Axonal transports of tripeptidyl peptidase II in rat sciatic nerves

Neurochemistry International 50 (2007) 236–242 www.elsevier.com/locate/neuint

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves Toshiyuki Chikuma a,*, Maki Shimizu a, Yukihiro Tsuchiya a, Takeshi Kato b, Hiroshi Hojo a a

Department of Hygienic Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida-shi, Tokyo 194-8543, Japan b Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama 236-0027, Japan Received 4 August 2006; accepted 14 August 2006 Available online 4 October 2006

Abstract Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12–72 h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72 h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts. # 2006 Elsevier Ltd. All rights reserved. Keywords: Tripeptidyl peptidase II; Double ligation technique; Immunohistochemistry; Rat sciatic nerve; Axonal flow

It has been reported that several types of neuropeptide function as neurotransmitters or modulators in the peripheral and central nervous systems. The expression of neuropeptide physiological function requires that newly synthesized neuropeptides in the cell body are transported to nerve terminals and released to synaptic clefts. Previously, we demonstrated that not only neuropeptides (e.g., substance P(SP) and cholecystokinin (CCK)) (Kato et al., 1987; Tozawa et al., 1990) but also enzymes required for the biosynthesis (e.g., processing endopeptidase, carboxypeptidase E (CPE) and peptidylglycine a-amidating monooxygenase (PAM)) (Tozawa et al., 1990; Yajima et al., 1994; Imaizumi et al., 2000) are transported to nerve terminals by anterograde axoplasmic flow. The functional level of a biologically active neuropeptide depends on the rates of synthesis and degradation. It has been generally considered that neuropeptides are degraded by enzymes in the extracellular space or synaptic clefts (Mackelvy

* Corresponding author. Tel.: +81 42 721 1564; fax: +81 42 721 1563. E-mail address: [email protected] (T. Chikuma). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.08.010

and Blumberg, 1986), and that these neuropeptide-degrading enzymes are also rapidly transported to nerve terminals. Neuropeptide-degrading enzymes may be removed from synaptic clefts by reuptake into dendrite where they are retrogradely transported to the cell body. Previously, we reported the axonal transport of some enzymes involved in the degradation of neuropeptides (Kato et al., 1987; Yamamoto et al., 2002; Yamamoto et al., 2003). However, the axonal transport of tripeptidyl peptidase II (TPP II; EC 3.4.14.10), a putative CCKinactivating serine peptidase, has not been investigated. CCK, and mainly the C-terminal octapeptide (CCK-8), is widely distributed in the mammalian central nervous system (Vanderhaeghen et al., 1980; Beinfeld et al., 1981). CCK-8 may act as a neurotransmitter or a neuromodulator and interacts with two different receptors, which have been cloned (Wank et al., 1992), with the CCKB receptor being the predominant subtype in the brain (Pelaprat et al., 1987). The psychological effects and some pharmacological activities of CCK-8 have been reported (Itoh and Lal, 1990; Singh et al., 1991; Smith and Gibbs, 1994; Vaccarino, 1994; Bradwejn et al., 1995; Wiesenfeld-Hallin and Xu, 1996; Benoliel et al., 1998;

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Desousa et al., 1999; Sebret et al., 1999; Tirassa et al., 1999; Beinfeld, 2003). TPP II was first reported as an extralysosomal tripeptidereleasing aminopeptidase in rat liver cytosol (Balow et al., 1983). TPP II is a ‘‘giant’’ protease, larger than the 26S proteasome, with a molecular weight of above 106 (Balow et al., 1983, 1986). It consists of multiple subunits, each with a molecular weight of 138 kDa. There is evidence that TPP II needs to be assembled into its oligomeric complex for maximal proteolytic activity (Tomkinson, 2000). It has been characterized as an intracellular serine peptidase present in different cell types and species and is considered to participate in general intracellular protein turnover (Balow and Eriksson, 1987; Tomkinson et al., 1987; Tomkinson, 1994). It was demonstrated that TPP II can compensate, at least in part, for the loss of the proteasome in proteasome-inhibitor-adapted cells (Glas et al., 1998; Geier et al., 1999). In addition to this general function, Rose et al. reported that a cholecystokinin-inactivating peptidase from the rat brain is also TPP II (Rose et al., 1988, 1996), which indicates a potential role of TPP II in the turnover of specific neuropeptides. In this study, we examined the axonal transport of TPP II in rat sciatic nerves using a double ligation technique. 1. Experimental procedures 1.1. Materials 1,10-Phenanthroline monohydrate and 3,30 -diaminobenzidine tetrahydrochloride were purchased from Wako (Tokyo, Japan). N-Ethylmaleimide (NEM), iodoacetic acid (IAA), diisopropylfluorophosphate (DFP), and p-chloromercuriphenylsulfonic acid (PCMS) were obtained from Sigma (St. Louis, MO, USA). Ala-Ala-Phe-MCA, Ala-Ala-Phe-chloromethylketone and 7amino-4-methylcoumarin (AMC) were obtained from the Peptide Institute (Osaka, Japan). Other materials and their sources were sodium pentobarbital (Dainabot, Tokyo, Japan), anti-human TPP II chicken antiserum (Immunsystem, Uppsala, Sweden), biotin-conjugated rabbit anti-chicken IgG (Rockland, PA, USA) and streptavidin conjugated horseradish peroxidase (streptavidin HRP) (Dako Cytomation, Denmark).

1.2. Ligation of rat sciatic nerves Male Wistar rats weighting 200–300 g were obtained from Sankyo Laboratory (Tokyo, Japan) and were housed with free access to food and water. A 12:12 light–dark cycle was maintained over 1 week. On the day of surgery, the animals were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.). Rat sciatic nerves were ligated at two places with surgical silk thread (4–0 gauge) (Figs. 1A and 2A). The animals were killed by decapitation between 0 and 72 h after the ligation. Each segment from the nerves was dissected on ice, and used as a source of TPP II activity or in the immunohistochemical study in additional experiments. All animal experiments were carried out in accordance with the 1980 Animal Experiment Guidelines of the Japanese Government, and have been approved by the Animal Experiment Committee of our university.

1.3. Sample preparation Each 3 mm segment was minced with scissors and homogenized by sonication in 100 ml of ice-cold phosphate-buffered saline (PBS). The homogenate was centrifuged at 100,000  g for 15 min and the supernatant obtained was used for the measurement of enzyme activity in each segment. On the other hand, each 5 mm segment of a sciatic nerve 48 h after ligation was minced with

Fig. 1. Distribution of TPP II activity in each segment of rat sciatic nerves 48 h after ligation. The supernatant of each segment was subjected to TPP II enzyme assay. (A) Schematic illustration of ligation in rat sciatic nerves, where arrows and ‘‘Ligation’’ indicate sites of ligation. (B) Distribution of TPP II activity in rat sciatic nerves. P1–P4, proximal segments; M1–M3, middle segments; D1– D3, distal segments. Data are mean  S.E.M. (bars) values from five to six animals. Statistical comparisons are indicated by solid lines. ANOVA was performed followed by the Student–Newman–Keuls test: ***p < 0.001. scissors, and homogenized in 31 ml of ice-cold PBS by sonication. After centrifugation at 100,000  g for 60 min, the supernatant obtained was used for Western blot analysis.

1.4. Assay for TPP II enzyme activity Enzyme activity was measured using Ala-Ala-Phe-MCA as the substrate by a previously reported method (Balow et al., 1986; Geier et al., 1999) with minor modification. In brief, the 100 ml reaction mixture consisted of 0.1 M sodium phosphate buffer (pH 6.8), 0.1 mM Ala-Ala-Phe-MCA, and enzyme in water. Incubation was carried out at 37 8C for the desired periods, and the reaction was terminated by adding 1 ml of 1 M sodium acetate buffer (pH 4.2). After centrifugation, the amount of AMC liberated from the substrate was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm using a CytoFluor II spectrophotometer (PerSeptive Biosystems, Framingham, MA).

1.5. Effects of various metal ions and inhibitors on enzymatic activity The supernatant of P1 and D1 segments in rat sciatic nerves was used for this experiment. Enzyme solution was preincubated with or without various chemicals (0.1 and 1 mM) in 0.1 M sodium phosphate buffer (pH 6.8) for 15 min at 25 8C. Immediately thereafter, 0.1 mM Ala-Ala-Phe-MCA (final concentration) was added to the reaction mixture and incubated at 37 8C for the desired periods. After stopping the reaction, fluorescence intensity was read as described above.

1.6. Western blot analysis To determine protein expression level, Western blot analysis of TPP II in rat sciatic nerves was performed. In brief, 20 ml of the supernatant was separated

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T. Chikuma et al. / Neurochemistry International 50 (2007) 236–242 in 0.3% hydrogen peroxide for 30 min, and washed three times in PBS for 10 min per wash, and then incubated with streptavidin HRP (1:150) for 45 min at room temperature. The sections were stained with 3,30 -diaminobenzidine in 50 mM Tris–HCl buffer (pH 7.6).

1.8. Statistical analysis One-way analysis of variance (ANOVA) was carried out to determine levels of significance in experiments. Multiple group comparisons were performed using the Student–Newman–Keuls test. Data are presented as mean  S.E.M. values.

2. Results 2.1. Axonal transport of TPP II 48 h after ligation

Fig. 2. Time-dependent changes in TPP II activity in supernatant of proximal (P) (*), middle (M) (&) and distal (D) (~) segments at 0, 12, 24, 48, and 72 h after ligation. (A) Schematic illustration of ligation in rat sciatic nerves, where arrows and ‘‘Ligation’’ indicate sites of ligation. (B) Distribution of TPP II activity in rat sciatic nerves. Data are mean  S.E.M. (bars) values from five animals. Statistical significance was determined by ANOVA followed by the Student–Newman– Keuls test compared with the M segment: **p < 0.01, ***p < 0.001.

by SDS-PAGE on an 8% separating gel and transferred to a poly(vinylidene difluoride) (PVDF) membrane. The membrane was blocked for 1 h at room temperature in PBS containing 0.05% Tween 20 (TPBS) and 5% skim milk, and then incubated with an anti-human TPP II chiken antibody (1:2000) for 1.5 h at room temperature. Subsequently, the membrane was washed four times in TPBS, and incubated for 30 min with biotin-conjugated rabbit antichiken IgG (1:8000). After washing the membrane four times in TPBS, it was incubated with streptavidin HRP (1:3000) for 30 min at room temperature, and resulting protein bands were visualized by staining with 3,30 -diaminobenzidine. The blotted membrane was subjected to densitometric image analysis using Image J 1.32 j software. Optical density (OD) was background-subtracted and analyzed. The final OD is expressed as the grand mean (S.E.M.) of individual means.

As shown in Fig. 1A, the proximal, middle and distal segments of rat sciatic nerves are abbreviated as P1–P4, M1– M3 and D1–D3 segments, respectively. The activities of TPP II in rat sciatic nerves 48 h after ligation are shown in Fig. 1B. In the P1 segment, the TPP II enzyme activity was 3.01  0.41 nmol/min/3 mm and increased 4.1-fold in comparison with that in the M1 segment (0.73  0.19 nmol/min/ 3 mm). The TPP II enzyme activity in the D1 segment (1.97  0.17 nmol/min/3 mm) also increased 2.4-fold in comparison with that in the M3 segment (0.83  0.21 nmol/ min/3 mm). Both the rates of anterograde axonal transport (P1/ M1) and the retrograde axonal transport (D1/M3) of TPP II activity were statistically significant ( p < 0.001). 2.2. Time-dependent axonal flow As shown in Fig. 2A, the proximal, middle and distal segments of rat sciatic nerves are abbreviated as P, M and D segments, respectively, and these segments were used as an enzyme source for the investigation of time-dependent axonal flow. Time-dependent changes in TPP II activity in segments P, M and D were studied between 0 and 72 h after ligation

1.7. Immunohistochemistry The animals were killed by decapitation 48 h after the ligation, and then sciatic nerves were removed. The sciatic nerves were permeabilized with icecold 4% paraformaldehyde in PBS for 2.5 h, equilibrated at 4 8C for at least 1 day in 30% sucrose in PBS, and frozen at 20 8C. The frozen sciatic nerves were sliced at 10 mm on a cryostat and mounted on a glass slide. The sliced sections were incubated in PBS containing 1% normal rabbit serum and 2% BSA for 30 min at room temperature to remove nonspecific immunoreaction, and incubated overnight at 4 8C with an anti-human TPP II chicken antibody (1:1000) dissolved in PBS containing 0.05% Triton X-100. The sections were washed three times in PBS for 10 min per wash and incubated for 2 h at room temperature with biotin-conjugated rabbit anti-chicken IgG (1:500). After washing three times in PBS for 10 min each wash, the sections were incubated

Fig. 3. Effects of pH on TPP II activity in supernatant of P1 (*) and D1 (&) segments of rat sciatic nerves: 0.1 M sodium phosphate buffer (pH 6.0–7.8) was used. Data are mean  S.E.M. (bars) values from four experiments. Incubation was carried out at 37 8C for 60 min.

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(Fig. 2B). The TPP II enzyme activity in segments P and D time-dependently increased at all time points examined. The statistical significance of the rates of anterograde axonal transport (P/M) and retrograde axonal transport (D/M) was found 12, 24, 48 and 72 h after ligation. The enzyme activity in segment M slightly increased until 48 h after ligation, but at 72 h the enzyme activity decreased.

Table 1 Effects of various metal ions and inhibitors on TPP II activity in rat sciatic nerves Reagent

2.4. Effects of various metal ions and inhibitors on enzymatic activity The effects of various metal ions and inhibitors on TPP II activity in segments P1 and D1 were examined at final concentrations of 0.1 and 1 mM. As shown in Table 1, several metals tested were found to inhibit the enzyme. In particular, Hg2+ and Cu2+ inhibited strongly the enzyme activity at 0.1 and 1 mM. Furthermore, 1,10-phenanthroline (a chelating agent), DFP (a serine protease inhibitor) and PCMS (a thiol protease inhibitor) also inhibited it strongly at a final concentration of 1 mM. Ala-Ala-Phe-chloromethylketone (AAP-CMK), a typical TPP II inhibitor, inhibited 85–90% of the enzyme activity. However, TPP II was only partially inhibited by thiol protease inhibitors (i.e., IAA, NEM), and a chelating agent (EDTA). TPP II activities in segments P1 and D1 showed similar patterns of inhibition by various metal ions and inhibitors. 2.5. Western blot analysis To determine whether TPP II is anterogradely and retrogradely transported at the protein level, Western blot analysis of sciatic nerves 48 h after the double ligation was performed. We chose this time because it enabled the appropriate accumulation of the enzyme, minimize the necrosis of neurons or connective tissues, and minimize the accumulation of macrophage and lysosomal enzymes at the ligation sites where necrosis occurred after a prolonged ligation of rat sciatic nerves (Kato et al., 1987; Kato et al., 1998). At 138 kDa, TPP II immunoreactivity in the P and D segments showed a denser band than that in the M segment (Fig. 4A). The quantitation of TPP II immunoreactivity showed that the immunoreactivities in P and D segments were 3.1- and 1.7-fold higher than that in the M segment, respectively (Fig. 4B). The data are consistent with the above-mentioned changes in the enzyme activity, suggesting that TPP II is anterogradely and retrogradely transported.

Final concentration (mM)

None

TPP II activity (% of control) P1

D1

100

100

HgCl2

1 0.1

6.6  0.8 7.4  1.1

5.8  0.8 5.6  0.5

CaCl2

1 0.1

74.3  2.6 105.9  2.1

43.5  3.1 75.1  1.0

ZnSO4

1 0.1

65.9  0.7 81.8  2.0

35.1  2.2 54.6  2.0

MgCl2

1 0.1

75.2  1.9 87.5  1.4

86.4  2.3 102.8  1.8

CuSO4

1 0.1

8.6  1.4 8.1  0.9

7.6  0 7.0  0.4

FeCl3

1 0.1

72.1  2.7 81.9  1.3

75.1  3.0 85.4  2.5

MnCl2

1 0.1

72.7  4.3 90.2  2.2

56.0  1.3 97.9  6.1

EDTA

1 0.1

15.2  1.0 17.1  1.0

28.4  0.5 23.2  3.6

1,10-Phe

1 0.1

9.3  1.3 34.6  3.0

8.9  1.1 19.9  4.1

IAA

1 0.1

69.4  0.9 78.4  2.1

71.2  1.9 73.8  2.7

NEM

1 0.1

31.0  0.2 70.4  6.6

30.2  0 65.0  3.9

PCMS

1 0.1

10.8  1.4 19.7  1.3

11.8  0.2 18.1  0.3

DFP

1 0.1

6.7  0.3 19.2  0.8

8.5  0.8 13.0  0.2

AAP-CMK

1 0.1

14.2  1.3 11.1  0.3

12.9  1.7 13.6  1.3

2.3. pH dependency To examine the pH dependency of TPP II enzyme activity, soluble fractions prepared from segments P1 and D1 were used. This investigation was performed in 0.1 M sodium phosphate buffer (pH 6.0–7.8). As shown in Fig. 3, relative TPP II activity in segment P1 was higher than that in segment D1. The catalytic activities of TPP II in segments P1 and D1 were maximum at pH values from approximately 6.5– 7.0.

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The assay for TPP II activity was carried out as described in Section 1. Data are mean  S.E.M. values determined in four separate experiments. 1,10-Phe, 1,10phenanthroline monohydrate; IAA, iodoacetic acid; NEM, N-ethylmaleimide; PCMS, p-chloromercuriphenylsulfonic acid; DFP, diisopropylfluorophosphate; AAP-CMK, Ala-Ala-Phe-chloromethylketone.

2.6. Immunohistochemistry To further clarify the axonal transport of TPP II in rat sciatic nerves, immunohistochemical analysis of the sciatic nerves was performed 48 h after double ligation. According to the abovementioned enzyme activity and Western blot analysis, immunoreactive TPP II apparently accumulated in the P1 and D1 segments but not in the M1 and M3 segments (Fig. 5A– D), and preimmune serum had no effect on staining (data not shown). These results indicate that TPP II is anterogradely and retrogradely transported. 3. Discussion In general, neuropeptides and processing enzymes are presumed to be transported by a rapid axonal flow. Previous

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Fig. 4. Distribution of TPP II immunoreactivity in supernatant of rat sciatic nerves 48 h after ligation. The supernatant (20 ml) of each 5 mm segment was used. (A) Western blot pattern of TPP II. (B) Quantitative assay of Western blotting was performed using Image J 1.32 j software. P, proximal segment; M, middle segment; D, distal segment. Data are mean  S.E.M. (bars) values from five animals. Values are expressed as a percentage of the activity in the M segment. Statistical significance was determined by ANOVA followed by Student–Newman–Keuls test: **p < 0.01.

studies showed that neuropeptides such as SP (Kato et al., 1987) and CCK (Tozawa et al., 1990; Cortes et al., 1991) are anterogradely transported in sciatic nerves. Koo et al. (1990) also reported an anterograde axonal transport of the amyloid

precursor protein (APP). We also reported a rapid anterograde axonal flow of PAM (Tozawa et al., 1990), CPE (Yajima et al., 1994) and processing endopeptidase (Imaizumi et al., 2000), which are neuron-specific and neuropeptide-processing enzymes. Moreover, putative neuropeptide-degrading enzymes, puromycin-sensitive aminopeptidase (PSA) and endopeptidase 24.15 (EP 24.15), are transported to nerve terminals by anterograde axoplasmic flow (Yamamoto et al., 2002; Yamamoto et al., 2003). Thus, the double ligation technique for rat sciatic nerves is relevant for studies of the axonal transport of neuropeptides and their metabolizing enzymes. The present study showed the biochemical properties of TTP II and its axonal transport in rat sciatic nerves using Ala-Ala-Phe-MCA as the substrate for TPP II. The identification of the peptidases responsible for the physiological inactivation of neuropeptides released into the extracellular space is a difficult but important task. The development of selective inhibitors protecting endogenous neuropeptides facilitates the identification of such peptidases as shown in the case of enkephalins. TPP II, a serine peptidase apparently involved in the inactivation of CCK-8, first releases the N-terminal tripeptide of CCK-8 and the resulting pentapeptide CCK-5 is further cleaved into inactive fragments. Purified TPP II displays a rather high specificity toward the sulfated octapeptide and CCK-5, but is still able to cleave a few other neuropeptides, although at a significantly lower rate. The role of TPP II as a major CCK-inactivating enzyme was finally established using butabindide, a rationally designed potent and

Fig. 5. Immunohistochemical staining of TPP II in rat sciatic nerves 48 h after ligation. (A) Proximal P1; (B) middle M1; (C) middle M3; (D) distal D1. Preabsorption with the TPP II immunogen peptide abolished staining (data not shown).

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selective TPP II inhibitor, which was shown to protect almost completely endogenous CCK-8 from hydrolysis in the brain slice model which, in its absence, was inactivated by about 85% (Rose et al., 1988, 1996). Facchinetti et al. (1998) have also reported that TPP II-like immunoreactivity is mostly detected in neurons in areas containing CCK terminals, as in the cerebral cortex or hippocampal formation, where pyramidal cell bodies and processes surrounded by CCK axons are immunoreactive. On the basis of these observations, we suppose that TTP II is transported from neuronal cell bodies to nerve terminals. However, the present biochemical and immunological data indicate that TPP II is accumulated in segments P1 and D1 after the ligation of rat sciatic nerves (Figs. 1B, 2B, 4 and 5), indicating the anterograde and retrograde axonal transport of the enzyme in rat sciatic nerves. Previously, we reported the retrograde axonal transport of deamidase, a putative neuropeptide-degrading enzyme, in addition to anterograde axonal transport in rat sciatic nerves (Yamamoto et al., 2003). Although we speculate that deamidase, a lysosomal enzyme, could be densely located in lysosomes in nerve terminals and retrogradely transported, TPP II is mainly detected in the extralysosomal fraction (Balow et al., 1983). A few possible explanations for this discrepancy are as follows: (1) The enzyme anterogradely transported is returned by retrograde axonal transport retaining its activity. (2) The enzyme anterogradely transported is different from the enzyme transported retrogradely. However, the property of the enzyme anterogradely transported is not significantly different from that of the retrogradely transported (Fig. 3 and Table 1). (3) TPP II may be associated with a different type of granule, and transported by retrograde flow, in comparison with anterograde axonal flow. In addition to its role in general protein turnover in the cytosol of most cells, a more specific role of TPP II can also be considered. Thus, a membrane-bound variant of TPP II has been identified as being responsible for the inactivation of the neuropeptide CCK. The amount of the membrane-bound variant of the enzyme varies among different tissues and constitutes about 4% of TPP II activity in the liver and 31% in the brain (Rose et al., 1996). TPP II activity was also detected in synaptosomal membranes from the human brain (Wilson et al., 1993). The noncompetitive N-methyl-[D]-aspartate (NMDA) receptor antagonist (+)MK-801 induces neurotoxicity and schizophrenia-like symptomatology. Arif et al. (2006) examined the involvement of somatostatin (SS) and the CCK peptidergic system in MK-801-induced schizophrenia-like model rat brain regions. SS mRNA and peptide expression levels significantly decreased in the posterior cingulate/retrosplenial cortices (PC/ RSC) and hippocampus but not in the frontal cortex 3 days after MK-801 treatment, whereas, CCK mRNA and peptide expression levels significantly decreased in all of the brain regions examined. Pretreatment with clozapine but not that with haloperidol completely recovered the changes in both mRNA and peptide expression levels of SS and CCK in these brain regions. One possible explanation for the peptide decrease is the activation of peptide-degrading enzymes following drug treatment as shown by our recent study that systemic treatment with MK-801 dose-dependently increases the activities of

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neuropeptide-degrading enzymes and lysosomal enzymes in rat brain regions (Ahmed et al., 2003). Among the enzymes with elevated activities, prolyl oligopeptidase and thimet oligopeptidase play important roles in the metabolism of many biologically active peptides and hormones in the CNS. Because the neuropeptide-degrading enzymes are considered as the prime molecules underlying the mechanism of peptide inactivation and regulation, it is reasonable to speculate that the increased activities of neuropetidases such as TPP II might in part play a role in the pathogenesis of negative schizophrenia via the metabolism of CCK. Although an in vivo substrate for TPP II and the function of axonally transported TPP II in rat sciatic nerves have not yet been elucidated, TPP II may be involved in the metabolism of neuropeptides and play a role in modulating neurotransmission. Acknowledgement We are very grateful to Dr. Akira Tanaka for valuable and helpful advise on the experiments and manuscript preparation. References Ahmed, M.M., Yamamoto, M., Chikuma, T., Rahman, M.K., Kato, T., 2003. Dose-dependent effect of MK-801 on the levels of neuropeptides processing enzymes in rat brain regions. Neurosci. Res. 47, 177–189. Arif, M., Ahmed, M.M., Kumabe, Y., Hoshino, H., Chikuma, T., Kato, T., 2006. Clozapine but not haloperidol suppresses the changes in the levels of neuropeptides in MK-801-treated rat brain regions. Neurochem. Int. 49, 304–311. Balow, R.-M., Ragnarsson, U., Zetterqvist, O., 1983. Tripeptidyl aminopeptidase in the extralysosomal fraction of rat liver. J. Biol. Chem. 258, 11622– 11628. Balow, R.-M., Tomkinson, B., Ragnarsson, U., Zetterqvist, O., 1986. Purification, substrate specificity, and classification of tripeptidyl peptidase II. J. Biol. Chem. 261, 2409–2417. Balow, R.-M., Eriksson, I., 1987. Tripeptidyl peptidase II in haemolysates and liver homogenates of various species. Biochem. J. 241, 75–80. Beinfeld, M.C., Meyer, D.K., Eskay, R.L., Jensen, R.T., Brownstein, M.J., 1981. The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res. 212, 51–57. Beinfeld, M.C., 2003. What we know and what we need to know about the role of endogenous CCK in psychostimulant sensitization. Life Sci. 73, 643– 654. Benoliel, J.-J., Becker, C., Mauborgne, A., Bourgoin, S., Hamon, M., Cesselin, F., 1998. Interactions between central opioidergic and cholecystokininergic systems in rats: possible significance for the development of opioid tolerance. Bull. Acad. Natl. Med. 2, 311–324. Bradwejn, J., Koszycki, D., Paradis, M., Reece, P., Hinton, J., Sedman, A., 1995. Effect of CI-988 on cholecystokinin tetrapeptide-induced panic symptoms in healthy volunteers. Biol. Psychiatry 38, 742–746. Cortes, R., Aman, K., Arvidsson, U., Terenius, L., Frey, P., Rehfeld, J.F., Walsh, J.H., Hokfelt, T., 1991. Immunohistochemical study of cholecystokinin peptide in rat spinal motoneurons. Synapse 9, 103–110. Desousa, N.J., Wunderlich, G.R., Cabo, C.D., Vaccarino, F.J., 1999. The expression of behavioral sensitization to amphetamine: role of CCKA receptors. Pharmacol. Biochem. Behav. 62, 31–37. Facchinetti, P., Rose, C., Rostaing, P., Triller, A., Schwartz, J.-C., 1998. Immunolocalization of tripeptidyl peptidase II, a cholecystokinin-inactivating enzyme, in rat brain. Neuroscience 88, 1225–1240. Geier, E., Pfeifer, G., Wilm, M., Lucchiari-Hartz, M., Baumeister, W., Eichmann, K., Niedermann, G., 1999. A giant protease with potential to substitute for some functions of the proteasome. Science 283, 978–981.

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