HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Appendix Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Di Nardo Di Maio, F. Articles by Felaco, M. Search for Related Content PubMed PubMed Citation Articles by Di Nardo Di Maio, F. Articles by Felaco, M.

Nitric Oxide Synthase in Healthy and Inflamed Human Dental Pulp

¹ Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, 78/A Üllôi út, Budapest, Hungary, 1082; and
² School of Dentistry, University of Chieti, 1 Via dei Vestini, Chieti, Italy, 66100;

^* corresponding author, mfelaco{at}unich.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Nitric oxide synthase (NOS) plays a significant role in the pathogenesis of pulpitis. In this study, we hypothesized the existence of endothelial (eNOS) and inducible (iNOS) enzyme isoforms in human dental pulp. Extracted third molar pulps were divided into groups based on clinical diagnosis: healthy, hyperemic, and irreversible pulpitis. We have localized the eNOS and iNOS by immunohistochemistry and have tested their mRNA expression by RT-PCR and protein levels by Western blots. eNOS is present in the endothelial cells and odontoblasts of the healthy pulp, but an elevation of eNOS mRNA and protein levels with a concomitant dilation of vessels was characteristic under pathological conditions. Healthy pulp tissue failed to exhibit any iNOS; however, acute inflammation enhanced the mRNA and protein levels of iNOS, mainly in the leukocytes. There are differences in localization and expression between eNOS and iNOS in healthy and inflamed dental pulp.

KEY WORDS: nitric oxide synthase • human dental pulp • inflammation • endothelial cells • odontoblast

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Dental pulp inflammation is characterized by changes in blood flow (Olgart et al., 1991), immunocompetent cell function (Bergenholtz et al., 1991), and neuronal activity (Närhi and Hirvonen, 1983). Several mediators—including histamine, prostaglandins, and neuropeptides—have been shown to be involved in one or more of these processes (Hirafuji et al., 1980; Grutzner et al., 1992), while all steps may involve nitric oxide (NO) (Nathan, 1992). NO is an intracellular messenger molecule with important cardiovascular, neurological, and immune functions (Nathan, 1992). It is produced by a group of isoenzymes collectively termed NO synthases (NOS) (Bredt et al., 1990). Three distinct isoforms of NOS have been cloned to date: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS (Nathan, 1992). The eNOS and nNOS are constitutive isoforms that can rapidly synthesize small amounts of NO following receptor stimulation (Carmignani et al., 2000). The iNOS is mainly involved in the inflammatory processes. Pro-inflammatory stimuli trigger resident and immigrant inflammatory cell populations induce iNOS (Nussler and Billiar, 1993). The iNOS produces large amounts of NO for sustained periods of time, which has a role in a non-specific immune response, acting as toxic agent in infections (Nathan, 1992; Nussler and Billiar, 1993; Moilanen et al., 1999; Carmignani et al., 2000).

Histochemical identification of nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d, one possible marker of NOS [Hope et al., 1991]) and immunohistochemical detection of NOS were used for localization of NOS in rodent, feline, canine, and human dental pulp and periodontal tissues, as well as in the rat pulp after tooth preparation (Kerezoudis et al., 1993b; Lohinai et al., 1997, 1998; Lohinai and Szabó, 1998; Law et al., 1999; Felaco et al., 2000a). To date, however, no data are available on the localization and expression of NOS in the inflamed human tooth pulp.

The aim of this study was to examine and to compare the eNOS and iNOS in human healthy and inflamed dental pulps. We have localized the eNOS and iNOS by immunohistochemistry, identified their mRNA expression by RT-PCR, and detected their protein levels by Western blot analysis in normal and pathological conditions.

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
The local Ethics Committee approved the experimental protocol. The patients participated in the approved protocol after providing informed consent. Third molars (n = 30) extracted from subjects aged between 20 and 30 yrs were divided into 3 groups. The first group consisted of healthy dental pulps (n = 10) removed for orthodontic reasons and used as controls. The second group contained teeth (n = 10) with hyperemia (the pain remained for 15–20 sec after cold stimulation with cryo-spray; VOCO, Nußloch, Germany). Dental pulps (n = 10) with irreversible pulpitis comprised the third group, verified by clinical history, radiographics, and thermal tests by cryo-spray. After extraction, the dental pulps were harvested by tooth fracturing, immediately frozen, and kept at -80°C for 24–48 hrs. Then longitudinal serial sections of about 7 µm were cut with a cryostat (Reichert-Jung Frigocut 2800, Nußloch, Germany). For histopathological analysis, 3 slides from 5 pulps in each group were stained with hematoxylin-eosin. We used these slides to measure the arteriolar diameter and to localize the odontoblasts and the inflammatory cells.

Biochemical Identification of eNOS and iNOS
The immunohistochemical localization of eNOS and iNOS was performed in 3-3 slides of 5 pulps from each group, with primary rabbit anti-eNOS (1:100) or primary rabbit anti-iNOS (1:100) antibodies (Santa Cruz Biotech Inc., Santa Cruz, CA, USA) as described previously (Felaco et al., 2000a). The eNOS and iNOS mRNAs were demonstrated from homogenizates of 5 dental pulps of each group by RT-PCR with 5'-TGTCTGTCTGCTGCTAG-3' (sense) and 5'-CTCTCCAGGCACTTCAGGC-3' (antisense) for human eNOS and 5'-AGTGATGGCAAGCACGACTTC-3' (sense) and 5'-TCTGTCACTCGCTCACCACGG-3' (antisense) for human iNOS primer pairs as published earlier (Innis et al., 1990; Felaco et al., 2000a). The eNOS and iNOS protein expressions were detected from equal amounts of protein (50 µg, obtained by homogenization of 5 third molar pulps from each group with lysis buffer [Sigma-Aldrich Co., St. Louis, MO, USA]) by Western blots with primary anti-eNOS or -iNOS antibodies (Santa Cruz Biotech Inc., Santa Cruz, CA, USA) as described in detail (Felaco et al., 2000a; Di Napoli et al., 2001).

Image Processing, Image Analysis, and Statistical Evaluation
The stained sections of the pulps were examined with a Leitz Dialux 22 (LEICA, Heidelberg, Germany) microscope. The quantitative evaluations of the immune reactions were performed by determination of the integrated optical density (IOD) changes by digital image analysis. Three investigated areas were randomly selected and recorded on 3-3 slides/5 pulps in each group. For data processing, each experimental frame was digitized into 512 x 512 pixels by a Sony videocamera connected to a LEICA Quantimet 500 plus (LEICA Cambridge Ltd, Cambridge, UK), and the change in IOD was determined with ISO Transmission Density (Kodak CAT 152-3406, Eastman Kodak Company, Rochester, NY, USA) as a standard.

We used the same analysis system to measure the diameter of 20 randomly chosen arterioles in the hematoxylin-eosin-stained coronal pulps of the studied groups. The examination was based on more than 1 slide/5 pulps/group. The blood vessels selected were of homogenous appearance, free of apparent artifact, and perpendicular to the plane of section (Law et al., 1999).

All results were expressed as mean ± standard deviation (SD). We performed repeated-measures ANOVA to compare means between groups. Probability of null hypothesis of < 5% (p < 0.05) was considered as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Histological analysis of the healthy pulp tissue showed a column-shaped, linear odontoblastic layer surrounding the usual connective tissue elements of the pulp, including blood vessels, nerve fibers, and lymphocytes. In the hyperemic group, vasodilation of the pulp arterioles (healthy vs. hyperemic pulp, p < 0.05; Fig. 1) and appearance of the inflammatory cells, mostly PMNs, were found. In irreversible pulpitis, the sections showed more pronounced vasodilation (hyperemic pulp vs. irreversible pulpitis, p < 0.05; Fig. 1) with altered odontoblastic stratum. Furthermore, leukocyte infiltration (primarily macrophages and plasma cells) was another common finding. PMN cells were also frequent but not dominant.

View larger version (85K): [in this window] [in a new window]   Figure 1. Hematoxylin-eosin staining of a healthy pulp tissue (A), hyperemic (B), and irreversible pulpitis (C) (oil immersion, magnification 100X). The diameters of the randomly selected arterioles were recorded only when they were perpendicular to the plane of section. The diameters of the arterioles showed a statistically significant increase in parallel with the development of an inflammatory pathological process. Data are presented as means ± SD of 5 pulps in each pulp condition, with 20 measurements in each group. The vascular diameter (A = 22 µm, B = 32 µm, C = 48 µm) differences were significant in all 3 groups (*p < 0.05 A vs. B, *p < 0.05 B vs. C, *p < 0.05 A vs. C).

View larger version (164K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of eNOS and iNOS in healthy and inflamed human dental pulp. eNOS in: (A) normal pulp, (B) hyperemic pulp, and (C) irreversible pulpitis. iNOS in: (D) normal pulp, (E) hyperemic, and (F) irreversible pulpitis. Magnification, 25X. eNOS was visualized in the endothelial cells (arrows), odontoblasts (arrowheads), and in some fibroblasts (*) as well. In control pulp, the immunohistochemical localization revealed a lack of iNOS immunoreactivity. However, in hyperemic pulp and irreversible pulpitis, iNOS immunopositivity was characteristic mainly in the accumulated leukocytes and in the area adjacent to that of dense leukocytic infiltration (arrows). Densitometric analyses of eNOS and iNOS immunoreactivity in the 3 groups are presented as integrated optical density (IOD) and expressed as means ± SD of 5 pulps in each group, with 9 independent measurements in each pulp. In Table 1 (Appendix), significant differences are shown in all 3 groups of eNOS and iNOS IOD as well (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).

In irreversible pulpitis from moderate to none, eNOS immunoreactivity could be identified in the central pulp tissue and odontoblasts (Fig. 2C). The central pulp of the inflamed teeth, especially the leukocytes and adjacent to the area of dense leukocyte infiltration, showed very high iNOS immunoreactivity (Fig. 2F). Furthermore, iNOS immunopositivity can be observed in the external stratum of the pulp, in the odontoblasts as well, mainly near the accumulated leukocytes.

Control sections incubated without primary or secondary antibodies failed to exhibit any immunoreactivity.

The IOD values of eNOS are the highest in the hyperemic pulp (healthy vs. hyperemic pulp, p < 0.05, hyperemic pulp vs. irreversible pulpitis, p < 0.05), but in irreversible pulpitis, they are also higher than in normal pulp (healthy pulp vs. irreversible pulpitis, p < 0.05; Appendix Table 1). The IODs of iNOS immunoreactivity are negligible in the normal pulp and are elevated in acute inflammatory processes. Significant differences were found among all 3 groups (healthy vs. hyperemic pulp, p < 0.05, hyperemic pulp vs. irreversible pulpitis, p < 0.05, healthy pulp vs. irreversible pulpitis, p < 0.05, Appendix Table 1).

eNOS and iNOS mRNA Levels by RT-PCR Analysis
To evaluate the transcription of eNOS (300 bp) and iNOS (340 bp) in healthy and inflamed human pulp tissue, we investigated their mRNA levels (Fig. 3). Though mRNA of eNOS was present in all 3 groups, the highest level was found in the hyperemic group (normal vs. hyperemic pulp, p < 0.05, hyperemic pulp vs. irreversible pulpitis, p < 0.05, normal pulp vs. irreversible pulpitis, p < 0.05, Appendix Table 2). In contrast, while healthy pulp tissue fails to exhibit any mRNA for iNOS, it shows a significant rise in hyperemia and irreversible pulpitis (healthy vs. hyperemic pulp, p < 0.05, hyperemic pulp vs. irreversible pulpitis, p < 0.05, healthy pulp vs. irreversible pulpitis, p < 0.05, Appendix Table 2).

View larger version (61K): [in this window] [in a new window]   Figure 3. Occurrence of mRNA of eNOS in 300 bp (upper part) and iNOS in 340 bp (lower part) by RT-PCR in human dental pulp in the healthy tissue (A), hyperemic (B), and irreversible pulpitis (C). The standard band is on the right lane (S 488 bp). The Fig. clearly shows the presence of eNOS 300 bp in all 3 groups, with the highest level in the hyperemic group. The presence of iNOS 340 bp in the healthy pulp is not detectable; however, a marked increase of iNOS 340 bp was found in hyperemic and irreversible pulpitis groups. 18S (488 bp) is the internal standard. Data are expressed as means ± SD of 5 pulps in each pulp condition. Significant differences of integrated optical density (IOD) of eNOS and also iNOS mRNA, indicated in Table 2 (Appendix), were found in all 3 groups (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).

View larger version (53K): [in this window] [in a new window]   Figure 4. Western blot analysis of eNOS (upper part) and iNOS (lower part) proteins obtained from human healthy (A) and hyperemic (B) dental pulp, as well as from the pulp with irreversible pulpitis (C). The pulpal proteins were stained by antibodies against human eNOS antigen (133 kD) and iNOS antigen (130 kD). ß-actin is used for control. All 3 groups contain the eNOS enzyme, with the highest amount in the hyperemic group. The iNOS protein was undetectable in the healthy group. A significant increase of iNOS enzyme was observed in the hyperemic and the irreversible pulpitis groups. The panels at right show the densitometric analysis of eNOS (upper part) and iNOS (lower part) protein bands. Data are expressed as means ± SD of 5 pulps in each group. The differences of integrated optical density (IOD) of eNOS and also iNOS bands, indicated in Table 3 (Appendix), were significant in all 3 groups (*p < 0.05, A vs. B; *p < 0.05, B vs. C; *p < 0.05, A vs. C in both cases).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

Evidence of NADPH-d reactivity, eNOS mRNA, and protein in pulpal vessels is co-existent with the role of NO as a mediator of blood-flow regulation (Kerezoudis et al., 1993b; Lohinai et al., 1997; Law et al., 1999; Felaco et al., 2000ab). Indeed, systemic infusion of the NOS inhibitors markedly reduces basal blood flow and can also inhibit cholinergic-induced vasodilation in the pulp (Kerezoudis et al., 1993a; Lohinai et al., 1995; Olgart et al., 1996). Furthermore, administration of a NO donor compound significantly decreases pulpal vascular resistance (Lohinai et al., 1995). Analysis of these data indicates that, apart from a NO-dependent basal vasodilator tone, the control of the dental pulp vascular tone can be regulated via a stimulated release of endogenous NO (Lohinai et al., 1995). At the onset of an inflammatory process, there is an increase in the pulpal eNOS with a concomitant vasodilation; later, however, the large amount of NO formed by up-regulated iNOS than by eNOS may contribute to the further dilation of the vessels observed in irreversible pulpitis.

The functions of NOS in the odontoblasts are still unknown (Felaco et al., 2000a). NO produced by eNOS in the odontoblasts may regulate the vascular tone of the adjacent vessels. Furthermore, NO may modulate nociceptive input in both directions, since low levels of peripherally generated NO are algestic, while high levels of NO were found to be analgestic. It is conceivable that, according to the hydrodynamic pain sensation theory, the dentinal fluid movement in the tubules induces shear stress of the odontoblasts, which may activate eNOS as the blood-flow alterations activate eNOS in the endothelial cells. Thus, the odontoblasts would operate as receptor cells and be responsible for dentin sensitivity. Through this mechanism, NO may act as a coupling mechanism between the dental sensory input and the blood flow increase of the pulp. On the other hand, NO may down-regulate sensitized nociceptors as well, since, in the management of dentin hypersensivity by potassium ion, the putative second messenger is NO produced by eNOS or iNOS in the odontoblasts (McCormack and Davies, 1996). The exact role of NO in the intradental sensory unit needs to be clarified in further studies. Furthermore, the large amount of NO synthesized by iNOS in the odontoblasts under pathological conditions may not only have analgestic effect but also may dilate local vessels, and/or it is possible that the iNOS-derived NO in caries is part of the first line of defense, in the hard dental tissues, against invading oral micro-organisms (Silva Mendez et al., 1999).

Inflammation models in the literature show that constitutive NOS accounts for the most NOS activity at the onset of the process (Kubes et al., 1991). It is likely that NO formed by constitutive eNOS plays an anti-inflammatory role in healthy pulp and during the early stages of inflammation in hyperemic pulp; this mechanism is aimed to limit the process by inhibiting leukocyte and platelet adhesion/aggregation of the endothelial surface and by increasing tissue perfusion (Kubes et al., 1991; Nathan, 1992). In contrast to the eNOS, analysis of our data suggests that iNOS is not present in healthy pulp, but is induced only in pathological processes. This observation is in accordance with the results obtained by Law et al.(1999), although their findings are based on experimental pulpitis induced in laboratory animals, while our data are human natural bacterial pulpitis cases. The progress of inflammation in the dental pulp is accompanied by a marked enhancement of total NOS activity, most of which is attributed to iNOS of the activated leukocytes.

The high rate of iNOS-produced NO formation can play a dual role in pulpitis. On the one hand, NO has beneficial effects, because NO is an antimicrobial agent, since it decreases the viability of cariogenic bacteria (Silva Mendez et al., 1999). Furthermore, NO acts as an immune modulator, and inhibits formation of microvascular thrombi (Albina and Henry, 1991; Nathan, 1992). On the other hand, NO has detrimental effects as well, because the exaggerated production of NO can cause toxic actions against the pulpal tissues (Lohinai and Szabó, 1998). Furthermore, the excessive vasodilation and increased vascular permeability elicited by local high NO can raise the intrapulpal hydrostatic pressure, because the pulp is a low-compliance system and is located in a closed and rigid dental chamber (Lohinai et al., 1995). Consequently, the increased intrapulpal pressure may compress the pulpal venules and thus significantly impair the pulpal perfusion that insults the pulp tissue and might even cause pulpal necrosis.

In conclusion, we have found that healthy and inflamed human dental pulps have different expressions and localizations of eNOS and iNOS: eNOS maintains pulpal homeostasis, while iNOS has a role only in inflammatory pathological processes.

	ACKNOWLEDGMENTS

 
This study was supported by the Italian Ministry of Education, by University and Scientific Research Grants (2001), and by Hungarian Research Grants from OTKA (#F-030448), ETT (#152/2000), and BLUE & Blue Co. (Hungary). The authors express their gratitude to Eva Ahsberg Borromeo, Ágnes Gara, and Ibolya Kocsis for their highly skilled excellent technical assistance.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received March 21, 2002; Last revision November 11, 2003; Accepted January 9, 2004

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Albina JE, Henry WL Jr (1991). Suppression of lymphocyte proliferation through the nitric oxide synthesizing pathway. J Surg Res 50:403–409.[ISI][Medline]

Bergenholtz G, Nagaoka S, Jontell M (1991). Class II antigen expressing cells in experimentally induced pulpitis. Int Endod J 24:8–14.[ISI][Medline]

Bredt DS, Hwang PM, Snyder SH (1990). Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768–770.[Medline]

Buga GM, Griscavage JM, Rogers NE, Ignarro LJ (1993). Negative feedback regulation of endothelial cell function by nitric oxide. Circ Res 73:808–812.[Abstract/Free Full Text]

Carmignani M, Volpe AR, Boscolo P, Qiao N, Di Gioacchino M, Grilli A, et al. (2000). Catecholamine and nitric oxide systems as targets of chronic lead exposure in inducing selective functional impairment. Life Sci 68:401–415.[ISI][Medline]

Di Napoli P, Antonio Taccardi A, Grilli A, Spina R, Felaco M, Barsotti A, et al. (2001). Simvastatin reduces reperfusion injury by modulating nitric oxide synthase expression: an ex vivo study in isolated working rat hearts. Cardiovasc Res 51:283–293.[Abstract/Free Full Text]

Felaco M, Di Maio FD, De Fazio P, D’Arcangelo C, De Lutiis MA, Varvara G, et al. (2000a). Localization of the e-NOS enzyme in endothelial cells and odontoblasts of healthy human dental pulp. Life Sci 68:297–306.[ISI][Medline]

Felaco M, Grilli A, Gorbunov N, Di Napoli P, De Lutiis MA, Di Giulio C, et al. (2000b). Endothelial NOS expression and ischemia-reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions. Biochim Biophys Acta 1524:203–211.[Medline]

Grutzner EH, Garry MG, Hargreaves KM (1992). Effect of injury on pulpal levels of immunoreactive substance P and immunoreactive calcitonin gene-related peptide. J Endod 18:553–577.[ISI][Medline]

Hirafuji M, Satoh S, Ogura Y (1980). Prostaglandins in rat pulp tissue. J Dent Res 59:1535–1540.[Free Full Text]

Hope BT, Michael GJ, Knigge KM, Vincent SR (1991). Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811–2814.[Abstract/Free Full Text]

Innis MA, Gelfand DH, Sninsky J, White TJ, editors (1990). PCR protocols, a guide to methods and applications. San Diego: Academic Press.

Kerezoudis NP, Olgart L, Edwall L (1993a). Differential effects of nitric oxide synthesis inhibition on basal blood flow and antidromic vasodilation in rat oral tissues. Eur J Pharmacol 241:209–219.[ISI][Medline]

Kerezoudis NP, Olgart L, Fried K (1993b). Localization of NADPH-diaphorase activity in the dental pulp periodontium and alveolar bone of the rat. Histochemistry 100:319–322.[ISI][Medline]

Kubes P, Suzuki M, Granger DN (1991). Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 88:4651–4655.[Abstract/Free Full Text]

Law AS, Baumgardner KR, Meller ST, Gebhart GF (1999). Localization and changes in NADPH-diaphorase reactivity and nitric oxide synthase immunoreactivity in rat pulp following tooth preparation. J Dent Res 78:1585–1595.[Abstract/Free Full Text]

Lohinai Z, Szabó C (1998). Role of nitric oxide in physiology and pathophysiology of periodontal tissues. Med Sci Monit 4:1089–1095.

Lohinai Z, Balla I, Marczis J, Vass Z, Kovách AG (1995). Evidence for the role of nitric oxide in the circulation of the dental pulp. J Dent Res 74:1501–1506.[Abstract/Free Full Text]

Lohinai Z, Székely AD, Benedek P, Csillag A (1997). Nitric oxide synthase containing nerves in the cat and dog dental pulp and gingiva. Neurosci Lett 227:91–94.[ISI][Medline]

Lohinai Z, Benedek P, Fehér E, Györfi A, Rosivall L. Fazekas A, et al. (1998). Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat. Br J Pharmacol 123:353–360.[ISI][Medline]

McCormack K, Davies R (1996). The enigma of potassium ion in the management of dentine hypersensitivity: is nitric oxide the elusive second messenger? Pain 68:5–11.[ISI][Medline]

Moilanen E, Whittle BJ, Moncada S (1999). Nitric oxide as a factor in inflammation. In: Inflammation: basic principles and clinical correlates. Gallin JI, Snyderman R, editors. Philadelphia: Lippincott Williams & Wilkins.

Närhi M, Hirvonen T (1983). Functional changes in cat pulp nerve activity after thermal and mechanical injury of the pulp. Proc Finn Dent Soc 79:162–167.[Medline]

Nathan C (1992). Nitric oxide as a secretory product of mammalian cells. FASEB J 6:3051–3064.[Abstract]

Nussler AK, Billiar TR (1993). Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 54:171–178.[Abstract]

Olgart L, Edwall L, Gazelius B (1991). Involvement of afferent nerves in pulpal blood-flow reactions in response to clinical and experimental procedures in the cat. Arch Oral Biol 36:575–581.[ISI][Medline]

Olgart L, Kostouros GD, Edwall L (1996). Local actions of acetylcholine on vasomotor regulation in rat incisor pulp. Acta Physiol Scand 158:311–316.[ISI][Medline]

Silva Mendez LS, Allaker RP, Hardie JM, Benjamin N (1999). Antimicrobial effect of acidified nitrite on cariogenic bacteria. Oral Microbiol Immunol 14:391–392.[ISI][Medline]

This Article Abstract Figures Only Full Text (PDF) Appendix Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Di Nardo Di Maio, F. Articles by Felaco, M. Search for Related Content PubMed PubMed Citation Articles by Di Nardo Di Maio, F. Articles by Felaco, M.