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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2001 as doi:10.1096/fj.00-0625fje. |
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* Institute of Clinical Pharmacology and Toxicology, Freie Universität Berlin, Benjamin Franklin Medical Center, Berlin, Germany;
Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; and
Department of Neurology, Jinan University Guangzhou, P.R. of China
2Correspondence: Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität Berlin, Garystrasse 5, 14195 Berlin, Germany. E-mail: paul{at}medizin.fu-berlin.de
SPECIFIC AIMS
In this study, we addressed the hypothesis that hypoxia can induce morphological changes in cultured astrocytes (AC) similar to those observed in vivo after ischemia or hypoxia of the brain. In addition, we investigated the role of the astrocytic endothelin (ET) system in mediating this response using northern blotting, RT-PCR, ET radioimmunoassay, calcium imaging, and ET receptor antagonists.
PRINCIPAL FINDINGS
1. Hypoxia triggers a morphological transformation of AC
In culture medium under serum-free conditions, AC are
characterized by a flat and polygonal cell shape (Fig. 1A
). We induced AC to acquire a stellate morphology similar to
their appearance in vivo by incubating cultures with 1 mM
dibutyryl-cAMP (DBcAMP) for 
24 h in serum-free medium.
Exposure of these cells to hypoxia for 24 h resulted in a
morphological transformation to the flat, polygonal form (Fig. 1E
). In control normoxic conditions, the stellate
cell shape was preserved (Fig. 1B
). To quantify the
morphological transformation, stellate cells were scored according to
the criteria by Kimelberg et al., i.e., cells having two or more
processes at least twice as long as the diameter of the cell body were
defined as stellate.
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2. ET-1 is a signaling substance to mediate the hypoxia-induced
change in morphology
Cultured AC constitutively release ET into the supernatant as
determined by radioimmunoassay. The peptide level in culture
supernatants of normoxic AC was 2.92 + 0.20 pg/mg protein
24 h after a medium change (n=4). Hypoxia induced an
increase in the level of ET to about 150%, namely, 4.23 +
0.56 pg/mg protein in the supernatants of AC exposed to 24 h of
hypoxia (n=4; P<0.005).
To test whether the increased level of ET could be an astrocytic
mediator of the hypoxia-induced morphological transformation, we
applied exogenous ET-1 to the supernatant and analyzed the
morphological appearance of AC. Addition of 100 nM synthetic ET-1 to
the culture media mimicked the morphological changes induced by hypoxia
(Fig, 1C). The involvement of the ET system was further
substantiated by the finding that PD142893 or PD145065 (5 µM), two
nonselective ET receptor antagonists, blocked the effects of hypoxia
(Fig. 1F
) or ET-1 (Fig. 1D
) on astrocyte
morphology.
To examine the molecular changes in the astrocytic ET system, we
performed Northern blots and RT-PCR. Prepro ET-1 mRNA was detectable in
extracts of cultured AC using Northern analysis. Exposure to hypoxia
for 24 h increased prepro ET-1 mRNA levels significantly to about
300% of baseline levels (Fig. 2
). Prepro ET-3 mRNA and ECE-1 mRNA were not detectable by Northern
blots. However, using RT-PCR, we were able to amplify both, prepro ET-3
and ECE-1 transcripts, suggesting a low baseline expression level of
these ET system components. ECE-2 mRNA was present in normoxic and
hypoxic AC without a significant difference in the mRNA level, as
determined by Northern analysis (Fig. 2)
.
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3. ET receptors are down-regulated on the trancriptional level, but
remain functional on hypoxic AC
Under normoxic conditions, ETA receptor
(ETAR) and ETB receptor
(ETBR) mRNAs were detectable in Northern blots of
total RNA from cultured AC. Hypoxia induced a significant
down-regulation of the ETAR and
ETBR mRNA, namely, to about 20% of their
baseline levels (Fig. 2)
. Both ETAR and
ETBR are G-protein-coupled receptors that lead to
a release of Ca2+ ions from internal stores. To
investigate, whether the down-regulation of ETAR
and ETBR mRNA detected by Northern blots affected
receptor functionality, we tested for the cells‘ ability to respond
with an increase in Ca2+ after application of ET.
Extracellular application of 100 nM of the nonselective ET isoform ET-1
triggered a [Ca2+]i
transient in 61 ± 36% of the hypoxic cells as compared to
96±9% of normoxic AC. To test for viability of individual cells, ATP
(100 µM) was delivered 3–5 min after the ET-application,
which generated an easily detectable
[Ca2+]i response. These
results demonstrate that ET receptors, although down-regulated on the
mRNA level, still mediate physiologic responses in the majority of
hypoxic AC.
CONCLUSIONS
Many authors have discussed the morphology of AC in a brain that has been subjected to ischemia or hypoxia. Loss of astrocytic processes during global cerebral ischemia was first recognized by Alzheimer in 1910. He noted that AC can undergo a process he termed ‘clasmatodendrosis’, which results in these cells assuming an amoeboid shape with a clear reduction in surface area. Recent ultrastructural studies revealed the disappearance of astrocytic processes from adjacent dendrites after hypoxia in rats. These changes after hypoxia are believed to promote neuronal deterioration by reducing astrocytic uptake of neurotransmitters and potassium. To isolate these astrocytic responses from the cerebral microenvironment and to investigate the underlying molecular mechanisms, it is important to establish an in vitro model. However, researchers had so far been unable to observe morphological changes in cultured AC subjected to hypoxia. In this study, we used cultured AC pretreated with DBcAMP. This substance induces a process-bearing, differentiated appearance of cultured AC. When subjected to hypoxia for 24 h, AC reassumed the flat, polygonal appearance of untreated cells. We therefore were able to generate an experimental model mimicking clasmatodendrosis in vitro showing that it can take place independent of the cerebral microenvironment.
AC produce components of the ET system. ET has been previously linked to morphological alterations in AC. Therefore, we sought to characterize the astrocytic ET system responses to hypoxia and their relation to the morphological transformation. Northern blotting and RT-PCR experiments, ET radioimmunoassay, and functional experiments on ET receptors indicate an activation of the astrocytic ET system in response to hypoxia. The amounts of ET-1 in the supernatants and of prepro ET-1 mRNA in cell extracts are increased in hypoxic cells. At the same time, ET receptors are down-regulated on the transcriptional level, but remain functionally active. Most important, when we applied ET receptor antagonists, we were able to prevent the morphological transformation of hypoxic AC. The morphological transformation of hypoxic AC, therefore, can be causally linked to the activation of the ET system and blocked by specific pharmacological intervention.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0625fje ; to cite this
article, use FASEB J. (March 28, 2001)
10.1096/fj.00-0625fje 
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