[Show abstract][Hide abstract] ABSTRACT: Diabetes induces changes in neurotransmitter release in central nervous system, which depend on the type of neurotransmitter and region studied. In this study, we evaluated the effect of diabetes (two and eight weeks duration) on basal and evoked release of [(14)C]glutamate and [(3)H]GABA in hippocampal and retinal synaptosomes. We also analyzed the effect of diabetes on the protein content of vesicular glutamate and GABA transporters, VGluT-1, VGluT-2 and VGAT, and on the α(1A) subunit of P/Q type calcium channels, which are abundant in nerve terminals. The protein content of vesicular glutamate and GABA transporters, and of the α(1A) subunit, was differently affected by diabetes in hippocampal and retinal synaptosomes. The changes were more pronounced in the retina than in hippocampus. VGluT-1 and VGluT-2 content was not affected in hippocampus. Moreover, changes occurred early, at two weeks of diabetes, but after eight weeks almost no changes were detected, with the exception of VGAT in the retina. Regarding neurotransmitter release, no major changes were detected. After two weeks of diabetes, neurotransmitter release was similar to controls. After eight weeks of diabetes, the basal release of glutamate slightly increased in hippocampus and the evoked GABA release decreased in retina. In conclusion, diabetes induces early transient changes in the content of glutamate and/or GABA vesicular transporters, and on calcium channels subunit, in retinal or hippocampal synaptosomes, but only minor changes in the release of glutamate or GABA. These results point to the importance of diabetes-induced changes in neural tissues at the presynaptic level, which may underlie alterations in synaptic transmission, particularly if they become permanent during the later stages of the disease.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE/AIM OF THE STUDY: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults in developed countries. Changes in metabolites and in metabolic pathways of the retina caused by hyperglycemia may compromise the physiology of the retina. Using nuclear magnetic resonance (NMR) spectroscopy, we aimed to investigate the effect of diabetes on the levels of intermediate metabolites in rat retinas and the metabolic pathways that could be affected.
Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/Kg, i.p.). Metabolic alterations were analyzed in streptozotocin-induced diabetic rat retinas by (1)H NMR spectroscopy. Glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose. Lactate production was evaluated by (1)H NMR spectroscopy using [U-(13)C]glucose.
Tissue levels of several metabolic intermediates were quantified, but no significant changes in the levels of most metabolites were detected, with the exceptions of glucose, significantly increased, and lactate, significantly reduced in diabetic rat retinas, as compared to age-matched controls. The cytosolic redox ratio, indirectly evaluated by lactate-to-pyruvate ratio, was significantly reduced in diabetic rat retinas, as well as glucose uptake. Parallel studies demonstrated that lactate production rates were significantly diminished, suggesting a reduction in the glycolytic flux.
These results suggest that diabetes may significantly decrease glycolysis in the retina since higher intracellular glucose levels do not translate into higher intracellular lactate levels or into higher rates of lactate production. These changes may alter the normal functioning of the retina during diabetes and may contribute for vision loss in DR.
No preview · Article · Nov 2010 · Current eye research
[Show abstract][Hide abstract] ABSTRACT: Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y(1) receptor agonist and selective antagonists for Y(1), Y(2), and Y(5) receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y(1) receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y(1) receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y(1) receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.
No preview · Article · Oct 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated the effects of biapigenin, a biflavone present in the extracts of Hypericum perforatum, in rat brain mitochondrial bioenergetics and calcium homeostasis. We found that biapigenin significantly decreased adenosine diphosphate (ADP)-induced membrane depolarization and increased repolarization (by 68 and 37%, respectively). These effects were blocked by atractyloside and bongkrekic acid, but not oligomycin. In the presence of biapigenin, an ADP-stimulated state 3 respiration was still noticeable, which did not happen in the presence of adenine nucleotide translocator (ANT) inhibitors. Taking in consideration the relevance of the ANT in the modulation of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium homeostasis was evaluated alone or in the presence of biapigenin. We found that biapigenin reduces mitochondrial calcium retention by increasing calcium efflux, an effect that was blocked by ADP plus oligomycin, an efficient blocker of the mPTP in brain mitochondria. Taken together, the results in this article suggest that biapigenin modulates mPTP opening, possibly by modulating ANT function, contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.
Full-text · Article · Aug 2009 · Neurotoxicity Research
[Show abstract][Hide abstract] ABSTRACT: Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.
No preview · Article · May 2009 · Journal of Neuroscience Research
[Show abstract][Hide abstract] ABSTRACT: Diabetic retinopathy (DR) is a leading cause of vision loss and blindness among adults between the age 20 to 74. Changes in ionotropic glutamate receptor subunit composition can affect retinal glutamatergic neurotransmission and, therefore, contribute to visual impairment. The purpose of this study was to investigate whether diabetes leads to changes in ionotropic glutamate receptor subunit expression at the protein and mRNA level in the rat retina.
Changes in the expression of ionotropic glutamate receptor subunits were investigated at the mRNA and protein levels in retinas of streptozotocin (STZ)-induced diabetic and age-matched control rats. Animals were euthanized one, four and 12 weeks after the onset of diabetes. Retinal protein extracts were prepared, and the receptor subunit levels were assessed by western blotting. Transcript levels were assessed by real-time quantitative PCR.
Transcript levels of most ionotropic glutamate receptor subunits were not significantly changed in the retinas of diabetic rats, as compared to age-matched controls but protein levels of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate, and N-methyl-D-aspartic acid receptors (NMDA) receptors were found to be altered.
The results provide evidence that diabetes affects the retinal content of ionotropic glutamate receptor subunits at the protein level. The possible implications of these changes on retinal physiology and visual impairment in DR are discussed.
[Show abstract][Hide abstract] ABSTRACT: Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.
Full-text · Article · Apr 2007 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Altered glutamatergic neurotransmission and calcium homeostasis may contribute to retinal neural cell dysfunction and apoptosis in diabetic retinopathy (DR). The purpose of this study was to determine the effect of high glucose on the protein content of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptor subunits, particularly the GluR2 subunit, because it controls Ca2+ permeability of AMPA receptor-associated channels. The effect of high glucose on the concentration of cytosolic free calcium ([Ca2+]i) was also investigated.
The protein content of GluR1, GluR2, GluR6/7, and KA2 subunits was assessed by Western blot. Cobalt staining was used to identify cells containing calcium/cobalt-permeable AMPA receptors. The [Ca2+]i changes evoked by KCl or kainate were recorded by live-cell confocal microscopy in R28 cells and in primary cultures of rat retina, loaded with fluo-4.
In primary cultures, high glucose significantly decreased the protein content of GluR1 and GluR6/7 subunits and increased the protein content of GluR2 and KA2 subunits. High glucose decreased the number of cobalt-positive cells, suggesting a decrease in calcium permeability through AMPA receptor-associated channels. In high-glucose-treated cells, changes in [Ca2+]i were greater than in control cells, and the recovery to basal levels was delayed. However, in the absence of Na+, to prevent the activation of voltage-sensitive calcium channels, the [Ca2+]i changes evoked by kainate in the presence of cyclothiazide, which inhibits AMPA receptor desensitization, were significantly lower in high-glucose-treated cells than in control cultures, further indicating that AMPA receptors were less permeable to calcium. Mannitol, used as an osmotic control, did not cause significant changes compared with the control.
The results suggest that elevated glucose may alter glutamate neurotransmission and calcium homeostasis in the retina, which may have implications for the mechanisms of vision loss in DR.
[Show abstract][Hide abstract] ABSTRACT: Several evidences suggest that glutamate may be involved in retinal neurodegeneration in diabetic retinopathy (DR). For that reason, we investigated whether high glucose or diabetes affect the accumulation and the release of [(3)H]-D-aspartate, which was used as a marker of the glutamate transmitter pool. The accumulation of [(3)H]-D-aspartate did not change in cultured retinal neural cells treated with high glucose (30 mM) for 7 days. However, the release of [(3)H]-D-aspartate, evoked by 50 mM KCl, significantly increased in retinal cells exposed to high glucose. Mannitol, which was used as an osmotic control, did not cause any significant changes in both accumulation and release of [(3)H]-D-aspartate. In the retinas, 1 week after the onset of diabetes, both the accumulation and release of [(3)H]-D-aspartate were unchanged comparing to the retinas of age-matched controls. However, after 4 weeks of diabetes, the accumulation of [(3)H]-D-aspartate in diabetic retinas decreased and the release of [(3)H]-D-aspartate increased, compared to age-matched control retinas. These results suggest that high glucose and diabetes increase the evoked release of D-aspartate in the retina, which may be correlated with the hypothesis of glutamate-induced retinal neurodegeneration in DR.
Full-text · Article · May 2006 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Kainate-induced epilepsy has been shown to be associated with increased levels of neuropeptide Y (NPY) in the rat hippocampus. However, there is no information on how increased levels of this peptide might modulate excitation in kainate-induced epilepsy. In this work, we investigated the modulation of glutamate release by NPY receptors in hippocampal synaptosomes isolated from epileptic rats. In the acute phase of epilepsy, a transient decrease in the efficiency of NPY and selective NPY receptor agonists in inhibiting glutamate release was observed. Moreover, in the chronic epileptic hippocampus, a decrease in the efficiency of NPY and the Y(2) receptor agonist, NPY13-36, was also found. Simultaneously, we observed that the epileptic hippocampus expresses higher levels of NPY, which may account for an increased basal inhibition of glutamate release. Consistently, the blockade of Y(2) receptors increased KCl-evoked glutamate release, and there was an increase in Y(2) receptor mRNA levels 30 days after kainic acid injection, suggesting a basal effect of NPY through Y(2) receptors. Taken together, these results indicate that an increased function of the NPY modulatory system in the epileptic hippocampus may contribute to basal inhibition of glutamate release and control hyperexcitability.
Full-text · Article · May 2005 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: The functionality of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors in chick embryo retina cells during development in vitro was studied by using Co(2+) uptake, and these data were correlated with the expression of the AMPA receptor subunit GluR4. We found that at 5 h in vitro only a small number of cells took up Co(2+) upon stimulation with 100 microM kainate or other AMPA receptor agonists, in the presence of cyclothiazide (CTZ), to inhibit desensitisation. The number of cells sensitive to kainate increased from 5 h in vitro to 3 days in vitro (DIV), and remained relatively constant until 14 DIV. When the cells were stimulated with (2S,4S)-4-methylglutamic acid (30 microM), a specific kainate receptor agonist, after inhibiting desensitisation with concanavalin A, we did not observe an increase in the number of cells responding, as compared to the control. The expression of the AMPA receptor subunit GluR4 during development was detected by immunofluorescence mainly at the perinuclear region of the cells, and the number of positive cells increased from 5 h in vitro to 7 DIV, and remained relatively constant until 14 DIV. The results suggest that AMPA receptors can be functionally active at early embryonic stages (5 h in vitro) in cultured retinal neurons, although in only a few cells, before synapse formation (E12). The localisation of GluR4 was well correlated with Co(2+) entry, since the strongest GluR4 immunoreactivity was found in the regions that showed the most intense labelling with Co(2+). Finally, we found that the expression levels of GluR4 at the neurites increased between 5 h in vitro and 7 DIV, near the period of synapse formation.
Full-text · Article · Feb 2003 · Developmental Brain Research
[Show abstract][Hide abstract] ABSTRACT: The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the NMDA receptors was followed by monitoring the changes in the intracellular free calcium concentration ([Ca2+](i)), in response to NMDA or to L-glutamate. The expression of the subunits NMDAR1, NMDAR2A/B and NMDAR2C in the retinospheroids and in chick retinas were determined by Western blot analyses. The changes in [Ca2+](i) in response to 400 microM NMDA increased from 5 h in vitro to 3 days in vitro (DIV) and remained constant until 14 DIV, whereas the [Ca2+](i) response to 500 microM L-glutamate increased from 5 h in vitro to 3 DIV and decreased slightly until 14 DIV. In the retinospheroids, the expression of the NMDAR1 and NMDAR2A/B subunits increased from 5 h in vitro until 14 DIV, whereas the NMDAR2C subunit increased from 5 h in vitro until 10 DIV and remained constant until 14 DIV. In the retinas, the expression of NMDAR1 increased from embryonic day 8 (E8) until E15, decreased until E18, and increased again until day 22 (post-hatched 1, PH1). The NMDAR2A/B increased from E8 until E18 and decreased slightly until PH1, whereas the NMDAR2C subunit increased from E8 until E15, remained constant until E18, and increased again until PH1. The results suggest that NMDA receptors are expressed and functionally active at early embryonic stages in the retina and in retinospheroids, before synapse formation (E12). However, the calcium responses to NMDA were relatively constant from 3 DIV until 14 DIV, showing no correlation with the increase in the expression of the studied NMDA receptor subunit during the same period. Also, the patterns of NMDA receptor subunits expressed in chick embryo retina cells cultured in vitro and in retina cells developing in vivo were similar.
No preview · Article · Apr 2002 · Molecular Brain Research
[Show abstract][Hide abstract] ABSTRACT: The activity and the subunit expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the AMPA/kainate receptors was monitored by following the changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to AMPA, kainate or to L-glutamate, and the expression of the receptor subunits GluR1, GluR2/3, GluR4 and GluR6/7 was determined in the retinospheroids and in chick retinas by immunodetection using polyclonal antibodies. The changes in [Ca(2+)](i) in response to 400 microM kainate increased from 5h in vitro to 3 days, and remained constant until day 14, whereas the [Ca(2+)](i) in response to 500 microM L-glutamate or 400 microM AMPA increased from 5h in vitro to 3 days, and thereafter decreased slightly until day 14. The [Ca(2+)](i) responses to kainate are mainly due to AMPA receptor stimulation, since the signals were abolished by LY303070, the AMPA receptor antagonist, and were not affected by MK-801, the NMDA receptor antagonist. In retinospheroids, the levels of expression of GluR1 subunit increased from 5h in vitro until day 7, then decreased until day 14. The levels of expression of GluR2/3 and GluR4 subunits increased from 5h in vitro until day 10, and remained constant until day 14. The levels of kainate receptor subunits GluR6/7 increased from 5h in vitro until day 3, and thereafter decreased slightly until day 14. In the retinas, the expression of GluR1 and GluR6/7 subunits increased from day 8 until day 15, and then decreased until day 22 (post-natal 1). The subunits GluR2/3 and GluR4 increased from day 8 until day 18, and remained constant until day 22. The results suggest that AMPA/kainate receptors are expressed at early embryonic stages, although at low levels and before synapse formation (E12). However, the AMPA receptors are not completely functional at the first stage studied since they do not respond to the agonist AMPA. Also, the patterns of AMPA/kainate receptor subunit expression in retinospheroids of chick embryo retina cells cultured in vitro and in retina cells developing in the embryo (in vivo) were similar, indicating that the AMPA/kainate receptor subunits expression in these primary cultures mimics their expression in the developing chick retina.
Full-text · Article · Mar 2002 · International Journal of Developmental Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The L-type voltage sensitive calcium channels (VSCC) of chick embryo retinospheroids were characterized during the development in vitro. Functionally, the activity of VSCC was characterized by continuously monitoring the changes in the intracellular free Ca2+ concentration (delta[Ca2+]i) with indo-1, in response to 30 mM KCl. The contribution of the L-type VSCC was evaluated using the L-type VSCC antagonist, nitrendipine. We also characterized the binding of [3H]nitrendipine to retinospheroid membranes during development, and determined the Kd and Bmax values. We observed that the changes in [Ca2+]i in response to 30 mM KCl increased from 159.46 +/- 6.62 nM at 0 days in vitro (DIV) retinospheroids to 704.4 +/- 59.9 nM at 14 DIV retinospheroids. Nitrendipine (2 microM) blocked the delta[Ca2+]i response by approximately 67% in all ages tested. No significant difference in the Kd values for the nitrendipine binding was observed during in vitro development of the retinospheroids. However, the Bmax increased from 27.99 +/- 1.95 fmol/mg protein in 0 DIV retinospheroids to 131.09 +/- 14.24 fmol/mg protein in 14 DIV retinospheroids, supporting the delta[Ca2+]i results. The results presented suggest that the increase in [Ca2+]i during development was due to an increase in the number of L-type channels. Therefore, the expression of L-type VSCC is developmentally regulated during retinogenesis in vitro and accompanies neuronal maturation, probably regulating the Ca2+ input crucial to the onset of important intracellular Ca2+-dependent functions.
No preview · Article · Jan 1998 · Developmental Brain Research
[Show abstract][Hide abstract] ABSTRACT: The Ca2+ stores of digitonin permeabilized chick embryo retina cells in culture were characterized, by using the fluorescence of Fluo-3 potassium salt to follow continuously the free [Ca2+] in the medium. After ATP dependent Ca2+ accumulation, the Ca2+ release was induced by several agents; 10 microM cyclic-ADP-ribose (cADPR), 40 microM Ins (1,4,5)P3 10 microM thapsigargin (Th), 25 microM ionomycin (Ion), 15 microM CCCP together with 4.5 micrograms/ml oligomycin (CCCP/Olig), 50 microM arachidonic acid (AA). Neither Ins(1,4,5)P3 nor cADPR were able to mobilize Ca2+ from internal stores in these cells, but Th and AA were effective in releasing Ca2+. Four major Ca2+ stores in chick embryo retina cells were distinguished: i) the thapsigargin sensitive Ca2+ store, most likely the ER; ii) the Ca2+ store sensitive to oligomycin and CCCP, most likely the mitochondrial Ca2+ store, iii) an AA sensitive Ca2+ store, which is distinct from the previous two; and, iv) the Ca2+ store only sensitive to ionomycin. The capacities of these different Ca2+ stores of the chick embryo retina cells, relative to the total intracellular stores, are: 63.3%, 14.1%, 8.2%, for the ER, the mitochondrial and for the AA sensitive Ca2+ stores, respectively.
No preview · Article · Feb 1997 · Cellular Signalling
[Show abstract][Hide abstract] ABSTRACT: The fluorescent Ca2+ indicator Indo-1 was used to study the effect of depolarization evoked by KCl or 4-aminopyridine (4-AP) on the intracellular free calcium concentration responses (delta[Ca2+]i) in rat striatal synaptosomes. Depolarization of the synaptosomes with [KCl] > 7.5 mM induced a rapid increase of the [Ca2+]i followed by a decay towards a plateau. The size of the [Ca2+]i response varied sigmoidally with the synaptosomal membrane potential, with a transition potential of -27.3 mV. Depolarization with 4-AP evoked a dose-dependent sustained increase of the [Ca2+]i. Nitrendipine, omega-Conotoxin GVIA (omega-CgTx) and omega-Agatoxin IVA (omega-Aga IVA) were used to evaluate the relative role of L-, N-, P- and possibly Q-type voltage-sensitive Ca2+ channels (VSCCs) on the [Ca2+]i changes evoked by each of the two depolarizing agents. Nitrendipine caused only about 10% inhibition of the effect of either agent on the [Ca2+]i, suggesting that the L-type VSCCs have a modest contribution. The omega-CgTx decreased the response to KCl and 4-AP by 15 and 30%, respectively, but the latter effect may be partially due to a non-specific effect on Na+ channels. The omega-Aga IVA reduced the response to 4-AP by 26.5%, and this effect was additive to that of omega-CgTx, further suggesting that the striatal nerve terminals possess P- and/or Q-type, in addition to N-type Ca2+ channels. Neomycin (0.35 mM), tentatively used as an antagonist of the P-type channels, had a potent effect, decreasing the response to K(+)-depolarization and to 4-AP by, respectively, 32.5 and 48.5%. It is suggested that at the concentration used the antibiotic also partially blocks VSCCs which do not belong to the L-, N-, P- or Q-type VSCCs. We conclude that striatal nerve endings are equipped with at least four to five pharmacologically distinct classes of VSCCs, which are sensitive to well known antagonists of the L-, N-, P-, and Q-type VSCCs.
No preview · Article · Jan 1996 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: In this study we investigated the release of Ca2+ in brain microsomes after Ca2+ loading by the Ca(2+)-ATPase or by the Na+/Ca2+ exchanger. The results show that in microsomes loaded with Ca2+ by the Ca(2+)-ATPase, Ins(1,4,5)P3 (5 microM) released 21 +/- 2% of the total Ca2+ accumulated, and that in the microsomes loaded with Ca2+ by the Na+/Ca2+ exchanger, Ins(1,4,5)P3 released 28 +/- 3% of the total Ca2+ accumulated. These results suggest that receptors of Ins(1,4,5)P3 may be co-localized with the Na+/Ca2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P3 receptors in the plasma membrane where the Na+/Ca2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P3 inhibited the Ca(2+)-ATPase by 33.7%, but that it had no significant effect on the Na+/Ca2+ exchanger.
No preview · Article · Dec 1992 · Cellular Signalling
[Show abstract][Hide abstract] ABSTRACT: 1. Introdução O tecido nervoso é composto por neurónios e por células da glia. Os neurónios possuem longos axónios e neurites, que interagem extensivamente entre eles e também com as células da glia. Estas células não sobrevivem durante o processo de homogeneização, os neurónios não permanecem intactos: os corpos celulares são dissociados dos prolongamentos, sendo ambos fragmentados em estruturas mais pequenas. Nas estruturas mais simples assim formadas as membranas plasmáticas conseguem resselar formando partículas osmóticamente activas. Algumas destas partículas formam-se a partir dos terminais nervosos, contendo os organelos existentes na região pré-sináptica; estas estruturas são designadas por sinaptossomas. Muitas vezes, estas estruturas podem também possuir pedaços da membrana pós-sináptica ainda associada. As preparações sinaptossomais incluem terminais nervosos axodendríticos e axossomáticos, que representam sinapses entre um axónio e uma dendrite, ou entre um axónio e um corpo celular, respectivamente. A partir do córtex cerebral de pequenos roedores é possível obter uma preparação sinaptossomal relativamente homogénea uma vez que, no córtex cerebral existe um grande número de terminais axodendríticos uniformes, que se destacam facilmente do axónio que na célula viva é responsável pela comunicação com o corpo celular. Além disso, a contaminação por mielina pode ser facilmente eliminada através de diversas centrifugações.