From the Department of Pharmacology, University of
Illinois at Chicago, Chicago, Illinois 60612, the
§ Department of Biochemistry, University of Chicago School
of Medicine, Chicago, Illinois 60637, and the
¶ Institut de Recherches Scientifiques Sur Le Cancer,
94801 Villejuif, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The presence of functional thromboxane A2 receptors in neonatal rat oligodendrocytes and human oligodendroglioma cells was investigated using immunocytochemistry, ligand affinity chromatography, radioligand binding analysis, immunoblot analysis, and calcium mobilization studies. Immunocytochemical studies revealed the presence of receptor protein on both oligodendrocytes and human oligodendroglioma cells. Ligand affinity chromatography allowed for the purification of a protein with an electrophoretic mobility (55 kDa) indistinguishable from human platelet thromboxane A2 receptors. This affinity purified protein was immunoreactive against a polyclonal anti-thromboxane A2 receptor antibody. Intact human oligodendroglioma cells specifically bound [3H]SQ29,548 with a KD of 4 nM and were found to have approximately 3500 binding sites per cell. Human oligodendroglioma cells also demonstrated calcium mobilization in response to receptor activation with U46619. These results demonstrate the presence of a functional thromboxane A2 receptor in oligodendrocytes and are consistent with previous observations indicating a high density of thromboxane A2 receptors in myelinated brain and spinal cord fiber tracts.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thromboxane A2
(TXA2)1 is a
bioactive arachidonic acid metabolite with thrombotic, vasospastic, and
bronchospastic properties (1-3). It has been implicated in a variety
of physiological and pathophysiological states (4). The receptor for
TXA2 has been purified (5, 6), cloned, and sequenced, and
the structure of the gene indicates that this receptor is a member of
the seven transmembrane domain, G-protein-coupled receptor superfamily. The receptor protein has been located in a variety of tissues including
blood platelets (7), vascular smooth muscle (8, 9), vascular
endothelium (10), placenta (11), and kidney (12) and appears to be
directly involved in the regulation of intracellular calcium levels
(13). In this regard, previous studies have provided evidence that the
TXA2 receptor protein is coupled to at least one G-protein,
i.e. Gq in platelets (14, 15) and astrocytes
(16), and appears to modulate phospholipase C- activity resulting in
activation of the inositol triphosphate second messenger system (17,
18).
Although highly specific agonists and antagonists have proven to be of significant value in characterizing TXA2 receptors in a number of different cell lines, little information is currently available concerning their existence or function in the central nervous system. To a large extent, this has been due to the presence of high lipid concentrations in central nervous system tissue. Thus, nonspecific binding has obscured the demonstration of receptor binding in solubilized brain, due to the lipophilic nature of TXA2 receptor ligands. In addition, the heterogeneity of cell types in the central nervous system has complicated attempts at receptor localization at a cellular level.
Based on these limitations, previous studies have employed cultured cell systems in an effort to investigate the presence of TXA2 receptors in the central nervous system. In these experiments, it was demonstrated that cultured rabbit astrocytes were capable of specifically binding the TXA2 ligand ONO NT-126 (20). These results, therefore, provided evidence that at least one cellular component of the central nervous system possesses TXA2 receptor activity. However, since astrocytes are ubiquitously distributed in the brain, it has not been possible to ascribe a specific or unique function for TXA2 in the various brain structures.
On the other hand, a separate approach has involved the use of TXA2 receptor antibodies. These experiments provided documentation that TXA2 receptors are in fact localized in discrete brain regions (21). Specifically, polyclonal antibodies against the complete receptor protein (TxAb), as well as against two decapeptide sequences (P1Ab and P2Ab) of the receptor, revealed a high density of TXA2 receptors in myelinated fiber tracts, notably in the striatum, spinal cord, and optic tract (Fig. 1). Given that astrocytes and vascular endothelial cells both possess TXA2 receptors (10, 20), the preferential labeling of myelinated fiber tracts indicates the existence of a highly concentrated pool of TXA2 receptors separate from either astrocytes or vascular endothelial cells. One possible source of this high density labeling pattern would be oligodendrocytes, the cell type exclusively responsible for myelin synthesis in the central nervous system.
|
To determine whether or not oligodendrocytes possess TXA2 receptors, the present experiments were undertaken using cultured neonatal rat oligodendrocytes (OLG) and human oligodendroglioma (HOG) cells (22). Immunocytochemistry, immunoblot analysis of solubilized membrane proteins, ligand affinity chromatography of TXA2 receptor protein, and radioligand binding studies provided results that demonstrate that both OLG and HOG cells possess thromboxane A2 receptor protein. The identification of this receptor in oligodendrocytes, in conjunction with its high level of expression in myelinated fiber tracts, suggests that thromboxane A2 may play an important role in modulating oligodendrocyte function.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Pregnant female Sprague-Dawley rats were obtained from Sasco (Madison, WI). Human oligodendroglioma cells were a generous gift from Dr. Glyn Dawson, Department of Biochemistry, University of Chicago. Poly-L-lysine, bovine pancreatic insulin, human apo-transferrin, cytosine-D-arabinofuranoside, deoxyribonuclease I (DNase I), sodium selenite, fura-2/AM, Pluronic F-127, CHAPS, polyclonal anti-glial fibrillary acidic protein (anti-GFAP), and anti-galactocerebroside (anti-GalCer) antibodies were purchased from Sigma. Mouse monoclonal anti-myelin basic protein (anti-MBP) was purchased from Boehringer Mannheim. Dulbecco's modified Eagle's medium (DMEM) supplemented with glucose (4.5 g/liter) and L-glutamine was purchased from Mediatech (Washington, D. C.). Fetal bovine serum (FBS) was purchased from BioWhittaker (Walkersville, MD). Chamber slides for immunocytochemistry were from Nunc Corp. (Naperville, IL), and 75-cm2 tissue culture flasks were obtained from Bellco Glass Co. (Vineland, NJ). Nylon mesh (30- and 100-µm pore) was purchased from Tetko, Inc. (Briarcliff Manor, NY). All other tissue culture plasticware was from Fisher. Trypsin and antibiotic/antimycotic solution was purchased from Life Technologies, Inc. 4-Chloro-1-naphthol (horseradish peroxidase color developing reagent) and the Bradford protein assay kit were from BioRad. Biotinylated goat anti-rabbit IgG (H + L), biotinylated goat anti-mouse IgG (H + L), FITC-conjugated goat anti-rabbit IgG, and the Vectastain ABC kit were from Vector Laboratories (Burlingame, CA). TxAb and P2Ab were produced as described previously (21, 23). [3H]SQ29,548 was obtained from NEN Life Science Products. SQ29,548 was obtained from Cayman Chemical Co. (Ann Arbor, MI), and BM13.177 (24) was a generous gift from Dr. K. Stegmeier, Boehringer Mannheim GmbH (Mannheim, Germany). Triton X-100-solubilized adult ovine oligodendrocyte membranes (25) were kindly provided by Dr. Sara Szuchet, Department of Neurology, University of Chicago (Chicago, IL).
Cell Culture
Mixed Glial Cultures-- Oligodendrocytes were prepared from 2-day-old Sprague-Dawley rat pups using a modification of the method of McCarthy and de Vellis (26). Protocols for animal use were approved by the University of Illinois at Chicago Animal Care Committee. Briefly, animals (approximately 15 per culture procedure) were sacrificed by decapitation and the cerebral hemispheres removed in a sterile manner. Meninges and large blood vessels were removed by rolling the tissue over sterile Whatman No. 1 filter paper to reduce fibroblast contamination. Pooled tissue was immersed in ice-cold, serum-free DMEM and minced prior to enzymatic dissociation with trypsin and 100 Kunitz DNase I (30 min at 37 °C). The pooled tissue was then mechanically disaggregated by gentle trituration through a narrow-bore Pasteur pipette and by filtration through 100-µm nylon mesh. The resultant mixed cell culture was distributed into 75-cm2 tissue culture flasks that had been treated with 0.1 mg/ml poly-L-lysine to facilitate attachment. Cells were plated at a density of approximately 2 cerebral hemispheres per flask and maintained in a medium consisting of DMEM supplemented with 10% FBS, penicillin, streptomycin, and amphotericin B. Mixed glial cultures were kept at 37 °C in an atmosphere of 95% air, 5% CO2. Culture media was changed every 3rd day for the 1st week and every other day for the 2nd week.
Purified Oligodendrocyte Cultures-- Oligodendrocytes were purified from mixed glial cultures on day 14 by mechanical dissociation involving shaking on a rotary shaker (220 rpm × 14 h at 37 °C). Once the OLG precursors had been dislodged, the cells were pooled, washed with warm DMEM/10% FBS, and dissociated into a single cell suspension by trituration through a narrow-bore Pasteur pipette. The cells were then filtered through 30-µm nylon mesh, counted, and plated in 100 × 20-mm poly-L-lysine-treated tissue culture dishes at a density of approximately 1 × 107 cells per plate. Cells were incubated in DMEM/ 10% FBS for 3 h at 37 °C in an atmosphere of 95% air, 5% CO2 to allow for attachment. Following this incubation, the media were changed to DMEM/3% FBS supplemented with biotin, insulin, transferrin, ARA-C, antibiotic/antimycotic solution, and sodium selenite (27). Oligodendrocytes were typically used for experiments by the 4th day following subculture.
Human Oligodendroglioma Cells-- A previously characterized line of human oligodendroglioma cells (HOG) was maintained by continuous cell culture (22). HOG cells from a 5th generation culture were initially distributed on 100 × 20-mm tissue culture dishes in DMEM/10% FBS supplemented with antibiotic/antimycotic solution. Cells were maintained at 37 °C in an atmosphere of 95% air, 5% CO2. The 5th generation culture was expanded with approximately 5 × 108 cells preserved in DMEM/10% FBS with 10% Me2SO4 in liquid nitrogen until needed. The remaining cells were passaged and used accordingly. Cells were not used after the 12th passage.
Membrane Solubilization
Solubilized oligodendrocytes (for immunoblot analysis and ligand affinity chromatography) were prepared by aspirating the culture medium and incubating the cells with serum-free DMEM for 15 min at 37 °C. Cells were then sequentially washed with warm serum-free DMEM (2 × 5 ml) and with ice-cold 25/5 buffer (25 mM Tris, 5 mM MgCl2, pH 7.4). Three ml of solubilizing buffer (50 mM Tris-HCl, 5 mM MgCl2, 2 mM CHAPS) supplemented with protease inhibitors (100 µM EDTA, 1.0 µM leupeptin, 1.0 mM phenylmethylsulfonyl fluoride) were added to each tissue culture dish, and the cells were incubated at 4 °C with constant rocking. Unlysed cells and cellular debris were removed by centrifugation. Protein concentration in pooled solubilization fractions was determined according to the method of Bradford (28). HOG cells were solubilized according to a previously described method (6).
Immunocytochemistry
Oligodendrocytes or HOG cells were plated onto Nunc 4- or 8-well polystyrene chamber slides that had been pretreated with poly-L-lysine (0.1 mg/ml). Cells were thoroughly washed with warm, serum-free DMEM and then fixed using 2% paraformaldehyde in phosphate-buffered saline for 1 h at 4 °C. Unreacted aldehyde groups were blocked with NH4Cl in Tris-buffered saline (TBS, 30 mM Tris, 120 mM NaCl, pH 7.4), and nonspecific antibody binding sites were blocked with 3% normal goat serum in DMEM. For the detection of intracellular epitopes, cells were permeabilized with 0.1% Triton X-100 for 3 min, followed by treatment with paraformaldehyde, NH4Cl, and normal goat serum. Cells were then incubated at room temperature with the appropriate dilutions of primary antibody (TxAb, P2Ab, anti-MBP, or anti-GalCer) or a pre-immune IgG control for 30 min. After washing with TBS, cells were then incubated with the appropriate secondary antibody (1:200). Both primary and secondary antibodies were diluted in 3% normal goat serum in TBS. Preparations for light microscopy were stained using a metal-enhanced DAB kit (Pierce) and mounted in glycerol. FITC-stained cells were visualized using a Jenval microscope with the appropriate fluorescence optics. Preabsorbtion of P2Ab was accomplished by overnight incubation of the antibody with 0.1 M P2 peptide (HAALFEWHAV) at 4 °C. Appropriate control experiments were performed by staining cells in the absence of a primary antibody, secondary antibody, or chromagen.
SDS-PAGE, Silver Staining, Immunoblot Analysis
Solubilized OLG or HOG cell membranes or ligand affinity purified TXA2 receptors were subjected to SDS-PAGE on 10% polyacrylamide gels, under nonreduced conditions, according to the method of Laemmli (29). For immunoblot analysis of TXA2 receptors, the proteins were transferred onto nitrocellulose membranes according to the method of Towbin (30). After transfer, nonspecific antibody binding sites were blocked with 3% gelatin in TBS for 2 h and then incubated with the appropriate primary antibody, diluted in 1% gelatin/TBS, overnight at room temperature. Bound antibody was detected by the Vectastain ABC kit method using a biotinylated goat anti-rabbit IgG (H + L) or goat anti-mouse IgG (H + L), diluted 1:500 in 1% gelatin/TBS, as the secondary antibody. Color bands were developed using 4-chloro-1-naphthol. Silver staining of polyacrylamide gels was performed according to the method of Morrissey (31).
Radioligand Binding Assay
Binding of [3H]SQ29,548 to intact HOG cells was evaluated using a vacuum filtration assay. Confluent layers of HOG cells were incubated for 30 min in warm, serum-free DMEM and then harvested using gentle trypsinization and mechanical dissociation with a stream of warm DMEM delivered through a Pasteur pipette. The monolayer was dissociated into a single cell suspension by gentle trituration. Maintenance of cell integrity, cell counts, and extent of dissociation were monitored by light microscopy and assessment of trypan blue uptake. Cells were diluted to a concentration of 1 × 107 cells/ml and aliquoted into glass tubes (0.5 ml/tube). Following a 5-min incubation at room temperature with either buffer (total binding) or 20 µM unlabeled SQ29,548 (nonspecific binding), the cells were incubated for 20 min at room temperature with various concentrations of [3H]SQ29,548. At the end of the incubation period, the cells were vacuum filtered through GF/C glass fiber filters, and the filters were rapidly washed (2 × 5 ml) with ice-cold 25/5 buffer. The receptor-bound radioligand was measured by liquid scintillation spectrometry. Specific binding was calculated as the difference between binding measured in the absence and in the presence of 20 µM unlabeled SQ29,548. Scatchard analysis of radioligand binding data was performed using the EBDA and LIGAND software packages (32).
Ligand Affinity Chromatography
TXA2 receptors were purified by the method of Kim et al. (6). Specifically, CHAPS-solubilized OLG or HOG cell membranes in buffer A (20 mM Tris, 10 mM CHAPS, 20% (v/v) glycerol, 550 mM KCl, 0.2 mM EGTA, 0.5 mg/ml asolectin) were incubated overnight at 4 °C with an affinity matrix consisting of the specific TXA2 receptor ligand SQ31,491 immobilized to Affi-Gel 102 (Bio-Rad). Unbound proteins were eluted as flow-through, and the affinity column was washed with 14 ml of buffer A. TXA2 receptors were eluted with buffer A containing 50 mM BM13.177 at a flow rate of 1 ml/8 min. Following elution of the first 1-ml fraction, flow was stopped for 30 min and restarted to elute the subsequent 1-ml fractions. Receptor content was assessed by immunoblot analysis utilizing TxAb (1:500) as a probe.
Measurement of Intracellular Ca2+ Mobilization
Cytosolic calcium levels were measured in HOG cells using the method of Lum and co-workers (33). HOG cells were grown on poly-L-lysine treated, 25-mm diameter glass coverslips in DMEM (phenol red-free) supplemented with 10% FBS until cells were >75% confluent. Cells were serum-starved for 2 days in DMEM supplemented with 0.2% lactalbumin enzyme hydrolysate and then loaded with 2 µM fura-2/AM for 45 min at room temperature. Pluronic F-127 (0.5% (w/v)) was employed as a dispersing agent for the AM ester. Following a 2 × wash with Hanks' buffered salt solution, coverslips were placed in a Sykes-Moore chamber (Bellco, Vineland, NJ). The chamber was placed onto the stage of an inverted Nikon Diaphot microscope that was equipped with quartz optics and coupled to a Deltascan microspectrofluorometer system (PTI, Inc., Princeton, NJ). An optically isolated group of 3-4 cells was illuminated by a 75-watt xenon arc lamp at alternating excitation wavelengths of 340 and 380 nm. The emitted light was passed through an interference filter at 510 nm and collected via a photomultiplier. Fluorescence intensity was measured at 10 points/s. Background autofluorescence (in the absence of fura-2) was determined at the beginning of each experiment and was subtracted automatically during data collection. At the end of each experiment, 10 µM ionomycin was added to obtain fluorescence of Ca2+-saturated fura-2, and 0.1 M EGTA was added to obtain fluorescence of free fura-2. The fluorescence ratio of excitation wavelengths 340 and 380 nm (R340/380) were computed using the PTI software. Statistical analysis was performed using the Student's t test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Morphologically, cultured oligodendrocytes demonstrated a complex network of cytoplasmic processes, as well as numerous intra-process connections characteristic of this cell type (Fig. 2, A and B). Identification of cells as differentiated oligodendrocytes was accomplished by immunohistochemical labeling of subcultured cells with anti-myelin basic protein (anti-MBP), an OLG specific marker (Fig. 2A). Solubilized oligodendrocytes from primary culture were also found to immunoreact with anti-MBP, producing an immunoreactive protein band at approximately 21.5 kDa (Fig. 2D). The degree of purity of OLG cultures was further established by the absence of labeling with anti-glial fibrillary acidic protein (anti-GFAP), an astrocyte-specific protein (Fig. 2B). Human oligodendroglioma (HOG) cells appeared flat and epithelioid in culture, demonstrating short, narrow cytoplasmic processes. These cells, which have been shown to possess some OLG-specific proteins, were immunoreactive against an anti-GalCer antibody (Fig. 2C).
|
To determine whether cultured OLG and/or HOG cells contained TXA2 receptors, immunohistochemical studies were undertaken using antibodies against the purified receptor (TxAb). Both cell types revealed a marked staining of TXA2 receptors in fixed, Triton X-100-permeabilized cells (Fig. 3, A and D). Unpermeabilized HOG cells demonstrated labeling that was restricted to the plasma membrane (Fig. 3C). On the other hand, no staining was observed when using pre-immune IgG in either cell type (Fig. 3, B and E).
|
Additional evidence for the existence of TXA2 receptors in HOG cells was obtained using a different polyclonal antibody (P2Ab), raised against a decapeptide sequence from the first extracellular loop of the receptor protein (residues 89-98). As can be seen in Fig. 4, A and B, incubation of HOG cells with P2Ab resulted in a positive staining pattern that was abolished by preabsorption of the antibody with the peptide antigen (P2). Collectively, these results indicate that both OLG and HOG cells possess TXA2 receptors.
|
Expression of TXA2 receptors was further established using SDS-PAGE and immunoblot analysis. Specifically, HOG and OLG cell membrane proteins were extracted by treating washed cells with CHAPS at 4 °C. Unlysed cells were removed by centrifugation, and soluble proteins were resolved by electrophoresis and blotted onto nitrocellulose. A human platelet solubilized membrane preparation (SMP) was immunoblotted in parallel as a positive control. It was found (Fig. 5, lanes 2-4) that TxAb (diluted 1:500) not only immunoreacted with TXA2 receptor protein in platelet SMP but also revealed positive immunoreactivity against the 55-kDa receptor protein in solubilized OLG cells and HOG cell membranes. This immunoreactivity was not observed when probing either cell type with the pre-immune IgG (Fig. 5, lanes 6-9). To confirm that the positive immunoreactivity seen in solubilized OLG and HOG cells was not due to expression of the receptor in the immature or transformed phenotypes, respectively, immunoblot analysis was performed on a Triton X-100 soluble membrane fraction prepared from adult ovine oligodendrocytes (Fig. 5, lane 5). This experiment demonstrated a single immunoreactive band present at 55 kDa.
|
To purify the TXA2 receptor present on oligodendrocytes, affinity chromatography was utilized. In this experiment, solubilized OLG proteins were incubated with an SQ31,491 ligand affinity column previously shown to purify TXA2 receptors from platelets (6). The column was then eluted with the receptor antagonist (BM13.177), and SDS-PAGE analysis of the wash and the elution fractions was performed. It can be seen (Fig. 6A) that while the final wash fraction (W4, lane 4) was essentially devoid of protein, competition with BM13.177 resulted in the elution of a major protein band at 55 kDa (lanes 5 and 6). Evidence that this 55-kDa protein represented the TXA2 receptor was obtained from immunoblot analysis which revealed positive reactivity with TxAb (Fig. 6B). Taken together, these results represent the first purification of TXA2 receptors from oligodendrocytes and provide evidence that this receptor protein has a molecular mass in the range of 55 kDa.
|
Because oligodendrocytes synthesize large quantities of lipids and proteolipids, which result in increased nonspecific binding of lipophilic TXA2 receptor ligands, the next series of experiments was undertaken to evaluate the capability of intact HOG cells to specifically bind the TXA2 antagonist [3H]SQ29,548. Using a modification of the protocol employed in detecting TXA2 binding sites in astrocytoma cells (19), these experiments revealed a single class of high affinity binding sites, with a dissociation constant (KD) of 4 ± 0.2 nM and a maximum binding of 34 ± 4 pM (Fig. 7). The calculated maximum binding (Bmax) of 34 pM corresponds to approximately 3500 binding sites per HOG cell. This KD of 4 nM is comparable to that obtained for the TXA2 receptor in human platelets (KD = 7.3 nM) (34) and 1321N1 human astrocytoma cells (KD = 10.9 nM) (19).
|
The final series of experiments investigated the ability of a TXA2 receptor agonist to modulate intracellular calcium levels in HOG cells. Cells grown on glass coverslips were loaded with fura-2/AM and exposed to various concentrations of U46619 in the presence or absence of the receptor antagonist, SQ29,548. It was found that concentrations of U46619 as low as 300 nM were capable of eliciting intracellular Ca2+ mobilization as visualized by an increase in the fluorescence ratio of excitation wavelengths 340 and 380 nm (R340/380). The time course of the U46619-induced calcium mobilization revealed a biphasic response (Fig. 8A). A challenge with 300 nM U46619 caused an initial rapid increase in calcium, which peaked by 19 ± 4 s. The peak rise in Ca2+ (224 ± 15% over base line, n = 3, p = 0.0002) was followed by a second phase of slow decrease to half-maximal (t1/2) within 30 s and a prolonged plateau phase at a higher R340/380. Pretreatment of HOG cells with 100 nM SQ29,548 for 3 min prior to agonist stimulation completely ablated the U46619-mediated response (Fig. 8B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent evidence suggests the existence of thromboxane A2 receptors in cells of the central nervous system. In this connection, radioligand binding assays have revealed the presence of TXA2 binding sites in cultured rabbit astrocytes (35), and electrophysiological experiments have demonstrated a TXA2 response in CA1 hippocampal neurons (36). In addition, association of TXA2 receptor protein with myelinated tracts in adult rat brain and spinal cord slices has been provided using immunohistochemical techniques (21). Based on this latter finding, the present series of experiments were undertaken to investigate the presence of the thromboxane A2 receptor in oligodendrocytes, the cell type responsible for central nervous system myelination.
In the initial studies, cultured OLG and HOG cells were immunocytochemically probed with antibodies directed against the purified TXA2 receptor (TxAb) and against a decapeptide segment from a putative extracellular receptor loop (P2Ab). These experiments provided evidence for specific receptor labeling on the plasma membranes of both cell types and receptor labeling of non-nuclear intracellular sites in permeabilized HOG cells. The molecular size of the labeled protein was next established by SDS-PAGE of CHAPS-solubilized OLG and HOG cells. The observed electrophoretic mobility of this protein (55 kDa) was indistinguishable from that of the TXA2 receptor purified from platelets.
The functional ability of this 55-kDa protein to bind TXA2 receptor ligand was investigated using affinity chromatography. In these studies, solubilized oligodendrocytes were incubated with a ligand affinity matrix. Elution of the column matrix resulted in the selective purification of receptor protein having an apparent molecular mass of 55 kDa.
Separate experiments evaluated the ability of intact HOG cells to bind a TXA2 receptor antagonist ([3H]SQ29,548). The results demonstrated that HOG cells exhibit approximately 3500 binding sites per cell with a KD of approximately 4 nM. These values are comparable to those observed in human platelets (i.e. Bmax = 2000 and KD = 5 nM).
Finally, experiments were conducted to investigate the functional significance of TXA2 receptor activation in these cells. Since the TXA2 receptor is known to be linked to increases in platelet (37) and astrocyte (53) cytosolic calcium, it was determined whether receptor activation leads to a similar effect in HOG cells. Utilizing fura-2 as an indicator of changes in intracellular calcium concentration, HOG cells were shown to respond to the TXA2 mimetic U46619, and this effect could be blocked by pretreatment of cells with the TXA2 receptor antagonist SQ29,548.
Collectively, these findings demonstrate that oligodendrocytes possess functional TXA2 receptors. However, in order for TXA2 to play a role in the modulation of OLG cell function or development, a source for this eicosanoid must be made available under physiological and/or pathological conditions. In this respect, oligodendrocytes may have access to TXA2 in at least three possible manners. First, although the identification of an exact in vivo source remains controversial, TXA2 has been shown to be generated by astrocytes (38), microglia (39), and vascular endothelial cells (10) in culture. With regard to astrocytes, a ubiquitously distributed glial cell, activation of phospholipase A2 and production of TXA2 by thromboxane synthase occurs in response to stimulation of cells by mediators such as platelet-activating factor (40) and ATP (41).
A second possible source of TXA2 may be as a consequence of central nervous system injury. In this case, compromise of the blood-brain barrier, either by mechanical trauma or by cerebrovascular insult, could potentially result in exposure of oligodendrocytes and myelin-ensheathed neurons to TXA2 which was produced by activated platelets or other blood system components (42). In addition, a dramatic increase in the generation of arachidonic acid metabolites and reactive lipid mediators, with subsequent breakdown of the blood-brain barrier, has been shown to occur following post-hypoxic or post-anoxic injury and reperfusion (43). In this regard, oligodendrocyte damage has been shown to occur following ischemic neuronal damage and the breakdown of the blood-brain barrier, and an apoptosis-like process has been implicated in ischemic oligodendrocytic death (44).
Finally, inflammatory processes within the brain parenchyma, which
trigger the activation of microglia, can lead to the production of
TXA2 (39). In addition, the cytokine interleukin-1 has
been shown to elicit TXA2 release from astrocytes (45).
Thus, a variety of physiological and pathophysiological conditions
could lead to the exposure of oligodendrocytes to TXA2.
With regard to a possible role for TXA2 in this cell type, it should be noted that oligodendrocytes are a highly specialized and terminally differentiated cell type, solely responsible for central nervous system myelination. Oligodendrocytes originate from a bipotential precursor cell (O-2A) (46) and undergo a complex differentiation process that directs the cells to synthesize large quantities of the protein, glycoprotein, and proteolipid components of myelin sheaths (47). At present, relatively little information is available concerning the endogenous or exogenous regulation of myelin protein synthesis and maintenance. However, recent evidence suggests that changes in intracellular calcium may modulate myelin basic protein levels. In these studies, it was found that treatment of cultured oligodendrocytes with the calcium ionophore, ionomycin, led to oligodendrocyte injury and vesicular demyelination, possibly through activation of calcium-dependent proteases (48, 49). In addition, the authors of these studies observed that oligodendrocytes were more sensitive to the effects of increases in [Ca2+]i than astrocytes. Thus, signal transduction mechanisms, e.g. TXA2 receptor activation, which lead to elevations in intracellular calcium, may serve to regulate myelin degradation.
Alternatively, TXA2 receptors may serve to modulate myelin homeostasis through a protein kinase C-mediated pathway. In this regard, it is commonly accepted that stimulation of TXA2 receptors leads to inositol triphosphate-mediated intracellular calcium mobilization and diacylglycerol generation. Furthermore, activation of protein kinase C by diacylglycerol in oligodendrocytes has been shown to lead to phosphorylation of myelin basic protein (50, 51). Since it has been postulated that phosphorylation of MBP may act as a myelinogenic signal, TXA2 receptor activation could play a role in elaboration and/or maintenance of myelin membranes.
In summary, the present results provide the first demonstration of functional thromboxane A2 receptors on neonatal rat oligodendrocytes and human oligodendroglioma cells. These findings, coupled with the high density expression of the receptor in myelinated tracts in situ, raise the possibility that thromboxane may play an important role in the regulation of oligodendrocyte function. Clearly, additional experiments are required to explore this interesting possibility.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully appreciate the assistance of Dr. Hazel Lum, Dept. of Pharmacology, University of Illinois at Chicago, for assistance with measurement of intracellular calcium, and Dr. Yan Qi, Dept. of Pharmacology, Northwestern University, Chicago, for assistance with oligodendrocyte primary culture.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL-24530, NATO Grant CRG-940595, and The Blowitz Foundation. This work was conducted under the auspices of the Association for United States-French Biomedical Cooperation. A preliminary report of this work was presented at a joint meeting of the International Society for Neurochemistry and the American Society for Neurochemistry, Boston, MA, July 1997.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Ave. (MC 868), Chicago, IL 60612. Tel.: 312-996-4983; Fax: 312-996-1225; E-mail: gcl{at}tigger.uic.edu.
1
The abbreviations used are: TXA2,
thromboxane A2; OLG, oligodendrocyte; HOG, human
oligodendroglioma; SMP, human platelet solubilized membrane
preparation; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE,
polyacrylamide gel electrophoresis; FBS, fetal bovine serum; MBP,
myelin basic protein; GFAP, glial fibrillary acidic protein, GalCer,
galactocerebroside; TBS, Tris-buffered saline; DAB, diaminobenzamide;
FITC, fluorescein isothiocyanate; U46619, 9,11-dideoxy-9,11
-epoxymethanoprostaglandin F2
;
DMEM, Dulbecco's modified Eagle's medium; H + L, heavy and light
chain; SQ29,548, [1S-[1
, 2
(5z), 3
,
4
]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|