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Effect of acute stress on NTPDase and 5′-nucleotidase activities in brain synaptosomes in different stages of development
Int. J. Devl Neuroscience 28 (2010) 175–182
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Effect of acute stress on NTPDase and 50 -nucleotidase activities in brain synaptosomes in different stages of development Anica Horvat *, Ivana Stanojevic´, Dunja Drakulic´, Natasˇa Velicˇkovic´, Snjezˇana Petrovic´, Maja Milosˇevic´ Laboratory of Molecular Biology and Endocrinology, ‘‘Vincˇa’’ Institute of Nuclear Sciences, P.O. Box 522, 11001 Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2009 Received in revised form 5 November 2009 Accepted 15 November 2009
The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto-nucleotidase activities at specific stages of postnatal development (15-, 30-, 60- and 90-day-old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72 h poststress. At 1 h after stress NTPDase and ecto-50 -nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short-term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long-term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72 h poststress. In adult rats only ATP hydrolysis was decreased after 24 h, indicating that ecto-ATPase was affected by stress. Ecto-50 -nucleotidase hydrolysing activity was decreased within 24 h in adult rats, while in 15- and 30-day old rats it decreased 72 h post-stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto-50 -nucleotidase was detected. Our results highlight the developmental-dependence of brain ecto-nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time-dependent ecto-nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats. ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Acute restraint stress NTPDase Ecto-50 -nucleotidase Postnatal development Synaptosomal plasma membranes Rat brain
1. Introduction Adenine nucleotides, including adenosine 50 -triphosphate (ATP) and adenosine, are important signalling molecules that exert immediate effects such as neurotransmission (Burnstock, 1972) and long-term trophic effects. In the central nervous system (CNS) ATP is released from neurons, glia, microglia, endothelial and blood cells (for review see Rathbone et al., 1999). As is the case for other classical neurotransmitters, ATP is stored in vesicles and released into the synaptic cleft shortly after neuronal stimulation. Under physiological conditions extracellular ATP concentrations are low. In contrast, in some pathological conditions large amounts of ATP may be released and cause cell death (Inoue, 2002). Extracellular ATP generates diverse physiological responses by activating two specific P2 receptor subtypes: metabotropic P2Y and ionotropic P2X
Abbreviations: ADP, adenosine 50 -diphosphate; AMP, adenosine monophosphate; ATP, adenosine 50 -triphosphate; CNS, central nervous system; NTPDase, nucleoside triphosphate diphosphohydrolase; SPM, synaptosomal plasma membrane. * Corresponding author. Tel.: +381 113443619; fax: +381 112455561. E-mail address: [email protected] (A. Horvat). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.11.005
receptors, both of which are widely expressed in the developing and mature nervous system (Abbracchio and Burnstock, 1994). Depending on the P2 receptor subtype and their involvement in different signalling pathways, these receptors trigger and mediate short-term processes that affect cellular metabolism, adhesion, activation, intracellular communication and migration. In addition, purinergic signalling has profound effects on long-term responses including cell proliferation, differentiation and apoptosis (for review see Majumder et al., 2007; Franke and Illes, 2006). P2 receptor-mediated signalling is terminated by the coordinated action of ecto-nucleotidases including ecto-nucleoside triphosphate diphosphohydrolase (NTPDases) and ecto-50 -nucleotidase. These enzymes hydrolyse terminal phosphate residues of extracellular nucleotides and convert ATP into adenosine (Zimmermann, 2000). In the brain, three membrane-bound NTPDases (NTPDase1, 2 and 3), differing in their preference for a substrate, have been described. NTPDase1 hydrolyses ATP and ADP equally well resulting in AMP formation whereas NTPDase2 almost exclusively hydrolyses ATP producing ADP. NTPDase3 degrades ATP to AMP with a transient ADP accumulation (Kukulski and Komoszynski, 2003; Lavoie et al., 2004). Ecto-50 -nucleotidase further hydrolyses AMP to adenosine. These enzymes might have a
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protective function keeping extracellular ATP, ADP and adenosine within physiological levels (Zimmermann, 1992). Adenosine has several functions within the CNS including down-regulation of neurotransmission and neuroprotective actions in some pathological conditions (Latini and Pedata, 2001) and is particularly well suited to function as a transcellular messenger to signal metabolic imbalance. The neuroprotective actions of adenosine are attributed to the activation of presynaptic A1 receptors, which reduce neurotransmitter release and depress the neuronal activity in the CNS. By activating pre-synaptic A1 receptors adenosine inhibits adenylyl cyclase activity, regulates phospholipase C-dependent intracellular calcium ion accumulation and induce changes in membrane ion channel function. Calcium ion uptake inhibition in the pre-synaptic terminal is protective and contributes to a reduction in release of different neurotransmitters, especially excitatory amino acid neurotransmitter reduction is relevant to adenosine’s neuroprotective effect (Phillips and Wu, 1981). In addition, extracellular adenosine can exert various trophic effects during development by activating in different extent three different types of P1 (A1, A2 and A3) receptors (for review see Cunha, 2001; Cunha, 2005). Activation of the stress response is essential for maintenance of vital functions. A few studies have reported that after exposure to stressors the concentrations of extracellular ATP (Bodin and Burnstock, 2001) and adenosine (Latini and Pedata, 2001; Fontella et al., 2004) change. In adult mammals both acute and chronic stress have been shown to modulate enzymes involved in nucleotide hydrolysis in blood serum, spinal cord and certain brain structures (Torres et al., 2002a,b; Fontella et al., 2004). As stress responsiveness changes significantly during postnatal development (Romeo et al., 2004; Romeo, 2005), and the expression and activities of ecto-nucleotidases in the CNS vary throughout development, the effect of acute restraint stress on synaptosomal plasma membrane NTPDase and ecto-50 -nucleotidase activities in female rats at different postnatal developmental stages was investigated. 2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were of analytical grade or better. 2.2. Animals Female Wistar Albino rats at different developmental stages 15, 30, 60 and 90 days of age (P15, P30, P60 and P90) were used in this study. Experimentally naive animals were housed in groups of 4–5 in perspex cages (65 cm 25 cm 15 cm) with sawdust on the floor under standard conditions: 12 h light/dark cycle in a humid animal facility, constant temperature (22 2 8C) and access to food and water ad libidum. The restraint-stress procedure was performed between 09:00 and 10:00. All animal treatments were approved by the Ethics Committee of the Serbian Association for the Use of Animals in research and education. All efforts were made to reduce the number of rats used in for the study. 2.3. Acute stress procedure Age-matched rats were divided into two groups: control (n = 3–5) and stressed (n = 9–15). Acute restraint stress was applied by placing the rats in plywood boxes (containing six 1 cm circular holes for breathing) causing immobilisation for 45 min. Thereafter, the rats were sacrificed 1 (n = 3–5), 24 (n = 3–5) and 72 (n = 3– 5) h later. After stressing, P15 rats were placed in cages with their mothers until they were sacrificed. Control rats were kept in their normal cages under regular conditions. The whole experiment was repeated 3 times. 2.4. Synaptosomal preparation After decapitation using a small animal guillotine (Harvard Apparatus, Holliston, MA, USA) the brain was rapidly removed for immediate synaptosomal plasma membrane (SPM) isolation starting with ice-cold isotonic medium (0.32 M sucrose, 5 mM Tris–HCl, pH 7.4). Synaptosomes were purified according to modified method of Cotman and Matthews (1971) described previously (Horvat et al., 2001). In brief,
the crude synaptosomal pellet (from pooled brain homogenates of same group) was layered onto a Ficoll gradient and synaptosomes were collected after centrifugation at 65,000 g at 4 8C for 55 min. 2.5. SPM preparation The method described by Towle and Sze (1983) was followed incorporating some slight modifications (Horvat et al., 1995). Purified synaptosomes were lysed in 5 mM Tris–HCl, pH 7.4 by homogenisation before storage at 20 8C overnight. The following day synaptosomal lysate was layered onto a discontinuous sucrose gradient (0.8, 1.0, 1.2 M) and SPM was collected from the 1.0 and 1.2 M interphase after centrifugation at 65,000 g at 4 8C for 2 h. SPM was washed using 5 mM Tris– HCl, pH 7.4 and pelleted by centrifugation at 15,000 g at 4 8C, for 20 min. SPM protein was determined according to Markwell et al. (1978) using bovine serum albumin as a standard. Samples of SPM were kept at 70 8C until use. 2.6. Enzyme assays ATP and ADP hydrolysis assays contained 50 mM Tris–HCl buffer pH 7.4 and 5 mM MgCl2 in a final volume of 200 ml. SPM (40 mg of protein for ATP and ADP and 80 mg for AMP) was added to this mixture and pre-incubated for 10 min at 37 8C. Reactions were initiated by ATP, ADP or AMP addition to a final concentration of 1.0 mM. Incubations continued for an additional 15 min for the ATP and ADP assays or 30 min for the AMP assay. Reactions were stopped with 22 ml of ice-cold 3 M trichloroacetic acid. Samples were chilled on ice before being incorporated into the inorganic phosphate (Pi) release assay (Pennial, 1966), using KH2PO4 as a reference standard. SPM purity was evaluated using several inhibitors (data not shown) and obtained results indicated no significant cross-contamination (<10%) with other sub-cellular fractions. In all enzyme assays the incubation times and protein concentrations were chosen in order to ensure reaction linearity. Other assay conditions including pH and cation concentrations had been adjusted to assure optimal enzyme activities. Enzyme specific activity was expressed as nmolPi/min/ mg of SPM protein. 2.7. Data analysis The data are presented as mean specific enzyme activity SEM from 3 to 4 independent SPM preparations (n 9 rats/developmental stage/time point) performed in triplicate. Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, considering p < 0.05 as significant.
3. Results Our unpublished results indicated no differences in ATP, ADP or AMP hydrolysis with respect to the estrus phase of adult female rats. With regard to these findings, the estrus phase did not checked before stress exposure. 3.1. Effect of stress on NTPDase activities Short-term restraint stress led to different effects on ectonucleotidase activity in brain SPMs isolated from rats of different ages. ATP hydrolysis decreased by 10% (F = 4.36, p < 0.05) in young (P15) and by 20% (F = 10.50, p < 0.01) in pubertal (P60) rats, compared with non-stressed age-matched rats. In contrast, a small but significant (F = 9.41, p < 0.01) increase of 11% was detected in adult (P90) rats, compared with non-stressed rats. No significant change in ATP hydrolysis was observed in pre-pubertal (P30) rats (Fig. 1). ADP hydrolysis in restraint-stressed P15, P30 and P60 rats decreased by 30% (F = 10.97, p < 0.01), 11% (F = 4.59, p < 0.05) and 40% (F = 55.25, p < 0.001), respectively compared to appropriate non-stressed age-matched rats. In contrast, a 25% (F = 9.41, p < 0.01) increase in ADP hydrolysis was detected in restraint-stressed P90 rats compared with non-stressed rats (Fig. 2). ATPase and ADPase activities in non-stressed (control) rats rose from P15 to P60. No further increase was seen in adult (P90) rats (Figs. 1 and 2). The ATP/ADP hydrolysis ratio was higher 1 h after stress in P15, P30 and P60 rats (but not in P90 rats) compared with non-stressed age-matched rats (Table 1). Long-term effects of acute restraint stress on ATP hydrolysis in SPMs isolated from rats at different ages are shown in Fig. 3. In P15
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3.2. Effect of stress on ecto-50 -nucleotidase activity
Fig. 1. ATP hydrolysis 1 h post-restraint stress. ATP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after acute restraint-stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity SEM from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
In P15 and P30 rats AMP hydrolysis decreased by 63% (F = 38.98, p < 0.001) and by 40% (F = 35.48, p < 0.001), respectively 1 h postrestraint stress, compared with non-stressed age-matched rats as the short-term effect. In contrast, an increase in AMP hydrolysis by 33% (F = 51.34, p < 0.001) was found in P90 rats, compared with non-stressed rats. According to obtained results, ecto-50 -nucleotidase hydrolysing activity in naive rats was augmented permanently during postnatal development (Fig. 5). The long-term effect of restraint stress on AMP hydrolysis was similar in P15 and P30 rats. AMP hydrolysis 24 h post-restraint stress was as that found in non-stressed rats and decreased by 52% and 23% (F = 27.43, p < 0.001 and F = 7.76, p < 0.05, respectively) in P15 and P30 rats, respectively 72 h post-restraint stress, compared with non-stressed age-matched rats. In P60 rats a significant increase in AMP hydrolysis was only detected 72 h (F = 5.46, p < 0.05) post-restraint stress, compared to non-stressed rats. In P90 rats hydrolysis of AMP decreased by 25% (F = 28.09, p < 0.001) 24 h and without changes 72 h post-restraint stress compared to non-stressed rats (Fig. 6). 4. Discussion
Fig. 2. ADP hydrolysis 1 h post-restraint stress. ADP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after restraint-stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity SEM from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
Table 1 ATP/ADP ratio in SPMs isolated from 15-, 30-, 60- and 90-day-old rats (P15, P30, P60 and P90) subjected to control (no stress) and rats of the same age 1, 24 and 72 h after restraint stress. Time after stress
Age (postnatal days) P15
Control 1 h % of control
3.9
P30 4.1
P60 4.4
P90 4.4
4.9** 125
4.6* 112
5.7*** 130
4.0 90
24 h % of control
3.8 97
4.4 107
4.7 107
3.7* 84
72 h % of control
4.1 105
4.4 107
4.6 104
4.7 107
The data are presented as the % of control: *p < 0.05, **p < 0.01, ***p < 0.001.
rats ATP hydrolysis 24 h post-restraint stress did not differ from that found in non-stressed rats. However, in P30 and P60 rats the decline in ATP hydrolysis lasted for 72 h post-restraint stress. In P90 rats ATP hydrolysis was found to be decreased 24 h postrestraint stress but returned to a level found in non-stressed rats by 72 h. ATP and ADP hydrolysis in rats of all ages demonstrated similar kinetics except in P90 rats where 24 h post-restraint stress only ATPase activity was decreased (Figs. 3 and 4). In P90 rats the ATP/ADP ratio decreased 24 h post-restraint stress (Table 1).
Our results, for the first time present short-term effects of acute restraint stress on SPM NTPDase and ecto-50 -nucleotidase activities in female rats, as well as developmental-dependent changes of these enzymes activities. In young rats both NTPDase and ecto-50 -nucleotidase activities were decreased. This contrasted to that seen in adult rats. Stress induced similar changes in ATP and ADP hydrolysis in SPMs. Given that ATP is the substrate of all three brain NTPDases1, 2 and 3, while ADP is the substrate for NTPDase1 and 3 (Zimmermann, 2000) and the ATP/ADP hydrolysis ratio was 1:0.25, one could assume that NTPDase3 activity was susceptible to stress. In addition, by an Western blotting method we confirmed the presence of NTPDase3 protein in brain SPM that showed a rat developmental-dependent profile (our unpublished results). Post ‘‘acute’’ CNS injuries (for example ischemia, hypoxia, mechanical stress and axotomy) allow the extracellular ATP concentration to increase into the millimolar range (Franke and Illes, 2006) causing competitive inhibition of ecto-50 -nucleotidase activity (Zimmermann, 1992; 2000). Taking these findings into account, the decrease in ecto-50 -nucleotidase activities in P15 and P30 rats, 1 h post-restraint stress may be an end result of stressinduced changes in the extracellular ATP concentration as a consequence of both increased ATP release and decreased NTPDase activity. Ecto-50 -nucleotidase plays a key role in many developmental processes and is transiently present at synapses. Furthermore, a difference in the protein structure of ecto-50 nucleotidase has been found during brain development (Schoen et al., 1993, 1988; Vogel et al., 1993). Together, these reported observations could explain the insensitivity of ecto-5‘-nucleotidase to stress in pubertal (P60) rats despite decreased ATP and ADP hydrolysis. In adult (P90) rats, stimulation of ecto-50 nucleotidase activity, as a result of a decreased extracellular ATP and ADP content caused by increased NTPDase activity, was most likely the explanation. The involvement of oxidative stress in ecto-nucleotidase inhibition in the developing brain is also plausible. It is known that restraint stress can induce oxidative stress involving the rapid generation of reactive oxygen and nitrogen species and/or lipid peroxidation (Atif et al., 2008). Other studies suggested the participation of oxidative stress in the inhibition of NTPDase and ecto-50 -nucleotidase activities in renal epithelial cells and in the brain (Vuaden et al., 2007; Matute et al., 2007). Oxidative stress can
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Fig. 3. The acute restraint-stress time-dependent effect on ATP hydrolysis. ATP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. Control values presented as the dotted line (100%). *p < 0.05; **p < 0.01; ***p < 0.001.
inhibit the activities of ecto-nucleotidase possibly by free radicals generation (Bavaresco et al., 2008). Stress-induced hormones may also signal to ecto-nucleotidase inhibition, as stress exposure causes a significant increase in serum corticosterone within 10–20 min, even in young (P18) rats (Velickovic et al., 2009). Such hormones, similar to other nonglucocorticoid-dependent factors, are able to alter the activity of plasma membrane enzymes involved in ATP hydrolysis including Na+, K+-ATPase and Ca2+-ATPase (Shaheen et al., 1996; Rodrigo et al., 2002; Bhargava et al., 2000, 2002). Also, glucocorticoids could modulate the expression and activity of ecto-nucleotidases in endothelial and mesangial cells as well as in a glioma cell line (Kapojos et al., 2004; Bavaresco et al., 2007). In the developing brain acute stress-induced decreased NTPDase activity results in an elevation of extracellular neurotoxic ATP within an hour. Massive extracellular ATP accumulation can be cytotoxic, as prolonged P2X receptor stimulation results in neuronal injury via increased intracellular calcium ion accumulation (Sorimachi et al., 2002) and enhanced cytotoxic effects of glutamate (Khakh and Henderson, 1998). In addition, decreased
NTPDase activity reduces AMP production, the substrate of ecto-50 nucleotidase, and consequently adenosine production ensues. An additional decrease in adenosine formation will be affected by stress-induced decrease in ecto-50 -nucleotidase activity that was observed in our experiments. Together these findings suggest that acute stress-mediated short-term effects on enzymes involved in extracellular ATP breakdown and P2 receptor stimulation are more pronounced in P60 rats, while effects on adenosine production and decreased P1 receptor stimulation are more marked in P15 rats. In adult (P90) rats short-term acute restraint stress induces higher adenosine production and P1 receptor activation by increasing NTPDase and ecto-50 -nucleotidase activities. Although, adenosine-mediated A2 receptor activation could cause increased neurotransmitter release (Cunha et al., 1992), activation of the A1 receptors, which are among the most widely distributed Gicoupled receptors in the mature brain would pre-synaptically inhibit neurotransmitter release or post-synaptically reduce neuronal excitability (Hass and Greene, 1988; Lamber and Teyler, 1991; Reppert et al., 1991). Due to the fact that neuronal cells are
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Fig. 4. The acute restraint-stress time-dependent effect on ADP hydrolysis. ADP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats, 1, 24 and 72 h after acute stress exposure. The data are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. Control values presented as the dotted line (100%). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5. AMP hydrolysis 1 h post-restraint stress. AMP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity (SEM) from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
particularly sensitive to cellular injury and have a high abundance of purinergic receptors that increase during development, our results suggest the existence of a physiological response to protect the adult CNS from damage caused by exposure to stressors. As a long-term effect of acute restraint-stress ATP and ADP hydrolysis rates reverted back to control values in P15 rats, 24 h post-restraint stress. At later developmental stages (P30 and P60 rats) restraint-stress-induced decreased NTPDase activity was persistent. Prolonged activation of P2 receptors as a result of decreased NTPDase activity can affect neurotransmission and synaptogenesis and can generally influence neuronal cell development causing P30 and P60 rats to be vulnerable to stress. In adult rats the ATP/ADP hydrolysis ratio decreased and parallelism in ATP and ADP responses disappeared 24 h postrestraint stress. This was probably due to a reduction in ectoATPase activity as only ATP hydrolysis was significantly decreased. In that way ATP accumulation was most likely the explanation for the reduction in ecto-50 -nucleotidase activity. In developing animals ecto-50 -nucleotidase activity was decreased 72 h postrestraint stress only during the pre-pubertal period. Long-term
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Fig. 6. The acute restraint-stress time-dependent effect on AMP hydrolysis. AMP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats, 1, 24 and 72 h after acute stress exposure. The data are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001
effects were actually most pronounced during early developmental stages. The decrease in ecto-50 -nucleotidase activity may have been the result of late translational/post-translational events that are different in developmental and adult stages and may be caused by prolonged effects of oxidative stress or other restraint stress-induced events involving glucocorticoids. The involvement of sex steroids in stress responsiveness of ecto-enzymes cannot be ruled out, especially when bearing in mind the observed differences in pre-pubertal and adult rats. Estradiol replacement therapy augments corticosterone-mediated responses to acute restraint stress in ovariectomised rats (by returning them back to the level in control rats) (Lunga and Herbert, 2004; Seale et al., 2004). Furthermore, decreased AMP hydrolysis in spinal cord synaptosomes from repeatedly stressed rats after estradiol deprivation has been observed (Fontella et al., 2005). Underlying mechanisms involved in restraint-stressinduced modulation of pre-synaptic extracellular nucleotide breakdown will be investigated in our further studies.
Given that P2 receptor expression is enhanced during maturation (Amadio et al., 2002), the toxic effects of ATP are more prominent in fully differentiated brains and therefore inhibition of ATP hydrolysis is more serious. As mentioned previously, in the mature brain adenosine A1 receptors are among the most widely distributed permitting adenosine to have a broad influence on neural function, to modulate neurotransmitter release and to confer protection against brain cell damage (Reppert et al., 1991). Our observed reduction in both ATP and AMP hydrolysis implying a higher extracellular ATP concentration as well as a lower adenosine concentration 24 h post-stress together indicates that this particular period after stress is critical for damage occurrence within the adult brain. Several previous studies have demonstrated the existence of different stress effects on ATP, ADP and AMP hydrolysis depending on synaptosomal source and stress duration. In synaptosomes from adult male rat brain structures chronic stress induced no change in ATP or ADP hydrolysis. In contrast, in spinal cord
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synaptosomes decreased ADP and increased AMP hydrolysis was observed. In addition, no change in ATP, ADP or AMP hydrolysis after acute stress was recorded in spinal cord synaptosomes (Torres et al., 2002a,b). Fontella and colleagues observed in adult male rat hippocampal synaptosomes increased ATP, ADP and AMP hydrolysis 22 h after acute stress. In comparison, chronic stress led to an increase only in ATP hydrolysis (Fontella et al., 2004). The discrepancies in the literature with respect to our results are probably due to different synaptosomal sources and our restraintstress procedure. In summary, our findings highlight differences between the young and adult rat purinergic system response to acute restraint stress. Furthermore, differences in acute stress-mediated shortand long-term effects on purinergic signalling are apparent. Acute stress influences nucleotide balance, their receptor activities and ecto-nucleotidase activity in such a manner that ultimately affect distinct physiological systems operative during different stages of development. Further studies focussing on the roles of glucocorticoids and sex steroids on stress-induced modulation of ectonucleotidase activities during postnatal development are currently ongoing. Acknowledgement This research was supported by project grant number 143044 from the Ministry of Sciences and Technological Development of Serbia. References Abbracchio, M.P., Burnstock, G., 1994. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacology and Therapeutics 64, 445–475. Amadio, S., D’Ambrosi, N., Cavaliere, F., Murra, B., Sancesario, G., Bernardi, G., Burnstock, G., Volonte`, C., 2002. P2 receptor modulation and cytotoxic function in cultured CNS neurons. Neuropharmacology 42, 489–501. Atif, F., Yousuf, S., Agrawal, S.K., 2008. Restraint stress-induced oxidative damage and its amelioration with selenium. European Journal of Pharmacology 600, 59–63. Bavaresco, C.S., Chiarani, F., Kolling, J., Ramos, D.B., Cognato, G.P., Bonan, C.D., Bogo, M.R., Sarkis, J.J.F., Nettoa, C.A., Wyse, A.T.S., 2008. Intrastriatal injection of hypoxanthine alters striatal ectonucleotidase activities: a time-dependent effect. Brain Research 6, 198–206. Bavaresco, L., Bernardi, A., Braganhol, E., Wink, M.R., Battastini, A.M.O., 2007. Dexamethasone inhibits proliferation and stimulates ecto-50 -nucleotidase/ CD73 activity in C6 rat glioma cell line. Journal of Neuro-Oncology 84, 1–8. Bhargava, A., Mathias, R.S., McCormick, J.A., Dallman, M.F., Pearce, D., 2002. Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1. Molecular Endocrinology 16, 1629–1637. Bhargava, A., Meijer, O.C., Dallman, M.F., Pearce, D., 2000. Plasma membrane calcium pump isoform 1 gene expression is repressed by corticosterone and stress in rat hippocampus. Journal of Neuroscience 20, 3129–3138. Bodin, P., Burnstock, G., 2001. Purinergic signalling: ATP release. Neurochemical Research 26, 959–969. Burnstock, G., 1972. Purinergic nerves. Pharmacological Reviews 24, 509–581. Cotman, C.W., Matthews, D.A., 1971. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochemica and Biophyssica Acta 249, 380–394. Cunha, R.A., 2001. Adenosine as a neuromodulator and as a homeostatic regulator in nervous system: different roles, different sources and different receptors. Neurochemistry International 38, 107–125. Cunha, R.A., 2005. Neuroprotection by adenosine in the brain: from A1 receptor activation to A2A receptor blockade. Purinergic Signalling 1, 111–134. Cunha, R.A., Johansson, B., Ribeiro, J.A., Sebastia˜o, A.M., 1992. Adenosine A2a receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus. Neuroscience Letters 196, 41–44. Fontella, F.U., Bruno, A.N., Balk, R.S., Ru¨cker, B., Crema, L.M., Correa, M.D., Battastini, A.M., Sarkis, J.J., Alexandre Netto, C., Dalmaz, C., 2005. Repeated stress effects on nociception and on ectonucleotidase activities in spinal cord synaptosomes of female rats. Physiological Behaviour 85, 213–219. Fontella, F.U., Bruno, A.N., Crema, L.M., Battastini, A.M.O., Sarkis, J.J.F., Netto, C.A., Dalmaz, C., 2004. Acute and chronic stress alter ecto-nucleotidase activities in synaptosomes from the rat hippocampus. Pharmacology, Biochemistry and Behaviour 78, 341–347. Franke, H., Illes, P., 2006. Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacology and Therapeutics 109, 297–324.
181
Hass, H.L., Greene, R.W., 1988. Endogenous adenosine inhibits hippocampal CA1 neurones: further evidence from extra- and intracellular recordings. Naunyn Schmiedeberg Archives Pharmacology 337, 561–565. Horvat, A., Nikezic´, G., Martinovic´, J.V., 1995. Estradiol binding to synaptosomal plasma membranes of rat brain regions. Experientia 51, 11–15. Horvat, A., Nikezic, G., Petrovic, S., Kanazir, D.T., 2001. Binding of estradiol to synaptosomal mitochondria: physiological significance. Cellular and Molecular Life Sciences 58, 636–644. Inoue, K., 2002. Microglial activation by purines and pyrimidines. Glia 40, 156–163. Kapojos, J.J., Van den Berg, A., Borghuis, T., Banas, B., Huitema, S., Poelstra, K., Bakker, W.W., 2004. Enhanced ecto-apyrase activity of stimulated endothelial or mesangial cells is downregulated by glucocorticoids in vitro. European Journal of Pharmacology 501, 191–198. Khakh, B.S., Henderson, G., 1998. ATP receptor-mediated enhancement of fast excitatory neurotransmitter release in the brain. Molecular Pharmacology 54, 372–378. Kukulski, F., Komoszynski, M., 2003. Purification and characterization of NTPDase 1 (ecto-apyrase) and NTPDase 2 (ecto-ATPase) from porcine brain cortex synaptosomes. European Journal of Biochemistry 270, 3447–3454. Lamber, N.A., Teyler, T.J., 1991. Adenosine depresses excitatory but not fast inhibitory synaptic transmission in area CA1 of the rat hippocampus. Neuroscience Letters 122, 50–52. Latini, S., Pedata, F., 2001. Adenosine in the central nervous system: release mechanisms and extracellular concentrations. Journal of Neurochemistry 79, 463–484. Lavoie, E.G., Kukulski, F., Levesque, S.A., Lecka, J., Sevigny, J., 2004. Cloning and characterization of mouse nucleoside triphosphate dihosphohydrolase-3. Biochemical Pharmacology 67, 1917–1926. Lunga, P., Herbert, J., 2004. 17b-Oestradiol modulates glucocorticoid, neural and behavioral adaptation to repeated restraint stress in female rats. Journal of Neuroendocrinology 16, 776–785. Majumder, P., Trujillo, C.A., Lopes, C.G., Resende, R.R., Gomes, K.N., Yuahasi, K.K., Britto, L.R.G., Ulrich, H., 2007. New insights of purinergic receptor signaling in neuronal differentiation, neuroprotection and brain disorders. Purinergic Signaling 3, 317–331. Markwell, M.A., Haas, S.A., Lieber, L., Tolbert, N.A., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry 87, 206–210. Matute, C., Alberdi, E., Domercq, M., Sanchez-Gomez, M.V., Perez-Samartin, A., Rodriguez-Antiguedad, A., Perez-Cerda, F., 2007. Excitotoxic damage to white matter. Journal of Anatomy 210, 693–702. Pennial, R., 1966. An improved method for determination of inorganic phosphate by the isobutanol-benzen extraction procedure. Analytical Biochemistry 14, 87–90. Phillips, J.W., Wu, P.H., 1981. The role of adenosine and its nucleotides in central synaptic transmission. Progress in Neurobiology 16, 187–239. Rathbone, M.P., Middlemiss, P.J., Gysbergs, J.W., Andrew, C., Herman, M.A.R., Reed, J.K., Ciccarelli, R., Di Iorio, P., Caciagli, F., 1999. Trophic effects of purines in neurons and glial cells. Progress in Neurobiology 59, 663–690. Reppert, S.M., Weaver, D.R., Stehle, J.H., Rivkees, S.A., 1991. Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord. Molecular Endocrinology 5, 1037–1048. Rodrigo, R., Rivera, G., Lucero, Y., Larraguibel, C., 2002. Effect of ethanol on regulation of (Na+K)-adenosine triphosphatase by aldosterone and dexamethasone in cultured renal papillary collecting duct cells. Endocrine 19, 301–304. Romeo, R.D., 2005. Neuroendocrine and behavioral development during puberty: a tale of two axes. Vitamins and Hormones 71, 1–25. Romeo, R.D., Lee, S.J., McEwen, B.S., 2004. Differential stress reactivity in intact and ovariectomized prepubertal and adult female rats. Neuroendocrinology 80, 387–393. Schoen, S.W., Graeber, M.R., Toth, L., Kreutzberg, G.W., 1988. Ecto-50 -nucleotidase in postnatal ontogeny of rat cerebellum. A marker for migrating nerve cells? Brain Research 39, 125–136. Schoen, S.W., Kreutzberg, G.W., Singer, W., 1993. Cytochemical redistribution of 50 nucleotidase in the developing cat visual cortex. European Journal of Neuroscience 5, 210–222. Seale, J.V., Wood, S.A., Atkinson, H.C., Harbuz, M.S., Lightman, S.L., 2004. Gonadal steroid replacement reverses gonadectomy induced changes in the corticosterone pulse profile and stress-induced hypothalamic–pituitary–adrenal axis activity of male and female rats. Joural of Neuroendocrinology 16, 989–998. Shaheen, A.A., Abd El-Fattah, A., Gad, M.Z., 1996. Effect of various stressors on the level of lipid peroxide, antioxidants and Na+, K(+)-ATPase activity in rat brain. Experientia 52, 336–339. Sorimachi, M., Yamagami, K., Wakamori, M., 2002. Activation of ATP receptor increases the cytosolic Ca(2+) concentration in ventral tegmental area neurons of rat brain. Brain Research 935, 129–133. Torres, I.L., Buffon, A., Dantas, G., Fu¨rstenau, C.R., Bo¨hmer, A.E., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002a. Chronic stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats. Pharmacology Biochemistry and Behaviour 74, 181–186. Torres, I.L., Buffon, A., Silveira, P.P., Duarte, M.Z., Bassani, M.G., Oliveira, S.S., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002b. Effect of chronic and acute stress on ectonucleotidase activities in spinal cord. Physiology and Behaviour 75, 1–5. Towle, A.C., Sze, P.Y., 1983. Steroid binding to synaptic plasma membrane: differential binding of glucocorticoid and gonadal steroids. Journal of Steroid Biochemistry 18, 135–143.
182
A. Horvat et al. / Int. J. Devl Neuroscience 28 (2010) 175–182
Velickovic, N., Djordjevic, A., Drakulic, D., Stanojevic, I., Secerov, B., Horvat, A., 2009. Cranial irradiation modulates hypothalamic–pituitary–adrenal axis activity and corticosteroid receptor expression in the hippocampus of juvenile rat. General Physiology and Biophysics 28, 219–227. Vogel, M., Zimmermann, H., Singer, W., 1993. Transient association of the HNK-1 epitope with 50 -nucleotidase during development of the cat visual cortex. European Journal of Neuroscience 5, 1423–1425.
Vuaden, F.C., De Paula Cognato, G., Bonorino, C., 2007. Lipopolysacharide alters nucleotidase activities from lymphocytes and serum of rats. Life Science 80, 1784–1791. Zimmermann, H., 2000. Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmiedeberg’s Archives of Pharmacology 362, 299–309. Zimmermann, H., 1992. Ecto-50 -nucleotidase: molecular structure and functional aspects. Biochemical Journal 285, 345–365.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Effect of acute stress on NTPDase and 50 -nucleotidase activities in brain synaptosomes in different stages of development Anica Horvat *, Ivana Stanojevic´, Dunja Drakulic´, Natasˇa Velicˇkovic´, Snjezˇana Petrovic´, Maja Milosˇevic´ Laboratory of Molecular Biology and Endocrinology, ‘‘Vincˇa’’ Institute of Nuclear Sciences, P.O. Box 522, 11001 Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2009 Received in revised form 5 November 2009 Accepted 15 November 2009
The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto-nucleotidase activities at specific stages of postnatal development (15-, 30-, 60- and 90-day-old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72 h poststress. At 1 h after stress NTPDase and ecto-50 -nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short-term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long-term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72 h poststress. In adult rats only ATP hydrolysis was decreased after 24 h, indicating that ecto-ATPase was affected by stress. Ecto-50 -nucleotidase hydrolysing activity was decreased within 24 h in adult rats, while in 15- and 30-day old rats it decreased 72 h post-stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto-50 -nucleotidase was detected. Our results highlight the developmental-dependence of brain ecto-nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time-dependent ecto-nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats. ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Acute restraint stress NTPDase Ecto-50 -nucleotidase Postnatal development Synaptosomal plasma membranes Rat brain
1. Introduction Adenine nucleotides, including adenosine 50 -triphosphate (ATP) and adenosine, are important signalling molecules that exert immediate effects such as neurotransmission (Burnstock, 1972) and long-term trophic effects. In the central nervous system (CNS) ATP is released from neurons, glia, microglia, endothelial and blood cells (for review see Rathbone et al., 1999). As is the case for other classical neurotransmitters, ATP is stored in vesicles and released into the synaptic cleft shortly after neuronal stimulation. Under physiological conditions extracellular ATP concentrations are low. In contrast, in some pathological conditions large amounts of ATP may be released and cause cell death (Inoue, 2002). Extracellular ATP generates diverse physiological responses by activating two specific P2 receptor subtypes: metabotropic P2Y and ionotropic P2X
Abbreviations: ADP, adenosine 50 -diphosphate; AMP, adenosine monophosphate; ATP, adenosine 50 -triphosphate; CNS, central nervous system; NTPDase, nucleoside triphosphate diphosphohydrolase; SPM, synaptosomal plasma membrane. * Corresponding author. Tel.: +381 113443619; fax: +381 112455561. E-mail address: [email protected] (A. Horvat). 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.11.005
receptors, both of which are widely expressed in the developing and mature nervous system (Abbracchio and Burnstock, 1994). Depending on the P2 receptor subtype and their involvement in different signalling pathways, these receptors trigger and mediate short-term processes that affect cellular metabolism, adhesion, activation, intracellular communication and migration. In addition, purinergic signalling has profound effects on long-term responses including cell proliferation, differentiation and apoptosis (for review see Majumder et al., 2007; Franke and Illes, 2006). P2 receptor-mediated signalling is terminated by the coordinated action of ecto-nucleotidases including ecto-nucleoside triphosphate diphosphohydrolase (NTPDases) and ecto-50 -nucleotidase. These enzymes hydrolyse terminal phosphate residues of extracellular nucleotides and convert ATP into adenosine (Zimmermann, 2000). In the brain, three membrane-bound NTPDases (NTPDase1, 2 and 3), differing in their preference for a substrate, have been described. NTPDase1 hydrolyses ATP and ADP equally well resulting in AMP formation whereas NTPDase2 almost exclusively hydrolyses ATP producing ADP. NTPDase3 degrades ATP to AMP with a transient ADP accumulation (Kukulski and Komoszynski, 2003; Lavoie et al., 2004). Ecto-50 -nucleotidase further hydrolyses AMP to adenosine. These enzymes might have a
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protective function keeping extracellular ATP, ADP and adenosine within physiological levels (Zimmermann, 1992). Adenosine has several functions within the CNS including down-regulation of neurotransmission and neuroprotective actions in some pathological conditions (Latini and Pedata, 2001) and is particularly well suited to function as a transcellular messenger to signal metabolic imbalance. The neuroprotective actions of adenosine are attributed to the activation of presynaptic A1 receptors, which reduce neurotransmitter release and depress the neuronal activity in the CNS. By activating pre-synaptic A1 receptors adenosine inhibits adenylyl cyclase activity, regulates phospholipase C-dependent intracellular calcium ion accumulation and induce changes in membrane ion channel function. Calcium ion uptake inhibition in the pre-synaptic terminal is protective and contributes to a reduction in release of different neurotransmitters, especially excitatory amino acid neurotransmitter reduction is relevant to adenosine’s neuroprotective effect (Phillips and Wu, 1981). In addition, extracellular adenosine can exert various trophic effects during development by activating in different extent three different types of P1 (A1, A2 and A3) receptors (for review see Cunha, 2001; Cunha, 2005). Activation of the stress response is essential for maintenance of vital functions. A few studies have reported that after exposure to stressors the concentrations of extracellular ATP (Bodin and Burnstock, 2001) and adenosine (Latini and Pedata, 2001; Fontella et al., 2004) change. In adult mammals both acute and chronic stress have been shown to modulate enzymes involved in nucleotide hydrolysis in blood serum, spinal cord and certain brain structures (Torres et al., 2002a,b; Fontella et al., 2004). As stress responsiveness changes significantly during postnatal development (Romeo et al., 2004; Romeo, 2005), and the expression and activities of ecto-nucleotidases in the CNS vary throughout development, the effect of acute restraint stress on synaptosomal plasma membrane NTPDase and ecto-50 -nucleotidase activities in female rats at different postnatal developmental stages was investigated. 2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were of analytical grade or better. 2.2. Animals Female Wistar Albino rats at different developmental stages 15, 30, 60 and 90 days of age (P15, P30, P60 and P90) were used in this study. Experimentally naive animals were housed in groups of 4–5 in perspex cages (65 cm 25 cm 15 cm) with sawdust on the floor under standard conditions: 12 h light/dark cycle in a humid animal facility, constant temperature (22 2 8C) and access to food and water ad libidum. The restraint-stress procedure was performed between 09:00 and 10:00. All animal treatments were approved by the Ethics Committee of the Serbian Association for the Use of Animals in research and education. All efforts were made to reduce the number of rats used in for the study. 2.3. Acute stress procedure Age-matched rats were divided into two groups: control (n = 3–5) and stressed (n = 9–15). Acute restraint stress was applied by placing the rats in plywood boxes (containing six 1 cm circular holes for breathing) causing immobilisation for 45 min. Thereafter, the rats were sacrificed 1 (n = 3–5), 24 (n = 3–5) and 72 (n = 3– 5) h later. After stressing, P15 rats were placed in cages with their mothers until they were sacrificed. Control rats were kept in their normal cages under regular conditions. The whole experiment was repeated 3 times. 2.4. Synaptosomal preparation After decapitation using a small animal guillotine (Harvard Apparatus, Holliston, MA, USA) the brain was rapidly removed for immediate synaptosomal plasma membrane (SPM) isolation starting with ice-cold isotonic medium (0.32 M sucrose, 5 mM Tris–HCl, pH 7.4). Synaptosomes were purified according to modified method of Cotman and Matthews (1971) described previously (Horvat et al., 2001). In brief,
the crude synaptosomal pellet (from pooled brain homogenates of same group) was layered onto a Ficoll gradient and synaptosomes were collected after centrifugation at 65,000 g at 4 8C for 55 min. 2.5. SPM preparation The method described by Towle and Sze (1983) was followed incorporating some slight modifications (Horvat et al., 1995). Purified synaptosomes were lysed in 5 mM Tris–HCl, pH 7.4 by homogenisation before storage at 20 8C overnight. The following day synaptosomal lysate was layered onto a discontinuous sucrose gradient (0.8, 1.0, 1.2 M) and SPM was collected from the 1.0 and 1.2 M interphase after centrifugation at 65,000 g at 4 8C for 2 h. SPM was washed using 5 mM Tris– HCl, pH 7.4 and pelleted by centrifugation at 15,000 g at 4 8C, for 20 min. SPM protein was determined according to Markwell et al. (1978) using bovine serum albumin as a standard. Samples of SPM were kept at 70 8C until use. 2.6. Enzyme assays ATP and ADP hydrolysis assays contained 50 mM Tris–HCl buffer pH 7.4 and 5 mM MgCl2 in a final volume of 200 ml. SPM (40 mg of protein for ATP and ADP and 80 mg for AMP) was added to this mixture and pre-incubated for 10 min at 37 8C. Reactions were initiated by ATP, ADP or AMP addition to a final concentration of 1.0 mM. Incubations continued for an additional 15 min for the ATP and ADP assays or 30 min for the AMP assay. Reactions were stopped with 22 ml of ice-cold 3 M trichloroacetic acid. Samples were chilled on ice before being incorporated into the inorganic phosphate (Pi) release assay (Pennial, 1966), using KH2PO4 as a reference standard. SPM purity was evaluated using several inhibitors (data not shown) and obtained results indicated no significant cross-contamination (<10%) with other sub-cellular fractions. In all enzyme assays the incubation times and protein concentrations were chosen in order to ensure reaction linearity. Other assay conditions including pH and cation concentrations had been adjusted to assure optimal enzyme activities. Enzyme specific activity was expressed as nmolPi/min/ mg of SPM protein. 2.7. Data analysis The data are presented as mean specific enzyme activity SEM from 3 to 4 independent SPM preparations (n 9 rats/developmental stage/time point) performed in triplicate. Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, considering p < 0.05 as significant.
3. Results Our unpublished results indicated no differences in ATP, ADP or AMP hydrolysis with respect to the estrus phase of adult female rats. With regard to these findings, the estrus phase did not checked before stress exposure. 3.1. Effect of stress on NTPDase activities Short-term restraint stress led to different effects on ectonucleotidase activity in brain SPMs isolated from rats of different ages. ATP hydrolysis decreased by 10% (F = 4.36, p < 0.05) in young (P15) and by 20% (F = 10.50, p < 0.01) in pubertal (P60) rats, compared with non-stressed age-matched rats. In contrast, a small but significant (F = 9.41, p < 0.01) increase of 11% was detected in adult (P90) rats, compared with non-stressed rats. No significant change in ATP hydrolysis was observed in pre-pubertal (P30) rats (Fig. 1). ADP hydrolysis in restraint-stressed P15, P30 and P60 rats decreased by 30% (F = 10.97, p < 0.01), 11% (F = 4.59, p < 0.05) and 40% (F = 55.25, p < 0.001), respectively compared to appropriate non-stressed age-matched rats. In contrast, a 25% (F = 9.41, p < 0.01) increase in ADP hydrolysis was detected in restraint-stressed P90 rats compared with non-stressed rats (Fig. 2). ATPase and ADPase activities in non-stressed (control) rats rose from P15 to P60. No further increase was seen in adult (P90) rats (Figs. 1 and 2). The ATP/ADP hydrolysis ratio was higher 1 h after stress in P15, P30 and P60 rats (but not in P90 rats) compared with non-stressed age-matched rats (Table 1). Long-term effects of acute restraint stress on ATP hydrolysis in SPMs isolated from rats at different ages are shown in Fig. 3. In P15
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3.2. Effect of stress on ecto-50 -nucleotidase activity
Fig. 1. ATP hydrolysis 1 h post-restraint stress. ATP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after acute restraint-stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity SEM from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
In P15 and P30 rats AMP hydrolysis decreased by 63% (F = 38.98, p < 0.001) and by 40% (F = 35.48, p < 0.001), respectively 1 h postrestraint stress, compared with non-stressed age-matched rats as the short-term effect. In contrast, an increase in AMP hydrolysis by 33% (F = 51.34, p < 0.001) was found in P90 rats, compared with non-stressed rats. According to obtained results, ecto-50 -nucleotidase hydrolysing activity in naive rats was augmented permanently during postnatal development (Fig. 5). The long-term effect of restraint stress on AMP hydrolysis was similar in P15 and P30 rats. AMP hydrolysis 24 h post-restraint stress was as that found in non-stressed rats and decreased by 52% and 23% (F = 27.43, p < 0.001 and F = 7.76, p < 0.05, respectively) in P15 and P30 rats, respectively 72 h post-restraint stress, compared with non-stressed age-matched rats. In P60 rats a significant increase in AMP hydrolysis was only detected 72 h (F = 5.46, p < 0.05) post-restraint stress, compared to non-stressed rats. In P90 rats hydrolysis of AMP decreased by 25% (F = 28.09, p < 0.001) 24 h and without changes 72 h post-restraint stress compared to non-stressed rats (Fig. 6). 4. Discussion
Fig. 2. ADP hydrolysis 1 h post-restraint stress. ADP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after restraint-stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity SEM from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
Table 1 ATP/ADP ratio in SPMs isolated from 15-, 30-, 60- and 90-day-old rats (P15, P30, P60 and P90) subjected to control (no stress) and rats of the same age 1, 24 and 72 h after restraint stress. Time after stress
Age (postnatal days) P15
Control 1 h % of control
3.9
P30 4.1
P60 4.4
P90 4.4
4.9** 125
4.6* 112
5.7*** 130
4.0 90
24 h % of control
3.8 97
4.4 107
4.7 107
3.7* 84
72 h % of control
4.1 105
4.4 107
4.6 104
4.7 107
The data are presented as the % of control: *p < 0.05, **p < 0.01, ***p < 0.001.
rats ATP hydrolysis 24 h post-restraint stress did not differ from that found in non-stressed rats. However, in P30 and P60 rats the decline in ATP hydrolysis lasted for 72 h post-restraint stress. In P90 rats ATP hydrolysis was found to be decreased 24 h postrestraint stress but returned to a level found in non-stressed rats by 72 h. ATP and ADP hydrolysis in rats of all ages demonstrated similar kinetics except in P90 rats where 24 h post-restraint stress only ATPase activity was decreased (Figs. 3 and 4). In P90 rats the ATP/ADP ratio decreased 24 h post-restraint stress (Table 1).
Our results, for the first time present short-term effects of acute restraint stress on SPM NTPDase and ecto-50 -nucleotidase activities in female rats, as well as developmental-dependent changes of these enzymes activities. In young rats both NTPDase and ecto-50 -nucleotidase activities were decreased. This contrasted to that seen in adult rats. Stress induced similar changes in ATP and ADP hydrolysis in SPMs. Given that ATP is the substrate of all three brain NTPDases1, 2 and 3, while ADP is the substrate for NTPDase1 and 3 (Zimmermann, 2000) and the ATP/ADP hydrolysis ratio was 1:0.25, one could assume that NTPDase3 activity was susceptible to stress. In addition, by an Western blotting method we confirmed the presence of NTPDase3 protein in brain SPM that showed a rat developmental-dependent profile (our unpublished results). Post ‘‘acute’’ CNS injuries (for example ischemia, hypoxia, mechanical stress and axotomy) allow the extracellular ATP concentration to increase into the millimolar range (Franke and Illes, 2006) causing competitive inhibition of ecto-50 -nucleotidase activity (Zimmermann, 1992; 2000). Taking these findings into account, the decrease in ecto-50 -nucleotidase activities in P15 and P30 rats, 1 h post-restraint stress may be an end result of stressinduced changes in the extracellular ATP concentration as a consequence of both increased ATP release and decreased NTPDase activity. Ecto-50 -nucleotidase plays a key role in many developmental processes and is transiently present at synapses. Furthermore, a difference in the protein structure of ecto-50 nucleotidase has been found during brain development (Schoen et al., 1993, 1988; Vogel et al., 1993). Together, these reported observations could explain the insensitivity of ecto-5‘-nucleotidase to stress in pubertal (P60) rats despite decreased ATP and ADP hydrolysis. In adult (P90) rats, stimulation of ecto-50 nucleotidase activity, as a result of a decreased extracellular ATP and ADP content caused by increased NTPDase activity, was most likely the explanation. The involvement of oxidative stress in ecto-nucleotidase inhibition in the developing brain is also plausible. It is known that restraint stress can induce oxidative stress involving the rapid generation of reactive oxygen and nitrogen species and/or lipid peroxidation (Atif et al., 2008). Other studies suggested the participation of oxidative stress in the inhibition of NTPDase and ecto-50 -nucleotidase activities in renal epithelial cells and in the brain (Vuaden et al., 2007; Matute et al., 2007). Oxidative stress can
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Fig. 3. The acute restraint-stress time-dependent effect on ATP hydrolysis. ATP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. Control values presented as the dotted line (100%). *p < 0.05; **p < 0.01; ***p < 0.001.
inhibit the activities of ecto-nucleotidase possibly by free radicals generation (Bavaresco et al., 2008). Stress-induced hormones may also signal to ecto-nucleotidase inhibition, as stress exposure causes a significant increase in serum corticosterone within 10–20 min, even in young (P18) rats (Velickovic et al., 2009). Such hormones, similar to other nonglucocorticoid-dependent factors, are able to alter the activity of plasma membrane enzymes involved in ATP hydrolysis including Na+, K+-ATPase and Ca2+-ATPase (Shaheen et al., 1996; Rodrigo et al., 2002; Bhargava et al., 2000, 2002). Also, glucocorticoids could modulate the expression and activity of ecto-nucleotidases in endothelial and mesangial cells as well as in a glioma cell line (Kapojos et al., 2004; Bavaresco et al., 2007). In the developing brain acute stress-induced decreased NTPDase activity results in an elevation of extracellular neurotoxic ATP within an hour. Massive extracellular ATP accumulation can be cytotoxic, as prolonged P2X receptor stimulation results in neuronal injury via increased intracellular calcium ion accumulation (Sorimachi et al., 2002) and enhanced cytotoxic effects of glutamate (Khakh and Henderson, 1998). In addition, decreased
NTPDase activity reduces AMP production, the substrate of ecto-50 nucleotidase, and consequently adenosine production ensues. An additional decrease in adenosine formation will be affected by stress-induced decrease in ecto-50 -nucleotidase activity that was observed in our experiments. Together these findings suggest that acute stress-mediated short-term effects on enzymes involved in extracellular ATP breakdown and P2 receptor stimulation are more pronounced in P60 rats, while effects on adenosine production and decreased P1 receptor stimulation are more marked in P15 rats. In adult (P90) rats short-term acute restraint stress induces higher adenosine production and P1 receptor activation by increasing NTPDase and ecto-50 -nucleotidase activities. Although, adenosine-mediated A2 receptor activation could cause increased neurotransmitter release (Cunha et al., 1992), activation of the A1 receptors, which are among the most widely distributed Gicoupled receptors in the mature brain would pre-synaptically inhibit neurotransmitter release or post-synaptically reduce neuronal excitability (Hass and Greene, 1988; Lamber and Teyler, 1991; Reppert et al., 1991). Due to the fact that neuronal cells are
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Fig. 4. The acute restraint-stress time-dependent effect on ADP hydrolysis. ADP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats, 1, 24 and 72 h after acute stress exposure. The data are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. Control values presented as the dotted line (100%). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5. AMP hydrolysis 1 h post-restraint stress. AMP hydrolysis in SPMs isolated from 15- (P15), 30- (P30), 60- (P60) and 90-day-old (P90) control (non-stressed) rats (open bars) and after stress exposure (filled bars). The data are presented as the mean of specific hydrolysis activity (SEM) from 3 to 4 independent SPM preparations each performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
particularly sensitive to cellular injury and have a high abundance of purinergic receptors that increase during development, our results suggest the existence of a physiological response to protect the adult CNS from damage caused by exposure to stressors. As a long-term effect of acute restraint-stress ATP and ADP hydrolysis rates reverted back to control values in P15 rats, 24 h post-restraint stress. At later developmental stages (P30 and P60 rats) restraint-stress-induced decreased NTPDase activity was persistent. Prolonged activation of P2 receptors as a result of decreased NTPDase activity can affect neurotransmission and synaptogenesis and can generally influence neuronal cell development causing P30 and P60 rats to be vulnerable to stress. In adult rats the ATP/ADP hydrolysis ratio decreased and parallelism in ATP and ADP responses disappeared 24 h postrestraint stress. This was probably due to a reduction in ectoATPase activity as only ATP hydrolysis was significantly decreased. In that way ATP accumulation was most likely the explanation for the reduction in ecto-50 -nucleotidase activity. In developing animals ecto-50 -nucleotidase activity was decreased 72 h postrestraint stress only during the pre-pubertal period. Long-term
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Fig. 6. The acute restraint-stress time-dependent effect on AMP hydrolysis. AMP hydrolysis in brain SPM isolated from P15, P30, P60 and P90 female rats, 1, 24 and 72 h after acute stress exposure. The data are presented as the mean (SEM) percentage of specific hydrolysis activity with respect to the control value from 3 to 4 independent SPM preparations performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001
effects were actually most pronounced during early developmental stages. The decrease in ecto-50 -nucleotidase activity may have been the result of late translational/post-translational events that are different in developmental and adult stages and may be caused by prolonged effects of oxidative stress or other restraint stress-induced events involving glucocorticoids. The involvement of sex steroids in stress responsiveness of ecto-enzymes cannot be ruled out, especially when bearing in mind the observed differences in pre-pubertal and adult rats. Estradiol replacement therapy augments corticosterone-mediated responses to acute restraint stress in ovariectomised rats (by returning them back to the level in control rats) (Lunga and Herbert, 2004; Seale et al., 2004). Furthermore, decreased AMP hydrolysis in spinal cord synaptosomes from repeatedly stressed rats after estradiol deprivation has been observed (Fontella et al., 2005). Underlying mechanisms involved in restraint-stressinduced modulation of pre-synaptic extracellular nucleotide breakdown will be investigated in our further studies.
Given that P2 receptor expression is enhanced during maturation (Amadio et al., 2002), the toxic effects of ATP are more prominent in fully differentiated brains and therefore inhibition of ATP hydrolysis is more serious. As mentioned previously, in the mature brain adenosine A1 receptors are among the most widely distributed permitting adenosine to have a broad influence on neural function, to modulate neurotransmitter release and to confer protection against brain cell damage (Reppert et al., 1991). Our observed reduction in both ATP and AMP hydrolysis implying a higher extracellular ATP concentration as well as a lower adenosine concentration 24 h post-stress together indicates that this particular period after stress is critical for damage occurrence within the adult brain. Several previous studies have demonstrated the existence of different stress effects on ATP, ADP and AMP hydrolysis depending on synaptosomal source and stress duration. In synaptosomes from adult male rat brain structures chronic stress induced no change in ATP or ADP hydrolysis. In contrast, in spinal cord
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synaptosomes decreased ADP and increased AMP hydrolysis was observed. In addition, no change in ATP, ADP or AMP hydrolysis after acute stress was recorded in spinal cord synaptosomes (Torres et al., 2002a,b). Fontella and colleagues observed in adult male rat hippocampal synaptosomes increased ATP, ADP and AMP hydrolysis 22 h after acute stress. In comparison, chronic stress led to an increase only in ATP hydrolysis (Fontella et al., 2004). The discrepancies in the literature with respect to our results are probably due to different synaptosomal sources and our restraintstress procedure. In summary, our findings highlight differences between the young and adult rat purinergic system response to acute restraint stress. Furthermore, differences in acute stress-mediated shortand long-term effects on purinergic signalling are apparent. Acute stress influences nucleotide balance, their receptor activities and ecto-nucleotidase activity in such a manner that ultimately affect distinct physiological systems operative during different stages of development. Further studies focussing on the roles of glucocorticoids and sex steroids on stress-induced modulation of ectonucleotidase activities during postnatal development are currently ongoing. Acknowledgement This research was supported by project grant number 143044 from the Ministry of Sciences and Technological Development of Serbia. References Abbracchio, M.P., Burnstock, G., 1994. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacology and Therapeutics 64, 445–475. Amadio, S., D’Ambrosi, N., Cavaliere, F., Murra, B., Sancesario, G., Bernardi, G., Burnstock, G., Volonte`, C., 2002. P2 receptor modulation and cytotoxic function in cultured CNS neurons. Neuropharmacology 42, 489–501. Atif, F., Yousuf, S., Agrawal, S.K., 2008. Restraint stress-induced oxidative damage and its amelioration with selenium. European Journal of Pharmacology 600, 59–63. Bavaresco, C.S., Chiarani, F., Kolling, J., Ramos, D.B., Cognato, G.P., Bonan, C.D., Bogo, M.R., Sarkis, J.J.F., Nettoa, C.A., Wyse, A.T.S., 2008. Intrastriatal injection of hypoxanthine alters striatal ectonucleotidase activities: a time-dependent effect. Brain Research 6, 198–206. Bavaresco, L., Bernardi, A., Braganhol, E., Wink, M.R., Battastini, A.M.O., 2007. Dexamethasone inhibits proliferation and stimulates ecto-50 -nucleotidase/ CD73 activity in C6 rat glioma cell line. Journal of Neuro-Oncology 84, 1–8. Bhargava, A., Mathias, R.S., McCormick, J.A., Dallman, M.F., Pearce, D., 2002. Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1. Molecular Endocrinology 16, 1629–1637. Bhargava, A., Meijer, O.C., Dallman, M.F., Pearce, D., 2000. Plasma membrane calcium pump isoform 1 gene expression is repressed by corticosterone and stress in rat hippocampus. Journal of Neuroscience 20, 3129–3138. Bodin, P., Burnstock, G., 2001. Purinergic signalling: ATP release. Neurochemical Research 26, 959–969. Burnstock, G., 1972. Purinergic nerves. Pharmacological Reviews 24, 509–581. Cotman, C.W., Matthews, D.A., 1971. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochemica and Biophyssica Acta 249, 380–394. Cunha, R.A., 2001. Adenosine as a neuromodulator and as a homeostatic regulator in nervous system: different roles, different sources and different receptors. Neurochemistry International 38, 107–125. Cunha, R.A., 2005. Neuroprotection by adenosine in the brain: from A1 receptor activation to A2A receptor blockade. Purinergic Signalling 1, 111–134. Cunha, R.A., Johansson, B., Ribeiro, J.A., Sebastia˜o, A.M., 1992. Adenosine A2a receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus. Neuroscience Letters 196, 41–44. Fontella, F.U., Bruno, A.N., Balk, R.S., Ru¨cker, B., Crema, L.M., Correa, M.D., Battastini, A.M., Sarkis, J.J., Alexandre Netto, C., Dalmaz, C., 2005. Repeated stress effects on nociception and on ectonucleotidase activities in spinal cord synaptosomes of female rats. Physiological Behaviour 85, 213–219. Fontella, F.U., Bruno, A.N., Crema, L.M., Battastini, A.M.O., Sarkis, J.J.F., Netto, C.A., Dalmaz, C., 2004. Acute and chronic stress alter ecto-nucleotidase activities in synaptosomes from the rat hippocampus. Pharmacology, Biochemistry and Behaviour 78, 341–347. Franke, H., Illes, P., 2006. Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacology and Therapeutics 109, 297–324.
181
Hass, H.L., Greene, R.W., 1988. Endogenous adenosine inhibits hippocampal CA1 neurones: further evidence from extra- and intracellular recordings. Naunyn Schmiedeberg Archives Pharmacology 337, 561–565. Horvat, A., Nikezic´, G., Martinovic´, J.V., 1995. Estradiol binding to synaptosomal plasma membranes of rat brain regions. Experientia 51, 11–15. Horvat, A., Nikezic, G., Petrovic, S., Kanazir, D.T., 2001. Binding of estradiol to synaptosomal mitochondria: physiological significance. Cellular and Molecular Life Sciences 58, 636–644. Inoue, K., 2002. Microglial activation by purines and pyrimidines. Glia 40, 156–163. Kapojos, J.J., Van den Berg, A., Borghuis, T., Banas, B., Huitema, S., Poelstra, K., Bakker, W.W., 2004. Enhanced ecto-apyrase activity of stimulated endothelial or mesangial cells is downregulated by glucocorticoids in vitro. European Journal of Pharmacology 501, 191–198. Khakh, B.S., Henderson, G., 1998. ATP receptor-mediated enhancement of fast excitatory neurotransmitter release in the brain. Molecular Pharmacology 54, 372–378. Kukulski, F., Komoszynski, M., 2003. Purification and characterization of NTPDase 1 (ecto-apyrase) and NTPDase 2 (ecto-ATPase) from porcine brain cortex synaptosomes. European Journal of Biochemistry 270, 3447–3454. Lamber, N.A., Teyler, T.J., 1991. Adenosine depresses excitatory but not fast inhibitory synaptic transmission in area CA1 of the rat hippocampus. Neuroscience Letters 122, 50–52. Latini, S., Pedata, F., 2001. Adenosine in the central nervous system: release mechanisms and extracellular concentrations. Journal of Neurochemistry 79, 463–484. Lavoie, E.G., Kukulski, F., Levesque, S.A., Lecka, J., Sevigny, J., 2004. Cloning and characterization of mouse nucleoside triphosphate dihosphohydrolase-3. Biochemical Pharmacology 67, 1917–1926. Lunga, P., Herbert, J., 2004. 17b-Oestradiol modulates glucocorticoid, neural and behavioral adaptation to repeated restraint stress in female rats. Journal of Neuroendocrinology 16, 776–785. Majumder, P., Trujillo, C.A., Lopes, C.G., Resende, R.R., Gomes, K.N., Yuahasi, K.K., Britto, L.R.G., Ulrich, H., 2007. New insights of purinergic receptor signaling in neuronal differentiation, neuroprotection and brain disorders. Purinergic Signaling 3, 317–331. Markwell, M.A., Haas, S.A., Lieber, L., Tolbert, N.A., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry 87, 206–210. Matute, C., Alberdi, E., Domercq, M., Sanchez-Gomez, M.V., Perez-Samartin, A., Rodriguez-Antiguedad, A., Perez-Cerda, F., 2007. Excitotoxic damage to white matter. Journal of Anatomy 210, 693–702. Pennial, R., 1966. An improved method for determination of inorganic phosphate by the isobutanol-benzen extraction procedure. Analytical Biochemistry 14, 87–90. Phillips, J.W., Wu, P.H., 1981. The role of adenosine and its nucleotides in central synaptic transmission. Progress in Neurobiology 16, 187–239. Rathbone, M.P., Middlemiss, P.J., Gysbergs, J.W., Andrew, C., Herman, M.A.R., Reed, J.K., Ciccarelli, R., Di Iorio, P., Caciagli, F., 1999. Trophic effects of purines in neurons and glial cells. Progress in Neurobiology 59, 663–690. Reppert, S.M., Weaver, D.R., Stehle, J.H., Rivkees, S.A., 1991. Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord. Molecular Endocrinology 5, 1037–1048. Rodrigo, R., Rivera, G., Lucero, Y., Larraguibel, C., 2002. Effect of ethanol on regulation of (Na+K)-adenosine triphosphatase by aldosterone and dexamethasone in cultured renal papillary collecting duct cells. Endocrine 19, 301–304. Romeo, R.D., 2005. Neuroendocrine and behavioral development during puberty: a tale of two axes. Vitamins and Hormones 71, 1–25. Romeo, R.D., Lee, S.J., McEwen, B.S., 2004. Differential stress reactivity in intact and ovariectomized prepubertal and adult female rats. Neuroendocrinology 80, 387–393. Schoen, S.W., Graeber, M.R., Toth, L., Kreutzberg, G.W., 1988. Ecto-50 -nucleotidase in postnatal ontogeny of rat cerebellum. A marker for migrating nerve cells? Brain Research 39, 125–136. Schoen, S.W., Kreutzberg, G.W., Singer, W., 1993. Cytochemical redistribution of 50 nucleotidase in the developing cat visual cortex. European Journal of Neuroscience 5, 210–222. Seale, J.V., Wood, S.A., Atkinson, H.C., Harbuz, M.S., Lightman, S.L., 2004. Gonadal steroid replacement reverses gonadectomy induced changes in the corticosterone pulse profile and stress-induced hypothalamic–pituitary–adrenal axis activity of male and female rats. Joural of Neuroendocrinology 16, 989–998. Shaheen, A.A., Abd El-Fattah, A., Gad, M.Z., 1996. Effect of various stressors on the level of lipid peroxide, antioxidants and Na+, K(+)-ATPase activity in rat brain. Experientia 52, 336–339. Sorimachi, M., Yamagami, K., Wakamori, M., 2002. Activation of ATP receptor increases the cytosolic Ca(2+) concentration in ventral tegmental area neurons of rat brain. Brain Research 935, 129–133. Torres, I.L., Buffon, A., Dantas, G., Fu¨rstenau, C.R., Bo¨hmer, A.E., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002a. Chronic stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats. Pharmacology Biochemistry and Behaviour 74, 181–186. Torres, I.L., Buffon, A., Silveira, P.P., Duarte, M.Z., Bassani, M.G., Oliveira, S.S., Battastini, A.M., Sarkis, J.J., Dalmaz, C., Ferreira, M.B., 2002b. Effect of chronic and acute stress on ectonucleotidase activities in spinal cord. Physiology and Behaviour 75, 1–5. Towle, A.C., Sze, P.Y., 1983. Steroid binding to synaptic plasma membrane: differential binding of glucocorticoid and gonadal steroids. Journal of Steroid Biochemistry 18, 135–143.
182
A. Horvat et al. / Int. J. Devl Neuroscience 28 (2010) 175–182
Velickovic, N., Djordjevic, A., Drakulic, D., Stanojevic, I., Secerov, B., Horvat, A., 2009. Cranial irradiation modulates hypothalamic–pituitary–adrenal axis activity and corticosteroid receptor expression in the hippocampus of juvenile rat. General Physiology and Biophysics 28, 219–227. Vogel, M., Zimmermann, H., Singer, W., 1993. Transient association of the HNK-1 epitope with 50 -nucleotidase during development of the cat visual cortex. European Journal of Neuroscience 5, 1423–1425.
Vuaden, F.C., De Paula Cognato, G., Bonorino, C., 2007. Lipopolysacharide alters nucleotidase activities from lymphocytes and serum of rats. Life Science 80, 1784–1791. Zimmermann, H., 2000. Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmiedeberg’s Archives of Pharmacology 362, 299–309. Zimmermann, H., 1992. Ecto-50 -nucleotidase: molecular structure and functional aspects. Biochemical Journal 285, 345–365.