SEARCH

SEARCH BY CITATION

Keywords:

  • astrocyte;
  • calcium;
  • GDNF;
  • ifenprodil;
  • IL-1β;
  • NMDA

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

J. Neurochem. (2011) 119, 686–696.

Abstract

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in neuroinflammatory and neuropathic pain conditions. Astrocytes produce and secrete GDNF, which interacts with its receptors to induce Ca2+ transients. This study aimed first to assess intracellular Ca2+ responses of astrocytes in primary culture when exposed to the neuroprotective and anti-inflammatory peptide GDNF. Furthermore, incubation with the inflammatory inducers lipopolysaccharide (LPS), NMDA, or interleukin 1-β (IL-1β) attenuated the GDNF-induced Ca2+ transients. The next aim was to try to restore the suppressed GDNF responses induced by inflammatory changes in the astrocytes with an anti-inflammatory substance. Ifenprodil, an NMDA receptor antagonist at the NR2B subunit, was tested. It was shown to restore the GDNF-evoked Ca2+ transients and increased the Na+/K+-ATPase expression. Ifenprodil seems to be a potent anti-inflammatory substance for astrocytes which have been pre-activated by inflammatory stimuli.

Abbreviations used
BBB

blood–brain barrier

GDNF

glial cell line-derived neurotrophic factor

HHBSS

Hank’s HEPES buffered saline solution

LPS

lipopolysaccharide

TLR4

Toll-like receptor 4

Astrocytes are the neural cells mainly responsible for the maintenance of brain homeostasis. They form highly organized anatomical domains which are integrated by extensive networks of gap junctions and interconnected in a glial information-transfer system. One signalling pathway in this system propagates Ca2+ waves (Blomstrand et al. 1999; Scemes and Giaume 2006). Regulation of Ca2+ waves may be a mechanism by which the astrocyte networks detect changes in the CNS microenvironment and regulate brain activities under various physiological and pathophysiological conditions. This ability, along with the expression of a wide array of receptors, transporters, and ion channels, means that astrocyte networks are ideally positioned to sense and modulate neuronal activity. The glial cell line-derived neurotrophic factor (GDNF) produced and secreted by astrocytes (Nicole et al. 2001; Kuno et al. 2006) has neuroprotective functions that enhance the survival of dopaminergic, motor, and primary sensory neurons (Lin et al. 1993; Airaksinen and Saarma 2002). GDNF regulates the permeability of the blood–brain barrier (BBB) and activates the barrier function of capillary endothelial cells (Igarashi et al. 2000; Abbott et al. 2006). GDNF has been discussed to play an important role in the modulation of nociceptive signals and dysfunction of this system may contribute to the development and/or maintenance of neuropathic pain states (Nagano et al. 2003; Wang et al. 2003; Dong et al. 2005). This is especially relevant because long-term neuropathic pain is considered to result from a low-grade neuroinflammation in the CNS (Saadé and Jabbur 2008). Inflammatory mediators may penetrate the BBB in central glial response to injury, and inflammatory cytokines, such as interleukin 1-β (IL-1β) released from glia, may modulate neuronal activity and facilitate pain transmission. High levels of IL-1β produced under conditions of injury, stress, or disease evoke increased NMDA receptor phosphorylation and sensitivity in inflammatory pain models (Viviani et al. 2003; Guo et al. 2007). GDNF has protecting capabilities for cortical neurons by reducing the NMDA-induced Ca2+ influx (Nicole et al. 2001; Wang et al. 2002). Different NMDA antagonists were shown to enhance the production of GDNF in astrocytes (Toyomoto et al. 2005; Wu et al. 2009).

Ifenprodil acts as an NMDA receptor antagonist at the NR2B subunit (Iwata et al. 2007). It is used clinically as a neuroprotective agent in head ischemia, Parkinson’s disease, and stroke (Kato et al. 2006). Ifenprodil also enhances the production of GDNF in cultured astrocytes (Toyomoto et al. 2005).

Parameters associated with neuroinflammation are down-regulation of Na+ transporters, changing of Ca2+ signalling in the astrocyte networks, and release of cytokines (Morita et al. 2003; Schmidt et al. 2007; Hansson et al. 2008; Delbro et al. 2009; Vallejo et al. 2010). With lipopolysaccharide (LPS), NMDA, and IL-1β as inflammatory inducers, we wanted to evaluate how astrocytes behave concerning GDNF-evoked Ca2+ signalling, after inflammatory stimulation. We hypothesized that when astrocyte function is influenced by one or several of the inflammation inducers; it is possible to reverse some of the cell’s inflammatory dysfunction with an anti-inflammatory substance. During inflammation, NMDA receptor phosphorylation is increased and in addition IL-1β release from astrocytes occurs. As ifenprodil was shown to reduce astrocytic swelling and polyamine levels we wanted to evaluate if ifenprodil has any capabilities to restore some parameters, which are affected in inflammatory activated astrocytes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

The experimental protocols were approved by the Ethical Committee in Gothenburg for Laboratory Animals (Nos. 232-2007; 255-2007; 205-2010; 211-2010). The in vitro model involved astrocytes co-cultured with brain endothelial cells (Hansson et al. 2008; Delbro et al. 2009). The biological rationale for co-culturing astrocytes with endothelial cells is that astrocytes are influenced by substances which are released from the capillary endothelial cells of the BBB (Huber et al. 2001). These interactions are essential for a functional neurovascular unit (Abbott et al. 2006; Willis and Davis 2008). 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 (Hansson et al. 2008; Delbro et al. 2009).

Chemicals

All chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) if not stated otherwise.

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, ∅ 20 mm, BergmanLabora, Stockholm, Sweden) as described by Hansson et al. (1984, 2008).

Microvascular endothelial primary cultures

Brain capillary fragments were isolated and endothelial cells cultured as described by Hansson et al. (2008), using a modified version of the method used by Abbott et al. (1992).

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 (Hansson et al. 2008; Delbro et al. 2009).

Calcium imaging

Astrocytes co-cultured with brain microvascular endothelial cells were incubated at 22°C with the Ca2+-sensitive fluorophore probe fura-2AM (Invitrogen Molecular Probes, Eugene, OR, USA) for 30 min [8 μL in 990 μL Hank’s HEPES buffered saline solution (HHBSS), 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 dimethyl sulfoxide and 10 μL pluronic acid (Molecular Probes, Leiden, the Netherlands). After incubation, the cells were rinsed three times with HHBSS before exposure to a substance. The antagonist was applied 3.5 min before the agonist. To determine the underlying Ca2+ source of the GDNF-evoked Ca2+ transients, internal stores were depleted by preincubation with a sarcoendoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 μM) and caffeine (20 mM) (Sharma and Vijayaraghavan 2001), or incubated with a Ca2+ free buffer (exchange of CaCl2 with MgCl2, and 1 mM EGTA). The experiments were performed at 22°C using a calcium imaging system and Simple PCI software (Compix Inc., Imaging Systems, Hamamatsu Photonics Management Corp., Cranberry Twp, PA, USA) and an inverted epifluorescence microscope (Nikon ECLIPSE TE2000-E) with a ×20 (NA 0.45) fluorescence dry lens and a Polychrome V, monochromator-based illumination system (TILL Photonics GMBH, CA, USA). The different substances were infused by a peristaltic pump (Instech Laboratories, Plymouth Meeting, PA, USA) at approximately 600 μL/min. One minute from the start of the experiment, the stimulating substance was pumped into the pump tubes for 30 s. The substance took approximately 60 s to reach the cells through the tubes. HHBSS 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 Res Digital Cooled CCD Camera (Hamamatsu Photonics Corp.).

The total areas under the transients, reflecting the amounts of Ca2+ released (Berridge 2007), were analysed to provide measures of the vigour of the Ca2+ responses. The amplitude was expressed as the maximum increase of the 340/380 ratio. The area under the Ca2+ peaks was calculated in Origin (Microcal Software Inc., Northampton, MA, USA).

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting

Cells were rinsed twice in phosphate-buffered saline and immediately lysed 20 min on ice in cold radioimmunoprecipitation 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% sodium dodecyl sulfate, 5 μg/mL aprotinin, and 5 μg/mL leupeptin, pH 7.4. The procedure was performed according to the process described by Persson et al. (2005). 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. The β-actin control was processed concomitantly with Na+/K+-ATPase blots but because of strength of signal issues, a blot depicting both molecules was not possible. However, β-actin signals were uniform across samples. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis were conducted using the Novex pre-cast gel system (Invitrogen) according to the manufacturer’s recommendations using 4–12% Bis–Tris 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, Sundbyberg, Sweden) in TBST (50 mM Tris–HCl, 150 mM NaCl and 0.05% Tween) for 60 min at 22°C. The membranes were then probed with primary antibodies over night (+4°C), washed 4 × 2 min with TBST, and subsequently probed with secondary horse-radish peroxidase-conjugated secondary antibodies for 60 min at 22°C, and finally washed several times in TBST. The primary antibodies used were Toll-like receptor 4 (TLR4; rabbit polyclonal) (Santa Cruz Biotechnology) diluted 1 : 500 and Na+/K+ ATPase (α-subunit) (mouse monoclonal) (Sigma) diluted 1 : 250. The secondary antibodies used were horse-radish peroxidase-conjugated donkey anti mouse or rabbit F(ab′)2 fragment (both from Jackson Immuno-Research, West Grove, PA, USA) diluted 1 : 10 000.

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 Inc., Waltham, MA, USA) and visualized with a FUJI Film LAS-3000 (Tokyo, Japan).

Protein determination

The protein determination assay was performed in accordance with the manufacturer’s instructions using a DC Protein Assay (Bio-Rad, Hercules, CA, USA), based with some modifications on the method used by Lowry et al. (1951). Both standard (0–4 mg/mL bovine serum albumin) and samples were mixed with the reagents, incubated for 15 min at 22°C, read at 750 nm with a Versa-max microplate reader, and analysed using SoftMax Pro 4.8, both from Molecular Devices (Sunnyvale, CA, USA).

Statistics

Differences between grouped mean values were identified using one-way anova with Dunnet’s multiple comparisons test. Error bars show standard error of the mean (SEM).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

GDNF elicits Ca2+ responses in astrocytes

Astrocytes were stimulated with GDNF (0.1–10 ng/mL = 10−12 to 10−10 M) and Ca2+-imaging experiments were performed on 15- to 17-day-old co-cultured astrocytes. The endothelial cell inserts were removed just before the experiments. In 0.1 ng/mL GDNF, all astrocytes responded (40/40); for 1 ng/mL GDNF, 32% responded (13/40 cells); and for 10 ng/mL GDNF, 50% responded (20/40 cells). The amplitudes and areas under the Ca2+ peaks (AUC) were calculated and the number of peaks was counted for each cell. The [Ca2+]i ratio was found to be highest at 0.1 ng/mL GDNF, in agreement with other studies (Nicole et al. 2001; Mills et al. 2007). In all further experiments, the cells were stimulated with 0.1 ng/mL GDNF (Fig. 1).

image

Figure 1.  GDNF-evoked Ca2+ transients. Astrocytes were stimulated with GDNF (0.1–10 ng/mL) and Ca2+ imaging experiments were performed on 15- to 17-day-old co-cultured astrocytes. In 0.1 ng/mL GDNF 100% of the astrocytes responded (40/40 cells), 1 ng/mL GDNF 32% responded (13/40 cells), and 10 ng/mL GDNF 50% responded (20/40 cells). The amplitudes and areas under the Ca2+ peaks were calculated and the number of peaks was counted for each cell. The cells were from four different coverslips, from two different seeding times. Ca2+ responses, expressed as max ratio increase of intensity at 340 and 380 nm, in astrocytes after stimulation with GDNF, the area under curve (AUC) of Ca2+ transients was calculated, number of peaks calculated, and responding cells is shown (%). Data are mean ± SEM.

Download figure to PowerPoint

To test if the Ca2+ elevation was of cross-membrane or of intracellular origin, GDNF was given in a Ca2+-free extracellular medium and this evoked Ca2+ transients in most of the cells. When the cells were incubated with thapsigargin, which is a depletor of intracellular Ca2+ stores (Sharma and Vijayaraghavan 2001), followed by caffeine, also used to complete the store depletion (Sharma and Vijayaraghavan 2001), the Ca2+ response was inhibited. With thapsigargin and caffeine in Ca2+-free buffer, no cells responded (Fig. 2), all indicating that Ca2+ elevation occurred for most cells due to efflux from the storage in ER.

image

Figure 2.  GDNF-evoked Ca2+ transients were dependent on intracellular Ca2+ stores. (a) 0.1 ng/mL GDNF elicited Ca2+ responses in astrocytes, in all cells tested (40/40; 100%). (b) GDNF in a Ca2+-free medium evoked Ca2+ transients in nearly all of the cells. (c) To determine the underlying Ca2+ source, internal stores were depleted with thapsigargin, followed by caffeine. The Ca2+ responses were inhibited. (d) All cells were inhibited with thapsigargin, caffeine, in Ca2+-free buffer. The amplitudes were expressed as the 340/380 ratio. The cells were from four different coverslips, from two different seeding times. Results shown are from a typical experiment.

Download figure to PowerPoint

Ifenprodil restores GDNF-evoked Ca2+ transients attenuated by LPS

Cells were incubated with LPS for 1 h leading to significantly decreased total area under the peaks (AUC, < 0.001) when compared with controls. Thirty-five of 60 astrocytes (58%) responded to GDNF, the others were not responding (Fig. 3).

image

Figure 3.  Ifenprodil restores GDNF-evoked Ca2+ transients attenuated by LPS. GDNF elicited Ca2+ responses in the astrocytes were used as control in all culture dishes. The area under curve (AUC) of Ca2+ transients was calculated, and number of responding cells is shown in %. (a) Cells incubated with LPS for 1 h, showed attenuated Ca2+ signalling. The total area under the peaks (AUC) were significantly decreased (< 0.001). (b) Thirty-five of 60 cells responded (58%), with lower amplitudes compared with control; the remaining cells were non-responding. 10−5 M ifenprodil restored the GDNF down-regulated Ca2+ transients, AUC was not significantly different from control, and 55 of 60 cells (92%) responded. 10−6 M ifenprodil did not fully restore the AUC (< 0.05), but nearly all cells responded (58 of 60; 97%). 10−7 M ifenprodil had no effect on the LPS treated cells (< 0.001), compared with control, and only 34 of 60 cells (57%) responded. The cells were from six different coverslips, and from three different seeding times. (c) The appearance of the Ca2+ transients is visualized. Results shown are from a typical experiment. Statistical analysis: the level of significance was analysed using one way anova followed by Dunnet’s multiple comparisons test. Data are mean ± SEM. *< 0.05, ***< 0.001; NS., non significant.

Download figure to PowerPoint

Cells were incubated with LPS for 1 h, treated the last 3.5 min with ifenprodil, 10−5, 10−6, or 10−7 M, and were then stimulated with GDNF. Ifenprodil, 10−5 M, restored the GDNF-evoked Ca2+ transients, which had been attenuated by LPS. AUC was not significantly different from control, and 55 of 60 cells (92%) responded. Ifenprodil, 10−6 M, did not fully restore the AUC, < 0.05, compared with control, but nearly all cells responded, 58 of 60 (97%). Ifenprodil, 10−7 M, had no effect on the LPS treated cells, < 0.001, compared with control, and only 34 of 60 cells (57%) responded (Fig. 3).

These results show that the GDNF-evoked Ca2+ transients, which were attenuated by LPS, were restored by ifenprodil in a concentration-dependent manner.

Ifenprodil restores GDNF-evoked [Ca2+]i transients attenuated by NMDA

GDNF-evoked Ca2+ transients were pre-stimulated with NMDA. The AUC was decreased compared with the control, < 0.001. Only half of the astrocytes responded, 20 of 40 cells (50%) (Fig. 4).

image

Figure 4.  Ifenprodil restores GDNF-evoked [Ca2+]i transients attenuated by NMDA. GDNF elicited Ca2+ responses in the astrocytes were used as control in all culture dishes. The area under curve (AUC) of Ca2+ transients was calculated, and number of responding cells is shown in %. (a) GDNF-evoked Ca2+ transients were pre-stimulated with NMDA. The AUC was decreased compared with the control, < 0.001. (b) Only half of the astrocytes responded, 20 of 40 cells (50%). When the astrocytes were pre-stimulated with ifenprodil (10−5 M) and NMDA, the GDNF-attenuated Ca2+ transients were restored to the control level, and nearly all cells responded, 36 of 40 (90%). Ifenprodil alone had no effect on the GDNF-evoked Ca2+ transients, 37 of 40 cells responded (92%). The cells were from four different coverslips, and from two different seeding times. (c) The appearance of the Ca2+ transients is visualized. Results shown are from a typical experiment. Statistical analysis: the level of significance was analysed using one way anova followed by Dunnet’s multiple comparisons test. Data are mean ± SEM. ***< 0.001; NS., non significant.

Download figure to PowerPoint

Ifenprodil, 10−5 M, restored the GDNF-evoked Ca2+ transients, which had been attenuated by NMDA. AUC was not significantly different from control, and 36 of 40 astrocytes (90%) responded, indicating that the GDNF-evoked Ca2+ transients were decreased by NMDA, and were restored by ifenprodil. Ifenprodil alone, had no effect on the GDNF-evoked Ca2+ transients, 37 of 40 cells responded (92%) (Fig. 4).

Astrocytes express the NR2B subunit of the NMDA receptor

Astrocytes stimulated with NMDA responded immediately, 39 of 40 cells (97%). Ifenprodil, 10−5 M, blocked the NMDA-evoked Ca2+ response (< 0.001), 22 of 40 cells were blocked, the others responded with low amplitudes (Fig. 5).

image

Figure 5.  NMDA-evoked Ca2+ responses in astrocytes. The NMDA-induced Ca2+ responses (10−4 M) were blocked with ifenprodil (10−5 M), demonstrating the presence of the NR2B subunit on astrocytes. (a) The area under curve (AUC) of Ca2+ transients was calculated and (b) number of responding cells giving in %. The cells were from four different coverslips and from two different seeding times. (c) Typical Ca2+ responses, expressed as max ratio increase of intensity at 340 and 380 nm, in astrocytes after stimulation with NMDA (= control), and after treatment with ifenprodil. Statistical analysis: the level of significance was analysed using one way anova followed by Dunnet’s multiple comparisons test. Data are mean ± SEM. ***< 0.001.

Download figure to PowerPoint

The result indicates that astrocytes co-cultured with endothelial cells express the NR2B subunit of the NMDA receptor.

Ifenprodil restores GDNF-evoked Ca2+ transients attenuated by IL-1β

GDNF-evoked Ca2+ transients were pre-stimulated with IL-1β (10 ng/mL). The AUC was decreased compared with the control, < 0.001. All cells responded (40/40 cells) (Fig. 6).

image

Figure 6.  Ifenprodil restores GDNF-evoked Ca2+ transients attenuated by IL-1β. GDNF elicited Ca2+ responses in the astrocytes were used as control in all culture dishes. The area under curve (AUC) of Ca2+ transients was calculated, and number of responding cells is shown in %. (a) Cells treated with IL-1β (10 ng/mL) showed attenuated GDNF_evoked Ca2+ transients. Thirty of 30 cells responded (100%), with lower amplitudes compared with control, and the total area under the peaks (AUC) was significantly decreased (< 0.001). Ifenprodil restored the GDNF down-regulated Ca2+ transients, AUC was even higher than the control, < 0.05, and 40 of 40 cells (100%) responded. (b) Number of responding cells giving in %. The cells were from four different coverslips and from two different seeding times. (c) The appearance of the Ca2+ transients is visualized. Results shown are from a typical experiment. Statistical analysis: the level of significance was analysed using one way anova followed by Dunnet’s multiple comparisons test. Data are mean ± SEM. *< 0.05; ***< 0.001; NS., non significant.

Download figure to PowerPoint

Cells were incubated with IL-1β and treated 3.5 min with ifenprodil (10−5), and were then stimulated with GDNF. Ifenprodil restored the GDNF attenuated Ca2+ transients, AUC was even higher than the control, < 0.05, and 40/40 cells (100%) responded (Fig. 6).

Ifenprodil increases the Na+/K+-ATPase expression

Astrocytes were incubated with LPS for 24 h. The expression of Na+/K+-ATPase visualized with western blot was down-regulated compared with control cells, < 0.05 (Fig. 7a).

image

Figure 7.  Ifenprodil increases the Na+/K+-ATPase expression. The expression of Na+/K+-ATPase is visualized with western blot. The value of integrated density for the recognized antigen in each lane is expressed as % of control. (a) Astrocytes were incubated with LPS for 24 h. The expression of Na+/K+-ATPase was down-regulated compared with control cells. Astrocytes were incubated with ifenprodil (10−5 to 10−7 M), 30 min before the cells were treated with ifenprodil and LPS for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes pretreated with 10−5 M ifenprodil compared with control. Ifenprodil, 10−6 or 10−7 M, had no effect compared with LPS treated cells. (b) Astrocytes were incubated with LPS and IL-1β for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes pretreated with 10−5 M ifenprodil. (c) Astrocytes were incubated with LPS and NMDA for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes pretreated with 10−5 M ifenprodil. = 3. Statistical analysis: the level of significance was analysed using one way anova followed by Dunnet’s multiple comparisons test. Data are mean ± SEM. **< 0.01, *< 0.05; NS, non significant. Western blot membrane from a typical experiment, respectively, is shown.

Download figure to PowerPoint

Astrocytes were incubated with ifenprodil (10−5–10−7 M), 30 min before the cells were treated with ifenprodil and LPS for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes treated with 10−5 M ifenprodil compared with control (= 3). Ifenprodil 10−6 or 10−7 M had no effect compared with LPS treated cells (Fig. 7a).

Astrocytes were incubated with LPS and IL-1β for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes pretreated with 10−5 M ifenprodil compared with control, < 0.05 (= 3). (Fig. 7b).

Furthermore, other astrocytes were incubated with LPS and NMDA for 24 h. The expression of Na+/K+-ATPase was up-regulated in astrocytes pretreated with 10−5 M ifenprodil compared with control, < 0.05 (= 3). (Fig. 7c).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

Regulation of Ca2+ dynamics by transmitters and soluble factors is a possible mechanism by which the astrocyte network detects changes in the CNS microenvironment, such as inflammatory processes, and regulates brain activities. The complexity of Ca2+ signalling has made it difficult to determine the physiological role of these phenomena although Ca2+ signalling is an important function of astrocytes. Intra- and intercellular Ca2+ signalling have been proposed to play important roles in information processing. It is a well-known element of signalling pathways implicated in activity dependent neuronal survival. Neurotrophic factors as GDNF induce a small and rapid increase in intracellular Ca2+, which plays an important role for neuronal survival (Pérez-García et al. 2004). Ca2+ signalling over long distances is analogous to, but much slower than, the propagation of action potentials in neurons (Cornell-Bell et al. 1990).

In the present study, GDNF was shown to induce Ca2+ transients, and the responses were better at low concentrations. When these Ca2+ transients were influenced by LPS, few astrocytes responded to GDNF. LPS is a potent inflammatory activator (Nakamura 2002), commonly used in experimental neuroinflammation (Zielasek and Hartung 1996). Attenuation in Ca2+ signalling when astrocytes were exposed to LPS has been shown earlier (Morita et al. 2003; Hansson et al. 2008; Delbro et al. 2009). When astrocytes are exposed to inflammatory stimuli the TLR4 is activated, which is expressed by astrocytes and LPS has been shown to be a TLR4 agonist (Kielian 2006; Forshammar et al. 2011). It has been proposed that activation of TLR4 drives inflammation that gives rise to symptoms and promotes BBB disruption through injury to endothelial cells (Krasowska-Zoladek et al. 2007). From observations in different cellular systems, a down-regulation of Na+ transporters is observed when cells were exposed to LPS, which implies dysfunction of the Na+/K+-ATPase activity. Astrocytes have strong resistance to Na+ influx only when Na+/K+-ATPase activity is maintained. Protein expression of Na+/K+-ATPase was reduced in astrocytes in primary culture after 8 h, as an indication that the Na+ transporters were down-regulated (Forshammar et al. 2011).

Astrocytic swelling is part of a cytotoxic response that characterizes brain damage and a number of mediators have been identified that initiate this process. Inflammatory stimuli induce elevation of extracellular K+, increased release of neurotransmitters such as glutamate and elevation in polyamine levels, which all induces swelling (Kempski et al. 1991; Hansson 1994; Kimelberg et al. 1995; Trout et al. 1995). The intracellular Ca2+ release in astrocytes is then decreased which also has an influence on the polymerization of the actin cytoskeleton (Hansson 1994; Allansson et al. 2001; Forshammar et al. 2011). Ifenprodil is a non-competitive antagonist and an inhibitor of the polyamine binding site on the NR2B subunit of the NMDA receptor (Chizh et al. 2001; Iwata et al. 2007), and it reduces both astrocytic swelling and polyamine levels (Trout et al. 1995). Addition of ifenprodil to astrocytes incubated with LPS, resulted in restoration of the attenuated GDNF-evoked Ca2+ transients as well as more cells responding in a concentration dependent manner. Changes of the NR2B subunit at the NMDA receptor have been observed in rats treated with LPS (Harréet al. 2008). It is possible that receptors like the NR2B subunit of the NMDA receptor are activated by inflammatory stimuli, which may obstruct the GDNF-induced Ca2+ signalling.

NMDA induces intracellular Ca2+ release in our model astrocytes. NMDA receptors are involved in numerous physiological and pathological processes, including synaptic plasticity, chronic pain, psychosis, ischemic insults, and several degenerative disorders. Agents that target and alter NMDA receptor function may thus have therapeutic benefit. Allosteric sites, which differ from agonist-binding and channel-permeation sites, can be modulated either positively or negatively. Astrocytes express both the NR1 and NR2 subunits of the NMDA receptor (Conti et al. 1996; Schipke et al. 2001; Verkhratsky and Kirchhoff 2007) in a relationship between polyamine synthesis and binding to the NMDA receptor (Trout et al. 1995). NR2B has been shown to be over-expressed during pain in mice, and blocking this subunit has been a focus in research on pain targets (Chizh et al. 2001). During pain states, enhanced extracellular concentrations of polyamines have been observed in the CNS (Chizh et al. 2001).

Interactions between GDNF and NMDA receptors have been demonstrated on neurons, showing that GDNF, through activation of the extracellular signal regulated kinases pathway, modulates the activity of the NMDA receptor by reducing the Ca2+ influx (Nicole et al. 2001). In the present study, the GDNF-evoked Ca2+ signalling was attenuated by NMDA. Ifenprodil blocked the NR2B subunit, and the GDNF-evoked Ca2+ transients were restored, and also a larger number of astrocytes responded.

The ability of IL-1β to influence astrocyte function not only depends on the expression of the appropriate receptors, but also on the activation of specific intracellular signalling pathways. Thus, the proinflammatory cytokine IL-1β mediates its effects on immunity and inflammation by interacting with the type 1 IL-1R receptor expressed on astrocytes particularly after injury, suggesting a specific association with inflammatory responses (Friedman 2001). IL-1β has been shown to modulate the voltage-dependent Na+ currents in an IL-1 receptor dependent manner in neurons (Liu et al. 2006). We show in the present study interactions between GDNF and IL-1β, and the GDNF-evoked Ca2+ transients were attenuated by IL-1β. We show that even in the interactions between GDNF and IL-1β, ifenprodil could restore the GDNF-evoked Ca2+ transients.

Ifenprodil has not only antagonistic effects on the NMDA receptor, but has also been shown to inactivate tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons (Tanahashi et al. 2007). This effect can be another mechanism by which ifenprodil demonstrated positive effects on the attenuated Ca2+ transients evoked by GDNF, and in combination with LPS, NMDA or IL-1β. The effects of ifenprodil seem rather specific for GDNF-evoked Ca2+ transients because they were not observed in response to endomorphin-1 or nicotine stimulation.

For in vitro cortical duct cells, proinflammatory cytokines as TNF-α and IL-1β have been shown to decrease the expression of Na+/K+-ATPase during severe experimental sepsis (Schmidt et al. 2007). Stimulation of inflammatory reactive receptors, such as TLR4, NMDA or IL-1R, seems to change the GDNF-evoked Ca2+ signalling and thereby disturb the Ca2+ transients in the astrocytic networks. One explanation can be that the Na+ transporters are down-regulated by LPS.

What, if something, can help us to identify new methods for treating neuroinflammation and regulating cellular balance? Because astrocytes are intimate co-players with neurons in the CNS, more knowledge of astrocyte responses to inflammatory activators may give new insight to our understanding of mechanisms underlying neuroinflammation and help us learn how to attenuate neuroinflammation and restore glial cell function. One strategy would be to block inflammatory active receptors or receptor sites.

In conclusion, using the co-cultured model of astrocytes, we have shown that ifenprodil restored the GDNF-evoked Ca2+ transients and increased the Na+/K+-ATPase expression. We conclude that this is a step in understanding astrocyte response and neuroinflammatory mechanisms. More study is needed to find means to attenuate neuroinflammation and restore glial cell function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

This work was supported by the Swedish Research Council (grant No 33X-06812), Edit Jacobson’s Foundation, Folksam’s Forskningsstiftelse, Arvid Carlsson’s Foundation, and the Sahlgrenska University Hospital (LUA/ALF GBG-11587), Gothenburg, Sweden. It is no conflict of interest.

Authors’ contributions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References

CL participated in all experimental analyses and read and revised the manuscript. AW carried out the cell cultures and the Ca2+ imaging experiments and read and revised the manuscript. UB carried out the cell cultures and the western blot experiments and read and revised the manuscript. BB participated in conceptual analysis of the data and preparation of the final draft. EH supervised and designed the study, drafted the manuscript, and read and revised the manuscript. All authors read and approved the final manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Authors’ contributions
  8. References