摘要:

AbstractAt subnanomolar concentrations, vasoactive intestinal peptide (VIP) can act as an astroglial mitogen and as a secretagogue for neurotrophic substances released from glia (Brenneman et al.: J Neurosci Res 25:386–394, 1990). Here we report that treatment with subnanomolar (0.1 nM) VIP, that does not produce an increase in intracellular cAMP levels, induced the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus in neonatal cortical astrocytes, as revealed by immunohistochemistry, Western blot analysis, and measurements of the enzyme activity. Western blot analysis of subcellular fractions, using PKC isotype‐specific antisera, showed PKC alpha as well as the two novel PKC isotypes, delta and zeta immunoreactivities, whereas PKC beta or gamma immunoreactivities were not detected. PKC alpha was associated predominantly with the cytosolic compartment, while PKC delta was found in the plasma membrane and in nuclear fractions. In contrast, PKC zeta was distributed ubiquitously within the major subcellular fractions. Treatment of the cells with 0.1 nM VIP caused a marked increase in nuclear PKC alpha and, to a lesser extent, PKC delta and PKC zeta immunoreactivities. Western blot analysis showed that a low (1 nM) concentration of phorbol, 12‐myristate, 13 acetate also caused the subcellular redistribution of PKC immunoreactivities from the cytoplasm to the nuclear fraction, similar to VIP treatment. Exposure of astrocytes to high concentrations (1 μM) of phorbol, 12‐myristate, 13 acetate resulted in the down‐regulation of PKC alpha and PKC delta, while distribution of PKC zeta immunoreactivities were only slightly altered. Measurements of Ca2+‐ and phospholipid‐dependent PKC activities also showed a VIP‐induced redistribution of PKC activity from the cytoplasmic to the nuclear fractions. These results suggest that PKC may be involved in the signal transduction process elicited by VIP binding to the high affinity VIP receptors present on cortical astrocytes. The observed changes in the nuclear localization of PKC alpha and PKC delta in response to subnanomolar VIP may play a role in mediating the cellular response(s) to this peptide during neurodevelopment. © 1994 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the Unite

ISSN:0360-4012    

DOI:10.1002/jnr.490390402

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

年代:1994

数据来源: WILEY

摘要:

AbstractMedia conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (<1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)‐induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole‐cell patch‐clamp technique. This effect is specific for CCm and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA‐induced currents because glutamate‐induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagnoist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist‐like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA‐induced current in GABA‐responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAAreceptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist like activity, glia cells could possibly modulate GABAAreceptor‐mediated responses. © 19

ISSN:0360-4012    

DOI:10.1002/jnr.490390403

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

年代:1994

数据来源: WILEY

摘要:

Abstractß/A4 peptides are known to induce neurodegeneration in cultures of rat brain cells and rat neural cell lines (Yankner et al: Science 250:279–282, 1990; Behl et al: Biochem Biophys Res Commun 186:944–950, 1992). The current data show that these peptides induce similar neurodegeneration in SH‐SY5Y neuroblastoma cells, extending characterization of ß/A4 toxicity to a human nerve cell line. Human SH‐SY5Y cells respond to aggregated ß/A4 with changes in cell shape, membrane blebbing, antigenic modification, loss of attachment to the substrate, and cell death. ß/A4 peptides require aggregation for maximum toxic effects, as cellular degeneration is evoked by aggregated ß/A4 1‐42 and 4‐41 cysteine but not by monomeric ß/A4 1‐40. Aged (pre‐aggregated) ß/A4 1‐40 also evoked neurodegeneration. Antigenic changes comprise upregulation of Alzheimer's‐type tau epitopes, recognized by the PHF‐1 and Alz‐50 monoclonals. These particular changes in tau support the connectivity between this in vitro model and mechanisms leading to neurodegeneration in Alzheimer's disease. A significant feature of the SH‐SY5Y response is that cells must be differentiated before they become sensitive to the degeneration evoked by ß/A4. Signaling pathways leading to ß/A4‐evoked neurodegeneration thus are under experimental control, becoming complete only when proliferating cells withdraw from the cell cycle and develop a postmitot

ISSN:0360-4012    

DOI:10.1002/jnr.490390404

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

年代:1994

数据来源: WILEY

摘要:

AbstractCNP (2′,3′‐cyclic nucleotide 3′‐phosphodiesterase) is the earliest myelination specific polypeptide to be synthesized by oligodendrocytes (OLs). When non‐myelinating “naive” cells are transfected with the rat CNP cDNA, CNP accumulates intracellularly in a punctate manner, as well as at the plasma membrane. Filopodia and processes, like those of OLs become elongated and more numerous, and are filled with this protein. Post‐translational isoprenylation of the terminal C‐T‐I‐I sequence with either farnesyl or geranylgeranyl is essential for this phenomenon. In contrast, the non‐isoprenylated C397S mutant is homogeneously distributed throughout the cytoplasm and does not markedly affect cellular morphology. We have sythesized CNP and the C397S mutant in vitro and have shown that isoprenylation is essential for the binding of newly synthesized CNP to myeli

ISSN:0360-4012    

DOI:10.1002/jnr.490390405

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

年代:1994

数据来源: WILEY

摘要:

AbstractIncreased synthesis and release of S100β protein from activated astrocytes has been implicated in the overgrowth of dystrophic neurites in neuritic plaques in Alzheimer disease (AD). To evaluate the quantitative relationships between tissues levels of S100β and the numbers of neuritic plaques, by Tau‐2 immunoreactive (Tau‐2+) labeling, in tissue sections of hippocampus and adjacent temporal cortex and measured the levels of S100β protein, by Western immunoblot labeling, in samples of analagous regions from contralateral hemisphere of the same patients. In AD, tissue levels of S100β (two‐ to fivefold that of controls) were significantly correlated with the number of Tau‐2+plaques (R = 0.82,P<.01). Dual‐label immunohis‐tochemical analysis showed that most S100β+cells were activated GFAP+astrocytes. These results were substantiated by a significant correlation between S100β and GFAP tissue levels (R = 0.81,P<.05). Many of the S100β+astrocytes were clustered around and within Tau‐2+plaques. Indeed, no Tau‐2+plaques were found without associated activated S100β+astrocytes. Our findings provide further evidence of a role for S100β protein in dysregulation of neurons that leads to apparently nonsensical growth of imperfect neurites in AD, a potential key element in early stages of neuritic plaque pathogeneis

ISSN:0360-4012    

DOI:10.1002/jnr.490390406

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

年代:1994

数据来源: WILEY

摘要:

AbstractLippolysaccharide (LPS) or a combination of interleukin (IL)‐1ß and interferon (IFN)‐γ cause transcriptional induction of a calcium‐independent nitric oxide synthase (NOS) in astrocytes and C6 glioma cells. LPS induction of NOS in C6 cells was evidenced by a small amount of nitrite accumulation as compared with cells exposed to IL‐1ß/IFN‐γ, but the maximal NOS activity achieved (as revealed by cGMP formation) was the same. The NOS activity induced by LPS in C6 cells was maximal at 4 to 8 hr and then rapidly decreased, while NOS activity induced by IL‐1ß/IFN‐γ slowly decreased after 4 hr. In addition, the effects of re‐presenting IL‐1ß/IFN‐γ to both astrocytes and C6 cells after maximal induction of activity of the inducible from of NOS were studied. The re‐addition of cytokines prolonged both NOS mRNA expression and also enzyme activity, suggesting effects at either the transcriptional (further induction) or translational level (mRNA stability). These results imply that the time course of NO production by induced astrocytes depends both upon the nature of the inducing stimulus and the frequency of the cells' exposure

ISSN:0360-4012    

DOI:10.1002/jnr.490390407

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe transforming growth factors beta (TGF‐β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well‐defined steps of cellular actions possibly controlled by TGF‐βs, and since TGF‐β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF‐β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post‐lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF‐β 1‐3 mRNA were determined for each segment. We demonstrate that TGF‐ß1 mRNA levels are higher than those of TGF‐ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF‐ß2 mRNA could not be detected. For TGF‐ß1‐3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF‐β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF‐β‐IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF‐β‐IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF‐β‐IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF‐β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF‐β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might invol

ISSN:0360-4012    

DOI:10.1002/jnr.490390408

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe protective action of Ca2+and a series of other divalent cations on heat inactivation (48°C, 30 min) of [125I]ω‐conotoxin (CTX) binding sites was investigated in membranes prepared from rat forebrain. Moreover, the influence of GABA (500 ω) on this protection was studied. Binding of [125I]CTX as well as its inhibitory action on K+(55 mM) stimulated, Ca2+‐dependent transmitter release were studied in rat cerebellar granule neurons cultured in the presence or absence of the GABAAreceptor agonist THIP (4,5,6,7‐tetratydroisoxazolo[5,4‐c]pyridin‐3‐ol). In cells cultured in the presence of THIP (150 ω) it was investigated whether the ability of THIP to inhibit evoked transmitter release could be influenced by CTX. Ca2+and other divalent cations could effectively protect against heat inactivation of [125I]CTX binding sites in rat forebrain membranes, but this protective action was not influenced by the presence of 500 ω GABA. The cultured cerebellar granule neurons exhibited specific binding sites for [125I] CTX, the number of which was independent of exposure of the cells to THIP during the culture period. Evoked transmitter release was inhibited by CTX with an IC50value of 13 nM. In neurons cultured in the presence of 150 ω THIP, THIP could dose‐dependently inhibit evoked transmitter release, but this inhibitory action was not influenced by CTX (20 nM). The results show that cerebellar granule neurons exhibit functionally meaningful CTX binding sites. An association between such sites and GABA receptors is not apparent. ©

ISSN:0360-4012    

DOI:10.1002/jnr.490390409

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

年代:1994

数据来源: WILEY

摘要:

AbstractHypertension has been linked to opening of the blood‐brain barier and may be related to the expression of the smooth muscle α‐actin gene in contractile cells at the brain microvasculature. However, the cellular origin (i.e., endothelial cells, pericytes, smooth muscle cells) of the α‐actin mRNA in the brain microvasculature is not clearly identified. Therefore, we investigated the abundance of actin mRNA by Northern blot analysis in isolated brain microvessels and in brain microvascular endothelia or pericytes in tissue culture. All samples showed the characteristic 2.1 kb transcript corresponding to cytoplasmic and δ isoform mRNA. The 1.7 kb transcript corresponding to smooth muscle α‐actin was detected in freshly isolated bovine brain microvessels, in primary cultures of brain microvasular pericytes, or endothelial cells; the latter cultures contain both endothelial cells and pericytes. The α‐actin mRNA was absent in a cloned bovine brain endothelial cell line. The relative abundance of the α/(+γ) actin transcript ratio was: cultured pericytes>freshly isolated microvessels>endothelial primary. The cellular distribution of the smooth muscle α‐actin immunoreactive protein was studied by immunocytochemistry in cytospun/methanol‐fixed isolated bovine brain microvessels with a monoclonal antibody directed to the amino‐terminal decapeptide of the smooth muscle α‐actin isoform. This antibody reacted strongly with precapillary arterioles of isolated microvessels, whereas no immunostaining was observed in either capillary endothelial cells or in pericytes. In conclusion, the αL‐actin mRNA is expressed in brain microvascular pericytes in tissue culture, but the immunoreactive α‐actin protein is not expressed in brain microvascular pericytes in vivo. These data suggest that either (1) α‐action gene expression is induced in capillary pericytes in tissue culture or (2) α‐action mRNA in brain capillary pericytes in vivo is subject to translational repression resulting in no detectable α‐actin protein under

ISSN:0360-4012    

DOI:10.1002/jnr.490390410

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

年代:1994

数据来源: WILEY

摘要:

AbstractIn previous studies, we have reported on the expression of β1 intergrins in type 1 astrocytes and their function in cell‐substratum attachment (Tawil et al., J Cell Biol 120:261–271, 1993). Here we extend those findings by providing evidence that type 1 astrocytes express integrins of the β3 and possibly β4 subclasses and that the former (αvβ3) functions in attachment by recognizing the peptide, Arg‐Gly‐Asp, in vitronectin. In addition, we have examined immunocyto‐chemically the expression of β1 integrins on type 2 astrocytes and oligodendrocytes. The pattern of expression of integrins on these two cell types is distinct from type 1 astrocytes; most notably type 1 astrocytes but not oligodendrocytes or type 2 astrocytes express α1β1 heterodimers. Since type 2 astrocytes and oligodendrocytes originate from a common precursor (0‐2A), the α1β1 heterodimer may be a functional marker which distinguishes O‐2A‐derived cells from those of the type 1 astrocyte lineag

ISSN:0360-4012    

DOI:10.1002/jnr.490390411

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

年代:1994

数据来源: WILEY