when they were subjected to behavioral sessions. 3. Acquistion was measured in training sessions and retention in test sessions 24 h after training. Beta-endorphin or salina (control) was injected ip immdiately after training. Rats were subjected to shuttle and step-down inhibitory avoidance sessions using footshock of 0.2 or 0.8 mA intensity. 4. Undernutrition during suckling caused hyperreactivity to 0.2 mA footshocks. Beta-endorphin caused amnesia to shuttle avoidance task only in normal rats trained with 0.8 mA. Foor-shocks. In the step-down inhibitory avoidance task, ß-endorphin was amnesic only for normal rats and only for 0.2-mA footshocks. Beta-endorphin was not amnesic in undernourished rats