RESUMO
Primary cultures of astrocytes from neonatal rat brain were incubated with tritiated serotonin. After fixation they were stained by immunofluorescence for the astrocyte-specific marker glial fibrillary acidic protein and processed for autoradiography. Silver grain density was increased over cells positive for glial fibrillary acidic protein and was reduced to background levels when sodium was omitted from the medium or the specific inhibitors of serotonin uptake fluoxetine and chlorimipramine were present. The results indicate that mammalian astrocytes can take up serotonin by a sodium-dependent, high-affinity system previously thought to be the exclusive property of serotonergic nerve endings.
Assuntos
Astrócitos/metabolismo , Serotonina/metabolismo , Animais , Astrócitos/análise , Astrócitos/efeitos dos fármacos , Autorradiografia , Transporte Biológico , Células Cultivadas , Clomipramina/farmacologia , Imunofluorescência , Fluoxetina/farmacologia , Proteína Glial Fibrilar Ácida/análise , Ratos , Sódio/farmacologiaRESUMO
We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I(Na)) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7-21 rats express low density I(Na) (9.5-15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC(50) value of 39.3 nM. The I(Na) expression correlated with a approximately 25 mV more depolarized resting membrane potential (RMP) as compared with non-I(Na)-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 microM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I(Na)-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I(Na) expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 microM TTX. Analysis of the I(Na) activation/inactivation curves yields an I(Na) "window current" at -40+/-20 mV, implying a persistent I(Na) component being active around the NG2 glia RMP of approximately -45 mV. According to the constant-field equation analysis, this active I(Na) component leads to a pNa/pK ratio of 0.14 at RMP which is approximately threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I(Na) component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.
Assuntos
Hipocampo/citologia , Potenciais da Membrana/fisiologia , Neuroglia/fisiologia , Canais de Sódio/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Microscopia Confocal , Neuroglia/efeitos dos fármacos , Neuroglia/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Four hr after either a single injection or continuous infusion of methotrexate (MTX) plus purified [3',5',9(n)-3H]MTX in cynomolgus or rhesus monkeys, 80 to 98% of the 3H radioactivity present in the plasma was found not to represent intact MTX. The percentage of 3H-containing MTX products in the urine after 4 hr was considerably less, although more variable. This variability seemed to be related to variability in the amount of the total dose excreted. Non-MTX products were also found in selected tissues and the percentage of intact MTX found 4 hr after i.v. injection varied from 2 to 26%. The percentage of intact MTX was routinely measured by comparing the values obtained using the dihydrofolate reductase assay with values based on the specific activity of [3',5',9(n)-3H]MTX. Results obtained by diethylaminoethyl column chromatography on a few samples, however, showed good agreement with results from the reductase assay. [3',5',9(n)-3H]MTX products appeared in peaks eluting from the diethylaminoethyl column both earlier and later than the MTX peak, with the earlier peaks being present in only small amounts in the urine. After continuous i.v. infusion, only 2% or less of the radioactivity found in the cerebrospinal fluid after 4 hr represented intact MTX, with the remaining radioactivity eluting much earlier than MTX. In contrast, after direct injection into the left lateral ventricel, all the 3H radioactivity in both cerebrospinal fluid and brain tissue represented intact MTX for up to 4 hr after injection. The appearance of MTX products in the plasma and selected tissues of these primates a short time after i.v. injection is compared to other work in experimental animals and man and suggests a greater metabolism of MTX than was previously suspected.
Assuntos
Metotrexato/metabolismo , Animais , Medula Óssea/metabolismo , Encéfalo/metabolismo , Gatos , Feminino , Haplorrinos , Infusões Parenterais , Injeções Intravenosas , Injeções Intraventriculares , Rim/metabolismo , Fígado/metabolismo , Macaca fascicularis , Macaca mulatta , Masculino , Metotrexato/administração & dosagem , Probenecid/farmacologia , Baço/metabolismo , Fatores de TempoRESUMO
Entrapment of methotrexate (MTX) plus [3',5', 9(n)-3H]methotrexate into positively charged liposomes greatly alters the subsequent distribution of [3H]MTX in a cynomologous monkey (Macaca irus) after a single i.v. injection ([3H]MTX; refers to total radioactivity derived from purified [3H]MTX). When [3H]MTX is incorporated into small, sonically disrupted liposomes, the level of the entrapped [3H]MTX in the total plasma volume was still 50% of the total injected dose after 4 hr, which is 100 times greater than the level found when the same amount of free MTX (1 to 4 mg) plus [3H]MTX was injected. When entrapped in larger mechanicanically disperesed liposomes, however, the plasma levels of liposome-entrapped [3H]MTX at 4 hr was only 6-fold greater than free [3H]MT. The liposome-entrapped MTX (refers to MTX measured by dihydrofolate reductase assay) did not show detectable breakdown in the plasma whereas free MTX showed up to 97% breakdown. Increased clearance of [3H]MTX entrapped in mechanically dispersed liposomes was complemented by its much greater uptake into tissues, especially spleen, compared with sonically disrupted liposomes. There was over a 160-fold increased uptake by the spleen of liposome-entrapped [3H] MTX relative to free [3H]MTX, whereas for sonically disrupted liposomes the comparable ratio was 20. Although this liposome-entrapped MTX showed significant breakdown, it was less than that found after injection of free MTX. In certain tissues, especially the small intestine, a reduced uptake of liposome-entrapped [3H]MTX WAS FOUND. Uptake of liposome-entrapped [3H]MTX into liposomes led to a much lower renal clearance of [3H]MTX, especially in the case of sonically disrupted liposomes. Possible reasons for these effects and the relationship of our findings to those of others are discussed.
Assuntos
Lipossomos/metabolismo , Metotrexato/metabolismo , Animais , Feminino , Haplorrinos , Macaca fascicularis , Masculino , Taxa de Depuração Metabólica , Metotrexato/sangue , Metotrexato/urina , PermeabilidadeRESUMO
We have shown previously that process-bearing GFAP+ astrocytes freshly isolated from rat hippocampus CA1 and CA3 regions are heterogeneous in ion channel expression and K(+) uptake capabilities, such that two distinct populations of astrocytes can be described (Zhou and Kimelberg, 2000). In the present study, we report that glutamate transporter (GT) currents can only be measured from one type of these freshly isolated hippocampal CA1 astrocytes [variably rectifying astrocytes (VRAs)] but were not detectable in the second type of astrocyte [outwardly rectifying astrocytes (ORAs)]. The GT currents showed a strict Na(+) dependency and high affinity for glutamate (EC(50) of 4 +/- 1.1 microm). The astrocytes lacking GT currents (ORAs) showed an AMPA receptor current density (55 pA/pF) that was 42-fold higher than VRAs (1.3 pA/pF). In contrast, the GABA(A) currents were of comparable current density in both types. The specificity of these differences makes it unlikely that they are attributable to preparative damage. Therefore, these findings strongly indicate that, within a single region of the hippocampus, GFAP+ astrocytes comprise a functionally diverse population that are qualitatively different in their functional glutamate transporter and quantitatively different in their functional AMPA receptor expression. This heterogeneity implies that GFAP+ astrocytes may participate in or modulate glutamate synaptic transmission differently.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Astrócitos/classificação , Astrócitos/metabolismo , Hipocampo/metabolismo , Ácido Caínico/análogos & derivados , Receptores de AMPA/biossíntese , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Fatores Etários , Sistema X-AG de Transporte de Aminoácidos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Separação Celular , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Canais Iônicos/biossíntese , Ácido Caínico/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores , Receptores de GABA-A/metabolismoRESUMO
Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either 3H-labelled dipalmitoyl phosphatidylcholine and [14C]cholesterol or with [14C]cholesterol and [3H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [3H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured. It was found that plasma radioactivity from [3H]methotrexate and [14C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [3H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [3H]methotrexate than negative liposomes and showed very low permeability to [3H]methotrexate in in vitro studies, even in the presence of high concentrations of serum. [14C]Cholesterol radioactivity was cleared more rapidly from plasma than 3H-radioactivity from liposome-entrapped [3H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. 3H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [14C]cholesterol during the first 3 h. The more rapid disappearance of [14C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes. Renal excretion of 3H from liposome-entrapped [3H]methotrexate was considerably less than that of 3H from free [3H]methotrexate. There was insignificant excretion, however, of 14C from cholesterol in the urine. Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to 3H-containing products in plasma and partially prevented its breakdown in tissues. These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drub, maintain high plasma levels and reduce its renal excretion.
Assuntos
Lipossomos , Metotrexato/metabolismo , Animais , Colesterol/sangue , Eritrócitos/metabolismo , Haplorrinos , Cinética , Metotrexato/sangue , Especificidade de Órgãos , SonicaçãoRESUMO
The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.
Assuntos
Adenosina Trifosfatases/metabolismo , Colesterol/farmacologia , Magnésio/farmacologia , Fosfatidiletanolaminas/farmacologia , Fosfatidilserinas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Concentração de Íons de Hidrogênio , Medula Renal/enzimologia , Cinética , Lipossomos/metabolismo , Microssomos/enzimologia , Potássio/farmacologia , Coelhos , Sódio/farmacologia , Sonicação , Temperatura , TermodinâmicaRESUMO
Chloride transport in primary cultures of astroglial cells from rat brain shows saturation kinetics, is inhibited by SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), and exchanges with HCO-3. These properties are similar to those of the anion exchange system in erythrocytes. Estimated of intracellular C1- concentration([C1-]i) in the primary cultures give values in the range of 31 to 43 mM, which are 3- to 5-times greater than predicted from equilibration with an average measured membrane potential of -70 mV, suggesting that these cells also actively accumulate Cl-.
Assuntos
Astrócitos/metabolismo , Cloretos/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Bicarbonatos/metabolismo , Transporte Biológico Ativo , Bucladesina/farmacologia , Células Cultivadas , Potenciais da Membrana , Ouabaína/farmacologia , RatosRESUMO
The uptake of ouabain-sensitive 86Rb+ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive 86Rb+ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive 86Rb+ uptake in both cell types. This increase ouabain-sensitive cation transport was reflected in increased levels of (Na++K)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same Km as 3T3 cells. These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.
Assuntos
Adenosina Trifosfatases/metabolismo , Divisão Celular , Transformação Celular Neoplásica , Ouabaína/farmacologia , Rubídio/metabolismo , Vírus 40 dos Símios , Transporte Biológico Ativo/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Cinética , Potássio/metabolismo , Potássio/farmacologia , Sódio/farmacologiaRESUMO
Astrocytic swelling occurs readily in ischemia and traumatic brain injury (TBI) as part of the cytotoxic or cellular edema response. Ischemia is known to produce large extracellular increases in both [K+] and excitatory amino acids (EAA) in vivo, and astrocytic swelling in vitro leads to marked release of EAA. In this study we compared the effect of swelling due to hypotonic media and high K+ medium on the uptake and release of EAA by rat primary astrocyte cultures in vitro. In both cases, there was a significant inhibition of uptake of [3H]L-glutamate and [3H]D-aspartate, and increased release of preloaded [3H]D-aspartate. The kinetics of the increased efflux was very different in response to hypotonic or high K+ media. In hypotonic medium there was a rapid initial release followed by a decline in the rate of release over time. This release was independent of whether Na+ was present. Upon exposure to high K+ medium there was a slow progressive increase in release of [3H]D-aspartate, which never showed any subsequent decline until the media was returned to normal [K+]. In high K+ media there was also an initial transient increase in [3H]D-aspartate release, which we attribute to reversal of the amino acid uptake system. The increased release due to hypotonic medium was not affected by a drop in temperature from 37 to 26 degrees C, while the increased release due to high K+ medium was completely inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ácido Aspártico/antagonistas & inibidores , Astrócitos/metabolismo , Encéfalo/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacocinética , Soluções Hipotônicas/farmacologia , Potássio/farmacologia , Animais , Ácido Aspártico/farmacocinética , Astrócitos/efeitos dos fármacos , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Isquemia Encefálica/complicações , Isquemia Encefálica/metabolismo , Ratos , TemperaturaRESUMO
Our initial paper discussed brain edema resulting from traumatic head injury and the need for specific and effective agents to treat the disorder and disclosed a novel approach for the discovery of a drug of this kind. The current study describes the synthesis of a series of [(2,3,9,9a-tetrahydro-3-oxo-9a-substituted-1H-fluoren-7-yl)oxy]alk anoic acids and their analogues. These compounds were evaluated in an in vitro cerebrocortical tissue slice assay for their relative potencies in inhibiting K+ + HCO3- induced swelling. Structural modification at a number of sites in the "lead" compound revealed that significant biological activity was inherent only within a very narrow range of structural types. The observation that nearly all the biological activity resided in one of the two enantiomers demonstrated the marked stereospecificity of the most active compounds. One of the most potent compounds, (R)-(+)-[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluoren -7-yl) oxy]acetic acid ((+)-5c), exhibited a dose-response relationship in the in vivo acceleration/deceleration brain edema assay, and the data from the two highest doses were statistically significant. Electron microscopic examination demonstrated that the perivascular astroglial swelling that arises from this procedure is abolished in the animals treated with (+)-5c. This compound is currently being evaluated for its clinical efficacy and safety in the treatment of traumatic head injury.
Assuntos
Edema Encefálico/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Gatos , Cloretos/metabolismo , Traumatismos Craniocerebrais/complicações , Relação Dose-Resposta a Droga , Cobaias , Neuroglia/ultraestrutura , Ratos , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Blunt and ischemic injuries of the brain have been shown to result in swelling that is predominantly limited to a single cell type, the astrocyte, within the complex cellular mosiac of cerebral gray matter. Evaluation of various diuretic (aryloxy)acetic acids in vitro using incubating cat brain slices and primary astrocyte cultures identified compounds with marked ability to inhibit brain tissue swelling. Some of the compounds significantly reduced the mortality and morbidity following acceleration/deceleration brain injury in anesthesized cats. A variety of (indanyloxy)alkanoic acids were synthesized which were analogous to the dually active (indanyloxy)acetic acids. Some of the 4-(indanyloxy)butanoic acids were found to be devoid of diuretic activity but to possess equal or greater activity than the dually active compounds in the in vitro and in vivo brain assays. Selected examples from both the (indanyloxy)acetic and 4-(indanyloxy)butanoic acid series showed marked chiral effects, with one enantiomer generally exhibiting a much greater activity than the other. A clinical study of severely head-injured patients treated with ethacrynic acid demonstrated a significantly improved outcome when compared to controls. These data suggest a clinical advantage for the nondiuretic (aryloxy)alkanoic acids which possess in vitro and in vivo activities in the cat brain assays that are comparable or superior to dually active compounds.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Ácidos Carboxílicos/síntese química , Animais , Astrócitos/efeitos dos fármacos , Bicarbonatos/farmacologia , Ácidos Carboxílicos/farmacologia , Gatos , Córtex Cerebral/metabolismo , Fenômenos Químicos , Química , Eletroencefalografia , Ácido Etacrínico/farmacologia , Técnicas In Vitro , Indanos/síntese química , Indanos/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The mammalian CNS is separated from the blood by tight junctions, collectively termed the blood-brain barrier (BBB). This imposes unique features of solvent and water movement into and out of the CNS. The basic equations for water fluxes driven by osmotic gradients are presented. The anatomy of the BBB and the physiology of the transport processes for cerebrospinal fluid production, extracellular fluid production and intercellular water and solute transport are then described. A quantitative analysis of the need for aquaporin-based water movements to accompany the known rates of CSF production is also presented. Finally, the mechanisms and roles of cellular and vasogenic edema in the CNS, especially in relation to aquaporins, are described.
Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/fisiologia , Homeostase/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Aquaporinas/metabolismo , Edema Encefálico/fisiopatologia , Líquido Cefalorraquidiano/metabolismo , Humanos , Pressão OsmóticaRESUMO
High-affinity uptake of serotonin (5-HT) by primary cultures of rat cortical astrocytes has been shown recently to be potently inhibited by tricyclic antidepressants in a manner similar to that described for brain synaptosomes [Katz and Kimelberg, J. Neurosci. 5, 1901 (1985)]. Since the high-affinity binding of [3H]imipramine (IMI) to brain membranes has been well correlated with the inhibition of synaptosomal 5-HT uptake, the binding of [3H]IMI to these astrocyte cultures was examined. No evidence for the existence of a high-affinity binding site was detected in either intact astrocytes or membranes prepared from astrocyte cultures. However, a very dense population of low-affinity binding sites was observed using both methods. This site was similar in affinity (0.606 microM for membranes and 0.959 microM for intact cells) to a low-affinity site observed with rat brain membranes (1.79 microM) but was present at a much greater density in astrocytes (1610 pmoles/mg protein for membranes and 672 for intact cells versus 53 pmoles/mg protein in brain), and may have prevented detection of the high-affinity site. Low-affinity binding to astrocytes was sodium independent, as was low-affinity binding to brain membranes. There was a poor correlation between the inhibitory potencies of the drugs tested against imipramine binding and 5-HT uptake. The binding of 15 nM [3H]IMI was nearly equipotently inhibited by all of the antidepressants tested with IC50 values ranging from 0.56 to 2.6 microM. Other receptor ligands such as 5-HT, chlorpheniramine, quipazine, atropine and benztropine were relatively weak inhibitors of [3H]IMI binding, whereas chlorpromazine was more potent than the tricycle antidepressants.
Assuntos
Astrócitos/metabolismo , Imipramina/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Células Cultivadas , Cinética , Ratos , Serotonina/metabolismo , TrítioRESUMO
Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats. As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention. However, the actual classification of these electrophysiologically complex glial cells remains unclear. They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100beta, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP). To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats. In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells). We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA. We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive. We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively. In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP. The expression of GFAP at the transcript level indicates GFAP (-)/GFAP mRNA (+) glial cells have an astrocytic identity. As single cell RT-PCR is able to detect both GFAP (-)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/fisiologia , Neuroglia/fisiologia , RNA Mensageiro/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Neuroglia/metabolismo , Técnicas de Patch-Clamp , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
The occurrence of astrocytic swelling in response to CNS trauma is reviewed. This response occurs rapidly and appears earlier than the reactive astrocytic response to neuronal and axonal injury. Astrocytic swelling is viewed as an exaggerated pathologic extension of normal astrocytic functions, such as regulation of extracellular ion levels and brain pH. Potential deleterious consequences of swelling, such as failure of ion homeostasis mechanisms, impaired uptake of neurotransmitters, and release of excitotoxic amino acids, are discussed. Possible pharmacologic interventions are reviewed showing that inhibition of such swelling or prevention of some of its potential deleterious consequences by an anion transport inhibitor, L-644,711, may have beneficial effects in head trauma.
Assuntos
Astrócitos/patologia , Edema Encefálico/fisiopatologia , Lesões Encefálicas/fisiopatologia , Animais , Edema Encefálico/etiologia , Lesões Encefálicas/patologia , Edema , Encefalopatia Hepática/patologia , Encefalopatia Hepática/fisiopatologia , Humanos , Modelos Neurológicos , Potássio/metabolismoRESUMO
Receptors for transmitters, as varied as those expressed by neurons, have been described on primary astrocyte cultures prepared from new-born rats and mice. A variety of functional effects and considerable cell-to-cell and regional heterogeneity have been observed for such receptors in vitro. The various systems available for studying the presence and properties of receptors on astrocytes in situ, and the results from these studies, are discussed. Much fewer studies using these more difficult systems have been done. So far, some resemblances and differences between in situ and in vitro work have been observed. More of these in situ studies, to supplement the ongoing in vitro work, are needed to enable us to determine unequivocally which receptors are present on astrocytes, and their functions in vivo. If there is cell-to-cell and CNS regional heterogeneity in vivo comparable to that seen in vitro, these analyses will be very complex. To illustrate the importance and variety of receptor-linked functions, a number of suggestions are made in this commentary, based on current proposals for the roles of astrocytes. However, it is argued that we need to have a more complete understanding of astrocyte functions in vivo, before we can really understand the functional significance of astrocyte receptors.
Assuntos
Astrócitos/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Humanos , Camundongos , Modelos Neurológicos , Plasticidade Neuronal , Neurotransmissores/farmacologia , Neurotransmissores/fisiologia , Ratos , Transdução de SinaisRESUMO
Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations.
Assuntos
Astrócitos/fisiologia , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Separação Celular , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/genética , Fatores de TempoRESUMO
We have examined the question of anion-transport systems in glia using primary astrocyte cultures prepared from neonatal rat brains. These studies show that these cells have exchange or cotransport systems for Cl- that appear to be electrically neutral, that is, SITS-sensitive Cl-/Cl- or Cl-/HCO3- anion exchange, and furosemide- and bumetanide-sensitive Na+ + K+ + 2Cl- cotransport. These inhibitors inhibit a major component of the total 36Cl- flux and the remaining Cl- flux may be conductive; however, this conductive flux makes a small contribution to Em relative to K+, since large changes in [Cl-]o do not usually affect Em, which is predominantly a K+ diffusion potential. We have also found an alpha-receptor-mediated depolarization that is affected by imposed changes in Ecl. The alpha-receptor-mediated depolarization seen at normal [Cl-]o could be partially due to increased Cl- conductance because [Cl-]i appears to be several-fold higher than it would be if it were in equilibrium with the membrane potential.
Assuntos
Ânions/metabolismo , Astrócitos/metabolismo , Proteínas de Transporte/metabolismo , Animais , Bicarbonatos/metabolismo , Encéfalo/metabolismo , Dióxido de Carbono/metabolismo , Células Cultivadas , Cloretos/metabolismo , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Proteínas de Membrana , Ratos , Simportadores de Cloreto de Sódio-PotássioRESUMO
Inhibitors of cell-swelling-activated anion channels, including the antiestrogenic compound tamoxifen (TAM), have been shown to attenuate the increase in excitatory amino acids (EAA) during ischemia. Since TAM enters the CNS we tested whether it provides protection from damage due to reversible middle cerebral artery occlusion (rMCAo) in rats. TAM (5 mg/kg, i.v.) infused 25 min before ischemia, potently reduced the total volume of the infarct from 328 +/- 34 mm3 to 41 +/- 21 mm3, a reduction of 87%, as measured by TTC staining. It was equally effective when infused starting at 1 h after reperfusion, i.e. 3 h after initiation of rMCAo. Protection of neurons was also found histologically. TAM had no effect on CBF as measured by hydrogen clearance. This appears to be the first report of a marked neuroprotective effect of TAM. Further studies are needed to determine whether its effects are due to inhibition of EAA release and/or other potential neuroprotective sites of action.