摘要:

AbstractBinding of3H‐L‐aspartate to membranes from retinal glial cells in primary culture was characterized. Binding kinetics showed a saturable, reversible binding to three populations of sites with KB= 40,200, and 1,300 nM. The first two were present at 1 day in vitro (DIV), whereas the latter two were observed at 12 DIV. The possibility of the 40 nM site being neuronal cannot be discarded, since some neurons are present at 1 DIV. In 12 DIV cultures, the presence or absence of sodium determined two different pharmacological patterns, comparable to those described for electrogenic glutamate transport in Müller cells, and QA metabotropic receptors in astrocytes, respectively. Results suggest that, as has been shown for some receptors in nerve tissue, the properties of glial cell receptors undergo age‐dependent changes. In turn, this could be related to changes in the function of neurotransmitter substances during devel

ISSN:0894-1491    

DOI:10.1002/glia.440040502

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractVery little information is available concerning the pharmacology of type 2 astroglia. During the past decade it has become apparent that two distinct lineages of astroglial cells can be defined in vitro. These two lineages are commonly referred to as type 1 and type 2 and are distinguished from each other on the basis of their morphological features and antigenic phenotypes. In contrast to type 1 astroglia, very little is known about the pharmacology of type 2 astroglia. The lack of information concerning the responsiveness of these cells stems primarily from difficulties encountered in isolating large numbers of type 2 astroglia free of other cell types. In the present study video‐ and photometer‐based imaging systems were used to monitor the influence of a series of neuroligands on the intracellular calcium levels of individual cerebral type 2 astroglia in order to assess their expression of calcium‐mobilizing receptors. The responses of 85 immunocytochemically identified cerebral type 2 astroglia to bradykinin (BK), norepinephrine (NE), histamine (HIST), carbachol (CARB), 2‐methyl‐thio ATP (2MT‐ATP), glutamate (GLUT), and serotonin (5‐HT) were analyzed. Approximately 50% of cerebral type 2 astroglia responded to BK, NE, HIST, CARB, and 2MT‐ATP whereas only 16% and 9% of the cells responded to GLUT and 5‐HT, respectively. The number of neuroligands that increased calcium in individual cells ranged from 0 to 6. These responses are quite similar to those previously demonstrated in cultured cerebral type 1 astroglia. No pattern of receptor co‐expression was observed for the different neuroligands tested. While variable, distinct differences were observed in the time course of the calcium responses elicited by different neuroligands. The present study demonstrates that cultured cerebral type 2 astroglia are heterogenous when judged by their ability to respond to neuroligands with a rise in in

ISSN:0894-1491    

DOI:10.1002/glia.440040503

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractFura‐2 and indo‐1 fluorescence measurements were used to examine intracellular Ca++concentration ([Ca++]i) and its modulation by voltage‐activated influx in murine cortical astrocytes in primary cell culture. Extracellular K+was increased from 5 to 50 mM to depolarize cells to determine if Ca++influx through voltage activated Ca++channels could alter [Ca++]i. In confluent 4 to 6 weeksin vitroastrocyte cultures 50 mM K+increased [Ca++]i3‐4‐fold (from 150 nM up to 550 nM); this increase was blocked by nifedipine and enhanced by BayK 8644 indicating that influx was through L‐type channels. However, in 1 to 2 weeksin vitroastrocyte cultures, high K+reduced [Ca++]i. L‐type channels were apparently present in these cells because high K+in combination with BayK 8644 increased [Ca++]i. Following pretreatment of 1 to 2 weeksin vitroastrocytes with dibutyryl cAMP (dbcAMP) high K+increased [Ca++]iin the absence of BayK 8644 indicating enhanced activity of Ca++channels in agreement with previous voltage‐clamp studies. Ca++influx through voltage‐activated channels in cultured cortical astrocytes can substantially increase [Ca++]iand these channels can be dynamically modulated by dihydropyridines. Immature astrocytes may express ‘silent’ or inactive Ca++channels or have a much low

ISSN:0894-1491    

DOI:10.1002/glia.440040504

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe cell‐attached and excised patch configurations of the patch clamp technique were used to study stretch‐activated ion channels in Muller glial cells that were obtained from postmortem adult human retinas and were maintained in culture. A stretch‐activated channel permeable to monovalent and divalent cations was found. Ion channels of this type have not been demonstrated previously in glial cells, though indirect evidence has suggested that such stretch‐activated channels may help mediate a compensatory response of glia to swelling. Consistent with a possible role for volume regulation is the finding that the activation of calcium‐permeable stretch‐sensitive channels is associated with an increase in the activity of calcium‐activated potassium channels. Activation of potassium channels to produce an efflux of potassium with a subsequent loss of anions and cell water could be an effective mechanism to decrease glia

ISSN:0894-1491    

DOI:10.1002/glia.440040505

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn the presence of Ba++, an increase in the bath HCO 3−at constant CO2(i.e., Variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO3−/CO2at constant bath pH was not significantly affected by the presence of 50 μmol/l strophanthidin. In the absence of Ba++, addition of HCO3−/CO2at constant bath pH produced a Na+‐dependent hyperpolarization. Therefore, CO2movements, electrogenic Na+/K+pump activity and changes in Ba++binding do not contribute significantly to the hyperpolarization induced by HCO3−. These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO3−is due to an electrogenic Na+/HCO3−cotransporter, which transports Na+, HCO3−(or its equivalent), and net negative charge across the glial membrane.To study the role of electrogenic Na+/HCO 3−cotransport in the regulation of pHi in glial cells, we used intracellular double‐barreled, pH‐sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pHi) was 7.32 (SD 0.03, n = 6) in HEPES‐buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO3−/CO2buffered solution. These values for pHiare more than 1.2 pH units alkaline to the pHipredicted from a passive distribution of protons; thus, these cells actively regulated pHi. Superfusion and with‐drawal of 15 mmol/l NH4+induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady‐steady‐state pHi. Recovery from acidification was stimulated by adding HCO3−/CO2at constant pH. In HCO3−/CO2‐buffered solutions, the recovery was Na+‐dependent, inhibited by 4‐acetamido‐4′‐isothiocyanato‐stilbene‐2,2′‐disulfonic acid (SITS), and associated with a hyperpolarization of the membrane. Thus it appears that the electrogenic Na+/HCO3−cotransporter helps maintain the relatively alkaline pHiof glial cells and also contributes to the ability of glial

ISSN:0894-1491    

DOI:10.1002/glia.440040506

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell‐type and the only vimentin‐positive population present. Virtually all passaged cells (<98%) contain vimentin‐positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin‐positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First‐passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin‐2 (IL‐2). In contrast, older cultures were highly responsive to IL‐2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina.The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies o

ISSN:0894-1491    

DOI:10.1002/glia.440040507

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractAstrocytes are found throughout the central nervous system. They interact closely with surrounding structures, their processes contributing to the glia limitans of the neural tube, and to the glial investment of blood vessels, and of the somas, axons, and synaptic structures of neurones. This paper presents evidence that astrocytes in the central nervous system also interact with each other in a dual way, adhering to their neighbours via their processes, and repelling the somas of those neighbours. We suggest that this interaction, which has been termed contact spacing, distributes astrocytes through the central nervous system, and forms the basis of their structural role.

ISSN:0894-1491    

DOI:10.1002/glia.440040508

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe report that platelet‐derived growth factor (PDGF) is a potent mitogen for oligodendrocyte type‐2 astrocyte (O‐2A) progenitor cells derived from the optic nerves of adult rats. Moreover, O‐2Aadultprogenitors cultured in PDGF express the range of properties we have described previously for O‐2Aadultprogenitors cultured in the presence of type‐1 astrocytes. Similarly, previous studies have demonstrated that PDGF is able to mimic the influence of type‐1 astrocytes on O‐2Aperinatalprogenitors. Specifically, O‐2Aadultprogenitors and O‐2Aperinatalprogenitors exposed to PDGF express differences in average cell cycle time (59 ± 5 h for O‐2Aadultprogenitors versus 20 ± 6 h for O‐2Aperinatalprogenitors), average rate of migration (4.1 ± 0.6 μm h−1versus 24.6 ± 5.4 μm h−1), morphology (unipolar versus bipolar), and antigenic phenotype (04+vimentin−versus 04−vimentin+). Thus, our present results indicate that a single signalling molecule secreted by type‐1 astrocytes produces markedly different cellular behaviours in

ISSN:0894-1491    

DOI:10.1002/glia.440040509

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractEpendymal cells in culture and in vivo were treated with mixture of galactocerebrosides. Galactocerebroside is the major glycolipid of myelin and in demyelinating diseases is found in cerebrospinal fluid. Morphological changes induced by this treatment were examined by microscopy at both optical and ultrastructural levels. In vitro, cilia, microvilli, and junctions between the cells disappeared, processes containing intermediate filaments developed, and the cells lost characteristics typical of ependymal cells and became more astrocyte‐like. As shown by vital staining with a fluorescent compound and by nuclear incorporation of bromodeoxyuridine, cells did not proliferate during the period of galactocerebroside treatment and the morphological transformation was restricted to the ependymal cells. In contrast, asialoganglioside‐GM1and sulfatides had no effect on ependymal cell morphology. Some of the in vitro observations could be reproduced in vivo. Junctions between ependymal cells disappeared and intercellular spaces appeared between these cells and the cerebral parenchyma at the basolateral side of the ependymal layer. At the apical side, morphological modifications of junctions and cilia were less evident. As these experimental conditions resemble those existing during demyelination the morphological changes described may account for perturbations of the physiological functions of the ependymal c

ISSN:0894-1491    

DOI:10.1002/glia.440040510

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe synthesis and release of plasminogen activators (PAs) in co‐cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS‐PAGE, zymography, and a two‐step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amilioride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10–14‐fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS‐PAGE followed by zymography. A PA ∼95 kDa was absent in co‐cultures of SCs + NCs but prominent by 4 days postdenervation; PA ∼50–70 kDa increased through 8 days postdenervation and PA ∼25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the

ISSN:0894-1491    

DOI:10.1002/glia.440040511

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY