Different response to exogenous l -arginine in nitric oxide production between hippocampus and striatum of conscious rats: a microdialysis study

Neuroscience Letters 366 (2004) 302–307

Different response to exogenous l-arginine in nitric oxide production between hippocampus and striatum of conscious rats: a microdialysis study Shuichi Hara a,∗ , Toshiji Mukai b , Kunihiko Kurosaki a , Hajime Mizukami a , Fumi Kuriiwa a , Takahiko Endo a a

b

Department of Forensic Medicine, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan Department of Legal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-0015, Japan Received 18 March 2004; received in revised form 20 May 2004; accepted 21 May 2004

Abstract We previously showed that systemic administration of a nitric oxide (NO) precursor, l-arginine (l-Arg), failed to reverse suppression by NO synthase (NOS) inhibitors of chemically induced shaking behavior in rats, leading to the hypothesis that exogenous l-Arg might be non-uniformly supplied to brain regions susceptible to NOS inhibitors. In the present study, therefore, we examined the effect of exogenous l-Arg on the extracellular levels of the oxidative nitric oxide (NO) products, nitrite (NO2 − ) and nitrate (NO3 − ), in two different brain regions, the hippocampus and the striatum, of conscious rats by means of in vivo brain microdialysis. The basal NO2 − levels in the two brain regions were comparable, while the NO3 − level was significantly lower in the hippocampus than the striatum. The addition of 10 mM l-Arg, but not d-Arg, to the perfusing solution significantly increased NO2 − and NO3 − in the hippocampus and NO2 − alone in the striatum. These increases were abolished by 1 mM N␻ -nitro-l-arginine, an NOS inhibitor. l-Arg at 1 mM was able to significantly increase NO2 − , but not NO3 − , in the hippocampus to a level comparable with that at 10 mM l-Arg, while it had no effect in the striatum. l-Arg (500 mg/kg, i.p.) induced a significant increase in NO2 − and NO3 − in the hippocampus, but not in the striatum. These results suggest that the striatum may have a lower ability to enhance NO production by utilising exogenous l-Arg than the hippocampus, despite higher basal NO production. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nitric oxide production; l-Arginine; Rat; Striatum; Hippocampus

It is well established that nitric oxide (NO) synthesized by NO synthase (NOS) is a messenger or modulator participating in physiological and pathological processes in the central nervous system [4,21,29]. Three isozymes have so far been identified: endothelial NOS (eNOS), neuronal NOS and inducible NOS [4,21,27,29]. l-Arg is the exclusive physiological substrate for all of the isozymes, and exists in the cells at a concentration far exceeding the Km value of each isozyme, indicating that the enzyme should be saturated with the substrate in the cell [27]. It is therefore surprising that supplementation of l-Arg enhances endothelial NO production both in humans [14,19] and in experimental animals [6] in vivo. This is known as the “arginine paradox”. Various ∗ Corresponding author. Tel.: +81 3 3351 6141x345; fax: +81 3 3353 7672. E-mail address: [email protected] (S. Hara).

hypotheses have been proposed to account for the paradox, including the ideas that supplementation of l-Arg may reverse inhibition of NOS by endogenous NOS inhibitors such as methylated arginines [27] and that the cationic amino acid transporter (CAT) co-localized with eNOS may directly deliver extracellular l-Arg to eNOS for NO production [13,18]. The latter hypothesis may be consistent with enhanced NO production, accompanying enhanced transport of extracellular l-Arg, in response to inflammatory mediators in macrophages [1,25], although the limited induction of the CAT does not always parallel the enhancement of NO production in arterial smooth muscle cells [7]. Systemic as well as central administration of l-Arg is able to enhance NO production in rat brain as well [24,31], but the mechanism of this enhancement is still unclear. We previously showed that paraquat (PQ), a herbicide that is one of the environmental neurotoxicants putatively

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.055

S. Hara et al. / Neuroscience Letters 366 (2004) 302–307

associated with idiopathic Parkinson’s disease [12,26], induced shaking behavior, which was associated with the central opioid system [8,10] and was suppressed by NOS inhibitors, such as 7-nitroindazole and N␻ -nitro-l-arginine (l-NA), but not N␻ -nitro-d-arginine [10], in rats. This strongly suggested that the shaking behavior might be mediated through stimulation of NO production in the brain. Since systemic administration of l-Arg reversed the suppression by NOS inhibitors of shaking behavior induced by a serotonin receptor antagonist [30], as well as other behaviors [17], we examined the effect of systemic l-Arg up to 500 mg/kg, which is sufficient to enhance NO production in rat cerebellum [24,31], on the suppression of the PQ-induced shaking behavior, but found that l-Arg had no effect [10,12]. We hypothesized that, although NOS inhibitors, which inhibit NOS to similar extents in various brain regions [15,23], influenced the central functions associated with NO, systemically administered l-Arg might be non-uniformly supplied to individual regions of the brain, resulting in a failure to reverse the inhibitors’ effect in a certain brain region(s); namely, the arginine paradox might not be equally applicable in all regions of the brain. In the present study, we examined whether exogenous l-Arg enhances NO production in the hippocampus and the striatum of conscious rats, as it has been reported to do in the cerebellum [24,31], by measuring the extracellular levels of the oxidative NO products, nitrite (NO2 − ) and nitrate (NO3 − ), with in vivo brain microdialysis. Male Sprague–Dawley rats, weighing 180–210 g, were purchased from Charles River Japan (Kanagawa, Japan). Animals were acclimated with free access to food and water in a facility with controlled temperature (22–24 ◦ C) and humidity (50–60%), on a 12-h light:12-h dark cycle (lights on between 06:00 and 18:00 h), for at least one week before all of the experiments. The total number of rats used was 83 (40 rats for the hippocampus and 43 rats for the striatum). The experimental protocol of this work was approved by the Tokyo Medical University Animal Care Committee and all experiments were performed in accordance with the Japanese Animal Research Association standards as defined in the Guideline for Animal Experiments and the Guiding Principle in the Use of Animals in Toxicology. Stereotaxic surgery and brain microdialysis were performed according to the methods previously reported [11,12]. The microdialysis probe with a 2 mm-long membrane (Eicom, Kyoto, Japan) was inserted into the hippocampus or the striatum (coordinates of the tip of the probe; −3.8 mm AP, 2.0 mm L, 4.0 mm DV and 0.2 mm AP, 3.0 mm L, 6.0 mm DV, respectively) [20] through the guide cannula (Eicom), which had been implanted under pentobarbital anesthesia (50 mg/kg, i.p.) at least 5 days before. The dialysis probe was perfused with a modified Ringer solution (147 mM NaCl, 3 mM KCl, 1.3 mM CaCl2 , 1 mM MgCl2 ) at a flow rate of 2 ␮l/min. When the perfusing solution was changed to the solution containing l-Arg, d-Arg or l-Arg plus l-NA (all from Sigma), and

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vice versa, a liquid-switch (SI-20; Eicom) was used. The location of the dialysis probe was verified after each experiment [12]. The assay of NO2 − and NO3 − in the perfused dialysate (20 ␮l) collected every 10 min was performed by means of an automated NO detector-HPLC system, which consisted of an HPLC pump system (ENO-10, Eicom), an automatic sample injector (AS-10; Eicom), a column oven (ATC-10; Eicom) and a Hitachi-L7420 flow-through spectrophotometer (Tokyo, Japan), as previously described [11,12]. Yamada and Nabeshima [31] observed unknown peaks which interfered with the determination of NO2 − and NO3 − when dialysate samples containing l-NA methyl ester were run on a similar HPLC system. In the present study, an unknown peak was eluted before NO2 − in the case of dialysate samples containing l-NA, but it did not interfere with the determination of NO2 − . Extracellular NO2 − and NO3 − levels and NOx as their sum were expressed as percentages of the respective basal levels, which were determined by averaging three consecutive dialysate samples in individual animals before various treatments. Data were expressed as means ± S.E.M. obtained from five to seven rats and analyzed by using one-way ANOVA followed by the Dunnett test for multiple-group comparisons or Student’s t-test for two-group comparisons. A P value of less than 0.05 was regarded as statistically significant. The basal level of extracellular NO2 − for the 10-min perfusion (20 ␮l) in the hippocampus was comparable to that in the striatum, while the basal level of extracellular NO3 − was significantly lower in the former than the latter (Fig. 1). The basal NOx level, therefore, was significantly lower in the hippocampus than the striatum (Fig. 1). Fig. 2 shows the changes in the extracellular NO2 − and NO3 − levels in the hippocampus and the striatum, when 1 or 10 mM l-Arg or 10 mM d-Arg was dissolved in the perfusing solution and applied to each brain region through

Fig. 1. The basal levels of extracellular NO2 − , NO3 − and NOx in the hippocampus (n = 40) and the striatum (n = 43). (*) Significantly different (P < 0.05) from the striatum by Student’s t-test.

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Fig. 2. Effect of application of l-Arg or d-Arg on extracellular NO2 − , NO3 − and NOx in the hippocampus (left) and the striatum (right). l-Arg or d-Arg was applied to each brain region by adding it to the perfusing solution for 10 min, as indicated with a horizontal solid bar in each graph. Each symbol (nothing, 䊐; l-Arg 1 mM, 䉱; l-Arg 10 mM, 䊉; d-Arg 10 mM, ) with a vertical bar indicates the mean ± S.E.M. of five to seven rats. Significantly different (P < 0.05) from the control (nothing) by one-way ANOVA followed by the Dunnett test: (#) 1 mM l-Arg vs. control; (*) 10 mM l-Arg vs. control; (†) 10 mM d-Arg vs. control.

the dialysis probe for 10 min. In the hippocampus, 10 mM l-Arg, but not d-Arg, induced a significant and prolonged increase in extracellular NO2 − and a significant, but transient, increase in NO3 − , as compared with the control. One millimolar l-Arg significantly increased extracellular NO2 − to a level comparable with that in the case of 10 mM l-Arg, but this increased NO2 − did not result in a significant increase in NOx . Extracellular NO3 − and NOx were slightly increased following the application of 1 mM l-Arg, and thereafter, decreased to a significantly lower level than the control by the end of the observation period. Such a decrease was also observed in the rats treated with 10 mM d-Arg. On the other hand, in the striatum, 10 mM l-Arg significantly increased extracellular NO2 − , as seen in the hippocampus, whereas a transient increase in extracellular NO3 − and NOx following the l-Arg application was too small to reach statistical significance (Fig. 2). One millimolar l-Arg and 10 mM d-Arg had no significant effect on either extracellular NO2 − or NO3 − (Fig. 2). The increase in extracellular NO2 − and NO3 − induced by 10 mM l-Arg in the hippocampus disappeared upon simultaneous application of 1 mM l-NA, an

NOS inhibitor, with l-Arg. l-NA abolished the increase in extracellular NO2 − in the striatum as well, while it decreased extracellular NO3 − and NOx , which were not increased by l-Arg (Fig. 3). When l-Arg at 500 mg/kg was intraperitoneally administered, extracellular NO2 − and NO3 − in the hippocampus were both significantly increased, as compared with those of the control treated with vehicle (saline), though the increases in NO2 − and NO3 − were prolonged and transient, respectively (Fig. 4). The changes in NOx seemed to be similar to those in NO3 − , but the increase in NOx due to l-Arg was slightly prolonged. In contrast, 500 mg/kg l-Arg had no effect on extracellular NO2 − and NO3 − (and therefore, NOx as well) in the striatum (Fig. 4). NOS is heterogenously distributed in rat brain [2,5,22], and its activities parallel the tissue contents of NOx in various brain regions of the rat [24]. Since the NOS activities [5,23,24] and the NOx contents [24] determined ex vivo in the hippocampus and the striatum are comparable, it is likely that NO production levels in the two brain regions are similar. In the present study, however, the basal NOx

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Fig. 3. Effect of l-NA on the increase in extracellular NO2 − , NO3 − and NOx in the hippocampus and the striatum. l-Arg (10 mM) and l-NA (1 mM) were simultaneously applied to each brain region by adding them to the perfusing solution for 10 min, as indicated with a horizontal solid bar in each graph. Each symbol (l-Arg alone for the hippocampus, ; l-Arg + l-NA for the hippocampus, 䊉; l-Arg alone for the striatum, ; l-Arg + l-NA for the striatum, 䉱) with a vertical bar indicates the mean ± S.E.M. of five or six rats. Significantly different (P < 0.05) from 10 mM l-Arg alone in each brain region by Student’s t-test: (#) the striatum; (*) the hippocampus.

determined by in vivo microdialysis was lower in the hippocampus than the striatum, indicating lower NO production in the former than the latter in the conscious rat brain in vivo. The direct application of l-Arg to the hippocampus and the striatum revealed that the response of the striatum in increasing the NO products was seemingly one-tenth of that of the hippocampus, both of these responses being mediated by NOS on the basis of their abrogation by the NOS inhibitor and lack of such a response to d-Arg, the enantiomer of l-Arg. It is unclear why l-Arg at 1 mM (but not 10 mM) or 10 mM d-Arg caused a decrease in NO3 − and NOx by the end of the observation period only in the hippocampus, but not in the striatum. Systemic administration of l-Arg

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Fig. 4. Effect of systemic administration of l-Arg on extracellular NO2 − , NO3 − and NOx in the hippocampus and the striatum. l-Arg at 500 mg/kg was intraperitoneally administered to rats, as indicated with an arrow in each graph. The control was given vehicle (saline) alone. Each symbol (control for the hippocampus, ; l-Arg for the hippocampus, 䊉; control for the striatum, ; l-Arg for the striatum, 䉱) with a vertical bar indicates the mean ± S.E.M. of six rats. (*) Significantly different (P < 0.05) from control for the hippocampus by Student’s t-test. No significant change was observed in the striatum.

also demonstrated the insensitivity of the striatum to l-Arg in increasing the NO products. It has been shown that the steady blood flow in the striatum is comparable with that of the hippocampus in urethane-anesthetized rats [9] and that the CAT-dependent uptake of exogenous l-Arg through the blood–brain barrier to the striatum is equivalent to that to the hippocampus in an in situ brain perfusion model of the rat [16]. These findings suggest that the striatum might be less responsive to exogenous l-Arg in enhancing NO production via NOS than the hippocampus, despite its higher basal NO production. Thus, systemic as well as central administration of l-Arg enhances NO production in rat brain, but the extent of the enhancement varies depending on the individual brain region. Since NOS inhibitors suppress NO production

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in various brain regions of the rats to similar extents [15,23], some phenomenon associated with a brain region that is insensitive to exogenous l-Arg, is resistant to the l-Arg, even though it is susceptible to NOS inhibitors. This is consistent with our previous finding that, although shaking behavior induced by PQ was suppressed by NOS inhibitors, systemic l-Arg failed to reverse the suppression [10,12]. The enhancement of NO production by exogenous l-Arg has been observed in rat cerebellum, where the application of 1 mM l-Arg, or intraperitoneal l-Arg at 500 mg/kg, is sufficient to induce a remarkable enhancement of NO production, as determined by in vivo microdialysis [31]. The high NOS activity of the cerebellum in rat brain [5,23,24] may be a possible explanation for the enhancement, but this is not the case for the hippocampus and the striatum, as described above. It is well-known as the so-called “arginine-paradox” that, although the concentration of intracellular l-Arg is saturating for NOS, additional exogenous l-Arg is able to enhance NO production mediated by NOS [27]. This paradox for eNOS could be at least partly explained by direct delivery of extracellular l-Arg to eNOS for NO production by CAT located in close proximity to the enzyme in arterial endothelial cells [13,18]. Although such a mechanism has not been confirmed in the brain, the different responses to exogenous l-Arg in enhancing NO production among brain regions might be dependent on not only the NOS activity, but also on extracellular l-Arg availability in each region; this is a hypothesis based on the differential mRNA expression of arginine transporters, such as CAT1, CAT2(B) and CAT3 [2,3,29]. We should not exclude possible participation of endogenous NOS antagonists, such as methylated arginines [27], in the region-dependent response to exogenous l-Arg in enhancing NO production in the brain, since heterogenous distribution of methylated arginines has been demonstrated [28]. These factors could all be implicated in the complex regulating system of NO production in the brain, as recently proposed for the so-called “Citrulline (Cit)-NO cycle”, which involves intracellular recycling of l-Arg for NO production from the NOS coproduct, l-Cit, intercellular trafficking of l-Arg and l-Cit among neurons, astrocytes and microglia, and uptake of the amino acids into the cells [2,29]; such a cycle could be associated with the responses to exogenous l-Arg in enhancement of NO production. The bulk of NOx in the hippocampus and the striatum consisted of NO3 − , in agreement with previous reports on various brain regions [24,31]. The finding that the basal extracellular NO3 − , but not extracellular NO2 − , was higher in the striatum than the hippocampus suggests that the oxidative pathways of NO to NO3 − might be favored in the striatum. It is of interest that exogenous l-Arg enhanced the formation of NO2 − more markedly and tonically than that of NO3 − in the two brain regions, although this did not always influence NOx . The possibility arises that some NO-related phenomenon may be overlooked by measuring NOx alone, since this may mask a significant change in NO2 − . In the cerebellum, however, the increase in extracellular NO3 − due

to exogenous l-Arg is more profound than that in extracellular NO2 − [31]. It is noteworthy that depolarization by 100 mM KCl increases extracellular NO2 − , but not extracellular NO3 − , in rat cerebellum, as determined by in vivo microdialysis, whereas stimulation of the glutamate receptors increases extracellular NO3 − , but not extracellular NO2 − [31]. In conclusion, the present study has demonstrated the differential enhancement of NO production by exogenous l-Arg in different brain regions of the rat in vivo. Variations in basal NO production do not appear to account for this difference; instead, complex mechanisms associated with various factors, including NOS, CATs and endogenous NOS antagonists, seem to be involved. These findings may have important implications for the use of exogenous l-Arg as a probe to investigate the role of NO in the brain.

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