RESUMO
Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, here we studied at embryonic day 12 the in vitro effect of two positive allosteric modulators of GABA binding, the barbiturate sodium pentobarbital and the neurosteroid allopregnanolone. In both cases an increase in E(max) values in membranes obtained from hypoxic embryos was observed. Studies of GABA-gated chloride influx showed that there were no differences in maximal chloride uptake between hypoxic and control membranes. We have already demonstrated that maximal density of GABA binding sites was decreased after hypoxia, suggesting that each of the remaining GABA(A) receptors display a greater chloride flux than controls. To further characterize GABA(A) receptor alterations, GABA-gated chloride influx modulated by the above barbiturate and neurosteroid was determined, finding that E(max) values were increased 60% and 42%, respectively. The increase in Cl(-) influx per receptor subsequent to hypoxic trauma, and the enhancement in the modulatory properties studied, may mediate neuronal damage by potential changes in subunit interaction at the GABA(A) receptor level.
Assuntos
Anestésicos/farmacologia , Moduladores GABAérgicos/farmacologia , Hipóxia/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Pentobarbital/farmacologia , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Animais , Embrião de Galinha , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5beta-pregnan-3beta-ol-20-one) on the GABA(A) receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 microM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 microM epipregnanolone decreased allopregnanolone EC(50) and E(max) values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K(d) and B(max) values for [3H]flunitrazepam and [3H]GABA binding. [3H]GABA binding modulation disclosed an increase in allopregnanolone EC(50) value with a decrease in its E(max) value. Diazepam EC(50) and E(max) values were enhanced, while low affinity sodium pentobarbital EC(50) value was reduced by epipregnanolone treatment. The investigation of the GABA(A) receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl(-) influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [3H]flunitrazepam and [3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA(A) receptor sites and function during chick optic lobe development.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Embrião de Galinha , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Flunitrazepam/metabolismo , Moduladores GABAérgicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Proteínas do Tecido Nervoso/química , Lobo Óptico de Animais não Mamíferos/embriologia , Pregnanolona/análogos & derivados , Receptores de GABA-A/química , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Membranas Sinápticas/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
Synaptosomes from the optic lobes of squid (Loligo forbesi) were prepared by homogenization and allowed to settle onto glass coverslips. Synaptosomes were loaded with Ca(2+) sensitive dyes (Fura-2 AM, Calcium Green-1 AM and Calcium Green-5N AM), visualized by light microscopy and Ca(2+) sensitive fluorescence signals recorded and analyzed. With Fura-2, resting Ca(2+) was found to be 80 nM (n = 10, SEM 5.7). Addition of K(+) (30 mM), caffeine (3 mM) and thapsigargin (10 microM) evoked transient increases in cytoplasmic Ca(2+). Addition of BAPTA-AM (20 microM) decreased intrasynaptosomal free Ca(2+). Similar results were obtained with Calcium Green-1 AM but not with Calcium Green-5N AM. We conclude that synaptosomes from the squid optic lobe posses intact membranes and mechanisms to regulate intrasynaptosomal free [Ca(2+)], as well as caffeine sensitive Ca(2+) stores. The results of this study are discussed with respect to the role of Ca(2+) in presynaptic protein synthesis.
Assuntos
Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Sinaptossomos/metabolismo , Animais , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Quelantes/farmacologia , Decapodiformes , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Compostos Orgânicos , Cloreto de Potássio/farmacologia , Sinaptossomos/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
In the present work, we studied the effect of zinc on GABA(A) receptor complex at three developmental stages of chick optic lobe (embryonic day 14, post-hatching day 1, and adulthood), in order to investigate the role of this cation in central nervous system (CNS) functional maturation. It was demonstrated that zinc exerts an inhibitory modulation of both GABA binding and GABA-gated chloride flux in a concentration-dependent manner with maximal effects at 100 microM zinc concentration. Maximal inhibition was higher at the embryonic stage and declined thereafter, disclosing minimal values at the adult stage. The effect of zinc on saturation GABA binding experiments performed at embryonic day 14 demonstrated that the cation decreased the maximal number of binding sites (B(max)) from 7. 53 +/- 1.06 pmol/mg protein to 4.63 +/- 0.53 pmol/mg protein, in the absence and presence of 100 microM zinc, respectively, while the dissociation constant (K(d)) remained unchanged. Analysis of the GABA concentration-effect curve at the embryonic stage revealed that the addition of 100 microM zinc decreased E(max) values for GABA stimulation of chloride uptake from 26.46 +/- 2.64% to 16.40 +/- 1. 96%, while EC(50) values were unaffected. In conclusion, our results suggest that zinc acts as a non-competitive inhibitor of both GABA binding and GABA responses during avian CNS development, with its effect inversely related to age.
Assuntos
Embrião de Galinha/efeitos dos fármacos , Moduladores GABAérgicos/farmacologia , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Zinco/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Embrião de Galinha/crescimento & desenvolvimento , Embrião de Galinha/metabolismo , Cloretos/metabolismo , Modelos Logísticos , Lobo Óptico de Animais não Mamíferos/embriologia , Lobo Óptico de Animais não Mamíferos/metabolismo , Análise de RegressãoRESUMO
It has been demonstrated in different vertebrate species that the GABAA receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [14C]progesterone metabolites as well as the characterization of their in vitro effects on the GABAA receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5 beta-pregnanedione, 5 beta-pregan-3 beta-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5 beta-pregnanedione was converted to 5 beta-pregnan-3 beta-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [3H]flunitrazepam binding by 5 beta-pregnan-3 beta-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5 beta-pregnan-3 beta-ol-20-one EC50 values were 0.195 +/- 0.049, 0.101 +/- 0.017, 0.147 +/- 0.009, and 0.569 +/- 0.114 microM, and Emax were 22.37 +/- 1.57, 23.67 +/- 4.02, 29.01 +/- 1.08, and 15.11 +/- 2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively. In conclusion, the biosynthesis of 5 beta-pregnan-3 beta-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABAA receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.
Assuntos
Encéfalo/metabolismo , Embrião de Galinha/efeitos dos fármacos , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Progesterona/metabolismo , Receptores de GABA-A/efeitos dos fármacos , Esteroides/biossíntese , Animais , Embrião de Galinha/crescimento & desenvolvimento , Embrião de Galinha/metabolismo , Moduladores GABAérgicos/farmacologia , Modelos Logísticos , Lobo Óptico de Animais não Mamíferos/embriologia , Lobo Óptico de Animais não Mamíferos/metabolismo , Pregnanolona/farmacologia , Ensaio RadioliganteRESUMO
Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABAA receptor complex. The presence of a 3 alpha-hydroxyl group and a 5 alpha-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [3H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3 alpha-hydroxy,5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 beta-hydroxy,5 beta-pregnan-20-one (3 beta,5 beta-P). Both steroids were found able to enhance [3H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3 alpha,5 alpha-P had greater potency (EC50 0.22 microM) and enhancing effect (Emax: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABAA receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3 alpha,5 alpha-P by its 3 beta,5 beta isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5 alpha-reduced progesterone metabolites present since early development, while 3 alpha,5 alpha-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5 beta-steroids in early avian development, we propose that 3 beta,5 beta-P, instead of the classical potent 3 alpha,5 alpha-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Pregnanolona/farmacologia , Ácido gama-Aminobutírico/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Embrião de Galinha , Isomerismo , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Lobo Óptico de Animais não Mamíferos/metabolismo , Receptores de GABA/efeitos dos fármacos , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismoRESUMO
[3H]SCH 23390 bound with high affinity (Kd = 0.6 nM) and in a saturable manner (Bmax = 130 fmol/mg protein) to membrane preparations of the chick optic lobe. Pharmacological experiments, using several dopaminergic ligands, revealed that [3H]SCH 23390 bound stereospecifically to dopaminergic receptors of the D1 type in this tissue. Other experiments revealed that dopamine was able to induce cyclic AMP accumulation in the optic lobe (ED50 = 3 microM), an effect that was blocked by fluphenazine, a potent D1 antagonist (IC50 = 1.8 microM). The developmental profile of tissue dopamine-dependent cyclic AMP accumulation, however, was quite different from the differentiation pattern of [3H]SCH 23390 specific binding sites. While [3H]SCH 23390 binding sites increased 4-fold after the 12th embryonic day (E12), dopamine-dependent cyclic AMP accumulation was maximal in earlier stages, decreasing progressively after E10. In tissues from embryos at E16 or older, no difference was observed between basal and dopamine-stimulated levels of cyclic AMP. These data suggest that D1 receptors are coupled to adenylate cyclase in a limited period of the development of the optic lobe and that D1 receptors not coupled to the enzyme can be a common feature in the CNS.
Assuntos
AMP Cíclico/metabolismo , Dopamina/farmacologia , Flufenazina/farmacologia , Lobo Óptico de Animais não Mamíferos/metabolismo , Receptores de Dopamina D1/fisiologia , Animais , Benzazepinas/metabolismo , Embrião de Galinha , Colforsina/farmacologia , Desenvolvimento Embrionário e Fetal , Cinética , Lobo Óptico de Animais não Mamíferos/efeitos dos fármacos , Lobo Óptico de Animais não Mamíferos/embriologia , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Serotonina/farmacologia , Sulpirida/farmacologiaRESUMO
The temporal course of the development of GABA receptor sites in chick optic lobe was studied as a parameter of neuronal differentiation in the central nervous system. At 10 days of incubation, specific [3H]GABA binding was of 0.08 pmol/optic lobe and increased 7-8 fold between 12 and 16 days of incubation, reaching at 16 days a value of 0.60 pmol/optic lobe. This coincides with the period of arrival of the retinal fibers to the optic lobe. After this stage, the number of GABA binding sites decreased to a value of 0.35 pmol/optic lobe at hatching. After hatching a new increase appeared which reached at 5 days post-hatching a value of 0.87 pmol/optic lobe. Scatchard analysis of the saturation binding data obtained at 16 days of incubation and at hatching revealed the presence of two binding sites: one with high affinity and the other with low affinity, while at 12 days of incubation, the earliest stage examined, only the low-affinity binding site appeared. The high-affinity binding site for [3H]GABA was inhibited by muscimol, GABA, and bicuculline (IC50: 0.006, 0.002 and 10 microM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 showed a marked increase in the amount of specific [3H]GABA binding after 16 days of incubation reaching a 3-fold increase at hatching. These results suggest that endogenous inhibitors of the higher affinity binding site, probably appear during this period.