RESUMO
In infants, the main cause of blindness is retinopathy of prematurity that stems in a hypoxic-ischemic condition. Caffeine is a psychoactive compound that at low to moderate concentrations, selectively inhibits adenosine A1 and A2A receptors. Caffeine exerts beneficial effects in central nervous system of adult animal models and humans, whereas it seems to have malefic effect on the developing tissue. We observed that 48-h exposure (during synaptogenesis) to a moderate dose of caffeine (30 mg/kg of egg) activated pro-survival signaling pathways, including ERK, CREB, and Akt phosphorylation, alongside BDNF production, and reduced retinal cell death promoted by oxygen glucose deprivation in the chick retina. Blockade of TrkB receptors and inhibition of CREB prevented caffeine protection effect. Similar signaling pathways were described in previously reported data concerning chemical preconditioning mechanism triggered by NMDA receptors activation, with low concentrations of agonist. In agreement to these data, caffeine increased NMDA receptor activity. Caffeine decreased the levels of the chloride co-transporter KCC2 and delayed the developmental shift on GABAA receptor response from depolarizing to hyperpolarizing. These results suggest that the caffeine-induced delaying in depolarizing effect of GABA could be facilitating NMDA receptor activity. DPCPX, an A1 adenosine receptor antagonist, but not A2A receptor inhibitor, mimicked the effect of caffeine, suggesting that the effect of caffeine occurs through A1 receptor blockade. In summary, an in vivo caffeine exposure could increase the resistance of the retina to ischemia-induced cell death, by triggering survival pathways involving CREB phosphorylation and BDNF production/TrkB activation.
Assuntos
Cafeína/farmacologia , Morte Celular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Embrião de Galinha , Galinhas , Isquemia/metabolismoRESUMO
Pancreas transplantation is an option to achieve better metabolic control and decrease chronic complications in patients with diabetes. Xenotransplantation becomes an important alternative. In this study, we show the clinical outcome of patients with type 1 diabetes transplanted with neonatal pig islets without immunosuppression. In a longitudinal study of 23 patients with type 1 diabetes, who received porcine islets between 2000 and 2004, we registered demographic and clinical characteristics every 3 months and chronic complications evaluation yearly. Porcine C-peptide was measured in urine samples under basal conditions and after stimulation with l-arginine. More than 50% were female, median current age was 20·8 years, median diabetes duration at transplantation 5·5 years, median current diabetes duration 11 years and median time post-transplantation 5·7 years. Their media of glycosylated haemoglobin reduced significantly after the first transplantation. Insulin doses remain with a reduction greater than 33% in more than 50% of the patients. Before transplantation, 14 of the 21 patients presented mild chronic complications and currently only two patients presented these complications. Porcine C-peptide was present in all urine samples under basal conditions and increased post-stimulation with l-arginine. These patients achieved an excellent metabolic control after the first transplantation. This could explain, as well as the remaining function of transplanted cells, the low frequency of chronic complications compared to patients with similar diabetes duration and age.
Assuntos
Complicações do Diabetes/prevenção & controle , Diabetes Mellitus Tipo 1/terapia , Transplante das Ilhotas Pancreáticas , Adolescente , Animais , Animais Recém-Nascidos , Peptídeo C/urina , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/fisiopatologia , Progressão da Doença , Intervalo Livre de Doença , Feminino , Seguimentos , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Suínos , Fatores de Tempo , Transplante Heterólogo , Adulto JovemRESUMO
Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A(1), A(3)) and excitatory (A(2a), A(2b)) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A(1) and A(2a) receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A(1) and A(2a) receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0-70 were used for ontogeny studies. Our results indicate that A(1) expression in the visual layers of the superior colliculus is higher until PND 28, while A(2a) expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5'-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A(1) and A(2a) receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.
Assuntos
AMP Cíclico/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Retina/crescimento & desenvolvimento , Colículos Superiores/crescimento & desenvolvimento , Adenina/análogos & derivados , Adenina/farmacologia , Agonistas do Receptor A1 de Adenosina , Agonistas do Receptor A2 de Adenosina , Inibidores de Adenosina Desaminase , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Animais Recém-Nascidos , Fármacos do Sistema Nervoso Central/farmacologia , Inibidores Enzimáticos/farmacologia , Peroxidase do Rábano Silvestre , Marcadores do Trato Nervoso , Ratos , Ratos Endogâmicos , Retina/anatomia & histologia , Retina/efeitos dos fármacos , Colículos Superiores/anatomia & histologia , Colículos Superiores/efeitos dos fármacos , Vias Visuais/anatomia & histologia , Vias Visuais/efeitos dos fármacos , Vias Visuais/crescimento & desenvolvimentoRESUMO
In the chick retina, the D1 dopaminergic system differentiates very early, as shown by receptor-mediated increases in intracellular cyclic AMP concentration and the presence of [(3)H]SCH23390-specific binding sites. Here, we characterized, by RT-PCR, the expression of defined D1 receptor subtypes D(1A), D(1B), and D(1D) during the development of the chick retina. Total RNA was extracted from retinas of 6-day-old embryos (E6) to 1-day-old hatched chickens and reverse-transcribed. The resulting cDNA was amplified using D(1A)-, D(1B)-, or D(1D)-specific primers, and the PCR-amplified products were analyzed by electrophoresis. The fragment corresponding to D(1A) receptor was detected in developing retina as early as E7, whereas the fragment corresponding to D(1B) was observed starting around E10. No PCR product corresponding to D(1D) was observed in the retina, although it was detected in chick brain. As synaptogenesis in chick retina begins after E11 and [(3)H]SCH 23390 D1 binding sites increase after this stage, the present results show that expression of D(1B) receptor increases during synaptogenesis, whereas D(1A) is the receptor subtype associated with the D1-like actions of dopamine early in retina development.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Dopamina D1/genética , Retina/embriologia , Retina/metabolismo , Animais , Benzazepinas/farmacocinética , Embrião de Galinha , Galinhas , RNA Mensageiro/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 microM carbachol was used, the estimated IC50 value for kainate was 0.2 microM and the maximal inhibition of approximately 50% was obtained with 1 microM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 microM veratridine, but not 50 mM KCl, inhibited approximately 65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 +/- 38.0 to 2044.5 +/- 299.9 cpm/mg protein, retinal response decreased to 861.6 +/- 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.
Assuntos
Carbacol/antagonistas & inibidores , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Agonistas Muscarínicos/farmacologia , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/metabolismo , Retina/embriologia , Retina/metabolismo , Veratridina/farmacologia , Animais , Embrião de Galinha , Agonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/farmacologia , Ácido Caínico/metabolismo , Cloreto de Potássio , Receptores Muscarínicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Canais de SódioRESUMO
Four subtypes of GABA carriers (GAT1-GAT4) that transport GABA in a sodium-dependent manner were identified so far. In this report, the sodium-dependent release of GABA was investigated in cultured chick retinal cells. Opening of voltage-sensitive sodium channels by veratridine or activation of non-NMDA glutamate receptors induced the release of GABA from cultured cells. The release of GABA was calcium-independent, but could be completely prevented by the substitution of sodium chloride by lithium or choline chloride in the extracellular medium, suggesting that GABA release could be triggered by multiple mechanisms that led to the flux of sodium into these cells. Pharmacological experiments revealed that, while GABA uptake was almost completely inhibited by the GAT-1 blockers NNC-711 (50 microM) or nipecotic acid (1 mM), the release of this amino acid was inhibited by NNC-711, but not by nipecotic acid. The incubation with beta-alanine (10 mM), a GAT-2/GAT-3 inhibitor, blocked 50% of GABA uptake but had no effect on the release. Our data suggest that sodium-dependent GABA release from cultured chick retina cells is mediated by a GAT-1 like transporter that shows some, but not all, the pharmacological properties of the GAT-1 carrier.
Assuntos
Proteínas de Transporte/fisiologia , Ácido Glutâmico/farmacologia , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos , Retina/metabolismo , Veratridina/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA , Retina/citologia , Retina/embriologia , Tetrodotoxina/farmacologia , TrítioRESUMO
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 muM carbachol was used, the estimated IC50 value for kainate was 0.2 muM and the maximal inhibition of ~50 percent was obtained with 1 muM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 muM veratridine, but not 50 mM KCl, inhibited ~65 percent of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 + 38.0 to 2044.5 + 299.9 cpm/mg protein, retinal response decreased to 861.6 + 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.
Assuntos
Animais , Embrião de Galinha , Carbacol/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Agonistas Muscarínicos/farmacologia , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/metabolismo , Retina/embriologia , Veratridina/farmacologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/farmacologia , Ácido Caínico/metabolismo , Cloreto de Potássio , Receptores Muscarínicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Canais de SódioRESUMO
In the present work we show the development of carbachol-induced accumulation of 3H-inositol phosphates (3H-InsPs) in the chick embryonic retina and its regulation by glutamate receptors. Although basal levels of 3H-InsPs increased during development, the retinal response to carbachol was high in the early developing stages and decreased after synaptogenesis in the retina. Eserine also stimulated the turnover of phosphoinositides in the embryonic but not in the mature retina. The effect of eserine could be blocked by atropine, suggesting that acetylcholine could be released from developing retina cells and further stimulate the turnover of InsPs in the embryonic tissue. Our data also show that muscarinic stimulation of turnover of 3H-InsPs could be blocked by stimulation of glutamatergic ionotropic receptors. Moreover, the effect of glutamate agonists did not seem to be mediated by the release of other neurotransmitters such as GABA, glycine, adenosine, or dopamine from the tissue because these neurotransmitters did not interfere with the retinal response to carbachol. These results suggest that muscarinic activation of phosphoinositide turnover occurs mainly in the embryonic retina and that activation of glutamate receptors can inhibit directly the muscarinic stimulation of hydrolysis of 3H-InsPs in this tissue.
Assuntos
Fosfatidilinositóis/metabolismo , Receptores de Glutamato/fisiologia , Receptores Muscarínicos/metabolismo , Retina/embriologia , Animais , Atropina/farmacologia , Carbacol/farmacologia , Embrião de Galinha , Ácido Glutâmico/farmacologia , Técnicas In Vitro , N-Metilaspartato/farmacologia , Fisostigmina/farmacologia , Pirenzepina/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Retina/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
GABA is a major inhibitory neurotransmitter in the central nervous system, including the retina. In the present paper we present evidence for the existence of two independent mechanisms for GABA release in cultured retina cells. Eight-day-old chick embryo retinas were dissociated and plated in 35-mm plastic dishes and cultured for 3 or 7 days at 37 degrees C. An increase of 3 to 5-fold in GABA release was observed in cultures of 3 or 7 days in vitro preloaded with 0.5 microCi [3H]GABA and stimulated with glutamate (100 microM) or veratridine (100 microM). Tetrodotoxin (1 microM) blocked the release induced by veratridine but not by glutamate. In contrast, the non-N-methyl-D-aspartate (NMDA) glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM) was able to inhibit GABA release promoted by glutamate but not by veratridine. These results indicate that depolarization of retinal cells by opening of voltage-dependent sodium channels or activation of non-NMDA glutamate receptors can trigger intracellular events that lead to calcium-independent GABA release.
Assuntos
Glutamatos/farmacologia , Neurotransmissores/farmacologia , Retina/citologia , Veratridina/farmacologia , Ácido gama-Aminobutírico/farmacocinética , 6-Ciano-7-nitroquinoxalina-2,3-diona , Animais , Células Cultivadas , Embrião de Galinha , Antagonistas GABAérgicos , Ácido Glutâmico , Quinoxalinas/farmacologia , Tetrodotoxina/farmacologia , Ácido gama-Aminobutírico/efeitos dos fármacosRESUMO
GABA is a major inhibitory neurotransmitter in the central nervous system, including the retina. In the present paper we present evidence for the existence of two independent mechanisms for GABA release in cultured retina cells. Eight-day-old chick embryo retinas were dissociated and plated in 35-mm plastic dishes and cultured for 3 or 7 days at 37 grade C. An increase of 3 to 5-fold in GABA release was observed in cultures of 3 or 7 days in vitro preloaded with 0.5 uCi[3H} GABA and stimulated with glutamate (100 uM) or veratridine (100 uM). Tetrodotoxin (1 uM) blocked the release induced by veratridine but not by glutamate. In contrast, the non-N-methyl-D-aspartate (NMDA)glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 uM) was able to inhibit GABA release promoted by glutamate but not by veratridine. These results indicate that depolarization of retinal cells byopening of voltage-dependent sodium channels or activation of non-NMDA glutamate receptors can trigger intracellular events that lead to calcium-independent GABA release
Assuntos
Embrião de Galinha , Células Cultivadas , Ácido gama-Aminobutírico/induzido quimicamente , Glutamatos , Retina/citologia , Tetrodotoxina , Veratridina/administração & dosagemRESUMO
1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.
Assuntos
Glutamato Descarboxilase/metabolismo , Retina/enzimologia , Ácido gama-Aminobutírico/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Glutamato Descarboxilase/biossíntese , Imuno-Histoquímica , Cinética , Retina/citologia , Retina/efeitos dos fármacosRESUMO
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.
Assuntos
Glutamato Descarboxilase/análise , Retina/química , Ácido gama-Aminobutírico/análise , Animais , Embrião de Galinha , Galinhas , Técnicas Imunoenzimáticas , Neurônios/química , Retina/embriologia , Retina/crescimento & desenvolvimento , Ácido gama-Aminobutírico/biossínteseRESUMO
[3H]SCH 23390 binds stereospecifically and with high affinity to D1 dopaminergic receptors in the developing chick retina. Autoradiographic experiments revealed that in retinas from 3-day-old chicken and embryos with 12, 14 and 16 days of development, specific labeling of [3H]SCH 23390 was mainly observed over the plexiform layers of the tissue, showing that dopaminergic D1 receptors are localized in retina cell neurites since the initial stages of neurite formation. The total number of [3H]SCH 23390 binding sites increased 5-fold during the differentiation of the retina, while the dopamine-dependent cyclic adenosine monophosphate (AMP) accumulation was significantly decreased. Consequently, the ratio between dopamine-dependent cyclic AMP accumulation and [3H]SCH 23390 binding sites decreased 10-fold as retina differentiated, indicating that a significant portion of D1 receptors in retinas from adult chicken are not effectively coupled to adenylate cyclase molecules.
Assuntos
Adenilil Ciclases/metabolismo , Neurônios/metabolismo , Receptores Dopaminérgicos/metabolismo , Retina/metabolismo , Envelhecimento/metabolismo , Animais , Autorradiografia , Benzazepinas/metabolismo , Benzazepinas/farmacologia , Embrião de Galinha , AMP Cíclico/metabolismo , Dopamina/fisiologia , Técnicas In Vitro , Indicadores e Reagentes , Membranas/efeitos dos fármacos , Membranas/metabolismo , Retina/embriologia , Retina/enzimologiaRESUMO
The present work shows the existence of adenosine-dependent cyclic adenosine monophosphate (AMP) accumulation in the chick optic tectum. When tecta from 18-day-old embryos were incubated with the phosphodiesterase inhibitor IBMX and RO 20-1724, the cyclic AMP level increased from 39.2 to 73.3 and 285.5 pmol/mg protein, respectively. The high level obtained with RO 20-1724 could be inhibited by increasing concentrations of IBMX or by adenosine deaminase, but not by dipyridamole. 2-Chloroadenosine promoted a dose-dependent cyclic AMP accumulation in tecta incubated with RO 20-1724 and adenosine deaminase. This effect was blocked by IBMX and varied substantially during the development of the tissue. The degree of stimulation increased after day 11 of incubation, attaining maximal levels on day 14. The effect of 2-chloroadenosine remained constant until day 18, a period when both the protein content and the basal cyclic AMP levels are increasing in the developing tectum. The cyclic AMP increase elicited by 2-chloroadenosine was greatly reduced in tecta from 20-day-old embryos and 2-day-old chicks. The putative transmitters glutamate and glycine and the neurotransmitter analogs isoproterenol and carbachol had no stimulatory effect on the cyclic AMP accumulation of tecta from 10- and 17-day-old embryos.