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RESEARCH REPORT |
1 Department of Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany;
2 Department of Operative Dentistry and Periodontology, University of Cologne, Germany;
3 Department of Anatomy I, University of Cologne;
4 Department of Anatomy II, University of Cologne;
5 Department of Pharmacology, Faculty of Medicine, 1 King’ College Circle, Toronto, ON, Canada; and
6 Department of Molecular and Cellular Sports Medicine, German Sports University, Cologne;
* corresponding author, yueksel.korkmaz{at}uni-duesseldorf.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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2-subunit/ß1-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC 2, and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10–5 M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10–4 M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.
KEY WORDS: nitric oxide • nitric oxide synthase • soluble guanylate cyclase • cyclic guanosine 3',5'-monophosphate • dentin-pulp complex
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The NO-sensitive enzyme-soluble guanylate cyclase (sGC) is a heme-containing, cytosolic heterodimeric enzyme consisting of an and a ß subunit. Four subunits of sGC have been cloned (1, ß1, 2, and ß2), and only 2 isoforms (1/ß1, 2/ß1) have been shown to exist at the protein level (Krumenacker et al., 2004). NO binds to the heme of the soluble guanylate cyclase (sGC), which catalyzes the increased production of cGMP. The cellular increase of cGMP leads to vasodilatation (Krumenacker et al., 2004) and neurotransmission (Burette et al., 2002).
Involvement of NO in the dental pulp circulation (Lohinai et al., 1995), localization of NOS I in the cat and dog pulpal nerve fibers (Lohinai et al., 1997), and existence of NOS III in endothelial cells of the human dental pulp (Felaco et al., 2000) have been detected. NADPH-diaphorase (Kerezoudis et al., 1993) and NOS III in odontoblasts (Felaco et al., 2000) indicate a role for NO in odontoblasts. NOS I in non-neuronal cells (Davis et al., 2001) and the involvement of NO-cGMP signaling molecules in neurotransmission have been described (Burette et al., 2002). The exact mechanism of neurotransmission in dentin is still not clear (Byers, 1984). The existence of NO-sensitive enzyme sGC in cells of the dentin-pulp complex is unknown.
The testing of the hypothesis that sGC can mediate the effect of NO in different types of cells in the dentin-pulp complex led us to determine the presence of NOS I-III, sGC, and cGMP in cells of the dentin-pulp complex, and to correlate alterations of cGMP staining intensities after treatment with the NO donor NONOate and NO inhibitor L-NAME in cells of the rat molar dentin-pulp complex.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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NADPH-diaphorase Histochemistry
The free-floating (20 µm) cryostat sections were stained by NADPH-diaphorase (Appendix 1).
Antibodies
The specificity of rabbit polyclonal antibodies to NOS I-III (Transduction Laboratories, Lexington, KY, USA) was confirmed by immunoblot analysis (Descarries et al., 1998) (Appendix 2). The rabbit anti-NOS III polyclonal antibody from Biomol (Hamburg, Germany) was characterized (Bloch et al., 2001). The sGC 2-subunit antibody was raised against the C-terminal peptide (H2N-CKKVSYNIGTMFLRETSL-COOH, amino acids 717–733) of the human 2-subunit, coupled by cysteine to keyhole-limpet hemocyanin (KLH) (Bamberger et al., 2001). The sGC ß1-subunit antibody was directed against the C-terminal peptide sequence of the rat ß1-subunit of sGC-coupled cysteine to KLH (H2N-PSRKNTGTEETNQDEN-COOH, amino acids 605–619) (Behrends et al., 2001). Immunoblot identity of fractions was verified with antibodies against the sGC 2-subunit (Bamberger et al., 2001) and the sGC ß1-subunit (Behrends et al., 2001). The rabbit anti-cGMP polyclonal antibody (Biogenesis Inc., Sandown, NH, USA) has been described previously (Bloch et al., 2001).
Immunohistochemistry
The free-floating cryostat sections (40 µm) were incubated for 48 hrs with primary antibodies [NOS I Transduction (1:1000) and Biomol (1:1000), NOS II from Transduction (1:1000) and Biomol (1:1500), NOS III from Transduction (1:1000), Biomol (1:1500), and Santa Cruz (1:800; Santa Cruz, CA, USA), the sGC 2- and ß1-subunits (1:800; Dr. S. Behrends/Alexis Biochemicals Corporation, San Diego, CA, USA), and cGMP (1:600; Biogenesis)], at 4°C. The sections were incubated for 1 hr with biotinylated anti-rabbit IgG (Vector Labs., Burlingame, CA, USA) and then with avidin-biotin peroxidase complex (1:100; Vector). The peroxidase reaction was visualized by 3,3'-diaminobenzidine (Sigma; St. Louis, MO, USA). Specificities of the antibodies were tested (Appendix 3).
Morphometric Quantification and Statistical Analysis
The quantification of relationship between lengths of dentinal tubules and lengths of labeled odontoblast processes for NOS I, NOS III, the sGC 2-subunit, the sGC ß1-subunit, and cGMP of each molar was assessed by the use of Optimas 6.0 morphometric software (Optimas Corporation, Bothell, WA, USA). Statistical analysis was performed by the Bonferroni post hoc test, and significance was considered at a p-value < 0.05 (N = 10).
Pre-embedding Immunoelectron Microscopy
The immunolabeled sections, which were identified by light microscopy for the sGC 2- and ß1-subunits, were post-fixed in 1% OsO4, dehydrated, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate.
Post-embedding Immunogold Staining
The rat molars were embedded in LR-White resin (London Resins, Reading, Berkshire, UK). Thin sections were mounted on formvar-coated nickel grids, and incubated with the rabbit anti-sGC ß1-subunit (1:100) and subsequently with 10 nm of gold-conjugated goat anti-rabbit IgG (1:25; Sigma). The immunogold labeling specificity was tested by calculation of the ratio of gold particles on the odontoblast processes to those on intertubular dentin matrix areas (N = 20).
Organ Bath Experiments and Immunohistochemistry of cGMP
The molars of 8 rats were dissected free from the roots with the use of forceps and placed in Tyrode’s solution (pH 7.4) of the following composition (in mM): CaCl2 x 2H2O, 1.8; MgCl2 x 6H2O, 1.1; KCl, 5.4; NaCl, 136.9; NaH2PO4, 0.4; glucose, 10.1; and NaHCO3, 23.8 (all solutions supplied from Merck, Darmstadt, Germany). The samples were treated with a 10-mL Tyrode’s solution containing either 10–5 M NO synthase donor spermine-NONOate (N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine) (Alexis), or 10–4 M NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (Alexis) at 37°C for up to 30 min. The molars were removed from the medium and treated by immunohistochemistry for cGMP. In all staining procedures, the visualization of the reaction products for cGMP was developed for 4 min.
Densitometry of the cGMP-staining Intensities and Statistical Analysis
The staining intensity for cGMP in blood vessels and odontoblasts was measured with use of the Optimas 6.00 image analysis program (Imaging Technology Inc., San Diego, CA, USA) (Appendix 4). Statistical comparisons were performed by the two-tailed Student’s t test for paired samples, with the software package SPSS for Windows, Version 10.0. Significance was considered at a p-value < 0.05 (N = 8).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) and in their initial processes (Fig. 1B). A subpopulation of odontoblasts revealed NOS II immunoreactivity (Fig. 1C). In the control section, NOS II was not detectable (Fig. 1D). NOS III was localized in odontoblasts and in their initial processes (Fig. 1E). The sGC 2-subunit was detected in odontoblasts (data not shown), while the sGC ß1-subunit was identified in odontoblasts (Fig. 1F) as well as in their peripheral processes, located near the dentino-enamel junction (Fig. 1G). cGMP was detected in odontoblasts and in their initial processes (Fig. 1H). In pulpal stroma cells, the sGC 2-subunit, the sGC ß1-subunit, and cGMP were identified (data not shown). Statistical analyses revealed a significance for localization of the sGC ß1-subunit in the peripheral odontoblast processes within dentin tubules (Fig. 1I). Localization of the sGC 2-subunit (Fig. 2A) and the sGC ß1-subunit (Figs. 2B, 2C) in odontoblasts and in their processes was identified at ultrastructural levels.
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), and immunoreactivity NOS III (Fig. 3B), and the sGC 2-subunit (data not shown). The sGC ß1-subunit was identified in endothelial cells as well as the smooth-muscle layer of blood vessels (Fig. 3C). cGMP was detected in blood vessels (Fig. 3D). NOS I (Fig. 3E) and the sGC 2-subunit (Fig. 3F) were identified in pulpal nerve fibers. The nerve fibers showing immunoreactivity to the sGC 2-subunit penetrated initial dentin tubules (Fig. 3G). At the ultrastructural level, the sGC-2-subunit-positive nerve endings were identified in close association with sGC 2-subunit-stained odontoblasts in predentin (Fig. 2A
). cGMP was detected in nerve fibers distributed perivascularly (Fig.3D), in the subodontoblastic plexus (Fig. 1H), and in predentin (Fig. 3H).
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; 70.93 ± 3.31), blood vessels (Fig. 4D; 63.84 ± 6.29)], odontoblasts (Fig. 4B; 88.60 ± 5.75) and blood vessels (Fig. 4E; 83.52 ± 8.09) revealed higher staining intensities for cGMP after treatment with NO donor spermine-NONOate. In the L-NAME-treated sections, the staining intensities of cGMP were significantly lower in odontoblasts (Fig. 4C; 61.74 ± 5.02) as well as blood vessels (Fig. 4F; 53.99 ± 4.19), compared with cGMP staining intensities in odontoblasts (Fig. 4A) and blood vessels (Fig. 4D) of the control sections. Fig. 4G shows that L-NAME elicited a significant decrease and NONOate induced a significant increase in the cGMP staining intensities in blood vessels and odontoblasts (N = 8; p < 0.05).
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, NOS III, Biomol) and sGC isoforms (Alexis) was further confirmed by production of the identical staining localization from antibodies against different epitopes of the signaling molecules (data not shown). Immunohistochemical controls of the pre-embedding immunoelectron microscopy (Fig. 2D), as well as post-embedding immunogold staining (data not shown), resulted in the disappearance of the specific reaction product. The ratio of gold particles on the peripheral odontoblast processes to those on intertubular dentin matrix areas confirmed the specificity of immunogold labeling (ratio 
10.80; N = 20). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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NADPH-diaphorase, and NOS I and NOS III in odontoblasts suggest a role for NO in the homeostasis of odontoblasts. The inconsistent existence of NOS II in odontoblasts indicates an activity-dependent basal expression of NOS II in odontoblasts. It has been reported that acute inflammation enhanced the mRNA levels of NOS II in human dental pulp (Di Nardo Di Maio et al., 2004).
NO is involved in osteoblast differentiation and proliferation (Hikiji et al., 1997). cGMP stimulates the differentation of osteoblast-like cells and the formation of mineralized nodules (Inoue et al., 1995). The sGC (2-subunit, ß1-subunit) detected in odontoblasts may indicate an involvement of NO-cGMP signaling in odontoblast differentiation and/or dentin biomineralization. Due to the important roles of NO in cellular differentiation (Peunova and Enikolopov, 1995), proliferation (Kuzin et al., 1996), and apoptosis (Dimmeler and Zeiher, 1997), a role of NO signaling in odontoblasts for these processes is suggested. sGC (2-subunit, ß1-subunit) and cGMP in pulpal stromal cells may indicate that NO, when released from endothelial cells, nerve fibers, and odontoblasts, diffuses to the stromal cells to activate sGC and to increase cGMP formation.
NOS III was detected in the endothelium, while the 2- and the ß1-subunits of the sGC and cGMP were found in endothelium and vascular smooth muscle. NO-cGMP signaling molecules are involved in vasodilatation (Krumenacker et al., 2004). It is likely that NO regulates the pulpal vasodilatation sGC-cGMP-dependent mechanism. NO synthesized from nerve fibers and odontoblasts could also reach endothelial cells and vascular smooth-muscle cells of the dental pulp to regulate blood vessels by activating sGC.
In contrast to our and previous findings on cat and dog dental pulp (Lohinai et al., 1997), NADPH-d staining (Kerezoudis et al., 1993) was not detectable in nerve fibers of the rat molar dental pulp. This could be explained by the denaturation of essential epitopes or cross-linking of proteins, depending on the fixation protocols and the use of different antibodies (Burette et al., 2002). NOS I, the sGC 2-subunit, and cGMP in nerve fibers indicate an involvement of NO-cGMP signaling molecules in modulation of the neurotransmission in the dentin-pulp complex in an autocrine manner.
The odontoblast processes may extend to the peripheral dentin, supporting an NO- and/or NO-cGMP-mediated modulation of neurotransmission from peripheral dentin up to nerve endings in the dentin-pulp complex. The localization of the sGC 2-subunit in nerve endings in initial dentin tubules indicates that sGC at these localizations may act as a receptor for neuromediator NO that may be generated by NOS I and NOS III in odontoblasts, and in their peripheral processes, in a paracrine manner. This assumption is corroborated by the fact that nerve endings have not been described in the outer two-thirds of dentin (Byers, 1984). The suggestion that stimulation of the release of NO by K+ is associated with analgesia through a modulation of dentin nociception (McCormack and Davies, 1996) may be explained by a cGMP-independent action of NO on channel proteins through S-nitrosylation. NO synthesized in odontoblasts by NOS I and NOS III may modulate dentin nociception via both cGMP-dependent and -independent mechanisms (Davis et al., 2001; Krumenacker et al., 2004).
Treatment of rat molars with NONOate is associated with an increased accumulation of cGMP in odontoblasts and blood vessels. This finding suggests that sGC is a critical mediator for the production of cGMP in the dentin-pulp complex. Incubation of molars with L-NAME resulted in a decrease in staining intensities of cGMP in odontoblasts and blood vessels, indicating a basal NO-sGC-dependent regulation. Analysis of these data shows an involvement of sGC in the regulation of odontoblasts and blood vessels, and indicates that sGC at these locations can act as a mediator of the effects of NO.
In conclusion, the correlation of the cellular localizations of NOS I, NOS III, sGC (2-subunit, ß1-subunit), and cGMP and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an involvement of NO-sGC-cGMP signaling molecules in odontoblast differentiation, dentin biomineralization, pulpal vascular regulation, and modulation of neurotransmission in the dentin-pulp complex.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received February 26, 2004; Last revision April 8, 2005; Accepted April 14, 2005
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