Naloxone and Ouabain in Ultralow Concentrations Restore Na⁺/K⁺-ATPase and Cytoskeleton in Lipopolysaccharide-treated Astrocytes

Abstract

Astrocytes respond to inflammatory stimuli and may be important modulators of the inflammatory response in the nervous system. This study aimed first to assess how astrocytes in primary culture behave in response to inflammatory stimuli concerning intracellular Ca²⁺ responses, expression of Toll-like receptor 4 (TLR4), Na⁺/K⁺-ATPase, actin filament organization, and expression of cytokines. In a cell culture model with lipopolysaccharide (LPS), astrocyte response was assessed first in the acute phase and then after incubation with LPS for 1–48 h. The concentration curve for LPS-stimulated Ca²⁺ responses was bell-shaped, and the astrocytes expressed TLR4, which detects LPS and evokes intracellular Ca²⁺ transients. After a long incubation with LPS, TLR4 was up-regulated, LPS-evoked Ca²⁺ transients were expressed as oscillations, Na⁺/K⁺-ATPase was down-regulated, and the actin filaments were disorganized. Interleukin-1β (IL-1β) release was increased after 24 h in LPS. A second aim was to try to restore the LPS-induced changes in astrocytes with substances that may have dose-dependent anti-inflammatory properties. Naloxone and ouabain were tested separately in ultralow or high concentrations. Both substances evoked intracellular Ca²⁺ transients for all of the concentrations from 10⁻¹⁵ up to 10⁻⁴ m. Neither substance blocked the TLR4-evoked Ca²⁺ responses. Naloxone and ouabain prevented the LPS-induced down-regulation of Na⁺/K⁺-ATPase and restored the actin filaments. Ouabain, in addition, reduced the IL-1β release from reactive astrocytes. Notably, ultralow concentrations (10⁻¹² m) of naloxone and ouabain showed these qualities. Ouabain seems to be more potent in these effects of the two tested substances.

Introduction

Infection, tissue damage, or other conditions can lead to a neuroinflammatory state. Lipopolysaccharide (LPS) exposure can be used experimentally as a model to generate an inflammatory response both in vitro and in vivo. Endotoxin, or LPS, is a potent inflammatory activator (1) in astrocytes and microglia, with stimulation of Toll-like receptor 4 (TLR4) (2). The expression of TLR4 is up-regulated in astrocytes under neuroinflammatory conditions (3). Activation of TLR4 leads to activation of nuclear factor-κB, a factor that regulates expression of genes involved in immune responses, including release of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (2).

Astrocytes in networks, positioned between the vasculature and synapses, monitor neuronal signaling, including rebuilding of synapses (4, 5). Astrocytes express almost the same repertoire of receptors and ion channels as neurons. They regulate synaptic transmission via a bidirectional communication with neurons, and they release gliotransmitters as well as factors, including cytokines, fatty acid metabolites, and free radicals (6–8).

The Ca²⁺ signaling in astrocytes over long distances is analogous to, but much slower than, the propagation of action potentials in neurons (9). These astrocytic Ca²⁺ waves (10, 11) can be evoked by transmitters released from neurons and glial cells, followed by activation of especially G protein-coupled receptors. Cytosolic Ca²⁺ plays a key role as a second messenger, and the control of Ca²⁺ signals is therefore critical. This involves coordination of Ca²⁺ entry across the plasma membrane (PM),² Ca²⁺ release from the endoplasmatic reticulum (ER), refilling of the ER stores, and extrusion across the PM (12). The Na⁺-Ca²⁺ exchanger, a Ca²⁺ transporter that controls the intracellular Ca²⁺ concentrations, is driven by the Na⁺ electrochemical gradient across the PM. This Na⁺ pump, Na⁺/K⁺-ATPase, indirectly modulates Ca²⁺ signaling (13), and inflammatory stimuli influence Ca²⁺ homeostasis in the astrocyte networks (5, 14–16).

The cytoskeleton seems to be important in this system for controlling of PM microdomains and the ER complex. The adaptor protein ankyrin B is associated with the Na⁺ pump and also with ER proteins, such as the inositol 1,4,5-trisphosphate (IP₃) receptor. The main cytoplasmic matrix of proteins, spectrin and actin, are attached to ankyrin B. An intact cytoskeleton is required for the propagation of astrocytic Ca²⁺ waves (17), and disruption of the cytoskeleton abolishes the Ca²⁺ oscillations by changing the balance between the Ca²⁺-regulating processes (18).

Parameters associated with neuroinflammation are down-regulation of Na⁺ transporters, changing of Ca²⁺ signaling in the astrocyte networks, and release of cytokines (16, 19–25). With LPS as an inflammatory inducer, we wanted to evaluate how astrocytes behave concerning Ca²⁺ signaling and cytokine release after inflammatory stimulation. We also wanted to evaluate how the astrocytes behave over time during incubation with LPS. We hypothesized that the astrocytes are influenced by the inflammation inducer LPS and that it is possible to restore some aspects of the cells to a homeostatic state using exposure to a substance that may have anti-inflammatory properties. We aimed to test this using naloxone, particularly in very low concentrations (26, 27). We further aimed to test ouabain in low and high concentrations and its effects of Na⁺/K⁺-ATPase activity (28) and evoked Ca²⁺ transients in astrocytes (28–30).

EXPERIMENTAL PROCEDURES

In this study, the in vitro model involved astrocytes co-cultured with brain endothelial cells (16, 24). The biological rationale for co-culturing astrocytes with endothelial cells is that the astrocytes are affected by substances released from the capillary endothelial cells of the BBB (31). These interactions are essential for a functional neurovascular unit (4, 32). The endothelial cells are not directly in physical contact with the astrocytes, and interaction in the model is brought about through the shared medium. The co-cultured astrocytes are morphologically differentiated with long, slender processes, and they exhibit greater Ca²⁺ responses and cytokine release than monocultured astrocytes (16, 24).

Chemicals

All chemicals were obtained from Sigma-Aldrich if not stated otherwise.

The experimental protocols were approved by the Ethical Committee in Gothenburg for Laboratory Animals (Nos. 232-2007, 255-2007, 205-2010, and 211-2010).

Primary Astrocytic Cultures

The primary astrocytic cultures were prepared from newborn rat cerebral cortices (Charles River, Sulzfeldt, Germany) and cultured on glass coverslips (nr 1, diameter 20 mm, BergmanLabora, Stockholm, Sweden) as described by Hansson et al. (24, 33).

Microvascular Endothelial Primary Cultures

Brain capillary fragments were isolated and endothelial cells cultured according to Hansson and co-workers (16, 24) using a modified version of the method used by Abbott and co-workers (34).

Astrocytes Co-cultured with Adult Rat Brain Microvascular Primary Cultures

The experimental astrocytes were obtained after co-cultivation with primary brain microvascular endothelial cultures and primary astrocytic cultures. Astrocytic cultures at 6 days in vitro were co-cultured with newly prepared microvascular cultures. The endothelial cells were grown in inserts above the astrocytic cultures. The cells from the two different cultures were never in contact. The cells were grown together for 9–11 days. At the time of the experiments, the astrocyte cultures were 15–17 days old, including the 9–11 days of co-cultivation. The endothelial cells were removed before the experimental procedure (16, 24).

Calcium Imaging

Astrocytes co-cultured with brain microvascular endothelial cells were incubated at room temperature with the Ca²⁺-sensitive fluorophore probe Fura-2/AM (Invitrogen Molecular Probes, Inc., Eugene, OR) for 30 min (8 μl in 990 μl of Hanks' HEPES-buffered saline solution, containing 137 mm NaCl, 5.4 mm KCl, 0.4 mm MgSO₄, 0.4 mm MgCl₂, 1.26 mm CaCl₂, 0.64 mm KH₂PO₄, 3.0 mm NaHCO₃, 5.5 mm glucose, and 20 mm HEPES, dissolved in distilled water, pH 7.4). The fluorophore probe was dissolved with 40 μl of DMSO and 10 μl of pluronic acid (Molecular Probes, Leiden, The Netherlands). After incubation the cells were rinsed three times with Hanks' HEPES-buffered saline solution before exposure to a substance. The antagonists were applied 3.5 min before the agonist. LPS, ouabain, or naloxone, were tested to verify if they induced Ca²⁺ transients on their own. To determine the underlying Ca²⁺ source of the LPS-, ouabain-, or naloxone-evoked Ca²⁺ transients, internal stores were depleted by preincubation with a sarcoendoplasmic reticulum Ca²⁺-ATPase inhibitor, thapsigargin (1 μm) and caffeine (20 mm) (35), or incubated with a Ca²⁺-free buffer (exchange of CaCl₂ with MgCl₂, and 1 mm EGTA). The experiments were performed at room temperature using a calcium imaging system and Simple PCI software (Compix Inc. Imaging Systems, Hamamatsu Photonics Management Corp., Cranberry Twp, PA) and an inverted epifluorescence microscope (Nikon ECLIPSE TE2000-E) with a ×20 (numerical aperture 0.45) fluorescence dry lens and a Polychrome V, monochromator-based illumination system (TILL Photonics GmbH). The various substances were infused into the cell baths using a peristaltic pump (Instech Laboratories, Plymouth Meeting, PA) at an approximate rate of 600 μl/min. One minute before the start of the recording sequence, the stimulating substance was injected into the pump tubes for 30 s. The substance took ∼80 s to reach the cells through the tubes. Hanks' HEPES-buffered saline solution continued to flow through the pump tubes and onto the cells throughout the experiment. The images were captured with an ORCA-12AG (C4742-80-12AG), high resolution digital cooled CCD camera (Hamamatsu Photonics Corp.).

The total areas under the transients (i.e. amounts of Ca²⁺ released) (36) were analyzed to provide measures of the vigor of the Ca²⁺ responses. The amplitude was expressed as the maximum increase of the 340/380 nm ratio. The area under the Ca²⁺ peaks was calculated in Origin (Microcal Software Inc., Northampton, MA).

Immunocytochemistry

The cells were fixed with 4% paraformaldehyde (Bie & Berntsen) for 10 min and washed twice with phosphate-buffered saline (PBS) (Invitrogen) containing 1% BSA (PBS-BSA). The cells were permeabilized with PBS-BSA containing 0.05% saponin (PBS-BSA-Sap) for 20 min. Thereafter, the cells were incubated for 1 h with a mixture of rabbit polyclonal antibody against TLR4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:100 and a monoclonal antibody against glial fibrillary acidic protein diluted 1:400 in PBS-BSA-Sap.

The cells were washed with PBS-BSA-Sap. for 3 × 5 min and then incubated with a mixture of FITC-conjugated F(ab′)₂ donkey anti-rabbit IgG and Texas Red-conjugated F(ab′)₂ donkey anti-mouse IgG secondary antibodies (Jackson Immunoresearch, Westgrove, PA), both diluted 1:150, and the nuclei marker Hoescht 33258 diluted 1:1000 in PBS-BSA-Sap. The cells were washed with PBS-BSA-Sap three times for 5 min each and finally rinsed with PBS. The coverslips were mounted on microscope slides with a fluorescent mounting medium (DAKO, Glostrup, Denmark) and viewed in a Nikon Eclipse 80i microscope. Pictures were taken with a Hamamatsu C5810 color-intensified 3CCD camera.

Viability Assay

A LIVE/DEAD viability assay kit (Invitrogen Molecular Probes) for mammalian cells was used, and assays were performed as follows.

The cells were gently washed twice with Dulbecco's PBS (Invitrogen). A mixture of 4 μm ethidium homodimer-1 (EthD-1) and 2 μm calcein AM diluted in Dulbecco's PBS was added, and the cells were incubated for 30–45 min at room temperature. Following incubation, 10 μl of fresh LIVE/DEAD reagent solution or Dulbecco's PBS was added to a clean microscope slide. Cells were then mounted upside down and immediately viewed in a Nikon Eclipse 80i microscope. Pictures were taken with a Hamamatsu C5810 color-intensified 3CCD camera.

SDS-PAGE and Western Blotting

Cells were rinsed twice in PBS and immediately lysed for 20 min on ice in cold radioimmune precipitation assay lysis buffer containing 50 mm Tris-HCl, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0,1% SDS, 5 μg/ml aprotinin, and 5 μg/ml leupeptin, pH 7.4. The procedure was done according to Persson et al. (37). Separate aliquots were taken for protein concentration determination. All samples were correlated for total protein contents, and an equal loading of 20 μg total protein of each sample was applied in each lane of the gel. β-Actin was used as control for equal loading.

SDS-PAGE was conducted using the Novex precast gel system (Invitrogen) according to the manufacturer's recommendations using 4–12% BisTris gels (Invitrogen) at 200 V for 50 min. The separated proteins were then transferred at 30 V for 60 min to a nitrocellulose membrane (Invitrogen) using NuPAGE transfer buffer (Invitrogen) supplemented with methanol and NuPage antioxidant. The membranes were rinsed twice with distilled water, and the proteins were visualized with Ponceau S solution (Sigma). Proteins were blocked with 5% fat-free skim milk (Semper AB) in TBST (50 mm Tris-HCl, 150 mm NaCl, and 0.05% Tween) for 60 min at room temperature. The membranes were then probed with primary antibodies, washed four times for 2 min each with TBST, subsequently probed with secondary horseradish peroxidase (HRP)-conjugated secondary antibodies, and finally washed several times in TBST. The primary antibodies used were TLR4 (rabbit polyclonal) (Santa Cruz Biotechnology, Inc.) diluted 1:500 and Na⁺/K⁺ ATPase (α-subunit) (mouse monoclonal) (Sigma) diluted 1:250. The secondary antibodies used were HRP-conjugated donkey anti-mouse or -rabbit F(ab′)₂ fragment (both from Jackson Immunoresearch) diluted 1:10000.

All primary and secondary antibodies were diluted in 5% fat-free skim milk in TBST. Protein was then detected with an enhanced chemiluminescence kit (PerkinElmer Life Sciences) and visualized with a Fuji Film LAS-3000 (Tokyo, Japan).

Cytokine Release

Co-cultured astrocytes were stimulated for 24 h with 10 ng/ml LPS diluted in unsupplemented minimum Eagle's medium to measure IL-1β release. The inserts with endothelial cells were removed just before incubation. When anti-inflammatory substances were used, the cells were incubated for 30 min with the antagonists before LPS treatment and remained in the medium throughout the experiment. Control (i.e. untreated) cells were kept in unsupplemented minimum Eagle's medium. Supernatants were collected for enzyme-linked immunosorbent assay (ELISA), and the cells underwent protein measurements.

Rat IL-1β (Nordic Biosite, Täby, Sweden) were used according to the manufacturer's instructions to measure the amounts of cytokine released. Between every incubation step, several washings were performed. The protein concentration was determined as described below. The amount of IL-1β release was correlated to the protein amount for each well.

Protein Determination

The protein determination assay was performed in accordance with the manufacturer's instructions using a DC Protein Assay (Bio-Rad), based with some modifications on the method used by Lowry et al. (38). Both a standard (0–4 mg/ml BSA) and samples were mixed with the reagents, incubated for 15 min at room temperature, read at 750 nm with a Versa-max microplate reader, and analyzed using SoftMax Pro 4.8, both from Molecular Devices (Sunnyvale, CA).

Actin Visualization

The astrocytic cytoskeleton was evaluated by staining of actin filaments after incubation with 10 ng/ml LPS for 0, 1, 4, 8, 24, or 48 h. Cultures were fixed with 4% paraformaldehyde and made permeable with PBS (Invitrogen) containing 1% BSA and 0.05% saponin followed by an Alexa^TM568-conjugated phalloidin probe (Invitrogen) diluted 1:40 in PBS supplemented with 1% BSA. The coverslips were rinsed three times in PBS and then mounted on microscope slides with Dako's fluorescent mounting medium (Dako Co., Glostrup, Denmark) before viewing with a fluorescence dry objective lens attached to an inverted Nikon Optiphot-2 microscope.

Statistics

The level of significance was analyzed using one-way analysis of variance followed by Dunnet's multiple comparisons test. Error bars show S.E.

RESULTS

Stimulation with LPS

Astrocytes were stimulated with LPS (0.01–100 ng/ml) and Ca²⁺ imaging experiments were performed on 15–17-day-old co-cultured astrocytes. The endothelial cell inserts were removed just before the experiments. In 0.01 ng/ml LPS, 47.5% of the astrocytes responded (19 of 40 cells); in 0.10 ng/ml LPS, 57.5% responded (23 of 40 cells); in 1.0 ng/ml LPS, 67.5% responded (27 of 40 cells); in 10 ng/ml LPS, 73.0% responded (29 of 40 cells); and in 100 ng/ml LPS, 25.0% responded (10 of 40 cells). The amplitudes and areas under the Ca²⁺ peaks were calculated, and the number of peaks was counted for each cell. The concentration curve showed a bell-shaped appearance visualized both with maximum ratio increase and amount of Ca²⁺ released (Fig. 1). The astrocytes were most sensitive to LPS at 0.10, 1.0, and 10 ng/ml. In all further experiments, the cells were stimulated with 10 ng/ml LPS.

FIGURE 1.

Stimulation with LPS. Astrocytes were loaded with the Ca²⁺ probe, Fura-2/AM, for 20 min and stimulated with LPS (0.01–100 ng/ml), with Ca²⁺ imaging experiments performed on 15–17-day-old co-cultured astrocytes. The endothelial cell inserts were removed just before the experiments. In 0.01 ng/ml LPS, 47.5% of the astrocytes responded (19 of 40 cells); in 0.10 ng/ml LPS, 57.5% responded (23 of 40 cells); in 1.0 ng/ml LPS, 67.5% responded (27 of 40 cells); in 10 ng/ml LPS, 73.0% responded (29 of 40 cells); and in 100 ng/ml LPS, 25.0% responded (10 of 40 cells). The amplitudes and areas under the Ca²⁺ peaks were calculated, and the number of peaks was counted for each cell. The concentration curve showed a bell-shaped appearance visualized both with maximum ratio increase and the amount of Ca²⁺ released. The cells were from four different coverslips, from two different seeding times. Ca²⁺ responses, expressed as maximum ratio increase of intensity at 340 and 380 nm, in astrocytes after stimulation with LPS, the area under the curve of Ca²⁺ transients was calculated, the number of peaks was calculated, and responding cells are shown (%). Data are mean ± S.E. (error bars). AUC, area under the curve.

To investigate if the Ca²⁺ elevation was cross-membrane or intracellular-dependent, LPS in a Ca²⁺-free medium evoked Ca²⁺ transients in half of the responding cells. All cells were blocked when the cells were incubated with thapsigargin, a depletor of intracellular Ca²⁺ stores (33), followed by caffeine, which was used to complete the store depletion (33). All cells were blocked with thapsigargin and Ca²⁺-free buffer (Fig. 2).

FIGURE 2.

LPS-evoked Ca²⁺ transients were dependent on intracellular Ca²⁺ stores. A, 10 ng/ml LPS elicited Ca²⁺ responses in astrocytes in all cells tested (40 of 40; 100%). B, LPS in a Ca²⁺-free medium evoked Ca²⁺ transients in half of the responding cells. C, to determine the underlying Ca²⁺ source, internal stores were depleted with thapsigargin, followed by caffeine, which was used to complete the store depletion. All cells were blocked. D, all cells were blocked with thapsigargin, caffeine, and Ca²⁺-free buffer. The amplitudes were expressed as the maximum increase of the 340/380 nm ratio. The cells were from four different coverslips, from two different seeding times. Results shown are from a typical experiment.

Astrocytes Express TLR4

Astrocytes express TLR4 visualized with Western blot (Fig. 3A). The astrocytes were stained for glial fibrillary acidic protein and for the TLR4, revealing that these cells do express TLR4 receptors. The cell nuclei are visualized with HO33258 (Fig. 3B). Astrocytes stimulated with LPS were blocked with the TLR4 antagonist LPS-RS (39) (Fig. 3C).

FIGURE 3.

Astrocytes express TLR4. A, astrocytes express TLR4 visualized with Western blot, the protein band expressed at 89 kDa. B, astrocytes were stained for the TLR4 receptor (green), revealing that these cells do express TLR4 receptors. The astrocytes were stained for glial fibrillary acidic protein (GFAP) (red), and the nuclei were visualized with HO33258 (blue). C, astrocytes stimulated with LPS were blocked with the TLR4 antagonist LPS-RS. These results show that astrocytes express TLR4 and that LPS-induced Ca²⁺ transients were blocked by the selective TLR4 antagonist LPS-RS.

Time-dependent Stimulation with LPS

For astrocytes incubated with LPS for 30 min or 1, 4, 6, or 24 h, the LPS-evoked Ca²⁺ transients were captured. At 6 and 24 h of exposure of LPS, the LPS-induced Ca²⁺ transients increased as well as the areas under curves; p < 0.001 at 6 h, p < 0.01 at 24 h (Fig. 4, A and B). The amplitudes in the control are the same as in Fig. 2A. The viability of the cells visualized with a live-dead kit shows very few dead cells with red nuclei in the figure. The viability did not change over time (Fig. 4C). TLR4 was up-regulated when the cells were incubated with LPS for 4 h and visualized with Western blot (Fig. 4D).

FIGURE 4.

Time-dependent stimulation with LPS. A, astrocytes were stimulated with 10 ng/ml LPS or incubated with 10 ng/ml LPS for 30 min or 1, 4, 6, or 24 h and then stimulated with LPS. The LPS-evoked Ca²⁺ transients were captured. The Ca²⁺ transients changed to oscillations. At 6- and 24-h exposure of LPS, the intracellular Ca²⁺ release increased. The areas under the curve (AUC) of Ca²⁺ transients are shown. n = 40 from four different coverslips, from two different seeding times. The level of significance was analyzed using one-way analysis of variance followed by Dunnet's multiple comparisons test. Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., nonsignificant. B, the appearance of the Ca²⁺ transients is visualized. One peak LPS-evoked Ca²⁺ transient was changed to Ca²⁺ oscillations after 1 h, and the Ca²⁺ oscillations increased from 6 h and were still dominant at 24 h. C, the viability of the cells, Fura-2-positive cells (green) made with a live-dead kit, shows very few dead cells, read nuclei. The viability does not change over time. D, expression of TLR4 was studied with Western blot. Cultures were incubated with LPS for 1, 4, 8, 24, or 48 h. The expression of TLR4 increased with time. TLR4 is seen as a band at 80 kDa. The TLR4 expression is also shown as integrated density.

Na⁺/K⁺-ATPase Expression Over Time

Expression of Na⁺/K⁺-ATPase was studied with Western blot for cultures incubated with LPS for 1, 4, 8, 24, or 48 h. The expression of Na⁺/K⁺-ATPase initially increased and decreased after 8 h of incubation (Fig. 5).

FIGURE 5.

Na⁺/K⁺-ATPase expressed by Western blot. Expression of Na⁺/K⁺-ATPase was studied with Western blot. Cultures were incubated with LPS for 1, 4, 8, 24, or 48 h. The expression of Na⁺/K⁺-ATPase initially increased and decreased after 8 h of incubation. The Na⁺/K⁺-ATPase expression is also shown as integrated density.

Actin Filaments

Untreated astrocytes stained with an Alexa^TM488-conjugated phalloidin probe were dominated by F-actin organized in stress fibers. After a 1- or 4-h incubation with LPS, respectively, small amounts of ring-formed actin filaments were observed. At 8 h, these ring structures became more pronounced, and the actin filaments were organized more diffusely. These rearrangements of the actin filaments became further apparent after the cells were incubated with LPS for 24 or 48 h (Fig. 6).

FIGURE 6.

Actin filaments. Astrocytes were stained with an Alexa^TM488-conjugated phalloidin probe. The untreated culture was dominated by F-actin organized in stress fibers. Cultures were incubated with LPS (10 ng/ml) for 1, 4, 8, 24, or 48 h. A more diffuse organization of the actin filaments was observed, and ring structures were more pronounced. The ring structures were most prominent at 8 h, and then a more diffuse fiber appearance appeared.

Stimulation with Naloxone

Astrocytes were stimulated with naloxone (10⁻¹⁵ to 10⁻⁴ m), and Ca²⁺ imaging was recorded where all astrocytes responded (40 of 40 cells in the respective concentrations). One peak was observed, and the areas under the curves were calculated. Naloxone-evoked Ca²⁺ transients were obtained in both low and high concentrations (Fig. 7A).

FIGURE 7.

Stimulation with naloxone. A, astrocytes were loaded with the Ca²⁺ probe, Fura-2/AM, for 20 min and stimulated with naloxone (10⁻¹⁵ to 10⁻⁴ m). All astrocytes responded, 100% (40 of 40 cells in the respective concentrations). One peak was observed, and the areas under the curves (AUC) were calculated. The concentration curve showed that naloxone evoked Ca²⁺ transients in low and high concentrations. n = 40 from four different coverslips, from two different seeding times. Data are mean ± S.E. (error bars). Naloxone-evoked Ca²⁺ transients were dependent on intracellular Ca²⁺ stores. B (top left) 10⁻¹² or 10⁻⁵ m naloxone elicited Ca²⁺ responses in astrocytes, in all cells tested (40 of 40; 100%). Top right, Ca²⁺-free medium evoked similar Ca²⁺ transients as controls. Bottom left, To determine the underlying Ca²⁺ source, internal stores were depleted with thapsigargin, followed by caffeine, which was used to complete the store depletion (40 of 40 cells; 100%). Bottom right, similar results were seen with thapsigargin, caffeine, and Ca²⁺-free medium. The amplitudes were expressed as the maximum increase of the 340/380 ratio. The cells were from four different coverslips, from two different seeding times. Results shown are from a typical experiment (10⁻¹² m). Naloxone did not block LPS-evoked Ca²⁺ transients. C, naloxone was not able to block LPS-induced Ca²⁺ transients visualized by stimulation with LPS. The same cells were treated with naloxone (10⁻⁵ m) 30 s before LPS was added to the medium.

To investigate if the Ca²⁺ elevation was cross-membrane or intracellular-dependent, thapsigargin and/or Ca²⁺-free buffer was used. Naloxone (10⁻¹² or 10⁻⁵ m) in a Ca²⁺-free medium evoked the same Ca²⁺ transients as control, suggesting that Ca²⁺ elevation occurs due to efflux from the storage in ER. All cells were blocked when the cells were incubated with thapsigargin followed by caffeine. All cells were blocked with thapsigargin, caffeine, and Ca²⁺-free buffer (Fig. 7B).

Naloxone did not block LPS-induced Ca²⁺ transients. This was visualized by first stimulation with LPS, after which the same cells were treated with naloxone (10⁻⁵ m) 30 s before LPS was added to the medium quality (Fig. 7C). Naloxone did not block TLR4, which detects LPS-evoked Ca²⁺ transients.

The main results here were that naloxone evoked Ca²⁺ transients in both low and high concentrations. Ca²⁺ was released from intracellular stores, and naloxone did not block the TLR4 Ca²⁺-evoked responses.

Stimulation with Ouabain

Astrocytes were stimulated with ouabain (10⁻¹⁵ to 10⁻³ m), and Ca²⁺ imaging experiments were performed. All astrocytes responded, 100% (40 of 40 cells in the respective concentrations). The Ca²⁺ transients were evoked partly as oscillations, and the areas under the curves were calculated. The concentration curve showed that ouabain evoked Ca²⁺ transients in both low and high concentrations (Fig. 8A).

FIGURE 8.

Stimulation with ouabain. A, astrocytes were loaded with the Ca²⁺ probe, Fura-2/AM, for 20 min and stimulated with ouabain (10⁻¹⁵ to 10⁻³ m). All astrocytes responded, 100% (40 of 40 cells in the respective concentrations). One peak was observed, and the areas under the curves (AUC) were calculated. The concentration curve showed that ouabain evoked Ca²⁺ transients in low and high concentrations. n = 40 from four different coverslips, from two different seeding times. Data are mean ± S.E. (error bars). Ouabain-evoked Ca²⁺ transients were dependent on intracellular Ca²⁺ stores. B (top left), 10⁻¹² or 10⁻⁵ m ouabain elicited Ca²⁺ responses in astrocytes, in all cells tested (40 of 40; 100%). Top right, Ca²⁺-free medium evoked similar Ca²⁺ transients as controls (40 of 40; 100%). Bottom left, to determine the underlying Ca²⁺ source, internal stores were depleted with thapsigargin, followed by caffeine, which was used to complete the store depletion. Bottom right, similar results were seen with thapsigargin, caffeine, and Ca²⁺-free medium. The amplitudes were expressed as the maximum increase of the 340/380 ratio. The cells were from four different coverslips, from two different seeding times. Results shown are from a typical experiment (10⁻¹² m). Ouabain did not block LPS-evoked Ca²⁺ transients. C, ouabain was not able to block LPS-induced Ca²⁺ transients visualized by stimulation with LPS. The same cells were treated with ouabain (10⁻⁵ m) 30 s before LPS was added to the medium.

To investigate if the Ca²⁺ elevation was cross-membrane or intracellular-dependent, thapsigargin and/or Ca²⁺-free buffer was used. Ouabain (10⁻¹² or 10⁻⁵ m) in a Ca²⁺-free medium evoked the same Ca²⁺ transients as control, an indicator that Ca²⁺ elevation occurred due to efflux from the storage in ER. All cells were inhibited when the cells were incubated with thapsigargin followed by caffeine. No Ca²⁺ transient amplitudes were seen when the cells were blocked with thapsigargin, caffeine, and Ca²⁺-free buffer (Fig. 8B).

Ouabain did not block LPS-induced-Ca²⁺ transients. This was visualized by first stimulation with LPS; thereafter, the same cells were treated with ouabain (10⁻⁵ m) 30 s before LPS was added to the medium (Fig. 8C).

The main results here were that ouabain evoked Ca²⁺ transients in both low and high concentrations. Ca²⁺ was released from intracellular stores, and ouabain did not block the TLR4 Ca²⁺-evoked responses.

Naloxone and Ouabain in Ultralow Concentrations Prevent LPS Down-regulation of Na⁺/K⁺-ATPase

Astrocytes were incubated with naloxone or ouabain (10⁻¹² and 10⁻⁵ m) 30 min before the cells were treated with naloxone or ouabain, respectively, and LPS for 1, 4, 8, 24, or 48 h. The expression of Na⁺/K⁺-ATPase visualized with Western blot was unaltered during the time curves for both naloxone and ouabain (Fig. 9). Naloxone as well as ouabain prevented LPS-induced down-regulation of Na⁺/K⁺-ATPase.

FIGURE 9.

Naloxone and ouabain prevented LPS down-regulated Na⁺/K⁺-ATPase expressed by Western blot. Expression of Na⁺/K⁺-ATPase was studied with Western blot. Cultures were preincubated with naloxone (10⁻¹² m (A) or 10⁻⁵ m (B)) or ouabain (10⁻¹² m (C) or 10⁻⁵ m (D)) for 30 min before the cultures were incubated with LPS (10 ng/ml) for 1, 4, 8, 24, or 48 h. The expression of Na⁺/K⁺-ATPase was not decreased after preincubation with naloxone or ouabain, respectively. In the ultralow concentrations, the results were more equable for both naloxone and ouabain.

Naloxone and Ouabain Restore Actin Filaments

When astrocytes were incubated with LPS for 24 h, the actin filaments were reorganized. In cells treated with naloxone or ouabain (10⁻¹² or 10⁻⁵ m), 30 min before the cells were incubated with both LPS and naloxone, respectively, the disrupted actin filaments were to a great extent restored. At 10⁻¹² m, the actin filaments were better restored for both naloxone and ouabain compared with 10⁻⁵ m (Fig. 10).

FIGURE 10.

Naloxone and ouabain restored actin filaments. Astrocytes were stained with an Alexa^TM488-conjugated phalloidin probe. The untreated culture was dominated by F-actin organized in stress fibers. Cultures were preincubated with naloxone (Nalox.; 10⁻¹² or 10⁻⁵ m) or ouabain (Ouab.; 10⁻¹² or 10⁻⁵ m) for 30 min before the cultures were treated with LPS (10 ng/ml) for 1, 4, 8, 24, or 48 h. Both naloxone and ouabain restored actin filaments.

Ouabain Reduces IL-1β Release

Astrocytes incubated with LPS for 24 h released IL-1β. Cells were treated with naloxone or ouabain (10⁻¹² or 10⁻⁵ m) 30 min before the cells were incubated with both LPS and naloxone or ouabain, respectively. Ouabain, at low or high concentrations, reduced the LPS-induced IL-1β increased release (Fig. 11).

FIGURE 11.

IL-1β release. Cultures were preincubated with naloxone (10⁻¹² or 10⁻⁵ m) or ouabain (10⁻¹² or 10⁻⁵ m) for 30 min before the cultures were treated with LPS (10 ng/ml) for 24 h. Ouabain in both low and high concentrations reduced the LPS-increased IL-1β. n = 4. The level of significance was analyzed using one-way analysis of variance followed by Dunnet's multiple comparisons test. Data are mean ± S.E. ***, p < 0.001; n.s., nonsignificant.

DISCUSSION

Regulation of Ca²⁺ dynamics by transmitters and soluble factors is a possible mechanism by which the astrocyte network detects changes in the CNS microenvironment and regulates brain activities, including inflammatory processes. The complexity of Ca²⁺ signaling has made it difficult to determine the physiological role of these phenomena, although this probably is an important function of astrocytes. During inflammation, TLR4 is activated, and astrocytes express TLR4, as demonstrated in the present study and by others (39, 40). LPS has been shown to be a TLR4 agonist (41), and this receptor is thought to be largely responsible for LPS-related signaling (2). It has been proposed that TLRs drive inflammation that gives rise to symptoms (42, 43). This study was focused on demonstrating that astrocytes react to inflammatory activation. LPS is frequently used as a model for the induction of inflammation both in vitro and in vivo. In this study, astrocyte-enriched cultures from newborn rat brain were co-cultured with endothelial cultures from adult rat brain. These astrocytes express TLR4, a feature that is further induced after incubation with LPS. This is in contrast to findings in astrocyte-enriched cultures with no co-cultivation, where TLR4 is expressed rather sparsely and is more difficult to induce. Although LPS normally does not readily pass the intact BBB, LPS itself can impair the integrity of the BBB and thereby affect the flux system between blood and brain (44). Therefore, although our findings in the present study must primarily be conceived as purely in vitro model data, they may have some bearing also for the understanding of in vivo systemic actions following the release of LPS (e.g. following brain trauma or sepsis), when a disruption of the BBB is prone to occur.

In this study, we found that acute stimulation of astrocytes with LPS affects the Ca²⁺ signaling; LPS-elicited Ca²⁺ transients were observed in a concentration-dependent bell-shaped distribution, where also the induced Ca²⁺ peak was blocked by the TLR4 antagonist LPS-RS. The Ca²⁺ for these transients originated from intracellular stores. Next, we observed that astrocyte behavior changed over time when they were incubated with LPS for longer time periods; the one peak LPS-evoked Ca²⁺ transient changed to Ca²⁺ oscillations after 1 h, and the Ca²⁺ oscillations increased in intensity from 6 h on and were still dominant at 24 h. It has been proposed that the size of the intracellular Ca²⁺ store in astrocytes is important when the Ca²⁺ response goes from transient to oscillatory and that this could be a property of reactive astrocytes (19). This has also been shown when astrocytes were stimulated by growth factors (19). A similar mechanism for converting this response pattern is shown in the present study when astrocytes are exposed to the inflammatory stimulus LPS. This can be one mechanism by which astrocytes detect changes and regulate brain activities such as processes of inflammation.

In renal inflammation, LPS induces down-regulation of sodium transporters, such as Na⁺/K⁺-ATPase, Na⁺-K⁺-2Cl⁻ co-transporter, Na⁺/H⁺ exchanger, and epithelial sodium channel (20). LPS also induces down-regulation of sodium transporters in liver cells (21), kidney cells (22), and intestinal epithelial cells (23). In vitro in cortical duct cells, proinflammatory cytokines, such as TNF-α and IL-1β, decreased the expression of Na⁺/K⁺-ATPase (20). From observations in different cellular systems, it can be surmised that inflammation (not only cytokine release) is important as well as down-regulation of Na⁺ transporters. Down-regulation of Na⁺ transporters means dysfunction of the Na⁺/K⁺-ATPase activity (20), which can lead to increased intracellular Na⁺ concentration. Astrocytes have strong resistance to Na⁺ influx only when Na⁺/K⁺-ATPase activity is maintained (45). This is why we evaluated protein expression for Na⁺/K⁺-ATPase in astrocytes after the cells were incubated with LPS over time. We found that the protein expression of Na⁺/K⁺-ATPase was reduced after 8 h, as an indication that the Na⁺ transporters were down-regulated.

The cytoskeleton controlling the PM microdomains and the ER complex is important with regard to calcium transients. The adaptor protein ankyrin B is associated with Na⁺/K⁺-ATPase and also with ER proteins, such as IP₃. The main cytoplasmic matrix proteins, spectrin and actin, are attached to ankyrin B. An intact cytoskeleton is required for the propagation of astrocytic Ca²⁺ waves (17), and disruption abolishes the Ca²⁺ oscillations by changing the balance between the Ca²⁺-regulating processes (18). We found that the actin filaments were disorganized after many h of LPS exposure. The actin filaments, which are normally organized in stress fibers, changed into ring structures after 1 h of treatment and were disrupted and disorganized at 24 h. We have observed similar patterns previously when astrocytes were exposed to ethanol, ammonium chloride, or lactate (46, 47). Others have seen morphological changes induced by LPS in cultured astrocytes (48, 49). In line with earlier published data, the present findings confirm that there is a connection between rearrangement of actin filaments and changes in astrocytic morphology and cell volume (46, 47).

Another notable finding in our study concerned substances that may have properties with anti-inflammatory qualities and up-regulation or restoration of markers related to inflammation: Na⁺/K⁺-ATPase protein expression, actin filament organization, Ca²⁺ oscillations, and IL-1β release. We have reasoned that when Na⁺/K⁺-ATPase is down-regulated, TLR4 is activated, and when Na⁺/K⁺-ATPase has a normal pump activity, TLR4 is not overexpressed, although it is still unclear if a direct causal connection exists between these processes. Our findings showed that TLR4 protein expression was up-regulated when the astrocytes were incubated with LPS in a time-dependent manner. LPS activated TLR4, leading to more intracellular Ca²⁺ time-dependent release. In a previous study (16), increased IL-1β production was noted, and this seems to be initiated through TLR4 activation (50). The Na⁺/K⁺-ATPase is an energy-transducing pump, and expression of these proteins was down-regulated with time. Inhibiting or modulating this pump has an influence on the intracellular Na⁺ concentration, which in turn results in an increases in intracellular Ca²⁺ concentration via the Na⁺-Ca²⁺ exchange (28).

We chose two substances to study in LPS-activated astrocytes: naloxone and ouabain. We opted to use them instead of more traditional anti-inflammatory agents because naloxone in ultralow concentrations has been suggested to have anti-inflammatory properties (27) in addition to its powerful effect as a μ-opioid receptor antagonist. Also, ultralow dose naloxone is effective in restoration of the antinociceptive effect of morphine in rats and in addition increases anti-inflammatory cytokine IL-10 expression (26).

Ouabain, a digitalis-derived glycoside is a well recognized Na⁺/K⁺-ATPase inhibitor, especially pronounced at high concentrations, and is also known to regulate intracellular Ca²⁺ release in cardiac myocytes, peritoneal macrophages, and astrocytes in high concentrations (13, 29, 51). Ouabain leads to increases in intracellular Ca²⁺ release in hippocampal astrocytes, which is IP₃ receptor-dependent (29). Ouabain also enhances LPS-down-regulated inducible nitric-oxide synthase activity in peritoneal macrophages (51). Interestingly, at low concentrations (nanomolar and picomolar), ouabain stimulates Na⁺/K⁺-ATPase activity (28) and activates complex signaling cascades in kidney cells (30). In subnanomolar concentrations, ouabain stimulates proliferation and differentiation of cardiac and smooth muscle cells (52).

In the present study, we observed that stimulation with naloxone evoked intracellular Ca²⁺ transients that were IP₃ receptor-dependent. The intracellular Ca²⁺ release was not concentration-dependent, and this was unexpected. Both at ultralow and high concentrations, Ca²⁺ transients had the same appearance. Stimulation with ouabain also evoked intracellular Ca²⁺ transients that were IP₃ receptor-dependent, and the intracellular Ca²⁺ release was not concentration-dependent. Ouabain and naloxone behaved in a similar way regarding Ca²⁺ transients in both ultralow and high concentrations. At high ouabain concentrations, Na⁺/K⁺-ATPase activity is inhibited, resulting in an elevation of intracellular Na⁺ concentration, and this in turn raises intracellular Ca²⁺ concentration via diminished Na⁺-Ca²⁺ exchange (28). At low concentrations, Na⁺/K⁺-ATPase is instead stimulated via activation of the Scr/epidermal growth factor receptor intracellular way, and this raises intracellular Ca²⁺ concentration via the phospholipase C/IP₃ receptor pathway (28). This means that intracellular Ca²⁺ release comes from intracellular Ca²⁺ stores but via two different mechanisms.

Because the LPS triggered astrocytes exert a lowered Na⁺/K⁺-ATPase activity, we found it relevant to investigate whether this down-regulation could be counteracted by naloxone and/or ouabain in ultralow concentrations. These two substances also had the ability to restore the actin filaments, which was even more pronounced at ultralow concentration. This shows the importance of a well working Na⁺ pump activity, which is coupled to the cellular cytoskeleton. Most notable here is that ultralow concentrations seem enough to induce the effects. In addition, ultralow concentrations of ouabain led to decreased IL-1β release. The mechanisms behind the reduced IL-1β release need to be further evaluated. The biosynthesis of IL-1β is complex and is regulated at multiple levels. In microglia, it has been shown that LPS stimulates TLR4, which induced IL-1β production depending on NF-κB, and that the purinergic receptor P2X7 is involved (53). However, ouabain did not inhibit the TLR4 Ca²⁺-evoked responses in our astrocytes. Furthermore, naloxone is known to reduce IL-1β synthesis in rats (27), which we were unable to reproduce in our astrocytes.

In conclusion, using a co-cultured model of astrocytes, we have shown that after a long incubation with LPS, TLR4 is up-regulated, LPS-evoked Ca²⁺ transients are expressed as oscillations, Na⁺/K⁺-ATPase is down-regulated, and the actin filaments are disorganized. Two substances with proposed anti-inflammatory properties in a low dose range, naloxone and ouabain, demonstrate the ability to limit these astrocytic LPS-induced alterations. Naloxone evokes intracellular Ca²⁺ transients but not in a concentration-dependent manner, and there is a similar pattern with ouabain, although neither blocks TLR4. Ultralow concentrations of naloxone prevent the LPS-induced down-regulation of Na⁺/K⁺- ATPase and restore the actin filaments. Ouabain demonstrates the same effect, although ouabain also reduces IL-1β release. Further study is warranted concerning modulation of the inflammatory response in astrocytes.