Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells.

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [3H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: approximately 4 microM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 microM SK&F 96365. DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]-thymidine incorporation with IC50 values of 20-30 microM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.     

ATP induces proliferation of retinal cells in culture via activation of PKC and extracellular signal-regulated kinase cascade

Both ATP and acetylcholine can induce the mobilization of intracellular calcium in the early developing chick embryo retina, a response that decreases during retinal development. In this study, the effects of these transmitters on the turnover of phosphoinositides and proliferation of developing retinal cells in culture were characterized. While ATP, UTP or carbachol were able to induce a >400% accumulation of phosphoinositides in retinal cell cultures, only ATP promoted a dose-dependent increase in [(3)H]-thymidine incorporation in cultured cells (EC(50)=8.6 microM), a response that was inhibited by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.1 or 0.25 mM). ADP, but not UTP or adenosine, also stimulated the proliferation of retinal cells (EC(50)=5.8 microM), indicating that activation of P2Y1 receptors mediates the proliferative response of retinal cells to ATP. The mitogenic effect of ATP was completely prevented by the PKC inhibitor chelerythrine chloride (0.5 microM) and the phospholipase C (PLC) inhibitor U73122 (0.5 microM). PD 98059 (25 or 50 microM), an inhibitor of the activation of extracellular signal-regulated kinases (ERKs) also blocked the increase in [(3)H]-thymidine incorporation induced by ATP. Moreover, the effect of ATP was pronounced in cultures obtained from retinas at embryonic days 6-8, but not at day 9. Since Müller and bipolar cells are the predominant cell types that proliferate at these embryonic stages, our data suggest that ATP, through activation of P2Y1 receptors coupled to phospholipase C, PKC and MAP kinases, affects DNA synthesis in one or both of these cell types in culture.     

ATP-induced proliferation of developing retinal cells: regulation by factors released from postmitotic cells in culture

ATP is an important mitogen in the developing retina and its proliferative response decreases as chick retinal cells differentiate in culture. Both non-stimulated or ATP-induced proliferative response was abolished if cycling cells were cocultured with cells from older embryos or cultured with conditioned medium (CM) from postmitotic cells. The effect of CM was dose-dependent and reversible, as removal of CM from the cultures restored both basal and ATP-induced incorporation of [3H]-thymidine. The effect of CM was also dependent on the developmental stage of the retina used to prepare the medium. As tissues from older embryos were used, inhibition of the basal and ATP-induced proliferative response of the cells increased. Similar inhibition of ATP-induced increase in [3H]-thymidine incorporation was observed using CM from purified glial cultures. Neither ARL 67156, an ecto-ATPase inhibitor, prevented nor TGF-beta1 and TGF-beta2 mimicked the inhibitory effect of conditioned medium. Incubation of cells with CM or ATP for 24 h completely abolished the formation of [3H]-phosphoinositides induced by ATP. These effects were blocked by the P2 receptor antagonist PPADS and were not observed with dialysed CM, suggesting that agonist-dependent desensitization of P2 receptors occurred in cultures incubated with CM. However, removal of small molecules such as nucleotides by dialysis did not affect the decline in the proliferative activity induced by CM, suggesting that desensitization is not responsible for the conditioned medium-dependent cell cycle arrest of early developing retinal cells in culture. These results suggest that factors released from postmitotic cells induce the arrest of retinal cells in the mitotic state, a phenomenon that is concomitant with agonist-dependent P2 receptor desensitization.     

Ca mobilization and capacitative Ca entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells

Release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) and capacitative Ca²⁺ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca²⁺ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca²⁺ pump of intracellular Ca²⁺ stores, and of SK&F 96365, an inhibitor of capacitative Ca²⁺ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [³H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [³H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC₅₀: 4 μM) as the blockade of capacitative Ca²⁺ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca²⁺ stores also inhibited [³H]-thymidine incorporation with IC₅₀ values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca²⁺ mobilization and capacitative Ca²⁺ entry in the regulation of DNA synthesis in the developing neural retina.     

Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l-arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [(3)H]-thymidine incorporation after drug treatment. Incubation for 24h with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [(3)H]-thymidine incorporation in a dose-dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8-bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S-nitrosylation mechanism. SNAP blocked the increase of [(3)H]-thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [(3)H]-Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [(3)H]-thymidine incorporation in retinas from 9-day-old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or "in vivo" through cyclic GMP and ERK-independent pathways.     

ATP controls cell cycle and induces proliferation in the mouse developing retina

Previous data suggest that nucleotides are important mitogens in the developing chick retina. Here, we extended the study on the mitogenic effect of ATP to newborn mouse retinal explants. Our results showed that P2Y₁ receptors were widely distributed in C57bl/6 mice retina and that the majority of PCNA positive cells co-localized with P2Y₁ receptor. To evaluate proliferation, retinal explants obtained from newborn mice were incubated with 0.5 μCi [³H]-thymidine or 3 μM BrDU 1 h before the end of culture. Our data showed that ATP induced a dose-dependent increase in [³H]-thymidine incorporation, an effect that was mimicked by ADP but not by UTP and was blocked by the P2 antagonist PPADS in a dose-dependent manner. The increase in [³H]-thymidine incorporation induced by ATP was only observed in explants cultured for 3 days or less and was mimicked by the ectoapyrase inhibitor ARL 67156. It corresponded to an increase in the number of BrdU⁺ cells in the neuroblastic layer (NL) of the tissue, suggesting that ATP, through activation of P2Y₁ receptors, induced proliferation of late developing progenitors in retinal explants of newborn mice. The increase in the number of BrdU⁺ cells was observed across the whole NL when explants were incubated with ATP for 24 h and no increase in the number of p-histone H3 labeled cells could be noticed at this time point. In longer incubations of 48 h with ATP or 24 h with ATP followed by a period of 24 h in fresh medium, an increase in the number of BrdU⁺ cells promoted by ATP was observed only in the middle and outer, but not in the inner NL. In these conditions, an increase in the number of p-histone H3 labeled cells was detected in the outer NL, suggesting that ATP induced cells to enter S and progress to G2 phase of the cell cycle in the first 24 h period of incubation. ATP also induced an increase and a decrease in the expression of cyclin D1and p27^kip1, respectively, in retinal progenitors of the NL. While the increase in the expression of cyclin D1 was observed when retinal explants were incubated for 3 h or longer periods of time, the decrease in the expression of p27^kip1 was noticed only after 6 h incubation with ATP. Both effects were blocked by the P2 receptor antagonist PPADS. These data suggest that ATP induces cell proliferation in retinal explants by inducing late developing progenitors to progress from G1 to S phase of cell cycle.     

Signal transduction pathways associated with ATP-induced proliferation of cell progenitors in the intact embryonic retina

ATP and ADP induce retinal cell proliferation through activation of PKC and extracellular signal-regulated kinases (ERKs). Here, we characterized the effect of purinergic agonists on the turnover of phosphoinositides and activation of ERKs during development of the chick embryo retina. When intact retinas were incubated with ATP, ADP or UTP, a dose-dependent accumulation of [³H]-phosphoinositides was observed (% of control, EC₅₀: 548 ± 20.5%, 0.18 mM; 314 ± 53.8%, 0.51 mM; 704 ± 139.9%, 0.018 mM, respectively). Only the response promoted by ADP was completely inhibited by the P2 receptor antagonists, PPADS and suramin. All the responses decreased with the progression of retinal development. Western blot assays revealed that ATP, ADP and UTP stimulated the phosphorylation of ERKs in the chick embryo retina very early during development (% of control: 174 ± 16; 199 ± 16.4 and 206 ± 37, respectively). The responses to ADP and UTP were transient and dose-dependent, showing EC₅₀ values of 0.12 mM and 0.009 mM. The response to ADP was inhibited by the antagonists PPADS and suramin and by U73122 and chelerythrine chloride, which block PLC and PKC, respectively. Conversely, chelerythrine chloride did not block the response induced by UTP. Immunohistochemical analysis revealed that ATP and ADP induced the phosphorylation of ERKs in cells of the neuroblastic layer of retinas from embryos at E8. Our data showed that ATP, ADP and UTP stimulate the turnover of InsPs and promoted the activation of ERKs in the chick embryo retina. ADP, through activation of P2Y₁ receptors, activated ERK pathway through PLC and PKC and UTP, via P2Y₄-like receptors, induced the phosphorylation of ERKs through a pathway that did not involve PKC.     

Involvement of the PI3K/AKT pathway in ATP-induced proliferation of developing retinal cells in culture

ATP induces the proliferation of chick retinal cells in culture through the activation of P2Y1 receptors, PKC and MAP kinases. Together with MAP kinases, the PI3K/AKT pathway has also been implicated as an important mediator in proliferative events during development. Here we investigated the participation of the PI3K/AKT signal pathway on ATP-induced proliferation of chick embryo retinal cells in culture. When retinal cultures obtained from 7-day-old embryos were cultivated for 1 day and treated with ATP, a transient and dose-dependent phosphorylation of both ERK and AKT was observed, an effect that could be mimicked by 500 μM ADP and blocked by 100 μM PPADS, a P2 receptor antagonist. Maximal stimulation of both enzymes was obtained with 100 μM ATP in 5 min, decreasing thereafter. Activation of these pathways by ATP seemed to be independent, since LY294002 and U0126, inhibitors of PI3K and MEK, did not block the activation of ERK and AKT, respectively, although each compound blocked its respective target. Moreover, when the cultures were incubated with ATP in the presence of LY294002, a decreased incorporation of [³H]-thymidine was observed, as compared to cultures treated only with ATP, a decline that was also obtained by incubating the cells with ATP plus 0.5 μM API-59CJ-Ome, an inhibitor of AKT. No decrease in cell viability was observed with this concentration of API-59CJ-Ome. An increase in cyclin D1 expression, that could be inhibited by 10 μM LY 294002 or 20 μM U0126, was observed when cells were incubated with 500 μM ADP. No effect of PI3K and MEK inhibitors was observed in the expression of p27kip1 in the cultures. These results suggest that, besides the involvement of the MAP kinases pathway, ATP-induced cell cycling of late developing retinal progenitors in culture also involves the activation of the PI3K/AKT pathway.     

Involvement of P2 purinoceptors and the nitric oxide pathway in [3H]purine outflow evoked by short-term hypoxia and hypoglycemia in rat hippocampal slices

The objective of this study was to study how the outflow of [3H]purines is altered during a brief period of ischemic-like conditions in superfused hippocampal slices and to show whether it is regulated by P2 purinoceptors and the nitric oxide (NO) pathway. The outflow of [3H]purines increased in response to 5 min of combined hypoxia/hypoglycemia. High performance liquid chromatography analysis verified the efflux of [3H]adenosine-triphosphate, [3H]adenosine-diphosphate, [3H]adenosine-monophosphate, [3H]adenosine, [3H]inosine, and [3H]hypoxanthine in response to ischemic-like conditions. The P2 receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic-acid-tetrasodium (PPADS) reduced significantly the [3H]purine efflux evoked by ischemic-like conditions, showing that P2 purinoceptors are involved in the initiation of purine outflow. The NO synthase inhibitor N-nitro-l-arginine-methyl-ester (l-NAME) attenuated significantly the [3H]purine outflow, evoked by ischemic-like conditions, while 7-nitroindazole (7-NI) caused only a mild decrease in the outflow. The NO donor sodium nitroprusside increased significantly the basal efflux of [3H]purines. In summary, a brief period of combined hypoxia/hypoglycemia induced the efflux of ATP in addition to the outflow of other purines. Since P2 receptor antagonists decreased the [3H]purine outflow evoked by ischemic-like conditions we propose that ATP, acting on P2 purinoceptors, is responsible for further efflux of purines after ischemic-like period. It seems likely that NO is also involved in the regulation of purine outflow, since inhibition of NO production attenuated the [3H]purine outflow, evoked by ischemic-like conditions, while exogenous NO facilitated the basal outflow.     

Purinergic nerves mediate the non-nitrergic relaxation of the human ileum in response to electrical field stimulation

There has been no direct functional evidence for a purinergic innervation of the human intestinal muscle. In the present study, the relaxant effects of electrical field stimulation (1 or 10 Hz for 20s), ATP, and isoprenaline were studied in organ bath experiments on precontracted circular muscle strips of the human ileum. Non-adrenergic, non-cholinergic relaxations in response to electrical field stimulation in the presence of a nitric oxide synthase inhibitor were significantly reduced by the P(2) purinoceptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 50 microM) or suramin (100 microM). A combination of the two antagonists yielded an approximately 70% inhibition at 1 Hz. The relaxant effect of exogenous ATP, but not that of isoprenaline, was inhibited by PPADS+suramin. It is concluded that purinergic nerves (through P(2) purinoceptors) play a mediating role in the non-nitrergic relaxation in the human ileum.     

Developmental regulation of adenosine A1 receptors, uptake sites and endogenous adenosine in the chick retina.

Although adenosine A1 receptors mediate the inhibition of dopamine-dependent stimulation of adenylate cyclase activity in the developing chick retina, their localization and function are unknown. We have examined the localization of these receptors, and of endogenous adenosine and adenosine uptake sites at several stages of chick retinal development. A1 receptors were already localized predominantly to plexiform regions by embryonic day 12 (E12) with no gross changes at subsequent stages. Adenosine immunoreactivity was absent from retina at E8 but was detected at E12 in the ganglion cell layer, as well as cells in the inner nuclear cell layer and photoreceptors. At more advanced developmental stages the immunoreactivity was greater, but displayed similar localizations. Uptake sites labeled with [3H]nitrobenzylthioinosine (NBI) were detected even earlier using binding and autoradiographic methods. [3H]NBI binding was saturable, and Scatchard analysis demonstrated a single class of sites with a Kd of 0.91 nM and Bmax of 298 fmol/mg protein in E15 retinal membranes. The binding was displaced by unlabeled NBI and dipyridamole. NBI binding sites differentiated earlier than adenosine A1 receptors or endogenous adenosine immunoreactivity, showing a diffuse distribution at E8, but predominating in the plexiform layers of more developed retinas. The results indicate that elements of a putative purinergic system differentiate at specific localizations early in retinal development.     

Involvement of P2 purinoceptors and the nitric oxide pathway in [H]purine outflow evoked by short-term hypoxia and hypoglycemia in rat hippocampal slices

The objective of this study was to study how the outflow of [³H]purines is altered during a brief period of ischemic-like conditions in superfused hippocampal slices and to show whether it is regulated by P₂ purinoceptors and the nitric oxide (NO) pathway. The outflow of [³H]purines increased in response to 5 min of combined hypoxia/hypoglycemia. High performance liquid chromatography analysis verified the efflux of [³H]adenosine-triphosphate, [³H]adenosine-diphosphate, [³H]adenosine-monophosphate, [³H]adenosine, [³H]inosine, and [³H]hypoxanthine in response to ischemic-like conditions. The P₂ receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic-acid-tetrasodium (PPADS) reduced significantly the [³H]purine efflux evoked by ischemic-like conditions, showing that P₂ purinoceptors are involved in the initiation of purine outflow. The NO synthase inhibitor N-nitro- -arginine-methyl-ester ( -NAME) attenuated significantly the [³H]purine outflow, evoked by ischemic-like conditions, while 7-nitroindazole (7-NI) caused only a mild decrease in the outflow. The NO donor sodium nitroprusside increased significantly the basal efflux of [³H]purines. In summary, a brief period of combined hypoxia/hypoglycemia induced the efflux of ATP in addition to the outflow of other purines. Since P₂ receptor antagonists decreased the [³H]purine outflow evoked by ischemic-like conditions we propose that ATP, acting on P₂ purinoceptors, is responsible for further efflux of purines after ischemic-like period. It seems likely that NO is also involved in the regulation of purine outflow, since inhibition of NO production attenuated the [³H]purine outflow, evoked by ischemic-like conditions, while exogenous NO facilitated the basal outflow.     

Apoptotic cell death of proliferating neuroepithelial cells in the embryonic retina is prevented by insulin

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT-mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor-deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl-3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S-phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl-3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.     

Extracellular ATP induces retinal photoreceptor apoptosis through activation of purinoceptors in rodents

We have previously demonstrated that photoreceptors express P2X₇ purinoceptors. These excitatory receptors are activated by extracellular adenosine 5′‐triphosphate (ATP) and have been implicated in neurodegeneration in other parts of the central nervous system (CNS). In this study we examined whether extracellular ATP could contribute to photoreceptor degeneration in rodents through excessive activation of P2 purinoceptors. Intravitreal injection of high concentrations of extracellular ATP into normal rat eyes induced extensive and selective apoptosis of photoreceptors within 18 hours of injection. Five days after injection the outer nuclear layer was severely degenerated and electroretinographic responses were impaired. Preinjection of the purinergic antagonist pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) protected against ATP‐mediated apoptosis. The initial phase of ATP‐induced photoreceptor death did not temporally coincide with retinal pigment epithelium degeneration or microglial activation, suggesting that cell death was due to direct activation of purinergic receptors on photoreceptors. Finally, we demonstrate that intravitreal injection of PPADS results in a 30% increase in photoreceptor survival in the rd1 mouse, a model of human recessive retinitis pigmentosa (RP). These findings highlight the importance of extracellular ATP in retinal neurodegeneration and provide a potential new avenue for therapeutic intervention in RP. J. Comp. Neurol. 513:430–440, 2009. © 2009 Wiley‐Liss, Inc.     

Neurogenesis of GABAergic cells in the retina of malnourished rats

The present study investigated how prenatal protein malnutrition affects the neurogenesis of GABAergic cells in the retina. Rats were treated with a multi-deficient diet, with only 8% of protein that was administered during the gestational and suckling periods. Pregnant mothers and pups from malnourished and control (fed with 22% protein) groups received a single intra-peritoneal injection of [3H]-thymidine at six developmental ages, from E14 to PN4, and the pups were sacrificed at PN18. Eyes were enucleated and cryosections of the retina were double labeled for GABA-immunocytochemistry and for autoradiography. The percentage of double labeled cells, in the retinal inner nuclear and ganglion cell layers, was determined for both groups. Qualitative and quantitative results showed that double labeled cells [GABA+/thymidine+] were present since E14, when mitotic activity for GABAergic cells starts, in both GCL and INL layers. The peak rate of GABAergic cell generation was reached in control animals injected with [3H]-thymidine at E18 in both central and peripheral sectors of the retina, but only at E20 in the malnourished group. The generation of cells of GABA phenotype showed a significant delay in both layers of the retina in the malnourished group. At PN4, close to the age that GABAergic mitotic activity ends in the control group, double labeled cells were significantly higher in the malnourished group. Our data showed a delay in GABAergic cell generation in the malnourished group when compared to the control group that might result in significant functional consequences in the developing retina.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

Opposite influence of the metabotropic glutamate receptor subtypes mGlu3 and -5 on astrocyte proliferation in culture

In non-synchronized, subconfluent secondary cultures of rat cortical astrocytes, the selective group-I metabotropic glutamate (mGlu) receptor agonist 3,5-dihydroxyphenylglycine (DHPG) increased [methyl-³H]-thymidine incorporation. This effect was mediated by the activation of the mGlu5 receptor, which was shown to be present by either RT-PCR or Western blot analysis. The mixed mGlu receptor antagonist (+)-α-methyl-4-carboxyphenylglycine reduced the increase in both intracellular Ca²⁺ and [methyl-³H]-thymidine incorporation produced by DHPG. In contrast, (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycylopropyl)glycine (DCG-IV), a potent and selective agonist of group-II mGlu receptors, reduced [methyl-³H]-thymidine incorporation in non-synchronized astrocyte cultures. The antiproliferative effect of DCG-IV was prevented by the selective group-II mGlu receptor antagonist (2S,1′S,2′S,3′R)-2-(2′-carboxy-3′-phenylcyclopropyl)glycine (PCCG-IV). The opposite effect of DHPG and DCG-IV on astrocyte proliferation was confirmed in cultures deprived of serum for 48 hours and then stimulated to proliferate with either epidermal growth factor (EGF) or the metabolically stable ATP analogue adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP). We conclude that activation of mGlu5 receptors enhances proliferation in cultured astrocytes, whereas activation of a receptor with pharmacological characteristics similar to those of mGlu2/3 receptors reduces proliferation. GLIA 21:390–398, 1997. © 1997 Wiley-Liss, Inc.     

Properties of dopamine agonist and antagonist binding sites in mammalian retina

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.     

Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain

The mechanism of control of GAD expression by GABA and excitatory amino acids (EAAs) was studied in chick and rat retina cultures using immunohistochemical and PAGE-immunoblot detection of the enzyme, as well as by measuring enzyme activity. Aggregate cultures were prepared with retina cells obtained from chick embryos at embryonic days 8-9 (E8-E9). Organotypical cultures were also prepared with retinas from E14 chick embryos, post-hatched chicken and P21 rats. GABA (1-20 mM) fully prevented GAD expression in aggregate and organotypical cultures from chick embryo retinas. A substantial, but not complete, reduction of GAD was also observed in organotypical cultures of post-hatched chicken and P21 rats, in which both forms of the enzyme (GAD65 and 67) were affected. The GABA effect was not mimicked by THIP (100 microM), baclofen (100 microM) or CACA (300 microM), agonists of GABAa, b and c receptors, respectively. NNC-711, a potent inhibitor of GABA transporters, reduced by 50% the inhibition of GAD activity promoted by GABA. Aggregates exposed to GABA and treated with glutamate (5 mM) or kainate (100 microM) displayed an intense GAD-like immunoreactivity in many cell bodies, but not in neurite regions. Immunoblot analysis revealed that the increase in GAD-like immunoreactivity by EAA corresponded to a 67-kDa protein. However, GAD activity was not detected. Treatment of aggregates or retina homogenates with SNAP, a NO producing agent (but not its oxidized form), reduced GAD activity by more than 60% indicating that the lack of enzyme activity in GAD-like immunoreactive cells, could be due to NO production by EAA stimulation.     

Identification of D1-dopamine receptor in chicken embryo retina with [125I]SCH 23982

The D1-dopamine receptor in chicken embryo retina was identified with the D1-dopamine receptor specific ligand, [125I]SCH 23982. Binding of [125I]SCH 23982 to both pre-hatched and post-hatched chicken retina was rapid, saturable and of high affinity. The dissociation constant and maximal binding capacity were 795 +/- 25 pM (mean +/- S.E.M., n = 3) and 32.2 +/- 3.8 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 13-day-old chicken embryo retina, and 785 +/- 58 pM (mean +/- S.E.M., n = 3) and 96.9 +/- 4.1 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 1-day-old post-hatched chicken retina. The binding properties of the D1-dopamine receptor in chicken retina were similar to those in rat striatum. The maximal binding capacity of the D1-dopamine receptor for [125I]SCH 23982 was increased concomitant with embryonic development, but without any changes in either affinity or pharmacological properties. Dopamine-stimulated adenylate cyclase activity in the retinal homogenates increased concomitant with embryonic development, diminished in the presence of 1 microM SCH 23390 (a D1-dopaminergic antagonist) but remained unaffected by 1 microM YM-09151-2 (a D2-dopaminergic antagonist).