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Effects of glucocorticoids in the gp120-induced inhibition of glutamate uptake in hippocampal cultures
Brain Research 972 (2003) 137–141 www.elsevier.com / locate / brainres
Research report
Effects of glucocorticoids in the gp120-induced inhibition of glutamate uptake in hippocampal cultures Sheila M. Brooke*, Robert M. Sapolsky Department of Biological Sciences, Gilbert Bldg., Rm 432, Stanford University, Stanford, CA 94305 -5020, USA Accepted 24 February 2003
Abstract Studies examining the development of AIDS Related Dementia have concentrated on neurotoxic properties of the HIV viral coat protein, gp120. We have previously shown that this neurotoxicity can be exacerbated by glucocorticoids (GCs), the stress hormones secreted by the adrenal. Moreover, GCs also worsen several of the mechanisms mediating gp120 neurotoxicity, such as increased calcium flux, ROS generation, and energy depletion. Gp120 interferes with the reuptake of glutamate in glia cultures, another possible mechanism by which it can be neurotoxic. This paper examines the role of GCs in exacerbating this phenomenon. It was found that while GCs do not exacerbate the decrease in reuptake of glutamate in glia cultures, they do enhance the decrease in mixed neuronal cultures and this latter effect appears to be energy-dependent. 2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Gp120; AIDS-related dementia; Glutamate uptake; Glucocorticoid
1. Introduction Twenty to fifty percent of the patients suffering from AIDS eventually develop AIDS Related Dementia (ARD), characterized by loss of cells in the CNS and decreased cognitive and motor function [25]. Loss of neurons and glia in the brain is in part attributed to protein elements of the HIV virus such as TAT, gp41, nef and gp120 [1–3,6]. Gp120 is thought to act primarily through the microglia to release neurotoxic elements such as TNF-a, ILB2, IL6, IL1, NO, quinolinic acid, arachidonic acid and glutamate, setting up cascades in the neurons that can result in increased calcium mobilization, reactive oxygen species (ROS) production, and decreased energy mobilization eventually leading to neuron death [19]. Glutamate release and persistence in the synaptic cleft is a major cause of neuronal death, especially in hippocampal neurons that have large numbers of receptors for this *Corresponding author. Tel.: 11-650-723-3260; fax: 11-650-7255356. E-mail address: [email protected] (S.M. Brooke).
excitatory amino acid (EAA). Glutamate’s primary route of inactivation is by reuptake, via the glia where it is converted to glutamine. Glia and neurons both have high affinity glutamate transporters, GLT1 and EAAC-1, that utilize sodium and potassium pumps that require sufficient energy reserves. Transporters on neurons occur in many regions of the brain including the hippocampus, and appear to aid in regulation of glutamate in the synapse, functioning when inhibitors of glia transporters are inactivated [13,16,18]. Two major mechanisms of gp120 neurotoxicity include the release of toxic elements from microglia exposed to gp120, and direct action of gp120 on chemokine receptors in neurons [19,20]. Gp120’s effect on the inactivation of glutamate may also play a role in neurotoxicity. It has been shown by several investigators that gp120 can inhibit the reuptake of glutamate in glial cultures, resulting in increased synaptic levels of glutamate, increased stimulation of glutamate receptors, and increased mobilization of free cytosolic calcium, all eventually resulting in increased neuron death [4,17,28]. With the increasing knowledge about the mechanisms
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underlying gp120-induced neurotoxicity has come an appreciation of potential modulators of toxicity such as glucocorticoids (GCs). GCs, the adrenal hormones released during stress, can exacerbate neuron death in conjunction with a wide variety of insults including EAAs, hypoxia– ischemia, and hypoglycemia (reviewed in Ref. [26]) as well gp120 neurotoxicity in hippocampal, cortical, and striatal cultures (reviewed in Ref. [10]). Hypothesized routes by which GCs increase the neurotoxicity of gp120 include increase in gp120-induced calcium mobilization, disruption of neuronal energetics, and increases in ROS accumulation and lipid peroxidation. GCs have also been shown to decrease the reuptake of glutamate in glia by disrupting energy supplies, and exacerbating glutamate uptake, but not release in mixed cultures [12,29]. This paper examines the effects of combining of gp120 and GCs on the reuptake of glutamate, using aspartate as a marker of glutamate uptake in hippocampal cultures. We report the somewhat surprising result that presence of neurons is an important factor in the extent of this inhibition.
2. Methods All chemical reagents unless otherwise noted were from Sigma (St Louis, MO, USA). Primary hippocampal mixed cultures were prepared from E18 prenatal rats according to previously described methods [7]. Following is a brief description of the procedure. After dissection, the tissue was treated with papain (Worthington Biochemical, Freehold, NJ, USA) according to the procedure recommended by the company. The cells were dissociated, filtered through an 80-mm cell strainer and resuspended in modified MEM media (UCSF Tissue Culture Facility, San Francisco, CA, USA) and supplemented with 10% horse serum (Hyclone, Logan, UT, USA). Cells were plated on 24-well plates treated with poly-D-lysine at a density of 1.6310 5 cells / cm 2 . These mixed cultures were used on days 10–12 when the percentage of neurons was 20–30%. Glia cultures were created from the mixed cultures by changing to cold glial media (MEM with 20% fetal calf serum) three times in the first 7 days of culture. This produces cultures with undetectable amounts of Map2 staining as a marker for neurons and with cells that morphologically resembled various types of glia cells (data not shown) [8]. Glutamate uptake protocol was based on published methods [17,28] using aspartate instead of glutamate. Aspartate is taken up using the same receptors with a similar Kd but is not further metabolized [14]. Twenty-four hours prior to the uptake studies the cells were treated with 1 mM corticosterone (CORT) or ethanol (0.01%) as control; this time-course is standard and commensurate with the time needed to reach the peak of the genomically-
mediated actions of this steroid hormone. The insult with gp120 IIIB (Immuno Diagnostics, Woburn, MA, USA) was administered by changing to KRPH buffer (136 mM NaCl, 1 mM CaCl 2 , 4.7 mM KCl, 1.25 mM Mg SO 4 , 4 mM NaH 2 PO 4 , 20 mM Hepes, 5 mM glucose) containing 200 pM gp120 in the experimental wells and KRPH buffer only in the control wells. Ninety minutes later glutamate uptake studies were done. One plate at a time, the cells were placed on a heating block at 37 8C and 1 mM of DL-threo-b hydroxyaspartate (THA) was added to the blanks, to block the glutamate transporters. A solution containing 0.25 mCi [ 3 H]D-aspartate (Perkin Elmer, Boston, MA, USA), and 1 mM D-aspartate in KRPH buffer was added to the cells. Five minutes later, the radioactive solution was removed and the wells washed four times with cold KRPH buffer containing THA (1 mM). After the last wash, the wells were aspirated dry and 1% Triton X-100 was added to lyse the cells. Duplicate aliquots of lysate were counted in a scintillation counter (Beckman). Pierce assay (Rockford, IL, USA) to determine protein levels in each well was also done. Experimental values were expressed as percentage of controls for each week’s experiments. Analysis was done by non-parametric and parametric two-way ANOVAs; SigmaStat (Jandel, San Rafael, CA, USA) software was used for statistical analysis. However data are presented as mean6standard error.
3. Results In Fig. 1, we confirmed the previous reports of gp120 and CORT each significantly decreasing the uptake of aspartate in glia cultures. The 18% reduction we observed with gp120 and the 22% decrease we observed in the presence of CORT are within previously reported ranges [15,17,28]. However CORT did not exacerbate the effect of gp120. Although the 21% decrease with this treatment was significantly different from control, it was not significantly different from CORT or gp120 alone. Since studies showing that CORT exacerbated gp120 neurotoxicity [7] were in mixed neuronal / glial hippocampal cultures, we then examined the effect of gp120 and CORT treatments on aspartate uptake in cultures with 20 to 30% neurons. Neither gp120 nor CORT alone had a significant effect on uptake; however, the addition of CORT to gp120 significantly exacerbated the latter’s action in media containing 5 mM glucose, resulting in a significant 15% decline (Fig. 2) from both control and gp120 alone. A number of studies suggest that the disruptive effects of glucocorticoids in the hippocampus are secondary to disruptive effects upon energetics (discussed below). Commensurate with that, the exacerbating effects of CORT were eliminated when cultures were supplemented with excess energy (20 mM glucose; Fig. 2).
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Fig. 1. Decrease in glutamate uptake as a percent of control in hippocampal glia cultures treated with corticosterone (CORT) (1 mM) for 24 h, gp120 (200 pM) for 90 min and gp1201CORT (corticosterone at 1 mM plus gp120 at 200 pM) at 5 mM glucose. ***Significantly different from control at P,0.001. N for each treatment528.
4. Discussion Results in this paper confirm previous findings with respect to the effects of both gp120 and GCs on glutamate transport into glia. However we were no longer able to obtain significant decreases with GCs and gp120 alone
(Fig. 2). We had not been able to find any reports of gp120 or GCs alone decreasing glutamate uptake in mixed cultures. An observation with respect to the former that was also noted in another recently published paper [4]. However, a combination of gp120 and GCs decreased the glutamate uptake in 5 mM glucose relative to control and
Fig. 2. Decrease in glutamate uptake as a percent of control in mixed hippocampal cultures treated with CORT (corticosterone 1 mM) for 24 h, gp120 (200 pM) for 90 min and gp1C (corticosterone at 1 mM plus gp120 at 200 pM) with glucose levels of 5 and 20 mM. *Significantly different from control at P,0.05, & significantly different from gp120 alone at P,0.05, and [ significantly different from gp1C at 5 mM glucose at P,0.05. N for each treatment526–35.
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gp120 treatments. In agreement with this finding, GCs exacerbate the cyanide-induced decrease in glutamate reuptake [12]. Glutamate, aspartate and EAAs in general are transported into cells by electrochemical gradients across the cell membrane that must be maintained at a negative potential. EAAs must be bound to one or more sodium ions for movement into the cell and potassium movement out of the cells is required to maintain equilibrium. The process is reversible and is highly energy-dependent, requiring one molecule of ATP for every molecule of glutamate taken up [27]. GCs and gp120 act in two different ways to decrease glutamate uptake. GCs are hypothesized to decrease the available energy necessary to maintain the sodium / potassium pumps that are part of the glutamate transporter system by disrupting the glucose transporter system in neurons and glia. In the short-term (hours), GCs initiate translocation of the transporters from the plasma membrane to intracellular storage sites and then over the longterm inhibit transcription of the transporter [11,29]. Gp120 acts on microglia and neurons to release arachidonic acid and superoxide that in turn inhibits glutamate uptake into glia [4,15]; these and other adverse effects of gp120 in the nervous system are specific, in that they are eliminated by denaturing the glycoprotein [7,10]. Whether GCs and gp120 are ultimately inhibiting glutamate uptake by altering Vmax and / or Km was not examined in this study, but either is a possibility. From our results we can hypothesize how these two independent mechanisms of interference with glutamate uptake may interact in hippocampal cultures. In glia cultures both GCs and gp120 can inhibit uptake as described. However in mixed cultures neurons that may not be affected directly by either treatment are able to transport some excess glutamate that accumulates in the presence of GCs and gp120 individually. It has also been reported that in the presence of neurons, astrocytes express both EAAT and GLT1 transporters, whereas only the latter are expressed in pure astrocyte cultures [27]. A recent report indicates that the uptake capacity of GLT1 and GLAST on astrocytes is enhanced in the presence of neurons [23]. Therefore, in the presence of GCs or gp120 alone there is no longer a reduction in uptake. However, we have previously demonstrated that only a combination of gp120 and GCs can disrupt mitochondrial potential in mixed cultures, therefore decreasing available energy supplies [9]. Because glutamate uptake has high-energy demand, when both gp120 and GCs are present at the same time, the disruption in energy production in the cells prevents the compensation due to excess transporter availability resulting in an overall decrease in the amount of uptake. We tested this hypothesis by increasing energy availability to the cultures by increasing glucose levels from 5 to 20 mM. With more energy available, the cells were able to
compensate for the disruption in energy supplies caused by the combination of GCs and gp120 and normal transport levels were restored (Fig. 2). It has previously been demonstrated that supplementation with glucose can decrease GCs exacerbation of gp120-induced neurotoxicity and calcium mobilization [9]. There is also a precedent with other insults such as EAAs, anti-metabolites, and hypoxia, for excess glucose reversing GCs exacerbation of neurotoxicity and calcium mobilization [26]. There is a body of literature indicating that astrocyte, astrocyte-rich and astrocyte-poor cultures act differently with respect to a number of parameters, for example neuron death in the presence of glutamate transporter inhibitor, and glutamine and the release of c-AMP [5,22,24]. This report therefore would appear to be another example of the importance of studying the neuron glia interaction rather than cell types individually. GCs exacerbation of gp120 neurotoxicity in mixed hippocampal cultures appears to occur by several mechanisms: calcium influx is increased, energy stores are disrupted, ROS generation increased and degradation decreased [10]. And these results indicate that GCs may also increase neurotoxicity by decreasing the efficiency of glutamate elimination from the synapse in mixed cultures. Gp120 may be a causal agent in the development of ARD [19] and GCs are used extensively in treatment of HIV related diseases such as Pneumocystis carinii pneumonia [21], a procedure that could possibly lead to excess neuron death in patients. A further understanding of the mechanisms of GCs exacerbation of gp120 neurotoxicity as presented could perhaps be useful information in the development of treatments for the prevention of ARD.
Acknowledgements This work was supported by Grant IH RO1 MH 53814.
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Research report
Effects of glucocorticoids in the gp120-induced inhibition of glutamate uptake in hippocampal cultures Sheila M. Brooke*, Robert M. Sapolsky Department of Biological Sciences, Gilbert Bldg., Rm 432, Stanford University, Stanford, CA 94305 -5020, USA Accepted 24 February 2003
Abstract Studies examining the development of AIDS Related Dementia have concentrated on neurotoxic properties of the HIV viral coat protein, gp120. We have previously shown that this neurotoxicity can be exacerbated by glucocorticoids (GCs), the stress hormones secreted by the adrenal. Moreover, GCs also worsen several of the mechanisms mediating gp120 neurotoxicity, such as increased calcium flux, ROS generation, and energy depletion. Gp120 interferes with the reuptake of glutamate in glia cultures, another possible mechanism by which it can be neurotoxic. This paper examines the role of GCs in exacerbating this phenomenon. It was found that while GCs do not exacerbate the decrease in reuptake of glutamate in glia cultures, they do enhance the decrease in mixed neuronal cultures and this latter effect appears to be energy-dependent. 2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Gp120; AIDS-related dementia; Glutamate uptake; Glucocorticoid
1. Introduction Twenty to fifty percent of the patients suffering from AIDS eventually develop AIDS Related Dementia (ARD), characterized by loss of cells in the CNS and decreased cognitive and motor function [25]. Loss of neurons and glia in the brain is in part attributed to protein elements of the HIV virus such as TAT, gp41, nef and gp120 [1–3,6]. Gp120 is thought to act primarily through the microglia to release neurotoxic elements such as TNF-a, ILB2, IL6, IL1, NO, quinolinic acid, arachidonic acid and glutamate, setting up cascades in the neurons that can result in increased calcium mobilization, reactive oxygen species (ROS) production, and decreased energy mobilization eventually leading to neuron death [19]. Glutamate release and persistence in the synaptic cleft is a major cause of neuronal death, especially in hippocampal neurons that have large numbers of receptors for this *Corresponding author. Tel.: 11-650-723-3260; fax: 11-650-7255356. E-mail address: [email protected] (S.M. Brooke).
excitatory amino acid (EAA). Glutamate’s primary route of inactivation is by reuptake, via the glia where it is converted to glutamine. Glia and neurons both have high affinity glutamate transporters, GLT1 and EAAC-1, that utilize sodium and potassium pumps that require sufficient energy reserves. Transporters on neurons occur in many regions of the brain including the hippocampus, and appear to aid in regulation of glutamate in the synapse, functioning when inhibitors of glia transporters are inactivated [13,16,18]. Two major mechanisms of gp120 neurotoxicity include the release of toxic elements from microglia exposed to gp120, and direct action of gp120 on chemokine receptors in neurons [19,20]. Gp120’s effect on the inactivation of glutamate may also play a role in neurotoxicity. It has been shown by several investigators that gp120 can inhibit the reuptake of glutamate in glial cultures, resulting in increased synaptic levels of glutamate, increased stimulation of glutamate receptors, and increased mobilization of free cytosolic calcium, all eventually resulting in increased neuron death [4,17,28]. With the increasing knowledge about the mechanisms
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underlying gp120-induced neurotoxicity has come an appreciation of potential modulators of toxicity such as glucocorticoids (GCs). GCs, the adrenal hormones released during stress, can exacerbate neuron death in conjunction with a wide variety of insults including EAAs, hypoxia– ischemia, and hypoglycemia (reviewed in Ref. [26]) as well gp120 neurotoxicity in hippocampal, cortical, and striatal cultures (reviewed in Ref. [10]). Hypothesized routes by which GCs increase the neurotoxicity of gp120 include increase in gp120-induced calcium mobilization, disruption of neuronal energetics, and increases in ROS accumulation and lipid peroxidation. GCs have also been shown to decrease the reuptake of glutamate in glia by disrupting energy supplies, and exacerbating glutamate uptake, but not release in mixed cultures [12,29]. This paper examines the effects of combining of gp120 and GCs on the reuptake of glutamate, using aspartate as a marker of glutamate uptake in hippocampal cultures. We report the somewhat surprising result that presence of neurons is an important factor in the extent of this inhibition.
2. Methods All chemical reagents unless otherwise noted were from Sigma (St Louis, MO, USA). Primary hippocampal mixed cultures were prepared from E18 prenatal rats according to previously described methods [7]. Following is a brief description of the procedure. After dissection, the tissue was treated with papain (Worthington Biochemical, Freehold, NJ, USA) according to the procedure recommended by the company. The cells were dissociated, filtered through an 80-mm cell strainer and resuspended in modified MEM media (UCSF Tissue Culture Facility, San Francisco, CA, USA) and supplemented with 10% horse serum (Hyclone, Logan, UT, USA). Cells were plated on 24-well plates treated with poly-D-lysine at a density of 1.6310 5 cells / cm 2 . These mixed cultures were used on days 10–12 when the percentage of neurons was 20–30%. Glia cultures were created from the mixed cultures by changing to cold glial media (MEM with 20% fetal calf serum) three times in the first 7 days of culture. This produces cultures with undetectable amounts of Map2 staining as a marker for neurons and with cells that morphologically resembled various types of glia cells (data not shown) [8]. Glutamate uptake protocol was based on published methods [17,28] using aspartate instead of glutamate. Aspartate is taken up using the same receptors with a similar Kd but is not further metabolized [14]. Twenty-four hours prior to the uptake studies the cells were treated with 1 mM corticosterone (CORT) or ethanol (0.01%) as control; this time-course is standard and commensurate with the time needed to reach the peak of the genomically-
mediated actions of this steroid hormone. The insult with gp120 IIIB (Immuno Diagnostics, Woburn, MA, USA) was administered by changing to KRPH buffer (136 mM NaCl, 1 mM CaCl 2 , 4.7 mM KCl, 1.25 mM Mg SO 4 , 4 mM NaH 2 PO 4 , 20 mM Hepes, 5 mM glucose) containing 200 pM gp120 in the experimental wells and KRPH buffer only in the control wells. Ninety minutes later glutamate uptake studies were done. One plate at a time, the cells were placed on a heating block at 37 8C and 1 mM of DL-threo-b hydroxyaspartate (THA) was added to the blanks, to block the glutamate transporters. A solution containing 0.25 mCi [ 3 H]D-aspartate (Perkin Elmer, Boston, MA, USA), and 1 mM D-aspartate in KRPH buffer was added to the cells. Five minutes later, the radioactive solution was removed and the wells washed four times with cold KRPH buffer containing THA (1 mM). After the last wash, the wells were aspirated dry and 1% Triton X-100 was added to lyse the cells. Duplicate aliquots of lysate were counted in a scintillation counter (Beckman). Pierce assay (Rockford, IL, USA) to determine protein levels in each well was also done. Experimental values were expressed as percentage of controls for each week’s experiments. Analysis was done by non-parametric and parametric two-way ANOVAs; SigmaStat (Jandel, San Rafael, CA, USA) software was used for statistical analysis. However data are presented as mean6standard error.
3. Results In Fig. 1, we confirmed the previous reports of gp120 and CORT each significantly decreasing the uptake of aspartate in glia cultures. The 18% reduction we observed with gp120 and the 22% decrease we observed in the presence of CORT are within previously reported ranges [15,17,28]. However CORT did not exacerbate the effect of gp120. Although the 21% decrease with this treatment was significantly different from control, it was not significantly different from CORT or gp120 alone. Since studies showing that CORT exacerbated gp120 neurotoxicity [7] were in mixed neuronal / glial hippocampal cultures, we then examined the effect of gp120 and CORT treatments on aspartate uptake in cultures with 20 to 30% neurons. Neither gp120 nor CORT alone had a significant effect on uptake; however, the addition of CORT to gp120 significantly exacerbated the latter’s action in media containing 5 mM glucose, resulting in a significant 15% decline (Fig. 2) from both control and gp120 alone. A number of studies suggest that the disruptive effects of glucocorticoids in the hippocampus are secondary to disruptive effects upon energetics (discussed below). Commensurate with that, the exacerbating effects of CORT were eliminated when cultures were supplemented with excess energy (20 mM glucose; Fig. 2).
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Fig. 1. Decrease in glutamate uptake as a percent of control in hippocampal glia cultures treated with corticosterone (CORT) (1 mM) for 24 h, gp120 (200 pM) for 90 min and gp1201CORT (corticosterone at 1 mM plus gp120 at 200 pM) at 5 mM glucose. ***Significantly different from control at P,0.001. N for each treatment528.
4. Discussion Results in this paper confirm previous findings with respect to the effects of both gp120 and GCs on glutamate transport into glia. However we were no longer able to obtain significant decreases with GCs and gp120 alone
(Fig. 2). We had not been able to find any reports of gp120 or GCs alone decreasing glutamate uptake in mixed cultures. An observation with respect to the former that was also noted in another recently published paper [4]. However, a combination of gp120 and GCs decreased the glutamate uptake in 5 mM glucose relative to control and
Fig. 2. Decrease in glutamate uptake as a percent of control in mixed hippocampal cultures treated with CORT (corticosterone 1 mM) for 24 h, gp120 (200 pM) for 90 min and gp1C (corticosterone at 1 mM plus gp120 at 200 pM) with glucose levels of 5 and 20 mM. *Significantly different from control at P,0.05, & significantly different from gp120 alone at P,0.05, and [ significantly different from gp1C at 5 mM glucose at P,0.05. N for each treatment526–35.
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gp120 treatments. In agreement with this finding, GCs exacerbate the cyanide-induced decrease in glutamate reuptake [12]. Glutamate, aspartate and EAAs in general are transported into cells by electrochemical gradients across the cell membrane that must be maintained at a negative potential. EAAs must be bound to one or more sodium ions for movement into the cell and potassium movement out of the cells is required to maintain equilibrium. The process is reversible and is highly energy-dependent, requiring one molecule of ATP for every molecule of glutamate taken up [27]. GCs and gp120 act in two different ways to decrease glutamate uptake. GCs are hypothesized to decrease the available energy necessary to maintain the sodium / potassium pumps that are part of the glutamate transporter system by disrupting the glucose transporter system in neurons and glia. In the short-term (hours), GCs initiate translocation of the transporters from the plasma membrane to intracellular storage sites and then over the longterm inhibit transcription of the transporter [11,29]. Gp120 acts on microglia and neurons to release arachidonic acid and superoxide that in turn inhibits glutamate uptake into glia [4,15]; these and other adverse effects of gp120 in the nervous system are specific, in that they are eliminated by denaturing the glycoprotein [7,10]. Whether GCs and gp120 are ultimately inhibiting glutamate uptake by altering Vmax and / or Km was not examined in this study, but either is a possibility. From our results we can hypothesize how these two independent mechanisms of interference with glutamate uptake may interact in hippocampal cultures. In glia cultures both GCs and gp120 can inhibit uptake as described. However in mixed cultures neurons that may not be affected directly by either treatment are able to transport some excess glutamate that accumulates in the presence of GCs and gp120 individually. It has also been reported that in the presence of neurons, astrocytes express both EAAT and GLT1 transporters, whereas only the latter are expressed in pure astrocyte cultures [27]. A recent report indicates that the uptake capacity of GLT1 and GLAST on astrocytes is enhanced in the presence of neurons [23]. Therefore, in the presence of GCs or gp120 alone there is no longer a reduction in uptake. However, we have previously demonstrated that only a combination of gp120 and GCs can disrupt mitochondrial potential in mixed cultures, therefore decreasing available energy supplies [9]. Because glutamate uptake has high-energy demand, when both gp120 and GCs are present at the same time, the disruption in energy production in the cells prevents the compensation due to excess transporter availability resulting in an overall decrease in the amount of uptake. We tested this hypothesis by increasing energy availability to the cultures by increasing glucose levels from 5 to 20 mM. With more energy available, the cells were able to
compensate for the disruption in energy supplies caused by the combination of GCs and gp120 and normal transport levels were restored (Fig. 2). It has previously been demonstrated that supplementation with glucose can decrease GCs exacerbation of gp120-induced neurotoxicity and calcium mobilization [9]. There is also a precedent with other insults such as EAAs, anti-metabolites, and hypoxia, for excess glucose reversing GCs exacerbation of neurotoxicity and calcium mobilization [26]. There is a body of literature indicating that astrocyte, astrocyte-rich and astrocyte-poor cultures act differently with respect to a number of parameters, for example neuron death in the presence of glutamate transporter inhibitor, and glutamine and the release of c-AMP [5,22,24]. This report therefore would appear to be another example of the importance of studying the neuron glia interaction rather than cell types individually. GCs exacerbation of gp120 neurotoxicity in mixed hippocampal cultures appears to occur by several mechanisms: calcium influx is increased, energy stores are disrupted, ROS generation increased and degradation decreased [10]. And these results indicate that GCs may also increase neurotoxicity by decreasing the efficiency of glutamate elimination from the synapse in mixed cultures. Gp120 may be a causal agent in the development of ARD [19] and GCs are used extensively in treatment of HIV related diseases such as Pneumocystis carinii pneumonia [21], a procedure that could possibly lead to excess neuron death in patients. A further understanding of the mechanisms of GCs exacerbation of gp120 neurotoxicity as presented could perhaps be useful information in the development of treatments for the prevention of ARD.
Acknowledgements This work was supported by Grant IH RO1 MH 53814.
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