RESUMO
Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPßS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.
Assuntos
Receptores Purinérgicos P2Y1/metabolismo , Retina/metabolismo , Fase S/fisiologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Apirase/farmacologia , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Relógios Circadianos/fisiologia , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Hexoquinase/farmacologia , Fotoperíodo , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2Y1/genética , Retina/citologia , Retina/enzimologia , Fase S/efeitos dos fármacos , Transdução de Sinais , Tionucleotídeos/farmacologia , Peixe-ZebraRESUMO
Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.
Assuntos
Difosfato de Adenosina/metabolismo , Morte Celular/fisiologia , Retina/citologia , Retina/metabolismo , Adenosina/metabolismo , Adenosina/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Fatores Etários , Animais , Antimetabólitos/toxicidade , Bromodesoxiuridina/toxicidade , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Hidrólise , Ouabaína/farmacologia , Comunicação Parácrina/fisiologia , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Peixe-ZebraRESUMO
Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of membrane-bound enzymes that hydrolyze extracellular di- and triphosphate nucleosides. E-NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E-NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Müller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Müller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto-ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated-signaling in vertebrate retinas.
Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Neurônios/enzimologia , Retina/citologia , Animais , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
In this study, we show that one single dose of gamma-irradiation at birth induces an inhibition of the cerebellar calcium dependent nitric oxide synthase (NOS) activity, probably correlated to the motor abnormalities and the disarrangement in the cerebellar cytoarchitecture observed in adult rats. This decrease in calcium dependent NOS activity could be associated with an increased protein kinase C (PKC) activity. PKC inhibition partially restores calcium dependent NOS activity, indicating that PKC activity could be negatively modulating the catalytic activity of calcium dependent NOS. These findings suggest that a decrease in nitric oxide (NO) production and the related increase in PKC activity could be intracellular events that participate in the onset of motor and cerebellar abnormalities induced by postnatal gamma-irradiation at early stages of life.