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Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons
Neuroscience Letters 579 (2014) 75–79
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons Kentaro Nishida a , Yuka Nomura a , Kanako Kawamori a , Yoshinori Moriyama b , Kazuki Nagasawa a,∗ a
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8530, Japan b
h i g h l i g h t s • VNUT was expressed by small-, medium- and large-sized dorsal root ganglion neurons. • VNUT predominantly detected in IB4-positive dorsal root ganglion neurons rather than NF200-positive ones. • VNUT suggested to be involved in pain signaling via formation of ATP-enriched vesicles in dorsal root ganglion neurons.
a r t i c l e
i n f o
Article history: Received 27 May 2014 Received in revised form 1 July 2014 Accepted 9 July 2014 Available online 17 July 2014 Keywords: Vesicular nucleotide transporter SLC17A9 ATP Dorsal root ganglion
a b s t r a c t ATP plays an important role in the signal transduction between sensory neurons and satellite cells in dorsal root ganglia (DRGs). In primary cultured DRG neurons, ATP is known to be stored in lysosomes via a vesicular nucleotide transporter (VNUT), and to be released into the intercellular space through exocytosis. DRGs consist of large-, medium- and small-sized neurons, which play different roles in sensory transmission, but there is no information on the expression profiles of VNUT in DRG subpopulations. Here, we obtained detailed expression profiles of VNUT in isolated rat DRG tissues. On immunohistochemical analysis, VNUT was found in DRG neurons, and was predominantly expressed by the small- and mediumsized DRG ones, as judged upon visual inspection, and this was compatible with the finding that the number of VNUT-positive DRG neurons in IB4-positive cells was greater than that in NF200-positive ones. These results suggest that VNUT play a role in ATP accumulation in DRG neurons, especially in small- and medium-sized ones, and might be involved in ATP-mediated nociceptive signaling in DRGs. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction ATP is an intracellular energy source and also plays an important role in intercellular signal transduction [3]. Intraplantar injection of ATP leads to excitation of primary sensory neurons, spontaneous pain behaviors, thermal hyperalgesia, and mechanical allodynia [10,11]. Thus, the ATP signaling pathway is important for pain transmission in the peripheral nervous system. At presynaptic terminals, to communicate with postsynaptic neurons and other cells through P2 receptors, ATP is reported to be released from neurons via exocytosis [3]. Previously, Sawada et al. elucidated that vesicular nucleotide transporter (VNUT)
∗ Corresponding author. Tel.: +81 75 595 4648; fax: +81 75 595 4756. E-mail address: [email protected] (K. Nagasawa). http://dx.doi.org/10.1016/j.neulet.2014.07.017 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
contributed to the uptake of ATP into vesicles in PC12 cells [19]. Furthermore, Jung et al. reported that VNUT-expressing lysosomal vesicles were localized within the soma and growth cones of cultured dorsal root ganglion (DRG) neurons, suggesting that VNUT is involved in the formation of ATP-containing vesicles in the peripheral nervous system [13]. DRG neurons consist of multiple types of primary sensory neurons with anatomical and physiological differences, and their subpopulations can be divided into myelinated and unmyelinated neurons (A and C fibers, respectively). Myelinated A fibers can be further classified into larger and smaller neurons (A and A␦ fibers, respectively). It is reported that large A fibers mediate innocuous tactile responses [15,17], while medium A␦ and small C-fibers mediate nociceptive responses [10]. Collectively, these three types of neurons in DRGs play different roles in sensory signaling, but the expression profiles of VNUT in subpopulations of DRG neurons have not been revealed yet.
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2.2. RT-PCR analysis
Fig. 1. Expression of mRNA for VNUT in rat DRGs and several other tissues. The mRNA expression levels of VNUT in rat DRGs, cerebral cortex (Cx), spinal cord (S.C.), kidney, small intestine, heart, lung and adrenal gland were determined by real-time PCR. The amount of mRNA was normalized as to the amount of mRNA for -actin. Each bar represents the mean ± SEM (N = 3).
Therefore, in this study, we examined the localization of VNUT in subpopulations of rat DRG neurons in detail. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (200–300 g; Japan SLC, Hamamatsu, Japan) were used in this study. All experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University, and were performed according to the Guidelines for Animal Experimentation of Kyoto Pharmaceutical University.
SD rats were perfused transcardially with saline under deep anesthesia (pentobarbital sodium, 25 mg/kg, i.p.), and then DRG tissues were isolated as described previously [16]. For reverse transcription (RT)-PCR, the DRGs were treated with an RNAlater® solution (Sigma-Aldrich, Saint-Louis, MO) at−20 ◦ C. Total RNA was extracted and reverse transcribed with a GeneEluteTM Mammalian Total RNA Kit (Sigma-Aldrich, Saint-Louis, MO) and a PrimeScriptTM RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturers’ protocols, respectively. Real-time quantitative RT-PCR was performed using a SYBR Premix Ex Taq Kit (Takara, Shiga, Japan). The primer sets used were as follows: rat Vnut (NM 001108613): 5 -AGCCTGATGCAGCCAATCC-3 (sense), 5 -AGGTGCCCAGGAGCAACATC-3 (antisense); and rat Actb (NM 031144): 5 -TGACCCTGAAGTACCCCATTG-3 (sense), 5 TGTAGAAAGTGTGGTGCCAAATC-3 (antisense). 2.3. Immunohistochemical analysis Animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 0.2% picric acid under deep anesthesia (pentobarbital sodium, 25 mg/kg, i.p.). The isolated DRGs were sectioned at 40 m thickness with a freezing microtome, and free-floating sections were immunoreacted with primary antibodies, rabbit anti-VNUT (1:200) [19], mouse anti-neurofilament 200 (1:500, #MCA1321 T; AbD Serotec, Kidlington, UK), and FITC-conjugated isolectin B4 (10 g/mL, #FL-1201; Vector Laboratories, CA) for 3 days at 4 ◦ C, followed by incubation for a day at 4 ◦ C with Alexa Flour® 594-conjugated donkey anti-mouse or -rabbit IgG antibodies (1:1000) and Alexa Flour®
Fig. 2. Immunohistochemical analysis of VNUT in rat DRGs. (A-C) Representative photomicrographs of immunostaining for VNUT in rat DRGs are shown (green). The nuclei were stained with Hoechst 33258 (blue). Scale bar = 50 m. (D) Distribution of VNUT-immunofluorescent intensity in DRG neuronal cell bodies (soma) vs. cell size for all DRG neurons. (E) Size distribution histogram of VNUT-positive DRG neurons and all DRG neurons. DRG neurons were classified as VNUT-positive when the cytoplasmic intensity was three SDs above the background. Each bar represents the mean ± SEM (N = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Distributions of VNUT in NF200- or IB4-positive DRG neurons. (A–H) Representative photomicrographs of double-immunostaining for VNUT (A, E; red), and a large-sized neuronal marker, NF200 (B, green), or a small-sized neuronal marker, IB4 (F, green) in rat DRGs (N = 3). The nuclei were stained with Hoechst 33258 (C, G; blue). Scale bar = 50 m. (I, J) Size distribution histogram of all neurons, VNUTpositive, and neuronal size marker-positive neurons (I, NF200; J, IB4). Panel K showed percentage of each cell marker-positive cell number per all VNUT-positive cell number. Each bar represents the mean ± SEM (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
488-conjugated donkey anti-rabbit IgG antibodies (1:1000; Life Technologies, Tokyo, Japan). Negative controls were prepared by omitting the primary antibodies, and none of them exhibited any fluorescence signals (data not shown). The sections were mounted on glass slides and then enclosed using a Prolong® antifade kit (Life Technologies, Tokyo, Japan). Photomicrographs were obtained under a confocal laser microscope (LSM510META; Carl Zeiss, Germany).
classified as VNUT-positive if their staining intensities were three times the standard deviation above the background excluding the nucleus. 2.5. Statistical analysis The data are shown as means ± SEM. 3. Results
2.4. Image analysis Image quantification and processing were performed with NIH Image J software. Background fluorescence for individual antibodies was determined from the corresponding negative controls. The region of interest (ROI) feature of Image J was used to measure cell area and fluorescence intensity. Area measurements were made by drawing the outlines of DRG neuronal cell bodies. Cells were
First, the mRNA for VNUT was detected in the isolated rat DRGs as in well-known VNUT-expressing tissues, and the expression level in DRGs was almost the same as that in the cortex, in which the VNUT expression levels are known to be relatively high (Fig. 1) [19]. On immunohistochemistry, VNUT-immunoreactivity was detected in the soma of the DRGs (Fig. 2). The correlation of VNUT fluorescent intensity of the soma of the DRGs vs. cell body size of DRG
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Table 1 Determination of VNUT-positive levels in rat DRG neurons. Soma size
VNUT-positive cell number/total cell number (% of VNUT-positive cell number as to total cell number)
Small Medium Large
28.2 ± 6.3/106.0 ± 9.8 (24.2 ± 6.3%) 15.5 ± 3.4/106.0 ± 9.8 (14.8 ± 3.0%) 3.5 ± 1.0/106.0 ± 9.8 (3.5 ± 1.2%)
Number of VNUT-positive cells and the total number of cells counted. Mean ± SEM (N = 6).
Table 2 Colocalization of VNUT immunoreactivity with NF200 or IB4 in rat DRG neurons. Cell marker
VNUT-positive cell number/cell marker-positive cell number (VNUT-positive neurons containing each cell marker)
NF200 IB4
7.0 ± 1.0/10.7 ± 2.4 (71.4 ± 14.9%) 17.0 ± 9.5/43.0 ± 6.8 (35.5 ± 14.8%)
Number of VNUT-positive cells and the total number of cells counted. Mean ± SEM (N = 3).
neurons, as judged on visual inspection, is shown in Fig. 2D. There was a positive correlation between VNUT fluorescent intensity and cell body size (n = 636, y = 0.020x + 60.895, p < 0.05). Based on a cell body size histogram of all DRG neurons (Fig. 2E), cells with cell body areas of less than 750 m2 , between 750 and 1750 m2 , over 1750 m2 were defined as small-, medium- and large-sized DRG neurons, respectively [1]. As shown Table 1, VNUT-positive cells were observed in small-, medium- and large-sized DRG neurons, 24.2 ± 6.3%, 14.8 ± 3.0% and 3.5 ± 1.2%, respectively, and indicating on visual inspection, differential expression profiles of VNUT in the subpopulations of DRG neurons that small- and medium-sized ones might preferably express VNUT. In the peripheral nervous system, NF200 is a marker for the myelinated larger A (large-sized neurons) and smaller A␦ fibers (medium-sized neurons) [4,14,18]. IB4-positive cells are known to be unmyelinated C fibers (small-sized neurons) [7,12]. As shown Fig. 3, NF200-positive cells were visually identified as large- and medium-sized neurons, and IB4-positive cells were visually identified as small-sized ones. VNUT-immunoreactivity was detected in both NF200- and IB4-positive cells (Fig. 3A–H), and a cell body size histogram of VNUT showed that VNUT-positive neurons were primarily counted in small-sized ones (Fig. 3I and J). To show the distribution more clearly, the numbers per VNUT-, NF200- and IB4-positive cells were calculated. In fact, of the total NF200-positive neurons, 71.4 ± 14.9% were VNUT-positive ones, whereas 35.5 ± 14.8% of the IB4-positive neurons were VNUTpositive (Table 2). As shown Fig. 3K, 18.7 ± 5.5% of the total VNUT-positive neurons were NF200-positive, whereas 32.9 ± 4.0% of that was IB4-positive. These findings suggested that VNUT was predominantly expressed by IB4-positive small- and NF200positive medium-sized DRG neurons, compared to in the case of NF200-positive large-sized ones. 4. Discussion The aim of this study was to clarify the localization of VNUT in subpopulations of rat DRGs, and we revealed that in isolated rat DRGs, VNUT was predominantly expressed by the small- and medium-sized DRG neurons. This finding suggests that VNUT causes the accumulation of ATP in DRG neurons, especially in the small- and medium-sized ones, and plays a role in the ATPmediated sensory signaling. Nociceptive and non-nociceptive signals initiated in skin and internal tissues are transmitted to the spinal cord via sensory fibers. It is generally accepted that nociceptive signals are carried
by specialized afferent fibers, unmyelinated C-fibers, and thinly myelinated A␦-fibers, and non-nociceptive signals by myelinated A-fibers [5]. In this study, we found that VNUT was predominantly expressed by small- and medium-sized DRG neurons, as compared to in large-sized ones. Since small- and medium-sized neurons are known to play roles in nociceptive signaling, it is suggested that VNUT might be involved in sensory signaling for nociceptive sensation by causing the accumulation of ATP in the small- and medium-sized neurons. In the peripheral and central nervous systems, ATP acts as a neurotransmitter via P2X3 homomeric or P2X2/3 heteromeric receptors in sensory neurons [8,20,21], via P2X4 receptors in spinal microglia [22], and via P2X7 receptors in satellite cells of DRGs [6]. An increase in the intercellular ATP concentration is caused by leakage from damaged tissues, lysosomal exocytosis in cultured astrocytes [24] and microglia [9], etc., and leads to activation of P2X receptors. On the other hand, intercellular ATP is degraded by NTPDase2, one of the ectoenzymes for nucleotides [2,23]. Through these systems, to maintain DRG signaling normally, the intercellular ATP level is strictly regulated, and an inbalance of generation and degradation of ATP induces dysfunction of sensory signaling. Recently, Jung et al. reported that the ATP was released from VNUTexpressed lysosomal vesicles in the cultured DRG neurons in vitro [13]. Together with our finding that VNUT was predominantly expressed by small- and medium-sized neurons, it is reasonable to consider that VNUT in DRGs plays an important role in the regulation of ATP signaling. 5. Conclusion The present study demonstrated that among subpopulations of DRG neurons, VNUT was predominantly expressed by the smalland medium-sized DRG neurons, and thus it was suggested that VNUT might play a role in nociceptive signaling in DRGs through the accumulation of ATP in the neurons. Acknowledgements Parts of this study were financially supported by a Grant-in-Aid for Young Scientists (B) (26860090) from the Ministry of Education, Science and Culture of Japan, the ‘Academic Frontier’ Project for Private Universities of the Japanese Ministry of Education, Culture, Sport, Science and Technology, and the Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research. References [1] E. Bergman, B. Ulfhake, Loss of primary sensory neurons in the very old rat: neuron number estimates using the disector method and confocal optical sectioning, J. Comp. Neurol. 396 (1998) 211–222. [2] N. Braun, J. Sevigny, S.C. Robson, K. Hammer, M. Hanani, H. Zimmermann, Association of the ecto-ATPase NTPDase2 with glial cells of the peripheral nervous system, Glia 45 (2004) 124–132. [3] G. Burnstock, Historical review: ATP as a neurotransmitter, Trends Pharmacol. Sci. 27 (2006) 166–176. [4] T. Chao, K. Pham, O. Steward, R. Gupta, Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia, J. Comp. Neurol. 506 (2008) 180–193. [5] X. Chen, J.D. Levine, Hyper-responsivity in a subset of C-fiber nociceptors in a model of painful diabetic neuropathy in the rat, Neuroscience 102 (2001) 185–192. [6] Y. Chen, X. Zhang, C. Wang, G. Li, Y. Gu, L.Y. Huang, Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons, Proc. Nat. Acad. Sci. U.S.A. 105 (2008) 16773–16778. [7] H.J. Cho, V. Staikopoulos, J.J. Ivanusic, E.A. Jennings, Hyperpolarizationactivated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons, Cell Tissue Res. 338 (2009) 171–177. [8] D.A. Cockayne, S.G. Hamilton, Q.M. Zhu, P.M. Dunn, Y. Zhong, S. Novakovic, A.B. Malmberg, G. Cain, A. Berson, L. Kassotakis, L. Hedley, W.G. Lachnit, G. Burnstock, S.B. McMahon, A.P. Ford, Urinary bladder hyporeflexia and
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[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
reduced pain-related behaviour in P2X3-deficient mice, Nature 407 (2000) 1011–1015. Y. Dou, H.J. Wu, H.Q. Li, S. Qin, Y.E. Wang, J. Li, H.F. Lou, Z. Chen, X.M. Li, Q.M. Luo, S. Duan, Microglial migration mediated by ATP-induced ATP release from lysosomes, Cell Res. 22 (2012) 1022–1033. S.G. Hamilton, A. Wade, S.B. McMahon, The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat, Br. J. Pharmacol. 126 (1999) 326–332. M. Hilliges, C. Weidner, M. Schmelz, R. Schmidt, K. Orstavik, E. Torebjork, H. Handwerker, ATP responses in human C nociceptors, Pain 98 (2002) 59–68. T. Ishikawa, M. Miyagi, S. Ohtori, Y. Aoki, T. Ozawa, H. Doya, T. Saito, H. Moriya, K. Takahashi, Characteristics of sensory DRG neurons innervating the lumbar facet joints in rats, Eur. Spine J. 14 (2005) 559–564. J. Jung, Y.H. Shin, H. Konishi, S.J. Lee, H. Kiyama, Possible ATP release through lysosomal exocytosis from primary sensory neurons, Biochem. Biophys. Res. Commun. 430 (2012) 488–493. K. Kobayashi, T. Fukuoka, H. Yamanaka, Y. Dai, K. Obata, A. Tokunaga, K. Noguchi, Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord, J. Comp. Neurol. 498 (2006) 443–454. C.N. Liu, P.D. Wall, E. Ben-Dor, M. Michaelis, R. Amir, M. Devor, Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury, Pain 85 (2000) 503–521. K. Nishida, E. Yasuda, K. Nagasawa, S. Fujimoto, Altered levels of oxidation and phospholipase C isozyme expression in the brains of theanine-administered rats, Biol. Pharm. Bull. 31 (2008) 857–860. M.H. Ossipov, D. Bian, T.P. Malan Jr., J. Lai, F. Porreca, Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats, Pain 79 (1999) 127–133.
79
[18] R. Ruscheweyh, L. Forsthuber, D. Schoffnegger, J. Sandkuhler, Modification of classical neurochemical markers in identified primary afferent neurons with Abeta-, Adelta-, and C-fibers after chronic constriction injury in mice, J. Comp. Neurol. 502 (2007) 325–336. [19] K. Sawada, N. Echigo, N. Juge, T. Miyaji, M. Otsuka, H. Omote, A. Yamamoto, Y. Moriyama, Identification of a vesicular nucleotide transporter, Proc. Nat. Acad. Sci. U.S.A. 105 (2008) 5683–5686. [20] V. Souslova, P. Cesare, Y. Ding, A.N. Akopian, L. Stanfa, R. Suzuki, K. Carpenter, A. Dickenson, S. Boyce, R. Hill, D. Nebenuis-Oosthuizen, A.J. Smith, E.J. Kidd, J.N. Wood, Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors, Nature 407 (2000) 1015–1017. [21] M. Tsuda, S. Koizumi, A. Kita, Y. Shigemoto, S. Ueno, K. Inoue, Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons, J. Neurosci. 20 (2000) RC90. [22] M. Tsuda, Y. Shigemoto-Mogami, S. Koizumi, A. Mizokoshi, S. Kohsaka, M.W. Salter, K. Inoue, P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury, Nature 424 (2003) 778–783. [23] H.O. Vongtau, E.G. Lavoie, J. Sevigny, D.C. Molliver, Distribution of ectonucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception, Neuroscience 193 (2011) 387–398. [24] Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.S. Gu, S. Duan, Regulated ATP release from astrocytes through lysosome exocytosis, Nat. Cell Biol. 9 (2007) 945–953.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons Kentaro Nishida a , Yuka Nomura a , Kanako Kawamori a , Yoshinori Moriyama b , Kazuki Nagasawa a,∗ a
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8530, Japan b
h i g h l i g h t s • VNUT was expressed by small-, medium- and large-sized dorsal root ganglion neurons. • VNUT predominantly detected in IB4-positive dorsal root ganglion neurons rather than NF200-positive ones. • VNUT suggested to be involved in pain signaling via formation of ATP-enriched vesicles in dorsal root ganglion neurons.
a r t i c l e
i n f o
Article history: Received 27 May 2014 Received in revised form 1 July 2014 Accepted 9 July 2014 Available online 17 July 2014 Keywords: Vesicular nucleotide transporter SLC17A9 ATP Dorsal root ganglion
a b s t r a c t ATP plays an important role in the signal transduction between sensory neurons and satellite cells in dorsal root ganglia (DRGs). In primary cultured DRG neurons, ATP is known to be stored in lysosomes via a vesicular nucleotide transporter (VNUT), and to be released into the intercellular space through exocytosis. DRGs consist of large-, medium- and small-sized neurons, which play different roles in sensory transmission, but there is no information on the expression profiles of VNUT in DRG subpopulations. Here, we obtained detailed expression profiles of VNUT in isolated rat DRG tissues. On immunohistochemical analysis, VNUT was found in DRG neurons, and was predominantly expressed by the small- and mediumsized DRG ones, as judged upon visual inspection, and this was compatible with the finding that the number of VNUT-positive DRG neurons in IB4-positive cells was greater than that in NF200-positive ones. These results suggest that VNUT play a role in ATP accumulation in DRG neurons, especially in small- and medium-sized ones, and might be involved in ATP-mediated nociceptive signaling in DRGs. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction ATP is an intracellular energy source and also plays an important role in intercellular signal transduction [3]. Intraplantar injection of ATP leads to excitation of primary sensory neurons, spontaneous pain behaviors, thermal hyperalgesia, and mechanical allodynia [10,11]. Thus, the ATP signaling pathway is important for pain transmission in the peripheral nervous system. At presynaptic terminals, to communicate with postsynaptic neurons and other cells through P2 receptors, ATP is reported to be released from neurons via exocytosis [3]. Previously, Sawada et al. elucidated that vesicular nucleotide transporter (VNUT)
∗ Corresponding author. Tel.: +81 75 595 4648; fax: +81 75 595 4756. E-mail address: [email protected] (K. Nagasawa). http://dx.doi.org/10.1016/j.neulet.2014.07.017 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
contributed to the uptake of ATP into vesicles in PC12 cells [19]. Furthermore, Jung et al. reported that VNUT-expressing lysosomal vesicles were localized within the soma and growth cones of cultured dorsal root ganglion (DRG) neurons, suggesting that VNUT is involved in the formation of ATP-containing vesicles in the peripheral nervous system [13]. DRG neurons consist of multiple types of primary sensory neurons with anatomical and physiological differences, and their subpopulations can be divided into myelinated and unmyelinated neurons (A and C fibers, respectively). Myelinated A fibers can be further classified into larger and smaller neurons (A and A␦ fibers, respectively). It is reported that large A fibers mediate innocuous tactile responses [15,17], while medium A␦ and small C-fibers mediate nociceptive responses [10]. Collectively, these three types of neurons in DRGs play different roles in sensory signaling, but the expression profiles of VNUT in subpopulations of DRG neurons have not been revealed yet.
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2.2. RT-PCR analysis
Fig. 1. Expression of mRNA for VNUT in rat DRGs and several other tissues. The mRNA expression levels of VNUT in rat DRGs, cerebral cortex (Cx), spinal cord (S.C.), kidney, small intestine, heart, lung and adrenal gland were determined by real-time PCR. The amount of mRNA was normalized as to the amount of mRNA for -actin. Each bar represents the mean ± SEM (N = 3).
Therefore, in this study, we examined the localization of VNUT in subpopulations of rat DRG neurons in detail. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (200–300 g; Japan SLC, Hamamatsu, Japan) were used in this study. All experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University, and were performed according to the Guidelines for Animal Experimentation of Kyoto Pharmaceutical University.
SD rats were perfused transcardially with saline under deep anesthesia (pentobarbital sodium, 25 mg/kg, i.p.), and then DRG tissues were isolated as described previously [16]. For reverse transcription (RT)-PCR, the DRGs were treated with an RNAlater® solution (Sigma-Aldrich, Saint-Louis, MO) at−20 ◦ C. Total RNA was extracted and reverse transcribed with a GeneEluteTM Mammalian Total RNA Kit (Sigma-Aldrich, Saint-Louis, MO) and a PrimeScriptTM RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturers’ protocols, respectively. Real-time quantitative RT-PCR was performed using a SYBR Premix Ex Taq Kit (Takara, Shiga, Japan). The primer sets used were as follows: rat Vnut (NM 001108613): 5 -AGCCTGATGCAGCCAATCC-3 (sense), 5 -AGGTGCCCAGGAGCAACATC-3 (antisense); and rat Actb (NM 031144): 5 -TGACCCTGAAGTACCCCATTG-3 (sense), 5 TGTAGAAAGTGTGGTGCCAAATC-3 (antisense). 2.3. Immunohistochemical analysis Animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 0.2% picric acid under deep anesthesia (pentobarbital sodium, 25 mg/kg, i.p.). The isolated DRGs were sectioned at 40 m thickness with a freezing microtome, and free-floating sections were immunoreacted with primary antibodies, rabbit anti-VNUT (1:200) [19], mouse anti-neurofilament 200 (1:500, #MCA1321 T; AbD Serotec, Kidlington, UK), and FITC-conjugated isolectin B4 (10 g/mL, #FL-1201; Vector Laboratories, CA) for 3 days at 4 ◦ C, followed by incubation for a day at 4 ◦ C with Alexa Flour® 594-conjugated donkey anti-mouse or -rabbit IgG antibodies (1:1000) and Alexa Flour®
Fig. 2. Immunohistochemical analysis of VNUT in rat DRGs. (A-C) Representative photomicrographs of immunostaining for VNUT in rat DRGs are shown (green). The nuclei were stained with Hoechst 33258 (blue). Scale bar = 50 m. (D) Distribution of VNUT-immunofluorescent intensity in DRG neuronal cell bodies (soma) vs. cell size for all DRG neurons. (E) Size distribution histogram of VNUT-positive DRG neurons and all DRG neurons. DRG neurons were classified as VNUT-positive when the cytoplasmic intensity was three SDs above the background. Each bar represents the mean ± SEM (N = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Distributions of VNUT in NF200- or IB4-positive DRG neurons. (A–H) Representative photomicrographs of double-immunostaining for VNUT (A, E; red), and a large-sized neuronal marker, NF200 (B, green), or a small-sized neuronal marker, IB4 (F, green) in rat DRGs (N = 3). The nuclei were stained with Hoechst 33258 (C, G; blue). Scale bar = 50 m. (I, J) Size distribution histogram of all neurons, VNUTpositive, and neuronal size marker-positive neurons (I, NF200; J, IB4). Panel K showed percentage of each cell marker-positive cell number per all VNUT-positive cell number. Each bar represents the mean ± SEM (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
488-conjugated donkey anti-rabbit IgG antibodies (1:1000; Life Technologies, Tokyo, Japan). Negative controls were prepared by omitting the primary antibodies, and none of them exhibited any fluorescence signals (data not shown). The sections were mounted on glass slides and then enclosed using a Prolong® antifade kit (Life Technologies, Tokyo, Japan). Photomicrographs were obtained under a confocal laser microscope (LSM510META; Carl Zeiss, Germany).
classified as VNUT-positive if their staining intensities were three times the standard deviation above the background excluding the nucleus. 2.5. Statistical analysis The data are shown as means ± SEM. 3. Results
2.4. Image analysis Image quantification and processing were performed with NIH Image J software. Background fluorescence for individual antibodies was determined from the corresponding negative controls. The region of interest (ROI) feature of Image J was used to measure cell area and fluorescence intensity. Area measurements were made by drawing the outlines of DRG neuronal cell bodies. Cells were
First, the mRNA for VNUT was detected in the isolated rat DRGs as in well-known VNUT-expressing tissues, and the expression level in DRGs was almost the same as that in the cortex, in which the VNUT expression levels are known to be relatively high (Fig. 1) [19]. On immunohistochemistry, VNUT-immunoreactivity was detected in the soma of the DRGs (Fig. 2). The correlation of VNUT fluorescent intensity of the soma of the DRGs vs. cell body size of DRG
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Table 1 Determination of VNUT-positive levels in rat DRG neurons. Soma size
VNUT-positive cell number/total cell number (% of VNUT-positive cell number as to total cell number)
Small Medium Large
28.2 ± 6.3/106.0 ± 9.8 (24.2 ± 6.3%) 15.5 ± 3.4/106.0 ± 9.8 (14.8 ± 3.0%) 3.5 ± 1.0/106.0 ± 9.8 (3.5 ± 1.2%)
Number of VNUT-positive cells and the total number of cells counted. Mean ± SEM (N = 6).
Table 2 Colocalization of VNUT immunoreactivity with NF200 or IB4 in rat DRG neurons. Cell marker
VNUT-positive cell number/cell marker-positive cell number (VNUT-positive neurons containing each cell marker)
NF200 IB4
7.0 ± 1.0/10.7 ± 2.4 (71.4 ± 14.9%) 17.0 ± 9.5/43.0 ± 6.8 (35.5 ± 14.8%)
Number of VNUT-positive cells and the total number of cells counted. Mean ± SEM (N = 3).
neurons, as judged on visual inspection, is shown in Fig. 2D. There was a positive correlation between VNUT fluorescent intensity and cell body size (n = 636, y = 0.020x + 60.895, p < 0.05). Based on a cell body size histogram of all DRG neurons (Fig. 2E), cells with cell body areas of less than 750 m2 , between 750 and 1750 m2 , over 1750 m2 were defined as small-, medium- and large-sized DRG neurons, respectively [1]. As shown Table 1, VNUT-positive cells were observed in small-, medium- and large-sized DRG neurons, 24.2 ± 6.3%, 14.8 ± 3.0% and 3.5 ± 1.2%, respectively, and indicating on visual inspection, differential expression profiles of VNUT in the subpopulations of DRG neurons that small- and medium-sized ones might preferably express VNUT. In the peripheral nervous system, NF200 is a marker for the myelinated larger A (large-sized neurons) and smaller A␦ fibers (medium-sized neurons) [4,14,18]. IB4-positive cells are known to be unmyelinated C fibers (small-sized neurons) [7,12]. As shown Fig. 3, NF200-positive cells were visually identified as large- and medium-sized neurons, and IB4-positive cells were visually identified as small-sized ones. VNUT-immunoreactivity was detected in both NF200- and IB4-positive cells (Fig. 3A–H), and a cell body size histogram of VNUT showed that VNUT-positive neurons were primarily counted in small-sized ones (Fig. 3I and J). To show the distribution more clearly, the numbers per VNUT-, NF200- and IB4-positive cells were calculated. In fact, of the total NF200-positive neurons, 71.4 ± 14.9% were VNUT-positive ones, whereas 35.5 ± 14.8% of the IB4-positive neurons were VNUTpositive (Table 2). As shown Fig. 3K, 18.7 ± 5.5% of the total VNUT-positive neurons were NF200-positive, whereas 32.9 ± 4.0% of that was IB4-positive. These findings suggested that VNUT was predominantly expressed by IB4-positive small- and NF200positive medium-sized DRG neurons, compared to in the case of NF200-positive large-sized ones. 4. Discussion The aim of this study was to clarify the localization of VNUT in subpopulations of rat DRGs, and we revealed that in isolated rat DRGs, VNUT was predominantly expressed by the small- and medium-sized DRG neurons. This finding suggests that VNUT causes the accumulation of ATP in DRG neurons, especially in the small- and medium-sized ones, and plays a role in the ATPmediated sensory signaling. Nociceptive and non-nociceptive signals initiated in skin and internal tissues are transmitted to the spinal cord via sensory fibers. It is generally accepted that nociceptive signals are carried
by specialized afferent fibers, unmyelinated C-fibers, and thinly myelinated A␦-fibers, and non-nociceptive signals by myelinated A-fibers [5]. In this study, we found that VNUT was predominantly expressed by small- and medium-sized DRG neurons, as compared to in large-sized ones. Since small- and medium-sized neurons are known to play roles in nociceptive signaling, it is suggested that VNUT might be involved in sensory signaling for nociceptive sensation by causing the accumulation of ATP in the small- and medium-sized neurons. In the peripheral and central nervous systems, ATP acts as a neurotransmitter via P2X3 homomeric or P2X2/3 heteromeric receptors in sensory neurons [8,20,21], via P2X4 receptors in spinal microglia [22], and via P2X7 receptors in satellite cells of DRGs [6]. An increase in the intercellular ATP concentration is caused by leakage from damaged tissues, lysosomal exocytosis in cultured astrocytes [24] and microglia [9], etc., and leads to activation of P2X receptors. On the other hand, intercellular ATP is degraded by NTPDase2, one of the ectoenzymes for nucleotides [2,23]. Through these systems, to maintain DRG signaling normally, the intercellular ATP level is strictly regulated, and an inbalance of generation and degradation of ATP induces dysfunction of sensory signaling. Recently, Jung et al. reported that the ATP was released from VNUTexpressed lysosomal vesicles in the cultured DRG neurons in vitro [13]. Together with our finding that VNUT was predominantly expressed by small- and medium-sized neurons, it is reasonable to consider that VNUT in DRGs plays an important role in the regulation of ATP signaling. 5. Conclusion The present study demonstrated that among subpopulations of DRG neurons, VNUT was predominantly expressed by the smalland medium-sized DRG neurons, and thus it was suggested that VNUT might play a role in nociceptive signaling in DRGs through the accumulation of ATP in the neurons. Acknowledgements Parts of this study were financially supported by a Grant-in-Aid for Young Scientists (B) (26860090) from the Ministry of Education, Science and Culture of Japan, the ‘Academic Frontier’ Project for Private Universities of the Japanese Ministry of Education, Culture, Sport, Science and Technology, and the Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research. References [1] E. Bergman, B. Ulfhake, Loss of primary sensory neurons in the very old rat: neuron number estimates using the disector method and confocal optical sectioning, J. Comp. Neurol. 396 (1998) 211–222. [2] N. Braun, J. Sevigny, S.C. Robson, K. Hammer, M. Hanani, H. Zimmermann, Association of the ecto-ATPase NTPDase2 with glial cells of the peripheral nervous system, Glia 45 (2004) 124–132. [3] G. Burnstock, Historical review: ATP as a neurotransmitter, Trends Pharmacol. Sci. 27 (2006) 166–176. [4] T. Chao, K. Pham, O. Steward, R. Gupta, Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia, J. Comp. Neurol. 506 (2008) 180–193. [5] X. Chen, J.D. Levine, Hyper-responsivity in a subset of C-fiber nociceptors in a model of painful diabetic neuropathy in the rat, Neuroscience 102 (2001) 185–192. [6] Y. Chen, X. Zhang, C. Wang, G. Li, Y. Gu, L.Y. Huang, Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons, Proc. Nat. Acad. Sci. U.S.A. 105 (2008) 16773–16778. [7] H.J. Cho, V. Staikopoulos, J.J. Ivanusic, E.A. Jennings, Hyperpolarizationactivated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons, Cell Tissue Res. 338 (2009) 171–177. [8] D.A. Cockayne, S.G. Hamilton, Q.M. Zhu, P.M. Dunn, Y. Zhong, S. Novakovic, A.B. Malmberg, G. Cain, A. Berson, L. Kassotakis, L. Hedley, W.G. Lachnit, G. Burnstock, S.B. McMahon, A.P. Ford, Urinary bladder hyporeflexia and
K. Nishida et al. / Neuroscience Letters 579 (2014) 75–79
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
reduced pain-related behaviour in P2X3-deficient mice, Nature 407 (2000) 1011–1015. Y. Dou, H.J. Wu, H.Q. Li, S. Qin, Y.E. Wang, J. Li, H.F. Lou, Z. Chen, X.M. Li, Q.M. Luo, S. Duan, Microglial migration mediated by ATP-induced ATP release from lysosomes, Cell Res. 22 (2012) 1022–1033. S.G. Hamilton, A. Wade, S.B. McMahon, The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat, Br. J. Pharmacol. 126 (1999) 326–332. M. Hilliges, C. Weidner, M. Schmelz, R. Schmidt, K. Orstavik, E. Torebjork, H. Handwerker, ATP responses in human C nociceptors, Pain 98 (2002) 59–68. T. Ishikawa, M. Miyagi, S. Ohtori, Y. Aoki, T. Ozawa, H. Doya, T. Saito, H. Moriya, K. Takahashi, Characteristics of sensory DRG neurons innervating the lumbar facet joints in rats, Eur. Spine J. 14 (2005) 559–564. J. Jung, Y.H. Shin, H. Konishi, S.J. Lee, H. Kiyama, Possible ATP release through lysosomal exocytosis from primary sensory neurons, Biochem. Biophys. Res. Commun. 430 (2012) 488–493. K. Kobayashi, T. Fukuoka, H. Yamanaka, Y. Dai, K. Obata, A. Tokunaga, K. Noguchi, Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord, J. Comp. Neurol. 498 (2006) 443–454. C.N. Liu, P.D. Wall, E. Ben-Dor, M. Michaelis, R. Amir, M. Devor, Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury, Pain 85 (2000) 503–521. K. Nishida, E. Yasuda, K. Nagasawa, S. Fujimoto, Altered levels of oxidation and phospholipase C isozyme expression in the brains of theanine-administered rats, Biol. Pharm. Bull. 31 (2008) 857–860. M.H. Ossipov, D. Bian, T.P. Malan Jr., J. Lai, F. Porreca, Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats, Pain 79 (1999) 127–133.
79
[18] R. Ruscheweyh, L. Forsthuber, D. Schoffnegger, J. Sandkuhler, Modification of classical neurochemical markers in identified primary afferent neurons with Abeta-, Adelta-, and C-fibers after chronic constriction injury in mice, J. Comp. Neurol. 502 (2007) 325–336. [19] K. Sawada, N. Echigo, N. Juge, T. Miyaji, M. Otsuka, H. Omote, A. Yamamoto, Y. Moriyama, Identification of a vesicular nucleotide transporter, Proc. Nat. Acad. Sci. U.S.A. 105 (2008) 5683–5686. [20] V. Souslova, P. Cesare, Y. Ding, A.N. Akopian, L. Stanfa, R. Suzuki, K. Carpenter, A. Dickenson, S. Boyce, R. Hill, D. Nebenuis-Oosthuizen, A.J. Smith, E.J. Kidd, J.N. Wood, Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors, Nature 407 (2000) 1015–1017. [21] M. Tsuda, S. Koizumi, A. Kita, Y. Shigemoto, S. Ueno, K. Inoue, Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons, J. Neurosci. 20 (2000) RC90. [22] M. Tsuda, Y. Shigemoto-Mogami, S. Koizumi, A. Mizokoshi, S. Kohsaka, M.W. Salter, K. Inoue, P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury, Nature 424 (2003) 778–783. [23] H.O. Vongtau, E.G. Lavoie, J. Sevigny, D.C. Molliver, Distribution of ectonucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception, Neuroscience 193 (2011) 387–398. [24] Z. Zhang, G. Chen, W. Zhou, A. Song, T. Xu, Q. Luo, W. Wang, X.S. Gu, S. Duan, Regulated ATP release from astrocytes through lysosome exocytosis, Nat. Cell Biol. 9 (2007) 945–953.