Expression and Distribution of Tartrate-resistant Purple Acid Phosphatase in the Rat Nervous System

Abstract

Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte–macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10–20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC₅₀ for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in α-motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.

Keywords

bone brain ganglion macrophage osteoclasts osteopontin protein phosphatase sensory sympathetic

Purple acid phosphatases (PAPs) are acid metallohydrolases that contain a binuclear Fe³⁺M²⁺ center in their active site, where M = Fe or Zn (Antanaitis and Aisen 1983; Averill et al. 1987; Doi et al. 1988). In mammals, these enzymes are also referred to as tartrate-resistant acid phosphatases (TRAPs) (EC 3.1.3.2) or Type 5 acid phosphatases (Vincent and Averill 1990). TRAPs are iron-containing monomeric glycoproteins with molecular weights of around 35,000 Da (Andersson et al. 1991). The deduced amino acid sequences of human, rat, and mouse TRAPs show a high degree of similarity with the mammalian members of the PAP family, e.g., uteroferrin (Uf) and bovine spleen PAP (Ketcham et al. 1989; Lord et al. 1990; Ek-Rylander et al. 1991; Cassady et al. 1993).

Mammalian PAPs exist in two interconvertible states: pink, reduced and enzymatically active, with a mixedvalent Fe(II)-Fe(III) cluster, and purple, oxidized and catalytically inactive, with the dinuclear pair as Fe(III)-Fe(III). The mammalian serine/threonine protein phosphatases calcineurin (Type 2B) (Griffith et al. 1995) and protein phosphatase Type 1 (PP-1) (Goldberg et al. 1995) also contain a di-nuclear metal center and reveal a striking similarity to the mammalian PAP enzyme in the coordination environment of the active site (Uppenberg et al. 1999).

In humans and rats, TRAP has been detected in a wide variety of tissues as a minor acid phosphatase isoenzyme. However, it is highly expressed in certain activated macrophages and bone-resorbing osteoclasts (Andersson and Marks 1989; Ek-Rylander et al. 1991; Yaziji et al. 1995). A role for TRAP in the resorption of bone and in cartilage breakdown in vivo is indicated by the skeletal alterations observed in TRAP knockout mice (Hayman et al. 1996). TRAP is a phosphoprotein phosphatase acting on phosphoproteins such as bone sialoprotein, osteopontin and osteonectin present in the bone matrix (Ek-Rylander et al. 1994; Andersson and Ek-Rylander 1995; Ljusberg et al. 1999). The role of PAP in other tissues is less well understood. The localization of splenic PAP to macrophage lysosomes (Schindelmeiser et al. 1987) and the catalytic generation of hydroxyl radicals by the enzyme (Hayman and Cox 1994) may point to a role in oxygen-dependent degradation of phagocytosed material. A co-localization of PAP with nitric oxide synthase-1 (NOS-1) in human urothelial cells may support the view that the PAP enzyme participates in free radical metabolism (Schindelmeiser et al. 1997).

Calcineurin, protein phosphatase-1 (PP-1) (Sim 1991), and fluoride-resistant acid phosphatase (FRAP) (Silverman and Kruger 1988) are some of the phosphatases that have been demonstrated in mouse brain. Recently, the TRAP/PAP isoenzyme was also detected by enzyme activity (Hayman et al. 2000) and at the mRNA level (Angel et al. 2000). In addition, histochemical TRAP activity was demonstrated in nerve cells of the olfactory bulb in the rat (Krizbai et al. 1997). In this report we provide evidence, based on the biochemical and histochemical expression of its mRNA, protein, and enzyme activity, that PAP is expressed in selected neurons and ganglion cells. The widespread, but not generalized, distribution of the PAP enzyme in the nervous system suggests participation of this enzyme in specific neuronal functions.

Materials and Methods

Experimental Animals

Five 3-week-old Sprague–Dawley rats of unspecified gender were used in each experiment. Rats were kept under controlled light/dark conditions with food and water available ad libitum. The rats were decapitated and tissues were dissected out for partial purification of TRAP and for RNA preparation, as described in the following sections.

Tissues for immunohistochemistry and in situ hybridization histochemistry were dissected out after perfusion via the ascending aorta with 4% paraformaldehyde in 0.1 M Sörensen's phosphate buffer, pH 7.4, immersed in the same fixative for 24 hr, and then embedded in paraffin. Sections (4.5 μm thick) were adhered to slides (SuperFrost; Menzel-Gläser, Braunschweig, Germany) and processed for immunohistochemistry or ISH histochemistry (see below). Before perfusion the animals were anesthetized with a lethal dose of sodium pentobarbital (60 mg/ml).

The use of animals in this study was approved by the local ethical committee of the Karolinska Institute (S 170/98).

Partial Purification of TRAP from Rat Neural Tissues and Bone

TRAP was purified from the rat brain, spinal cord, trigeminal ganglion, and bone essentially as described previously (Ek-Rylander et al. 1997). The dissected tissues were homogenized in 0.15 M KCl containing 0.1% Triton X-100. The homogenates were centrifuged at 3200 × g for 15 min. Protaminsulfate (5%; Sigma, St Louis, MO) was added drop-wise to the supernatants under continuous stirring, to a final concentration of 0.5%. The pH was adjusted to 6.5 with acetic acid, and insoluble material was allowed to precipitate for 30 min. The samples were then centrifuged as above. All procedures up to this point were carried out at 4C. The supernatants were loaded on a CM-cellulose column (4 ml) (CM-52; Whatman, Maidstone, UK) previously equilibrated with 0.1 M sodium acetate buffer, pH 6.5. The column was washed with three bed volumes of 0.15 M sodium acetate, pH 6.5, and the TRAP enzyme was then eluted with 0.4 M sodium acetate, pH 6.5. All steps were performed in the presence of the protease inhibitors E-64 (10 μg/ml), Pefabloc (1 mg/ml), pepstatin A (10 μg/ml) (Boehringer Mannheim; Mannheim, Germany), and EDTA (5 mM). Further purification of the samples was performed for electrophoresis and immunoblotting analysis. The samples were loaded on a phenyl–Sepharose column (Pharmacia Biotech; Uppsala, Sweden) previously calibrated with 0.05 M sodium acetate, pH 5.0, and 144 g/liter ammonium sulfate. Protein was eluted with a gradient from 0.05 M sodium acetate, pH 5.0, containing 144 g/liter ammonium sulfate, to 0.05 M sodium acetate, pH 5.0.

Assay Procedures

The TRAP enzyme activity in each purified sample was assayed in 96-well plates using p-nitrophenylphosphate (pNPP) as substrate in an incubation medium (200 μl) containing (final concentrations): 2.5 mM pNPP (ditris salt; Sigma), 0.1 M sodium acetate buffer, pH 5.8, 0.2 M KCl, 0.1% Triton X-100, 10 mM sodium tartrate, and the reducing agents ascorbic acid (1 mM) and FeCl3 (100 μM). The p-nitrophenol liberated after 1-hr incubation at 37C was converted into p-nitrophenolate by the addition of 50 μl of 0.9 M NaOH, and the absorbance was measured at 405 nm using a Spectramax250 spectrophotometer (Molecular Devices; Sunnyvale, CA). One unit (U) of TRAP activity corresponds to 1 μmol of p-nitrophenol liberated per minute at 37C. To oxidize the reduced enzyme, 10 mM hydrogen peroxide (H₂O₂) was added, and after incubation for 10 min at room temperature (RT), the substrate (pNPP) was added. The concentration of inhibitor required to reduce enzyme activity to 50% of the activity in the absence of inhibitor (IC50) for the oxyanions molybdate, tungstate, and phosphate was calculated using linear regression analysis. K_m for pNPP was calculated using the method of Lineweaver–Burke.

Electrophoresis and Immunoblotting Analysis

Partially purified TRAP (30 mU) from each tissue was subjected to SDS-PAGE in 12% slab minigels according to the procedure described by Laemmli (1970). Proteins were transferred to immuno-PVDF membranes (Bio-Rad; Hercules, CA) using 150 mA for 2 hr. The membranes were rinsed in 20 mM Tris-HCl, pH 7.5, containing 500 mM NaCl and 0.05% Tween-20 (TBST) and then incubated overnight at 4C in 10% dried non-fat milk in TBST. The membranes were then incubated with polyclonal rabbit antirat TRAP antibodies (Ek-Rylander et al. 1997) diluted 1:100 in TBST containing 0.5% dried non-fat milk for 1 hr at RT. After three washes for 10 min each in TBST, the membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch Laboratories, West Grove, PA). The membranes were washed six times for 10 min in TBST and developed for 1 min with Western Blot Chemiluminescence Reagent Plus (NEN Life Science; Boston, MA) according to the manufacturer's protocol. The membranes were then exposed to HyperfilmECL (Amersham Life Science; Poole, UK) using intensifying screens.

Total RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted according to the method of Chomczynski and Sacchi (1987). For RT-PCR analysis, tissues were dissected under aseptic conditions and immediately frozen in liquid nitrogen and then stored at −70C. mRNA was purified using an Oligotex mRNA Mini Kit (QIAGEN; Hilden, Germany). For nested PCR, total RNA was extracted using an RNeasy Mini Kit (QIAGEN).

The RT reaction was performed on 1 μg of mRNA or 5 μg of total RNA (for nested PCR) with 200 U Superscript II RNase H^– Reverse Transcriptase (Gibco; Paisley, UK) at 37C, using oligo d(T)_12–18 (Gibco) as primer, according to the manufacturer's protocol. Negative controls included reactions in which the reverse transcriptase was excluded from the reaction mixture.

PCR amplification was performed on aliquots of cDNA corresponding to 200 ng mRNA or 800 ng total RNA (for nested PCR) using 2.5 U Taq polymerase in the presence of 1.5 mM MgCl₂ and 1 × Q-Solution according to the manufacturer's protocol (QIAGEN). The 5′- TRAP primer (positions 32–47; 5′-TTCTGTTCCAGGAGCTT-3′) and 3′-TRAP primer (positions 600–616; 3′-CAGTCGGTCGTCGGACT-5′) were added (150 ng/reaction) (positions calculated from the rat TRAP cDNA sequence available at GenBank/EMBL Data Bank accession no. M76110). The expected size of the TRAP cDNA fragment is 586 bp.

For nested PCR, the 5′-TRAP primer 5′-CGCCAGAACCGTGCAGA-3′ (positions 160–176) was included in the second amplification, generating a band with a predicted size of 356 bp. The cDNA was amplified in a Perkin–Elmer Cetus Instruments DNA ThermalCycler, running 25, 30, 35, or 40 cycles under the following conditions: 94C, 1 min; 56C, 1 min; 72C, 1 min. In the nested PCR, cDNA was amplified in a Perkin–Elmer Cetus Instruments DNA GeneAmp PCR system 9700 running 40 cycles twice under the following conditions: 94C, 30 sec; 56C, 30 sec; 72C, 30 sec. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide, 0.15 μg/ml.

PCR analysis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was performed on all of the samples as an internal control for amplification of equal amounts of RNA. The primers 5′-CTGAACGGGAAGCTCACTGG-3′ and 3′-TGAGGTCCACCACCCTGTTG-5′corresponding to positions 664–683 and 976–957 in the rat GAPDH cDNA sequence (Fort et al. 1985) were used to generate a fragment of the predicted size of 312 bp.

Sequencing of TRAP cDNA PCR Product

Twenty ng of PCR product from the trigeminal ganglion RT-PCR reaction using 5′-TRAP primer (positions 34–49; 5′-TTCTGTTCCAGGAGCTT-3′) and 3′-TRAP primer (positions 602–618; 3′-CAGTCGGTCGTCGGACT-5′) was ligated into a pCR 2.1 vector and then transformed into TOP 10 F′ cells using a TA Cloning Kit (Invitrogen; Leek, The Netherlands) according to the manufacturer's protocol. Cells were plated onto Luria-Bertani-41 (LB) plates containing ampicillin (50 g/ml) and 5-bromo-4-chloro-3-indolyl · β-d-galactopyranoside (X-gal) (40 μl; 40 mg/ml) and screened for colonies containing vector with insert using blue/white selection.

Plasmids from selected colonies were miniprepared according to the protocol of Maniatis (Fritsch et al. 1989), with the exceptions that 400 μg lysozyme was added in the first step and that the plasmid DNA was precipitated twice in isopropanol. The accuracy of the insert was confirmed using PCR with the same primers as for nested PCR, and then sequenced at Cybergene (Huddinge, Sweden).

Immunohistochemistry

Paraffin sections of brain, spinal cord, spinal ganglia, trigeminal ganglion, celiac–superior mesenteric ganglion complex, superior cervical ganglion, and long bones were deparaffinized and rehydrated, and then incubated in a microwave oven in citrate buffer, pH 6.0 (1.8 mM citric acid, 9.8 mM sodium citrate) at 1100 W for 3 min and then at 160 W for another 12 min. After cooling to RT, immunostaining was achieved using 200 μl polyclonal antiserum, diluted 1:500, against TRAP as previously described (Ek-Rylander et al. 1997; Nordahl et al. 1998). After washing with 50 mM Tris-HCl, pH 7.6, 150 mM NaCl (TBS) three times for 10 min, the sections were exposed to 200 μl biotinylated goat anti-rabbit IgG (E0432; DAKO, Copenhagen, Denmark) diluted 1:300 for 30 min at RT and then washed as above. To amplify the signal, the sections were incubated with Strept-ABComplex/HRP (K0355; DAKO) according to the manufacturer's protocol for 30 min at RT. After washing, the sections were incubated for 5 min at RT with the substrate diaminobenzidine (DAB; Sigma) (600 μg/ml) in TBS containing 0.03% H₂O₂.

Incubation of parallel sections with primary antibodies preabsorbed with 1 μg purified PAP antigen or with normal swine serum (X0901; DAKO) diluted 1:500 served as controls.

Some of the sections were counterstained with Ehrlich hematoxylin before dehydration and mounting.

The localization of TRAP immunoreactivity and the anatomic structures were identified by using a stereotaxic atlas (Paxinos and Watson 1986; Paxinos et al. 1999a, b).

DIG Labeling of RNA Probe

The plasmid pT7T3 19U (Pharmacia) containing rat TRAP cDNA fragment of 832 bp (Ek-Rylander et al. 1991) was linearized with HindIII (for the sense probe) or with EcoRI (for the antisense probe). The labeling was performed with digoxygenin-UTP (DIG RNA Labeling Kit; Boehringer Mannheim) according to the manufacturer's protocol, with the exception that T3 RNA polymerase was used instead of SP6 polymerase.

In Situ Hybridization Histochemistry

Paraffin sections prepared as described above were heated overnight at 55C. After deparaffinization and rehydration, the sections were washed in PBS (0.145 M NaCl, 0.01 M Na2PO₄). After treatment with pepsin (1.5 mg/ml) (Boehringer Mannheim) in 0.02 M HCl at 37C for 20 min and washing in PBS, the sections were postfixed in 2% paraformaldehyde in PBS for 5 min at RT and then washed in PBS. The sections were prehybridized in 2 × SSC (1 × SSC = 150 mM NaCl, 15 mM sodium citrate)/50% formamide at 42C for 2 hr. Hybridization was performed in 30 μl of a solution containing 2 × SSC, 2 × Denhardt's, 1 mM EDTA, 0.25 mg/ml tRNA, 100 mg/ml dextran sulfate, 50% formamide, and 2.5 ng/ml probe at 42C overnight. The sections were washed twice in 2 × SSC for 10 min and then three times in 0.1 × SSC for 15 min at RT. The detection was performed using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) according to the manufacturer's protocol. After counterstaining with Ehrlich hematoxylin, the sections were dehydrated and mounted. Sections exposed to DIG-labeled sense probes served as negative controls.

Results

Partial Purification and Characterization of TRAP Activity from Rat Neural Tissue

TRAP was partially purified from trigeminal ganglion, spinal cord, brain and, as a comparison, from rat bone, according to an established procedure (Ek-Rylander et al. 1997). Addition of reducing agents, such as 1 mM sodium ascorbate and 0.1 mM FeCl₃, converts the inactive enzyme to the active Fe(III)Fe(II) mixed-valent form. The yields of activated TRAP enzyme activity (U/g tissue) varied significantly among the tissues (Table 1). The recoveries from trigeminal ganglia, spinal cord, and brain amounted to 10, 4, and 0.4%, respectively, compared to the recovery of TRAP activity from bone. After oxidation with H₂O₂, virtually no TRAP activity was detectable in any of the enzyme preparations analyzed (Table 1). The K_m of the isolated TRAP enzyme for hydrolysis of the substrate, pNPP, was in the range of 0.23–0.56 mM and was not significantly different between the various tissues. These values are similar to those previously determined for highly purified preparations of recombinant rat TRAP (Ek-Rylander et al. 1997).

To examine the sensitivity to different oxyanion inhibitors of TRAP from the trigeminal ganglion, spinal cord, brain, and bone, the IC₅₀ values for the known PAP/TRAP inhibitors molybdate, tungstate, and phosphate were determined (Table 1). The IC₅₀ values were derived from linear regression analysis. The sensitivity of TRAP enzyme activity for these inhibitors was similar in the different tissues, and the rank order of potency was molybdate>tungstate>phosphate.

Table 1

Biochemical properties of TRAP partially purified from rat long bones and neural tissue

	Bone	Trigeminal ganglia	Spinal cord	Brain
U/g tissue (reduced)	7.1 ± 3.8	0.75 ± 0.26	0.31 ± 0.14	0.03 ± 0.01
Reduced/unreduced	28	14	17	17
% activity after
reduction/oxidation	0.3	0	0	0.7
K_m	0.23 ± 0.04	0.56 ± 0.10	0.56 ± 0.19	0.38 ± 0.01
IC₅₀ (μM) MoO₄ ^2–	39 ± 8	58 ± 22	30 ± 14	10 ± 4
IC50 (μM) WO₄ ^2–	239 ± 63	241 ± 70	163 ±70	21 ± 2.6
IC50 (μM) PO₄ ^3–	1030 ± 200	1620 ± 680	1250 ± 110	900 ± 100

Identification of TRAP Protein and mRNA in Neural Tissue

Immunoblotting analysis of partially purified TRAP (30 mU) from the trigeminal ganglion, spinal cord, and brain showed the appearance of a protein band with the expected size of 35–37 kD in the absence of β-mercaptoethanol as the disulfide reductant (Figure 1). Recombinant rat TRAP (100 ng) expressed from a Baculovirus vector was used as a positive control (Ek-Rylander et al. 1997). To show the presence and relative abundance of TRAP mRNA in bone, trigeminal ganglion, spinal cord and brain, RT-PCR was performed and aliquots were analyzed after 25, 30, 35, and 40 cycles (Figure 2A). A PCR product of 586 bp, corresponding to the theoretical size, was detected, and the relative expression levels were consistent with those observed for enzymatic activity in the different tissues (Table 1). Sequencing of the 586-bp product obtained from the trigeminal ganglion showed complete identity with the bone TRAP cDNA sequence. A larger fragment, 1078 bp, was detected after amplification of cDNA from spinal cord and brain. The levels of GAPDH mRNA was similar in the different samples, ensuring that amplification of equal amounts of RNA had been achieved (Figure 2B). No product was amplified when reverse transcriptase had been omitted, demonstrating the absence of DNA contamination of the different RNA preparations.

Figure 1

Western blotting analysis of TRAP purified from neural tissue and bone in the absence of reducing agents. Thirty mU of purified TRAP enzyme activity from trigeminal ganglia, spinal cord, and brain was applied to a 12% non-reducing SDS-PAGE, blotted to PVDF, and incubated with polyclonal rabbit antibodies to TRAP. Baculovirus-expressed rat TRAP (100 ng) was used as a positive control. Pre-stained SDS-PAGE standards were electrophoresed in separate lanes.

Figure 2

RT-PCR analysis of TRAP mRNA expression in neural tissue and bone. (A) Aliquots were removed at different cycles of the PCR reaction to compare the levels of TRAP in neural tissues and bone. Arrows mark the larger-sized band, whereas TRAP marks the band of the expected size. (B) GAPDH was used as an internal control for the PCR reaction. (C) Nested PCR of the 40-cycle reaction in A.

Nested RT-PCR of cDNA from bone, trigeminal ganglion, spinal cord, and brain was performed with 40 cycles, resulting in a PCR product of 356 bp corresponding to the theoretical size (Figure 2C).

Immunohistochemical Localization of TRAP

TRAP-like immunoreactivity was encountered in neuronal cell bodies in both the central (Figures 3–7) and peripheral (Figures 8 and 9) nervous systems of untreated rats. The signal was seen in the cytoplasm of the cells but was not uniformly distributed. Instead, the signal appeared in aggregated structures (see Figures 5 and 7–9). Immunoreactive material could also be seen in neuronal processes close to the cell soma (Figure 5A). A very weak immunoreactive signal was observed in nerve fibers in the dorsal horn of the spinal cord. TRAP-immunoreactive material was also seen in glial cells, e.g., in the white matter of corpus callosum (Figures 3A–3C).

Central Nervous System. Wide distribution of TRAP-like immunoreactivity was observed in neurons in the rat brain and spinal cord. A large number of neuron populations were labeled but with a signal of variable intensity. The overall distribution is shown in the set of color micrographs (Figures 3A–3C) representing different levels of the brain. A strong signal was noted in the neurons in the septum and diagonal bands (Figure 3A), ventral pallidum, cerebral cortex, and piriform cortex (Figures 3A–3C). Intensely stained cells were also observed in the triangular septal nucleus. In the cerebral cortex, the most intense staining was seen in neurons in Layers V and VIb, somewhat less intense in Layer VIa, less in Layers II and III, and very weak in Layer I (Figures 3A–3C). All of the pyramidal cells in hippocampal fields CA3 and CA4 (Figures 3B–3C, 6, and 7B) were intensely immunoreactive, whereas in the CA1 (Figures 6 and 7A) and CA2 (Figure 6) fields a strong signal was limited to scattered neurons. A strong signal could be demonstrated in neurons in the hilar region of the hippocampus, whereas the majority of the granule cells of the dentate gyrus exhibited a weaker signal (Figures 6 and 7B). A few scattered granule cells in the dentate gyrus had a stronger TRAP immunoreactivity (Figure 7B). Strong TRAP-like immunoreactivity could be seen in neurons in the exterior globus pallidus (Figures 3B and 8D) and the caudate nucleus (Figures 3B and 8B), particularly in larger cells (Figure 8B), and in the interior globus pallidus (Figures 3B and 8A) and medial habenula (Figure 3B). Neurons in the thalamus and hypothalamus were intensely stained, with particularly strong labeling in the reticular, rhomboid, anterodorsal, and ventral posterolateral thalamic nuclei (Figure 3B). Moderate staining was observed in the dorsal nucleus of the hippocampal commissure, ventromedial hypothalamic nucleus, and basolateral amygdaloid nucleus. Weak labeling was observed in the dorsomedial and ventrolateral laterodorsal thalamic nuclei. There was intense TRAP-like immunoreactivity in neurons of the substantia nigra pars compacta and reticulata (Figures 3C and 8C), neurons of the Darkschewitsch nucleus, the lateral mammillary nucleus, the magnocellular red nucleus, and the postcommissurial magnocellular nucleus (Figure 3C).

In the pons and medulla oblongata, intensely stained neurons were present, e.g., in the dorsal part of the central gray and the deep gray layer of the superior colliculus (Figure 5A), the spinal trigeminal nucleus, the inferior olive, and the parvocellular reticular nucleus alpha (Figure 4B). A strong signal was seen in the neurons in the spinal cord (Figure 4C), particularly within α-motor neurons in the ventral spinal cord (Figures 4C and 5B). A very weak signal was noted in nerve fibers in the dorsal horn of the spinal cord. In the olfactory bulb, intense staining of nerve cells was observed in the mitral cell layer and external plexiform layer (Figure 4A). Scattered cells were also detected in the internal plexiform and granular layers and in the glomerular layer. Medium immunoreactivity could be seen in the mammillary nucleus. In the cerebellum, intense labeling was observed in the Purkinje cells, and in scattered neurons in the molecular and granular layer (Figures 4B, 9A, and 9B).

Figure 3

Micrographs of frontal sections of the rat brain at 0.7 mm anterior bregma (A) and 2.1 (B) and 4.8 mm (C,D) posterior to bregma after incubation with antiserum to TRAP (A–C) or TRAP antiserum preabsorbed with antigen (D). All sections were counterstained with Ehrlich's hematoxylin. Intensely TRAP-immunoreactive cells are observed in the septum and diagonal bands and in the piriform cortex (A). Similarly, strong labeling can be seen in all of the pyramidal cells of the hippocampal fields CA3 and CA4 (B,C) and in scattered cells in the CA1 and CA2 fields (C), whereas only a weak signal is seen in the granule cells of the dentate gyrus (B,C). In the cerebral cortex, neurons in Layers V and VIb are most strongly labeled (B). Strong TRAP-like immunoreactivity is also seen in neurons in the interior and exterior globus pallidus and in the reticular, rhomboid, and anterodorsal thalamic nuclei (B). Neurons with intense labeling can also be seen in the substantia nigra pars compacta, the magnocellular red nucleus, the postcommissurial magnocellular nucleus, the lateral mammillary nucleus, and the Darkschewitsch nucleus (C). No labeling is observed in the section incubated with control serum (D). ad, anterodorsal thalamic nucleus; ca3, pyramidal cell layer of CA3 in the hippocampus; cpu, caudate putamen; dk, Darkschewitsch nucleus; egp, exterior globus pallidus; igp, internal globus pallidus; lm, lateral mammillary nucleus; mhb, medial habenula; pir, piriform cortex; rh, rhomboid thalamic nucleus; rt, reticular thalamic nucleus; sn, substantia nigra; vdb, ventral diagonal band; I, V, and VIb, layers of the cerebral cortex. Bar = 500 μm.

Figure 4

Micrographs of sections of the rat olfactory bulb (A), medulla oblongata (B), and the thoracic spinal cord (C,D) after incubation with antiserum to TRAP (A–C) or hybridization with an antisense cRNA probe to TRAP mRNA (D). The sections in A and B were counter-stained with Ehrlich's hematoxylin. Strong TRAP-like immunoreactivity can be seen in neurons in the mitral cell layer and external plexiform layer of the olfactory bulb and in scattered cells in the internal granular and plexiform layers and the glomerular layer (A). TRAP immunoreactivity of varying intensity is found in many of the nuclei in the medulla oblongata, e.g., the PCRtA, IO, MVe and Sp5 (B). Both TRAP-like immunoreactivity (C) and TRAP mRNA (D) can be seen in nerve cells in the gray matter of the spinal cord. Gl, glomerular layer; EPl, external plexiform layer; IGr, internal granular layer; IO, inferior olive; Mve, medial vestibular nucleus; PCRtA, parvicellular reticular nucleus alpha; Sp5, spinal trigeminal nucleus. Bars: A,C,D = 250 μm; B = 500 μm.

Figure 5

Micrographs of sections of rat brain stem (A), spinal cord (B,C), and cerebral cortex (D,E) after incubation with antiserum to TRAP (A,B,D,E) or hybridization with an antisense cRNA probe to TRAP mRNA (C). Neurons with intense immunoreactivity for TRAP are observed in the trochlear nucleus and with medium level of labeling in the CG, the DpG, and the Pn (A). TRAP-like immunoreativity can be seen as rounded or irregularly shaped structures in the cytoplasm of neuronal cells, particularly the large α-motor neurons (B). A similar pattern of intense labeling for TRAP mRNA can be seen after ISH histochemistry. Note also the immunoreactive material and hybridization signal in the first part of the axon (arrows in B and C, respectively). (C) Higher magnification of part of Figure 4 D. TRAP-immunoreactive neurons are observed in all layers of the cerebral cortex (D). Both pyramidal cells (short arrows pointing to the same neurons in D and E) and granule cells (long arrows pointing to the same cell in D and E). (E) Higher magnification of part of D. 4, trochlear nucleus; Aq, aqueduct; CG, central gray; DpG, deep gray layer of the superior colliculus; Pn, pontine nucleus. Bars: A = 500 μm; B–D = 50 μm; E = 25 μm.

Figure 6

Micrograph montage of a frontal section of the rat hippocampus after incubation with antiserum against TRAP. Intense TRAP-like immunoreactivity can be seen in pyramidal cells of the hippocampal fields CA3 and CA4, in scattered pyramidal cells in the CA1 and CA2 fields, and in neurons in the hilar region. Weaker staining is seen in the majority of pyramidal cells in the CA1 and CA2 fields and in the granule cells in the dentate gyrus. Scattered TRAP-positive cells can also be seen in the hippocampal fields stratum oriens and radiatum and the molecular layer of the dentate gyrus. CA1, CA2, and CA3, pyramidal cell layers of the CA1, CA2, and CA3 fields of the hippocampus; DG, dentate gyrus of the hippocampus. Bar = 50 μm.

Peripheral Nervous System. TRAP-like immunoreactivity was observed in all neuronal cell bodies in both sensory ganglia, and paravertebral and prevertebral sympathetic ganglia (Figures 10 and 11). Medium to large nerve cells in sensory ganglia, including the trigeminal ganglion (Figures 10A and 10C) and dorsal root ganglia (Figures 11C and 11F), were intensely labeled, whereas smaller neurons exhibited a weaker staining intensity. Similarly, in the superior cervical ganglion (Figures 11A and 11D) and the celiac ganglion (Figures 11B and 11E), larger neurons were more intensely stained than smaller nerve cells, although the difference in cell size was not as marked as in the sensory ganglia.

Controls. No signal could be seen in sections incubated with primary antiserum preabsorbed with the antigen (see Figures 3D, 9C, and 10D) or after omission of the primary antiserum (not shown).

Histochemical Localization of TRAP mRNA by In Situ Hybridization

Central Nervous System. The distribution of TRAP mRNA expression closely resembled that of the TRAP-like immunoreactivity in the spinal cord (Figures 4D and 5C). In the cerebellum, a strong signal was seen in Purkinje cells (Figures 9D and 9E), and intense labeling was also observed in some cells in the molecular layer (Figures 9D and 9E).

Figure 7

Micrographs of sections of the CA1 field (A), CA4 field (B), and dentate gyrus (B) of rat hippocampus after incubation with antiserum to TRAP. Intense immunoreactivity for TRAP can be seen in scattered cells in the pyramidal cell layer of the CA1 field and the granule cell layer of the dentate gyrus (arrowheads on some of these cells in A and B), whereas the majority of the cells in these layers are weakly stained (arrows on some in A and B). Many intensely stained pyramidal cells are observed in the CA4 field (B). Bar = 50 μm.

Peripheral Nervous System. In the trigeminal ganglion, TRAP mRNA expression was most marked in small to medium-sized neurons, whereas the larger neurons were weakly labeled (Figure 10B and 10E).

Controls. No signal could be seen after hybridization with labeled sense probe (Figure 9F) or after treatment with RNase before the hybridization (not shown).

Discussion

This article describes the characterization and distribution of TRAP (or PAP) in the central and peripheral nervous systems of the rat. The TRAP enzyme is commonly isolated as the purple and catalytically inactive Fe(III)Fe(III) variant (Andersson and Gräslund 1995). TRAP is activated in the presence of reducing agents, forming the mixed-valent Fe(II)-Fe(III) variant, and oxidation of the reduced enzyme with H₂O₂ leads to a transition of the active mixed-valent enzyme to an inactive Fe(III)Fe(III) species. These properties are rather unique for the di-iron PAPs (Averill et al. 1987; Vincent and Averill 1990) and can therefore serve as useful specificity criteria for this class of acid phosphatases. Another characteristic property of the PAP enzymes is their sensitivity to tetrahedral oxyanion inhibitors, such as molybdate, arsenate, and tungstate (David and Que 1990). On the basis of the specific redox properties and the sensitivity to these inhibitors, the enzymatic data together strongly suggest that a diiron form of PAPs is expressed in the rat nervous system.

After the demonstration of TRAP enzymatic activity, it was of interest to investigate whether the TRAP enzyme could be demonstrated at the protein and mRNA levels, to ascertain that the TRAP enzyme activity indeed reflects the presence of TRAP protein. Furthermore, the levels of TRAP enzyme activity and TRAP mRNA, respectively, were compared among different tissues using a TRAP assay and RT-PCR. The results from these experiments show that TRAP enzyme activity and mRNA levels correlate well. In the morphological studies there was some discrepancy with regard to the granular layer of the cerebellar cortex. The granule cells showed TRAP-like immunoreactivity but no or very weak signal for TRAP mRNA. This could represent low sensitivity of the ISH histochemical method used or a fast turnover of the TRAP mRNA in these cells.

Figure 8

Micrographs of sections of the rat globus pallidus (A,D), caudate nucleus (B), and substantia nigra (C) after incubation with antiserum to TRAP. Intense TRAP immunoreactivity is observed in neurons of the IGP (A) and in the SNC and SNR (C). Scattered neurons in the caudate nucleus (arrowheads on some in B) and a majority of the neurons in the EGP (D) are also strongly TRAP-positive. In the caudate nucleus (B), the intensely labeled neurons are larger than the weakly labeled neurons (arrows on some), which are more numerous. IGP, interior globus pallidus; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. Bars = 50 μm.

The PAP enzymes expressed in bone and spleen are monomeric proteins ranging in size between 34 and 37 kD, depending largely on differences in the post-translational modifications (Baumbach et al. 1984, 1991; Saunders et al. 1985). PAP variants with this characteristic molecular weight were detected by Western blotting analysis in the partially purified extracts from the trigeminal ganglion, spinal cord, and brain. Moreover, the demonstration of a longer mRNA transcript from the spinal cord and brain may indicate that part of the 35-kD TRAP protein originates from a different transcript, due to alternative promoter usage (Reddy et al. 1995).

Figure 9

Micrographs of sections of the rat cerebellum after incubation with antiserum to TRAP (A,B) or TRAP antiserum preabsorbed with antigen (C), or after hybridization with an anti-sense (D,E) or sense (F) cRNA probe to TRAP mRNA. The section shown in C was counter-stained with Ehrlich's hematoxylin. Strong TRAP-like immunoreactivity (A,B) and TRAP mRNA expression (D,E) can be seen in the Purkinje cells (arrow in B on one Purkinje cell) of the cerebellum. Somewhat weaker signals are observed in neurons in the molecular and granular layer (B,E). No signal can be seen with the control serum (C) or the sense probe (F). Gr, granular layer; Mo, molecular layer; wm, white matter. Bars: A,D = 100 μm; B,C,E,F = 50 μm.

Figure 10

Micrographs of sections of the rat trigeminal ganglion after incubation with antiserum to TRAP (A,C) or TRAP antiserum preabsorbed with antigen (D), or after hybridization with an antisense cRNA probe to TRAP mRNA (B,E). The sections shown in C and D were counterstained with Ehrlich's hematoxylin. TRAP-like immunoreactivity (A) is seen in all neurons in the trigeminal ganglion, although a higher intensity of the immunoreactivity is generally observed in medium to large neurons (A,C). A higher TRAP mRNA expression is, on the other hand, generally seen in smaller neurons (B,E). Bars = 50 μm.

Figure 11

Micrographs of sections of the rat superior cervical ganglion (A,D), celiac ganglion (B,E), and a lumbar spinal ganglion (C,F) after incubation with antiserum to TRAP. The sections shown in B,C,E, and F were counterstained with Ehrlich's hematoxylin. All neurons in both sympathetic (A,B,D,E) and sensory (C,F) ganglia are TRAP-immunoreactive, although larger neurons are more intensely labeled, which is most obvious in the spinal ganglion (C,F). Bars = 50 μm.

Immunohistochemical analysis showed a widespread distribution of TRAP-like immunoreactivity in both the central and peripheral nervous systems. In view of the suggested close evolutionary relationship between the PAP/TRAPs and the protein phosphatases PP1, PP2A, and PP2B (or calcineurin) (Barford et al. 1998), it was of interest to compare the distribution of TRAP-like immunoreactivity with previously published data on the distribution of these protein phosphatases. Calcineurin has a widespread distribution, with several similarities to that of the TRAP-like immunoreactivity, e.g., a strong labeling of cortical Layers V and VIb (Goto et al. 1986). It was noted (Goto et al. 1986) that large cells in the cerebral cortex are more strongly stained for calcineurin than small cells, which is also a hallmark for TRAP-like immunoreactivity. Such a pattern was particularly obvious in the dorsal root ganglia, where large neurons were more intensely stained than the small-sized neurons (see also below). Differences in the distribution of calcineurin and TRAP immunoreactivities include, e.g., that neuronal cell bodies in the globus pallidus and substantia nigra were TRAP-positive but calcineurin-negative, and that calcineurin was detected in neuronal somata, dendrites, and axons, whereas TRAP was predominantly localized to somata.

The isoforms of PP-1 are also extensively distributed in the brain (da Cruz e Silva et al. 1995; Ouimet et al. 1995), with the highest expression of mRNA in the hippocampal pyramidal neurons of CA1–CA4 and in the granule cells of the dentate gyrus. Relatively high levels were also found in the thalamus and medial habenula, red nucleus, occulomotor nucleus, and in the granular layer of the cerebellum. The distribution patterns of the PP-1 isoforms therefore show considerable similarities to the distribution of TRAP immunoreactivity, but there are also notable differences. The globus pallidus, which showed high levels of TRAP immunoreactivity, is virtually devoid of PP-1α and PP-1γ1 and contains small amounts of PP-1β mRNA (da Cruz e Silva et al. 1995). Furthermore, unlike TRAP, a specific concentration of PP-1α and PP-1γ1 immunoreactivity to dendritic spines has been observed in the caudate nucleus and hippocampus (Ouimet et al. 1995).

The distribution of PP-2A in the rat brain (Saitoh et al. 1989) shows several similarities to the distribution of TRAP, exemplified by a strong immunoreactivity in the neocortex and caudate nucleus. However, unlike TRAP, PP-2A-like immunoreactivity is as abundant in dendrites as in neuronal cell somata.

FRAP enzyme activity has been shown to occur in the nervous system, and the localization of TRAP to neurons of sensory ganglia coincides with that of FRAP. However, FRAP is restricted to small sensory neurons (see Knyihar–Csillik and Csillik 1981), whereas TRAP immunoreactivity occurs in all ganglion cells, with the strongest signal in the large sensory ganglion cells. However, the signal for TRAP mRNA was found to be stronger in smaller neurons in the trigeminal ganglion.

The TRAP-immunoreactive material had a very distinct pattern, particularly in the large motor neurons in the spinal cord and in sensory ganglia, where the immunoreactive material was seen as aggregated round structures all over the cytoplasm. Electron microscopic analysis will be necessary to identify these structures, but it is possible that, similarly to the subcellular localization in osteoclasts, the TRAP-immunoreactive material may reside in lysosome-like structures (Reinholt et al. 1990a, b). However, it may also occur in large vesicles.

A role for TRAP/PAP enzymes in the formation of oxygen-derived free radicals has been indicated by several studies (Sibille et al. 1987; Hayman and Cox 1994). In particular, TRAP/PAP enzyme activity has been shown to generate hydroxyl radicals from super-oxide by an iron-catalyzed Fenton reaction under certain in vitro conditions (Sibille et al. 1987). In addition, a role for TRAP in nitric oxide (NO) metabolism has recently been indicated (Collin–Osdoby et al. 1998). Thioglycollate-elicited peritoneal macrophages from mice with a null mutation in the TRAP gene have significantly elevated production of both super-oxide and NO, corroborating an involvement of TRAP in the formation of reactive oxygen species (ROS) (Hayman et al. 1998). A co-localization of TRAP with nitric oxide synthase-1 (NOS-1) in human urothelial cells may also support the view that the TRAP enzyme participates in free radical metabolism (Schindelmeiser et al. 1997). NOS is abundantly expressed in the nervous system (Vincent and Kimura 1992). The distribution pattern of cells immunoreactive for NOS in the pyramidal cell layer of the hippocampus (Valtschanoff et al. 1993) suggests co-localization with TRAP. Thus, both NOS and TRAP are mainly found in pyramidal cells of the CA3 field, and both NOS- and TRAP-positive neurons are sparser in the CA1 region. The fact that NOS immunoreactivity, unlike TRAP, is abundant in dendrites and axons emanating from these neurons does not exclude a co-localization, but may merely reflect that TRAP is not transported to the neuronal processes. Whether or not NOS and TRAP are synthesized by the same cells, there may still be an interaction of NO with the TRAP enzyme. NO is known to diffuse into the target cells and bind to iron, a major target being the soluble guanylate cyclase, on stimulation of which the levels of cGMP are increased (Arnold et al. 1977; Ignarro 1990). Conceivably, NO could also bind to the binuclear Fe3+/Fe2+ center of the TRAP active site and modulate TRAP enzyme activity.

Copper–zinc superoxide dismutase (Cu–Zn SOD), an enzyme that may function both as a free radical scavenger and as a generator of ROS, has a widespread distribution in the rat brain (Moreno et al. 1997) and, similarly to TRAP, occurs in many nerve cell populations, but at varying levels. The localization of Cu–Zn SOD in, e.g., the dopaminergic neurons of the substantia nigra, suggests a role in removing superoxide anion generated by dopamine metabolism (Zhang et al. 1993). The significance of a putative co-localization between Cu–Zn SOD and TRAP is unknown but may indicate that both enzymes are involved in and, depending on the stimulus, may shift the balance in generation of ROS.

In bone, TRAP has been ascribed a role in bone resorption on the basis of its localization to osteoclasts (Andersson and Marks 1989; Ek-Rylander et al. 1991; Yaziji et al. 1995) and its ability to dephosphorylate bone matrix phosphoproteins such as bone sialoprotein and osteopontin (OPN) (Ek-Rylander et al. 1994). OPN is known as an anchor for the binding of osteoclasts to the bone via integrin receptors (Reinholt et al. 1990a, b; Flores et al. 1992), suggesting that one potential physiological function of TRAP is to regulate osteoclast attachment to or mobility on bone.

Interestingly, OPN mRNA expression was recently demonstrated in neurons in the rat brain (Shin et al. 1999; Ichikawa et al. 2000; Ju et al. 2000), with a distribution pattern partly overlapping that of TRAP. OPN mRNA expression was observed in nerve cell bodies mainly in the olfactory bulb, brainstem, and cerebellum, i.e., in areas where TRAP is found. The lack of signal for OPN in the diencephalon and part of the telecephalon (Shin et al. 1999), areas where TRAP is abundant may reflect that the levels of OPN expression are too low for detection by the ISH histochemistry technique. The possibility exists that OPN is a substrate for dephosphorylation by TRAP also in neuronal cells, although the function of OPN in brain neurons is thus far unknown.

Notably, there was an induction of OPN mRNA in the rat striatum and hippocampus after global forebrain ischemia (Lee et al. 1999) and in association with ischemic axonal death in periventricular leukomalacia (Tanaka et al. 2000). The induction appears to occur mainly in microglial cells in the rat brain and in foam cells in the human brain. Whether TRAP synthesis is also induced after brain ischemia needs further analysis. However, the fact that TRAP is expressed by macrophages and osteoclasts appears to indicate that TRAP is inducible in cells of similar ontogenic origin, i.e., microglia, and we have yet to provide evidence that neuronal TRAP is inducible.

In conclusion, the present data demonstrate that an enzymatically active TRAP protein occurs in the rat nervous system. Whether or not there are specific molecular forms of TRAP in the nervous system awaits further analysis. The distribution of TRAP within the nervous system appears to indicate that a large number of neuronal cell types have the capacity to synthesize this enzyme but, depending on the levels, that it may contribute differentially to neuronal survival and response to oxidative stress. Co-localization studies to identify, by neurotransmitter content or glial markers, which populations of cells express different levels of TRAP will be the starting point for understanding whether TRAP may be involved in pathological processes such as Alzheimer's disease.

Footnotes

Acknowledgements

Supported by grants from the Swedish Medical Research Council (10363), the Research Funds of Karolinska Institutet, and EC Biotechnology Program 6.1 (contract B104-CT-98-0385).

We are grateful for the expert technical assistance of Maria Norgård.

References

Andersson

Ek-Rylander

(1995) The tartrate-resistant purple acid phosphatase of bone osteoclasts—a protein phosphatase with multivalent substrate specificity and regulation. Acta Orthop Scand 66:189–194.

Andersson

Ek-Rylander

Minkin

(1991) Acid phosphatases. In Rifkin

Gay

, eds. Biology and Physiology of the Osteoclast. Boca Raton, CRC Press, 55–80.

Andersson

Marks

Jr (1989) Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts. J Histochem Cytochem 37:115–117.

Andersson

Gräslund

(1995) Diiron-oxygen proteins. Adv Inorg Chem 43:359–408.

Angel

Walsh

Forwood

Ostrowski

Cassady

Hume

(2000) Transgenic mice overexpressing tartrate-resistant acid phosphatase exibit an increased rate of bone turnover. J Bone Miner Res 15:103–110.

Antanaitis

Aisen

(1983) Uteroferrin and the purple acid phosphatase. Adv Inorg Biochem 5:111–136.

Arnold

Mittal

Katsuki

Murad

(1977) Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci USA 74:3203–3207.

Averill

Davis

Burman

Zirino

Sanders–Loehr

Loehr

Sage

Debrunner

(1987) Spectroscopic and magnetic studies of the purple acid phosphatase from bovine spleen. J Am Chem Soc 109:3760–3767.

Barford

Das

Egloff

M-P

(1998) The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164.

10.

Baumbach

Saunders

PTK

Bazer

Roberts

(1984) Uteroferrin has N-asparagine-linked high-mannose-type oligosaccharides that contain mannose 6-phosphate. Proc Natl Acad Sci USA 81:2985–2989.

11.

Baumbach

Saunders

PTK

Ketcham

Bazer

Roberts

(1991) Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro . Mol Cell Biochem 105:107–117.

12.

Cassady

King

Cross

NCP

Hume

(1993) Isolation and characterization of the genes encoding mouse and human type-5 acid phosphatase. Gene 130:201–207.

13.

Chomczynski

Sacchi

(1987) Single-step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.

14.

Collin–Osdoby

Rothe

Sunyer

Osdoby

(1998) Nitric oxide (NO) supression of osteoclast bone resorption involves an inhibition of tartrate-resistant acid phosphatase (TRAP) activity. Proc ASMBR-IBMS Second Joint Meeting 1998; s550.

15.

da Cruz e Silva

Fox

Ouimet

Gustafsson

Watson

Greengard

(1995) Differential expression of protein phosphatase 1 isoforms in mammalian brain. J Neurosci 15:3375–3389.

16.

David

Que

Jr (1990) Anion binding to uteroferrin. Evidence for phosphate coordination to the iron(III) ion of the dinuclear active site and interaction with the hydroxo bridge. J Am Chem Soc 112:6455–6463.

17.

Doi

Antanaitis

Aisen

(1988) The binuclear iron centers of uteroferrin and the purple acid phosphatases. Struct Bond 70:1–26.

18.

Ek-Rylander

Barkhem

Ljusberg

Öhman

Andersson

(1997) Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase. Biochem J 321:305–311.

19.

Ek-Rylander

Bill

Norgård

Nilsson

Andersson

(1991) Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone. J Biol Chem 266:24684–24689.

20.

Ek-Rylander

Flores

Wendel

Heinegård

Andersson

(1994) Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. J Biol Chem 269:14853–14856.

21.

Flores

Norgård

Heinegård

Reinholt

Andersson

(1992) RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin. Exp Cell Res 201:526–530.

22.

Fort

Piechaczyk

El Sabrouty

Dani

Jeantour

Blanchard

(1985) Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nuclear Acid Res 13:1431–1442.

23.

Fritsch

Maniatis

Sambrook

(1989) Small-scale preparations of plasmid DNA. In Nolan

, ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1.25–1.28.

24.

Goldberg

Huang

H-B

Kwon

J-G

Greengard

Nairn

Kuriyan

(1995) Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 376:745–753.

25.

Goto

Matsukado

Mihara

Inoue

Miyamoto

(1986) The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay. Brain Res 397:161–172.

26.

Griffith

Kim

Sintchak

Thomson

Fitzgibbon

Fleming

Caron

Hsiao

Navia

(1995) X-ray structure of calcineurin inhibited by the immunophilinimmunosuppressant FKBP12-FK506 complex. Cell 82:507–522.

27.

Hayman

Bune

Bradley

Rashbass

Cox

(2000) Osteoclastic tartrate-resistant acid phophatase (Acp 5): its localization to dendritic cells and diverse murine tissues. J Histochem Cytochem 48:219–228.

28.

Hayman

Bune

Cox

(1998) Characterisation of the role of tartrate-resistant acid phosphatase in murine growth plates and macrophage systems. Proc ASMBR-IBMS Second Joint Meeting 1998;s554.

29.

Hayman

Cox

(1994) Purple acid phosphatase of the human macrophage and osteoclast—characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells. J Biol Chem 269:1294–1300.

30.

Hayman

Jones

Boyde

Foster

Colledge

Carlton

Evans

Cox

(1996) Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Development 122:3151–3162.

31.

Ichikawa

Itota

Nishitani

Torii

Inoue

Sugimoto

(2000) Osteopontin-immunoreactive primary sensory neurons in the rat spinal and trigeminal nervous system. Brain Res 863:276–281.

32.

Ignarro

(1990) Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling. Pharmacol Toxicol 67:1–7.

33.

Kim

Cha

Kim

Lee

Chung

Chun

(2000) Ganglion cells of the rat retina show osteopontin-like immunoreactivity. Brain Res 852:217–220.

34.

Ketcham

Roberts

Simmen

RCM

Nick

(1989) Molecular cloning of the type 5, iron-containing, tartrate-resistant acid phosphatase from human placenta. J Biol Chem 264:557–563.

35.

Knyihar-Csillik

Csillik

(1981) FRAP: histochemistry of the primary nociceptive neuron. Prog Histochem Cytochem 14:1–137.

36.

Krizbai

Joó

Pestean

Preil

Bötcher

Wolff

(1997) Localization and biochemical characterization of acid phosphatase isoforms in the olfactory system of adult rats. Neuroscience 76:799–807.

37.

Laemmli

(1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T₄ . Nature 227:680–685.

38.

Lee

Shin

Choi

Kim

Cha

Chun

Lee

Kim

(1999) Transient upregulation of osteopontin mRNA in hippocampus and striatum following global forebrain ischemia in rats. Neurosci Lett 271:81–84.

39.

Ljusberg

Ek-Rylander

Andersson

(1999) Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases. Biochem J 343:63–69.

40.

Lord

Cross

NCP

Bevilacqua

Rider

Gorman

Groves

Moss

Sheer

Cox

(1990) Type 5 acid phosphatase—sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage. Eur J Biochem 189:287–293.

41.

Moreno

Nardacci

Ceru

(1997) Regional and ultrastructural immunolocalization of copper-zinc superoxide dismutase in rat central nervous system. J Histochem Cytochem 45:1611–1622.

42.

Nordahl

Andersson

Reinholt

(1998) Chondroclasts and osteoclasts in bones of young rats: comparison of ultrastructural and functional features. Calcif Tissue Int 63:401–408.

43.

Ouimet

da Cruz e Silva

Greengard

(1995) The α and γ1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines. Proc Natl Acad Sci USA 92:3396–3400.

44.

Paxinos

Carrive

Wang

P-Y

(1999a) In Paxinos

, ed. Chemoarchitectonic Atlas of the Rat Brainstem. San Diego, CA, Academic Press.

45.

Paxinos

Kus

Ashwell

KWS

Watson

(1999b) In Paxinos

, ed. Chemoarchitectonic Atlas of the Rat Forebrain. San Diego, CA, Academic Press.

46.

Paxinos

Watson

(1986) The Rat Brain in Stereotaxis Coordinates. New York, Academic Press.

47.

Reddy

Hundley

Windle

Alcantara

Linn

Leach

Boldt

Roodman

(1995) Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. J Bone Miner Res 10:601–606.

48.

Reinholt

Hultenby

Oldberg

Heinegård

(1990a) Osteopontin—a possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA 87:4473–4475.

49.

Reinholt

Mengarelli Widholm

Ek-Rylander

Andersson

(1990b) Ultrastructural localization of a tartrate-resistant acid ATPase in bone. J Bone Miner Res 5:1055–1061.

50.

Saitoh

Yamamoto

Ushio

Miyamoto

(1989) Characterization of polyclonal antibodies to brain protein phosphatase 2A and immunohistochemical localization of the enzyme in rat brain. Brain Res 489:291–301.

51.

Saunders

PTK

Renegar

Raub

Baumbach

Atkinson

Bazer

Roberts

(1985) The carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver. J Biol Chem 260:3658–3665.

52.

Schindelmeiser

Munstermann

Mayer

Holstein

Davidoff

(1997) Occurence of enzymes of free radical metabolism suggests the possible cytotoxic capacity of the transitional epithelium of the human ureter. Cell Tissue Res 287:351–356.

53.

Schindelmeiser

Münstermann

Witzel

(1987) Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen. Histochemistry 87:13–19.

54.

Shin

Cha

Chun

Chung

Lee

(1999) Expression of osteopontin mRNA in the adult rat brain. Neurosci Lett 273:73–76.

55.

Sibille

J-C

Doi

Aisen

(1987) Hydroxyl radical formation and iron-binding proteins—stimulation by the purple acid phosphatases. J Biol Chem 262:59–62.

56.

Silverman

Kruger

(1988) Acid phosphatase as a selective marker for a class of small sensory ganglion cells in several mammals: spinal cord distribution, histochemical properties, and relation to fluoride-resistant acid phosphatase (FRAP) of rodents. Somatosens Res 5:219–246.

57.

Sim

(1991) The regulation and function of protein phosphatases in the brain. Mol Neurobiol 5:229–246.

58.

Tanaka

Ozawa

Inage

Deguchi

Itoh

Imai

Kohsaka

Takashima

(2000) Association of osteopontin with ischemic axonal death in periventricular leukomalacia. Acta Neuropathol (Berl) 100:69–74.

59.

Uppenberg

Lindqvist

Svensson

Ek-Rylander

Andersson

(1999) Crystal structure of a mammalian purple acid phosphatase. J Mol Biol 290:201–211.

60.

Valtschanoff

Weinberg

Kharazia

Nakane

Schmidt

(1993) Neurons in rat hippocampus that synthesize nitric oxide. J Comp Neurol 331:111–121.

61.

Vincent

Averill

(1990) An enzyme with a double identity: purple acid phosphatase and tartrate-resistant acid phosphatase. FASEB J 4:3009–3014.

62.

Vincent

Kimura

(1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46:755–784.

63.

Yaziji

Janckila

Lear

Martin

Yam

(1995) Immunohistochemical detection of tartrate-resistant acid phosphatase in non–hematopoietic human tissues. Am J Clin Pathol 104:397–402.

64.

Zhang

Damier

Hirsch

Agid

Ceballos–Picot

Sinet

Nicole

Laurent

Javoy–Agid

(1993) Preferential expression of superoxide dismutase messenger RNA in melanized neurons in human mesencephalon. Neuroscience 55:167–175.