Elizabeth Giestal de Araujo

Trophic Factors Produced by Retinal Cells Increase the Survival of Retinal Ganglion Cells In Vitro

The naturally occurring neuron death of normal development has been shown to depend on trophic factors produced and released by target cells. It has also been shown that the afferent supply and local interactions play a role in the control of this degenerative phenomenon. We studied the effect of trophic factors produced by intrinsic retinal cells on the survival of retinal ganglion cells in vitro. Retinae of newborn hooded rats were retrogradely labelled with horseradish peroxidase injected into the superior colliculus to permit the identification of retinal ganglion cells in culture. We tested the effect of conditioned media either from aggregates or from explants of retinal cells from neonatal rats on the survival of ganglion cells in vitro. Our results showed that both conditioned media increased the survival of these cells. The trophic activity was dose‐dependent, was maintained after dialysis against a 12 kDa membrane, was abolished by heating at 56°C for 30 min, and was not found in conditioned medium from cerebral cortical explants. Conditioned medium obtained without fetal calf serum presented the same trophic effect. These results suggest that the local control of developmental neuron death by intrinsic retinal cells may be mediated by neurotrophic factors.

Interleukin-6 increases the survival of retinal ganglion cells in vitro.

Abstract Interleukin-6 is a pleiotropic cytokine that mediates cellular communication both in physiological and pathological states. In this work, we demonstrate that 50 ng/mL IL-6 increases the survival of retinal ganglion cells (RGCs) after 48 h in culture. This effect was blocked by an intracellular Ca +2 chelator, by inhibition of ryanodinic receptors and by an inhibitor of L-type Ca +2 channels. IL-6 effect is mediated by PKC, tyrosine kinase, PI3-kinase and MEK activity. The blockade of polypeptide release also abolished the effect of IL-6. These results suggest a role for this cytokine during the development of the central nervous system (CNS).

Neuronal Cell Survival : The Role of Interleukins

One of the central issues in neuroscience today is the study of the mechanisms of neuronal survival. Since the discovery of nerve growth factor (almost 60 years ago), many groups have clearly demonstrated the central role of neurotrophins on the regulation of neuronal cell survival during developmental stages as well as during adult life. However, neurotrophins are not alone in regulating neuronal survival, and many groups have demonstrated the effect of different cytokines on this phenomenon. In this brief review we will address the effect of interleukins (IL), particularly IL‐2, IL‐6, and IL‐4, on the survival of neuronal cells.

Electrophysiology of phagocytic membranes: Induction of slow membrane hyperpolarizations in macrophages and macrophage polykaryons by intracellular calcium injection

SummarySome electrophysiological characteristics of macrophages and macrophage polykaryons of foreign body granuloma have been investigated. Cells were obtained from implants of small coverslips in the subcutaneous tissue or in the peritoneal cavity of rats and mice. Transmembrane potentials ranged from −5 to −40 mV. Input resistances ranged from 5 to 120 MΩ, being significantly higher in mice polykaryons. Approximately 10% of the cells exhibited spontaneous slow membrane hyperpolarizations (SH) indistinguishable from those observed in macrophages. SH responses were invariably evoked by iontophoretic injection of calcium ions into the cytoplasm of mice macrophages or macrophage polykaryons. The amplitude of these responses increased with the amount of current carried by calcium ions into the cells. The maximum amplitude of the calcium-induced SH responses is a linear function of the logarithm of [K+]0 (from 3 to 40mm). The slope of the regression line is 43 mV for a 10-fold increase in [K+]0. Substituting sodium chloride by sodium isethionate or by choline chloride does not interfere with the occurrence of SH. The assumption that the SH is solely a consequence of an increase in the membrane conductance to K+ was used to calculate the potassium equilibrium potential (EK). TheEK value is also a linear function of the logarithm of [K+]0 (from 3 to 40mm). The slope of the regression line is 46 mV for a 10-fold increase in [K+]0. These results constitute evidence of the calcium dependence of K+ permeability during SH both in macrophages and macrophage polykaryons. Macrophage polykaryons are a more convenient model than macrophages for the study of the mechanisms underlying the SH responses and their possible physiological implications.

Interleukin-2 and interleukin-4 increase the survival of retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon observed during the normal development of the nervous system. The neuroprotective effects of cytokines produced by neuronal, glial or infiltrating cells on neurons have been extensively studied. In this work we studied the role of interleukin (IL)-2 and IL-4 on the survival of retinal ganglion cells (RGC) after 48 h in culture. Our results demonstrate that the effect of both ILs was dose-dependent and the treatment with either IL-2 (50 U/ml) or IL-4 (5 U/ml) induced a 2-fold increase in RGC survival. The effect of IL-4, but not of IL-2, was totally abolished by either 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or by 1 μM telenzepine, an inhibitor of M1 muscarinic receptor. Our results suggest that both cytokines could play an important role during the development of retinal tissue as well as during retina trauma.

Neuronal Cell Survival

One of the central issues in neuroscience today is the study of the mechanisms of neuronal survival. Since the discovery of nerve growth factor (almost 60 years ago), many groups have clearly demonstrated the central role of neurotrophins on the regulation of neuronal cell survival during developmental stages as well as during adult life. However, neurotrophins are not alone in regulating neuronal survival, and many groups have demonstrated the effect of different cytokines on this phenomenon. In this brief review we will address the effect of interleukins (IL), particularly IL‐2, IL‐6, and IL‐4, on the survival of neuronal cells.

The effect of PKC activation on the survival of rat retinal ganglion cells in culture.

Abstract Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept “in vitro” for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 30 μM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or 25 μM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells “in vitro” may be mediated by a mechanism that involves PKC activation.

Cholinergic activity modulates the survival of retinal ganglion cells in culture: the role of M1 muscarinic receptors.

The control of natural cell death is mediated by neurotrophins released by target, afferent and glial cells. In the present work we show that treatment of retinal cells ‘in vitro’ for 48 h with 25 μM carbamylcholine induced a two‐fold increase in retinal ganglion cells survival. This effect was dose‐dependent and mediated by M1 receptors since it could be blocked by 1 μM telenzepine (a M1 receptor antagonist) and mimicked by 200 μM oxotremorine (a M1 receptor agonist). The effect of carbamylcholine was abolished by 10 μM BAPTA‐AM (an intracellular Ca2+ chelator), 30 μM dantrolene (an inhibitor of ryanodinic receptors), 500 nM H‐89 (an inhibitor of PKA), 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 50 μM PD‐98059 (a MEK inhibitor). Treatment with 10 μM genistein (an inhibitor of tyrosine kinase), 25 μM LY‐294002 (a PI‐3 kinase blocker), 30 nM brefeldin‐A (a blocker of polypeptides release), 50 nM K‐252a (a Trk receptor inhibitor) and 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) totally inhibited the effect of carbamylcholine. Taken together our results indicate that muscarinic activity controls the survival of retinal ganglion cells through a mechanism involving the release of polypeptides and activation of Irk receptors.

Ouabain induces an increase of retinal ganglion cell survival in vitro: the involvement of protein kinase C.

Abstract Ouabain is a steroid derivative that can regulate many cellular events such as growth and proliferation. It modulates Na + ,K + -ATPase activity leading to the activation of different intracellular pathways through protein–protein interactions that have been characterized during the last few years. The aim of this work was to study the role of ouabain in rat retinal ganglion cell survival after 48 h in culture. Our results demonstrated that ouabain significantly induced an increase in retinal ganglion cell survival. The effect was dose-dependent and was maximal with 3.0 nM. The blockade of protein kinase C activity by 1.25 μM chelerythrine chloride abolished the ouabain effect, indicating an involvement of this intracellular pathway. None of the protein kinase inhibitors usually employed in the study of ouabain-driven intracellular pathways (PD98059, Ly294002, herbimycin, and genistein) was able to influence neuronal survival induced by ouabain. The data presented suggest that ouabain may be the trigger of an intracellular pathway responsible for neuronal survival.

Ca2+ Mobilization Induced by Ouabain in Thymocytes Involves Intracellular and Extracellular Ca2+ Pools

Abstract— Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na+/K+‐ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na+/Ca2+ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb‐c mouse thymocytes loaded with FURA‐2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na+/Ca2+ exchanger and on voltage‐dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na+/K+ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na+/K+‐ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.