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Nitric Oxide Synthase and PGE2 Reciprocal Interactions in Rat Dental Pulp: Cholinoceptor Modulation
Basic Research—Biology
Nitric Oxide Synthase and PGE2 Reciprocal Interactions in Rat Dental Pulp: Cholinoceptor Modulation Enri Borda,*† César Furlan,* Betina Orman,* Silvia Reina,* and Leonor Sterin-Borda*† Abstract In this study we determined the effect of cholinoceptor agonist pilocarpine on the stimulation of nitric oxide synthase (NOS) and on prostaglandin E2 (PGE2) generation upon rat dental pulp. By reverse transcriptase/ polymerase chain reaction (RT-PCR) we identified several products corresponding to m1, m2, m3, and m4 muscarinic acetylcholine receptors (mAChRs). The stimulation of M1, M2, M3, and M4 mAChRs by pilocarpine increases NOS activity and PGE2 generation. There is a correlation (correlation coefficient ⫽ 0.05) between NOS activity and PGE2 generation through the activation of phosphoinositide by phospholipase C (PLC), phospholipase A2 (PLA2), and cyclooxygenase 1 (COX-1). Exogenous PGE2 restored NOS activity inhibited by indomenthacin (INDO), whereas nitric oxide (NO) donor restored PGE2 generation inhibited by NG-methyl-Larginine acetate salt (L-NMMA). These data indicate that both NO and PGE2 interact with their own respective biosynthetic pathways modulating NOS and COX activities. Results could contribute to understanding the involvement of NO and PGE2 in healthy dental pulp given that cellular signals through the parasympathetic system modulate the function of the dentin–pulp complex. (J Endod 2007;33:142–147)
Key Words Dental pulp, muscarinic acetylcholine receptor, nitric oxide, nitric oxide synthase, pilocarpine, prostaglandin E2
From the *Pharmacology Unit, School of Dentistry, University of Buenos Aires; and †Argentine National Research Council (CONICET), Buenos Aires, Argentina. This work was supported by University of Buenos Aires (UBACYT, O-014) and Argentine National Research Council (PIP, 05680), Buenos Aires, Argentina. Address requests for reprints to Dr. Enri S. Borda, Pharmacology Unit, School of Dentistry, University of Buenos Aires, M.T. de Alvear 2142– 4° “B,” 1122 AAH Buenos Aires, Argentina. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.11.009
T
he dental pulp is a loose mesenchymal tissue characterized by its particular location and almost entirely enclosed in dentine, a mineralized tissue. Because of its topography the tissue has low interstitial compliance and limited capacity to expand during fluid volume changes (1). Innervation of dental pulp includes autonomic unmyelinated (cholinergic and adrenergic) and sensory nerve fibers that may also subserve dentinal fluid dynamics and regulate pulp blood flow (2). A striking feature of dental pulp innervation is its high density relative to that of other tissues in the body (3, 4). Dentists have developed many strategies to prevent infection because inflammation frequently leads to necrosis and subsequent loss of dental pulp (2). Several studies indicate a parasympathetic innervation in the dental pulp (5, 6) distributed around small blood vessels. Mediator release by parasympathetic nerves controls the pulpal blood flow, thus changing the diameter of blood vessels (7). The physiological role of parasympathetic vasomotor regulation in dental pulp is a controversial issue (2). Response to muscarinic acetylcholine receptors (mAChR) might vary according to the multiple subtypes of mAChR. Therefore, it is of great importance to determine their presence and function to define the real role played by mAChR in healthy dental pulp. It has been well established that the activation of mAChR triggers the release of two proinflammatory mediators, nitric oxide (NO) and prostaglandin E2 (PGE2) (8). NO, formed by NO synthases (NOS), is an important intercellular messenger in vascular, immune, and neuronal cells. At least three isoforms of NOS have been cloned: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (9). The eNOS and nNOS release small amounts of NO for a short period of time after receptor stimulation. In contrast, iNOS is expressed in response to proinflammatory stimuli and produces a large amount of NO for sustained periods of time (10). Prostaglandins (PGs) are biologically active lipids synthesized by the body from the precursor arachidonic acid, which in turn might generate NO (11). These bioactive autacoids have been found in many mammalian cells and some PGs have been identified as mediators of inflammation (12, 13). Biosynthesis of PGs occurs in rat dental pulp organ (13) and the role of PGs in mediating pulp inflammation has been shown. In later research (14, 15), PGE2 has been strongly implicated in pulpal inflammation, particularly in vascular permeability and vasodilation. There is a significant body of experimental evidence suggesting a relationship between NO biosynthesis and prostaglandin generation. NO donors have been reported to stimulate or inhibit prostaglandin biosynthesis in a variety of cellular or broken cellular systems. These effects may be mediated either by altered transcription of cyclooxygenase (COX) genes (16) by inhibition or stimulation of COX activity or by inactivation of downstream metabolizing enzymes that convert prostaglandin endoperoxides to stable eicosanoid products (17). The present works aims to characterize mAChR subtypes and the modulatory effect of cholinergic agonist pilocarpine on healthy dental pulp. The NO/PGE2 pathways were also determined. Figure 1 provides an introductory scheme showing parasympathetic fibers, ACh release, mAChR on target cells, and NO/PGE2 physiological effects on rat dental pulp.
Materials and Methods Animals Male Wistar rats from the Pharmacologic Bioterium (School of Dentistry, University of Buenos Aires) weighing 220 –260 g were used all through the experiments. The
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Basic Research—Biology mRNA Isolation and cDNA Synthesis Total RNA was extracted from rat dental pulp by homogenization using guanidinium isothiocyanate-phenol-chloroform extraction (18). Total RNA was extracted following the technique previously described (19) and adapted for rat dental pulp. A 20-L reaction mixture contained 2 ng of mRNA, 20 units of RNase inhibitor, 1 mM dNTPs, and 50 units of Moloney murine leukemia virus RT (Promega, Madison, WI, USA). First-strand cDNA was synthesized at 37°C for 60 minutes.
Figure 1. Muscarinic receptor-mediated vasodilatation in rat dental pulp. The arrival of an action potential at the cholinergic nerve terminal triggers the release of acetylcholine (ACh). Once released from the nerve terminal ACh elicits cellular response by activating post-synaptic receptors located on endothelium blood vessels, fibroblasts, and macrophages. The stimulation of mAChR on endothelium triggers the activation of nitric oxide synthase (NOS) that in turn catalyzes nitric oxide (NO) synthesis. The NO diffuses into the smooth muscle where it mediates relaxation. On the other hand, activation of mAChR on fibroblasts and macrophages causes the release of PGE2, which also causes the vessel smooth muscle to relax.
animal experiments were approved by the local Animal Ethics Committee at University of Buenos Aires. The animals were subjected to ambient environmental conditions (23–25°C, 12-hour dark/light cycle) and maintained with unrestricted access to water and food. The animals were killed by cervical dislocation.
Exposure of Dental Pulp Exposure of healthy rat dental pulp of upper incisors (left and right) was performed. After incisor teeth were removed horizontally with diamond burs to expose the pulp chamber at the pulp horn, they were carefully extracted and immediately kept at room temperature in Krebs Ringer bicarbonate (KRB) solution in the presence of CO2 in oxygen until different experimental assays could be carried out. Two teeth were used to harvest pulp for each assay.
PCR Procedures NOS isoform-mRNA levels were determined by a method that involves simultaneous co-amplification of both the target cDNA and a reference template (MIMIC) with a single set of primers. MIMICs for m1, m2, m3, and m4 mAChRs and glyceraldehyde-3-phosphate dehydrogenase (g3pdh) (Table 1) were constructed using a PCR MIMIC construction kit. Each PCR MIMIC consists of a heterologous DNA fragment with 5=- and 3=-end sequences that were recognized by a pair of genespecific primers. Sizes of PCR MIMIC were distinct from those of native targets. Sequences of oligonucleotide primer pairs used for construction of MIMIC and amplification of mAChR isoforms and g3pdh mRNA were as reported previously (20). Aliquots were taken from pooled first-strand cDNA from the same group and constituted one sample for PCR. A series of 10-fold dilutions of known concentrations of the MIMIC were added to PCR amplification reactions containing the first-strand cDNA. PCR MIMIC amplification was performed in 100 L of a solution containing 1.5 mM MgCl2, 0.4 M primer, dNTPs, 2.5 U Taq DNA polymerase, and 0.056 M Taq Start antibody (Clontech Laboratories, MountainView, CA, USA). After initial denaturation at 94°C for 2 minutes, the cycle condition was 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 45 seconds for enzymatic primer extension at 72°C for 45 cycles for mAChR subtypes. The internal control was the mRNA of the housekeeping gene g3pdh. PCR amplification was performed with initial denaturation at 94°C for 2 minutes, followed by 30 cycles of amplification. Each cycle consisted of 35 seconds at 94°C, 35 seconds at 58°C, and 45 seconds at 72°C. Samples were incubated for an additional 8 minutes at 72°C before completion. PCR products were subjected to electrophoresis according to previously described procedures (19). Different mAChR subtypes and mRNA levels were normalized with the levels of g3pdh mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis. Prostaglandin E2 (PGE2) Assay Rat dental pulp (10 mg) preparations were incubated for 60 minutes in 0.50 mL of KRB gassed with 5% CO2 in oxygen at 37°C. Pilocarpine were added 30 minutes before the end of the incubation period and blockers 30 minutes before the addition of different pilocarpine concentrations. Dental pulp was then homogenized into a 1.5-mL polypropylene microcentrifuge tube. Thereafter, all procedures used were those indicated in the protocol of Prostaglandin E2 Biotrak Enzyme Immuno Assay (ELISA) System (Amersham Biosciences, Piscataway, NJ, USA). The PGE2 results were expressed as picograms/well (pg/well).
TABLE 1. Oligonucleotides of primers for PCR Gene Product m1 m2 m3 m4 g3pdh
Sense
Anti-sense
Predicted Size (bp)
5=-GGGAGC TGGCCG CCCTGC-3= 5=-GATGAA AACACA GTTTCC ACTTC-3= 5=-GTGGTG TGATGA TTGGTC TG-3= 5=-ACCCAG ACCACC AAGGAA CGGCCA-3= 5=-ACCACA GTCCAT GCCATC AC-3=
5=-GCCTTT CTTGGT GGGCCT C-3= 5=-ATGATG AAAGCC AACAGG ATAGCC-3= 5=-TCTGCC GAGGAG TTGGTG TC-3= 5=-GGTACC TCACGG TGTCTG GGAGAC-3= 5=-TCCACC CACCC TGTTGC TGTA-3=
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Figure 2. RT-PCR product of m1, m2, m3, and m4 obtained from rat dental pulp (8 teeth). Tissues were prepared as described in Materials and Methods and then mRNA isolation and DNA synthesis were extracted and determined.
Determination of Nitric Oxide Synthase (NOS) Activity NOS activity was measured in rat dental pulp by production of [U-14C]-citrulline from [U-14C]-arginine according to the procedure previously described for brain slices (21). Briefly, after 20 minutes in preincubation KRB solution, tissues were transferred to 500 mL of prewarmed KRB equilibrated with 5% CO2 in O2 in the presence of [U-14C]arginine (0.5 mCi). Drugs were added and incubated for 20 minutes under 5% CO2 in O2 at 37°C. Tissues were then homogenized with an Ultraturrax in 1 mL of medium containing 20 mM HEPES (pH 7.4), 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM leupeptin, and 0.2 mM phenylmethylsulphonyl fluoride (PMSF) at 4°C. After centrifugation at 20,000 g for 10 minutes at 4°C, supernatants were applied to 2-mL columns of Dowex AG 50 WX-8 (sodium form); [14C]-citrulline was eluted with 3 mL of water and quantified by liquid scintillation counting. Measurement of Total Labeled Inositol Phosphates (InsP) Preparations of rat dental pulp were incubated for 120 minutes in 0.5 mL of KRB gassed with 5% CO2 in O2 with 1 Ci [myo-3H]-inositol ([3H]-MI) (sp. act. 15 Ci mmol⫺1) from Dupont/New England Nuclear (Boston, MA, USA). LiCl (10 mM) was added for inositol monophosphate accumulation. Pilocarpine was added 30 minutes before the end of the incubation period and the blockers 30 minutes before the addi-
Figure 4. Correlation in the modulatory effect of pilocarpine on NOS activity and PGE2 generation. NOS was plotted as a function of PGE2 (Pearson r: 0.9912; p value: 0.0010; alpha: 0.05). Values are the mean of six experiments of each group (12 teeth). For other details see legend of Fig. 1.
tion of the agonist. Water-soluble InsP was extracted after a 120-minute incubation and the results were expressed as area per milligrams of wet weight tissue (area/mg wet weight).
Drugs Pilocarpine, AF-DX 116, pirenzepine, 4-DAMP, tropicamide, atropine, U-73122, L-arginine, NG-methyl-L-arginine acetate salt (L-NMMA), indomethacin (INDO), and prostaglandin E2 (PGE2) were purchased from Sigma Chemical Company (St. Louis, MO, USA); 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA) was obtained from Tocris Cookson (Ellisville, MO, USA). Stock solutions were freshly prepared in the corresponding buffers. Statistical Analysis Student’s t test for unpaired values was used to determine the levels of significance. Analysis of variance (ANOVA) and Student–Newman–
Figure 3. (A) Increase in NOS activity of rat dental pulp by increasing concentrations of pilocarpine (). (B) Increase in PGE2 generation of rat dental pulp by increasing concentrations of pilocarpine (). The inhibitory action of 1 ⫻ 10⫺7 M pirenzepine (), 1 ⫻ 10⫺7 M AF-DX 116 (Œ), 1 ⫻ 10⫺6 M 4-DAMP (□), and 1 ⫻ 10⫺6 M tropicamide (〫) are also shown. Values represent the mean ⫾ SEM of nine experiments in each group (18 teeth). No effects were observed with M1, M2, M3, and M4 antagonists alone at the concentration used.
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Figure 5. Effect of 1 ⫻ 10⫺6 M INDO (Œ), 1 ⫻ 10⫺5 M L-NMMA (), 1 ⫻ 10⫺6 M U-73122 (□), and 5 ⫻ 10⫺5 M OBAA (〫) on the dose–response curve of pilocarpine () upon NOS activity (A) and PGE2 generation (B). Values are mean ⫾ SEM of seven experiments in each group (14 teeth). For other details see legend of Fig. 1; p ⬍ 0.001 between pilocarpine alone vs. pilocarpine in the presence of inhibitory agents.
Keuls test were used when pairwise a multiple comparison procedure was necessary. Differences between means were considered significant if p ⬍ 0.05.
Results As can be seen in Fig. 1, using specific oligonucleotide primers, RT-PCR amplified products showed bands of the predicted size for m1, m2, m3, and m4 mAChRs detected in the tissue, indicating that all four mAChR subtypes are expressed in the rat dental pulp. Figure 2 shows that pilocarpine from 1 ⫻ 10⫺10 to 1 ⫻ 10⫺7 M induced a concentration-dependent stimulation on NOS activity (A) and PGE2 generation (B). The selective M1, M2, M3, and M4 mAChR antagonists could antagonize the increased NOS activity and PGE2 generation, shifting the dose–response curves of pilocarpine to the right (Fig. 2A and B). The orders of potency for reducing NOS activation and PGE2 generation are similar: AF-DX 116 ⬎ pirenzepine ⬎ 4-DAMP ⫽ tropicamide.
Figure 3 demonstrated that under identical experimental conditions a significant correlation (correlation coefficient ⫽ 0.05) between pilocarpine-stimulated NOS activity and PGE2 generation was found. These results indicated that M1, M2, M3, and M4 mAChR activation induces an increase in NO as a result of the increase in PGE2 generation or vice versa. To determine whether NOS activity and PGE2 generation are dependent on each other, dental pulp preparations were incubated with different inhibitors of the enzymatic pathways known to be involved in the PGE2 and NO production by activation of mAChR subtypes. Figure 4A and B show that the inhibition of: COX-1 by indomethacin (INDO) (1 ⫻ 10⫺6 M), phospholipase A2 (PLA2) by OBAA (5 ⫻ 10⫺6 M), nitric oxide synthase (NOS) by L-NMMA (1 ⫻ 10⫺5 M), and phospholipase C (PLC) by U-73122 (1 ⫻ 10⫺6 M) attenuated the stimulatory action of different pilocarpine concentrations upon both NOS activity and PGE2 generation. Moreover, 1 ⫻ 10⫺8 M exogenous PGE2 restored NOS activity inhibited by INDO (Fig. 5A). On the other hand, 1 ⫻ 10⫺5 M
Figure 6. (A) Histograms shows basal, pilocarpine (1 ⫻ 10⫺8 M) alone, pilocarpine in the presence of INDO (1 ⫻ 10⫺6 M), and pilocarpine plus INDO restored by adding PGE2 (1 ⫻ 10⫺8 M) upon rat dental pulp. (B) Histograms show basal, pilocarpine alone (1 ⫻ 10⫺8 M), pilocarpine in the presence of L-NMMA (1 ⫻ 10⫺5 M), and pilocarpine plus L-NMMA restored by adding PGE2 (1 ⫻ 10⫺8 M). Values are mean ⫾ SEM of six experiments performed by duplicate in each group (12 teeth); p ⬍ 0.001 between pilocarpine alone vs. pilocarpine ⫹ INDO or pilocarpine ⫹ L-NMMA.
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Figure 7. Proposed model for the mechanisms where pilocarpine up-regulates NO/PGE2 pathway on healthy dental pulp. Pilocarpine acting on M1/M2 and M3/M4 mAChRs activates PLC mediating production of inositol-triphosphate (IP3). IP3 triggers intracellular release of calcium (Ca⫹⫹). Free Ca⫹⫹ activates nitric oxide synthase (NOS) with an increment of nitric oxide (NO). NO in turn stimulates cyclooxygenase 1 (COX-1) with increased production of PGE2. Free Ca⫹⫹ also activates phospholipase A2 (PLA2) with increased production of PGE2. On the other hand, PGE2 activated PLC increases NOS activity using free Ca⫹⫹ as an enzyme cofactor. Gray arrows indicate the site where the inhibition agents act.
L-arginine restored PGE2 generation inhibited by L-NMMA (Fig. 5B). Also, 1 ⫻ 10⫺8 M pilocarpine increased total phosphoinositol accumulation (area/mg wet weight: basal: 52 ⫾ 5, n ⫽ 6, pilocarpine: 105 ⫾ 8, n ⫽ 6) and 1 ⫻ 10⫺7 M atropine (60 ⫾ 6, n ⫽ 5) and 1 ⫻ 10⫺6 M U-73122 (49 ⫾ 5, n ⫽ 5) inhibited its accumulation, thus confirming participation of PLC in pilocarpine effects.
Discussion The current study provides pharmacological evidence for the existence of functional mAChR subtypes in isolated rat dental pulp and indicates that both NOS activity and PGE2 generation are involved in the mAChR mediated stimulatory effect of pilocarpine. Our findings demonstrate that the pilocarpine effect is related to M1/M2 and M3/M4 subtypes, given that the specific M1, M2, M3, and M4 cholinoceptor antagonists blocked the mAChR effect. Furthermore, our current results show greater effectiveness of M1/M2 when compared to M3/M4 antagonists to inhibit the pilocarpine-dependent increases in NOS activity and PGE2 generation. 146
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The mechanism by which pilocarpine-induced stimulation of NOS activity and PGE2 generation on dental pulp appears to occur secondarily to the activation of a number of enzymatic pathways commonly associated with receptor signaling. Here we provide evidence for a functional link between NO and PGE2 generation because INDO (COX-1 inhibitor), OBAA (PLA2 inhibitor), U-73122 (PLC inhibitor), and L-NMMA (NOS inhibitor) abrogated the increase of both NOS activity and PGE2 generation by pilocarpine. The fact that addition of a low concentration of PGE2 reversed the INDO-inhibited pilocarpine-increased NOS activity indicates a direct action of this prostanoid on NOS activity. On the other hand, exogenous L-arginine was able to reverse the inhibitory action of L-NMMA on pilocarpine-induced PGE2 generation, indicating that NO subserves PGE2 generation triggered by mAChR activation. Prostaglandins and NO represent some of the most relevant local mediators that participate under basal conditions, in the modulation of many cellular functions. In vivo studies demonstrated that the NOdependent pathway plays an important role in regulating the basal vasodilator tone and blood circulation in dental pulp (22). Furthermore, the substance P released from the sensory nerve endings produces vasodilatation, thus inducing NO production from endothelial cells (23). The biosynthesis and release of NO and prostaglandins share a number of similarities. Under normal circumstances the constitutive isoforms of these enzymes (constitutive NOS and COX) are found in virtually all organs. Accumulated evidence indicates that the cross talk between NO and prostaglandins at many levels exists (24), although this point still remains to be proved in dental pulp. An important link between NOS and COX pathways has been shown through studies that raised the possibility that COX enzymes represent important endogenous “receptor” targets for modulating the multifaceted roles of NO (16). On the other hand, NO biosynthesis may also modulate arachidonic acid release and COX metabolite generation (17). The current data indicate that the activation of NOS correlated with PGE2 generation. Moreover, both NO and PGE2 interact with their own respective biosynthetic pathways by modulating molecular events underlying NOS and COX activities. Based on the reciprocal interactions between NO and PGE2 (25), we observed that PLC and PLA2 are implicated in the pilocarpine effect, as was demonstrated in intact cells and membrane preparations (25). It has been well established that activation of PLC and PLA2 constitutes an important function of G-protein– coupled receptors (26). Therefore, pilocarpine by stimulating mAChR activates PLC, which hydrolyzes PIP2 into IP3 with a subsequent increase in the level of intracellular calcium, in turn inducing the direct activation of PLA2 (27). In fact, our results show that pilocarpine is able to increase the accumulation of inositol phosphates by PLC coupled to mAChR activation. The molecular mechanism involved in both NOS and COX activation may play a crucial role in understanding the reciprocal interaction between NO and COX. NO gas directly increases COX-1 activity of microsomal seminal vesicles, as well as recombinant COX-1. The activation of COX-1 leads to a remarkable sevenfold increase in PGE2 formation (16). In this context, there is a significant body of experimental evidence that suggests that NO and/or NOS activity stimulate COX-1 activity and production of prostanoids, mainly PGE2 (28). Whether PGE2 stimulated NOS activity is an open question. Our study demonstrates a stimulatory action of PGE2 on NOS activity. The mechanism by which PGE2 stimulates NOS activity appears to involve an increase in the hydrolysis of phosphoinositides by PLC, where calcium/calmodulin could act as an enzyme cofactor as was reported from PGE2-activated NOS on the submandibulary gland (29). Our finding suggests that pilocarpine—through its mAChR subtypes—increases basal levels of NO and PGE2, modulating their own release by affecting NOS and COX activities, respectively.
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Basic Research—Biology Figure 6 shows a diagram that ties together the various systems studied and Fig. 7 proposes a model for the mechanisms where pilocarpine activating mAChR up-regulates the NOS/ PGE2 pathway in dental pulp. In conclusion, this paper indicates a modulatory role of the parasympathetic system in dental pulp. The dense parasympathetic nerve supply of dental pulp might not only exert neuronal control, but it may also modulate the large terminal arteriolar diameter (30), suggesting an important role of neuronal regulation on tone vessel (3). NO/PGE2 might represent a signaling-dependent modulation of odontoblasts, blood vessels, and parasympathetic nerve fibers in the dentin–pulp complex.
Acknowledgment We thank Mrs. Elvita Vannucchi and Fabiana Solari for outstanding technical assistance. Also, the assistance of art designer Mr. Alejandro Thornton is gratefully acknowledged.
References 1. Bletsa A, Berggreen E, Fristad I, Tenstad O, Wiig H. Cytokine signalling in rat pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-induced acute inflammation. J Physiol 2006;573:225–36. 2. Olgart L, Kostouros GD, Edwall L. Local actions of acetylcholine on vasomotor regulation in rat incisor pulp. Acta Physiol Scand 1996;158:311– 6. 3. Zhang JQ, Nagata K, Iijima T. Scanning electron microscopy and immunohistochemical observations of the vascular nerve plexuses in the dental pulp of rat incisor. Anat Rec 1998;251:214 –20. 4. Iijima T, Zhang JQ. Three-dimensional wall structure and the innervation of dental pulp blood vessels. Microsc Res Tech 2002;56:32– 41. 5. Akai M, Wakisaka S. Distribution of peptidergic nerves. In: Inoqui R, Kudo T, Olgart LM, eds. Dynamic aspects of dental pulp. Cambridge, UK: Chapman & Hall, 1990:337– 48. 6. Luthman J, Luthman G, Hökfelt T. Occurrence and distribution of different neurochemical markers in the human dental pulp. Arch Oral Biol 1992;37:193–208. 7. Kim S, Dörscher-Kim JE, Lipowsky HH. Quantitative assessment of microcirculation in the rat dental pulp in response to alpha and beta adrenergic agonists. Arch Oral Biol 1989;100:1387–95. 8. Perez Leiros C, Rosignoli F, Genaro AM, Sales ME, Sterin-Borda L, Borda E. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in the rat salivary gland. J Auton Nerv Syst 2000;79:99 –107. 9. Liaudet L, Soriano FG, Szabo C. Biology of nitric oxide signaling. Crit Care Med 2000;28:N37–N52. 10. Shouthan GJ, Szabo C. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem Pharmacol 1996;51:383–94.
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11. Genaro AM, Stranieri GM, Borda E. Involvement of the exogenous nitric oxide signaling system in bradykinin receptor activation in rat submandibular salivary gland. Arch Oral Biol 2000;45:725–9. 12. Waterhouse PJ, Whitworth JM, Nunn JH. Development of a method to detect and quantify prostaglandin E2 in pulp blood from cariously exposed, vital primary molar teeth. Int Endod J 1999;32:381–7. 13. Antila R, Pohto P. In vitro studies on the prostaglandin system in tooth pulp. Proc Finn Dent Assoc 1984;80:245–52. 14. Dewhirst FE, Goodson JM. Prostaglandin synthase inhibition by eugenol, guaiacol and other dental medicaments. J Dent Res 1974;53:199. 15. Okiji T, Morita I, Zunada I, Murota S. Involvement of arachidonic acid metabolites increases in vascular permeability in experimental dental pulp inflammation in the rat. Arch Oral Biol 1989;34:523– 8. 16. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman G. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 1993;90:7240 – 4. 17. Mollace V, Muscoli C, Masini E, Cuzzocrea S, Salvemini D. Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors. Pharmacol Rev 2005;57:217–52. 18. Chomozynski P, Saachi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Ann Biochem 1987;162:156 –9. 19. Sterin-Borda L, Ganzinelli S, Berra A, Borda E. Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels. Neuropharmacology 2003;45:260 –9. 20. Orman B, Reina S, Borda E, Sterin-Borda L. Signal transduction underlying carbachol-induced PGE2 generation and COX-1 mRNA expression of rat brain. Neuropharmacology 2005;48:757– 65. 21. Borda T, Genaro AM, Sterin-Borda L, Cremaschi G. Involvement of endogenous nitric oxide signaling system in brain muscarinic acetylcholine receptor activation. J Neural Transm 1998;105:193–204. 22. Lohinai Z, Balla I, Marczis J, Vass Z, Kovach AG. Evidence for the role of nitric oxide in the circulation of the dental pulp. J Dent Res 1995;74:1501– 6. 23. Karabucak B, Walsch H, Jou YT, Simchon S, Kim S. The role of endothelial nitric oxide in the substance P induced vasodilation in bovine dental pulp. J Endod 2005;31:733– 6. 24. Clancy R, Varenika B, Huang W, et al. Nitric oxide synthase/COX cross-talk: nitric oxide activates COX-1 but inhibits COX-2 derived prostaglandin production. J Immunol 2000;165:1582–7. 25. Wallace JL, Del Soldato P. The therapeutic potential of NO-NSAIDs. Fundam Clin Pharmacol 2003;17:11–20. 26. Chakraborti S. Phospholipase A2 isoforms: a perspective. Cell Signal 2003;15:637– 65. 27. Pochet S, Métioui M, Grosfils K, Gomez-Muñoz A, Marino A, Dehaye JP. Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells. Cell Signal 2002;15:103–13. 28. Marnett LJ, Wrigth TL, Crews BC, Tannenbaum SR, Morrow JD. Regulation of prostaglandin biosynthesis by nitric oxide by targeted deletion of iNOS. J Biol Chem 2000;5:13427–30. 29. Borda E, Heisig G, Busch L, Sterin-Borda L. Nitric oxide synthase/prostaglandin E2 cross-talk in rat submandibular gland. Prost Leuk Ess Fatty Acids 2002;67:39 – 44. 30. Kerezoudis NP, Fried K, Olgart L. Haemodynamic and inmunohistochemical studies of rat incisor pulp after denervation and subsequent re-innervation. Arch Oral Biol 1995;40:815–23.
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Nitric Oxide Synthase and PGE2 Reciprocal Interactions in Rat Dental Pulp: Cholinoceptor Modulation Enri Borda,*† César Furlan,* Betina Orman,* Silvia Reina,* and Leonor Sterin-Borda*† Abstract In this study we determined the effect of cholinoceptor agonist pilocarpine on the stimulation of nitric oxide synthase (NOS) and on prostaglandin E2 (PGE2) generation upon rat dental pulp. By reverse transcriptase/ polymerase chain reaction (RT-PCR) we identified several products corresponding to m1, m2, m3, and m4 muscarinic acetylcholine receptors (mAChRs). The stimulation of M1, M2, M3, and M4 mAChRs by pilocarpine increases NOS activity and PGE2 generation. There is a correlation (correlation coefficient ⫽ 0.05) between NOS activity and PGE2 generation through the activation of phosphoinositide by phospholipase C (PLC), phospholipase A2 (PLA2), and cyclooxygenase 1 (COX-1). Exogenous PGE2 restored NOS activity inhibited by indomenthacin (INDO), whereas nitric oxide (NO) donor restored PGE2 generation inhibited by NG-methyl-Larginine acetate salt (L-NMMA). These data indicate that both NO and PGE2 interact with their own respective biosynthetic pathways modulating NOS and COX activities. Results could contribute to understanding the involvement of NO and PGE2 in healthy dental pulp given that cellular signals through the parasympathetic system modulate the function of the dentin–pulp complex. (J Endod 2007;33:142–147)
Key Words Dental pulp, muscarinic acetylcholine receptor, nitric oxide, nitric oxide synthase, pilocarpine, prostaglandin E2
From the *Pharmacology Unit, School of Dentistry, University of Buenos Aires; and †Argentine National Research Council (CONICET), Buenos Aires, Argentina. This work was supported by University of Buenos Aires (UBACYT, O-014) and Argentine National Research Council (PIP, 05680), Buenos Aires, Argentina. Address requests for reprints to Dr. Enri S. Borda, Pharmacology Unit, School of Dentistry, University of Buenos Aires, M.T. de Alvear 2142– 4° “B,” 1122 AAH Buenos Aires, Argentina. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.11.009
T
he dental pulp is a loose mesenchymal tissue characterized by its particular location and almost entirely enclosed in dentine, a mineralized tissue. Because of its topography the tissue has low interstitial compliance and limited capacity to expand during fluid volume changes (1). Innervation of dental pulp includes autonomic unmyelinated (cholinergic and adrenergic) and sensory nerve fibers that may also subserve dentinal fluid dynamics and regulate pulp blood flow (2). A striking feature of dental pulp innervation is its high density relative to that of other tissues in the body (3, 4). Dentists have developed many strategies to prevent infection because inflammation frequently leads to necrosis and subsequent loss of dental pulp (2). Several studies indicate a parasympathetic innervation in the dental pulp (5, 6) distributed around small blood vessels. Mediator release by parasympathetic nerves controls the pulpal blood flow, thus changing the diameter of blood vessels (7). The physiological role of parasympathetic vasomotor regulation in dental pulp is a controversial issue (2). Response to muscarinic acetylcholine receptors (mAChR) might vary according to the multiple subtypes of mAChR. Therefore, it is of great importance to determine their presence and function to define the real role played by mAChR in healthy dental pulp. It has been well established that the activation of mAChR triggers the release of two proinflammatory mediators, nitric oxide (NO) and prostaglandin E2 (PGE2) (8). NO, formed by NO synthases (NOS), is an important intercellular messenger in vascular, immune, and neuronal cells. At least three isoforms of NOS have been cloned: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (9). The eNOS and nNOS release small amounts of NO for a short period of time after receptor stimulation. In contrast, iNOS is expressed in response to proinflammatory stimuli and produces a large amount of NO for sustained periods of time (10). Prostaglandins (PGs) are biologically active lipids synthesized by the body from the precursor arachidonic acid, which in turn might generate NO (11). These bioactive autacoids have been found in many mammalian cells and some PGs have been identified as mediators of inflammation (12, 13). Biosynthesis of PGs occurs in rat dental pulp organ (13) and the role of PGs in mediating pulp inflammation has been shown. In later research (14, 15), PGE2 has been strongly implicated in pulpal inflammation, particularly in vascular permeability and vasodilation. There is a significant body of experimental evidence suggesting a relationship between NO biosynthesis and prostaglandin generation. NO donors have been reported to stimulate or inhibit prostaglandin biosynthesis in a variety of cellular or broken cellular systems. These effects may be mediated either by altered transcription of cyclooxygenase (COX) genes (16) by inhibition or stimulation of COX activity or by inactivation of downstream metabolizing enzymes that convert prostaglandin endoperoxides to stable eicosanoid products (17). The present works aims to characterize mAChR subtypes and the modulatory effect of cholinergic agonist pilocarpine on healthy dental pulp. The NO/PGE2 pathways were also determined. Figure 1 provides an introductory scheme showing parasympathetic fibers, ACh release, mAChR on target cells, and NO/PGE2 physiological effects on rat dental pulp.
Materials and Methods Animals Male Wistar rats from the Pharmacologic Bioterium (School of Dentistry, University of Buenos Aires) weighing 220 –260 g were used all through the experiments. The
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Basic Research—Biology mRNA Isolation and cDNA Synthesis Total RNA was extracted from rat dental pulp by homogenization using guanidinium isothiocyanate-phenol-chloroform extraction (18). Total RNA was extracted following the technique previously described (19) and adapted for rat dental pulp. A 20-L reaction mixture contained 2 ng of mRNA, 20 units of RNase inhibitor, 1 mM dNTPs, and 50 units of Moloney murine leukemia virus RT (Promega, Madison, WI, USA). First-strand cDNA was synthesized at 37°C for 60 minutes.
Figure 1. Muscarinic receptor-mediated vasodilatation in rat dental pulp. The arrival of an action potential at the cholinergic nerve terminal triggers the release of acetylcholine (ACh). Once released from the nerve terminal ACh elicits cellular response by activating post-synaptic receptors located on endothelium blood vessels, fibroblasts, and macrophages. The stimulation of mAChR on endothelium triggers the activation of nitric oxide synthase (NOS) that in turn catalyzes nitric oxide (NO) synthesis. The NO diffuses into the smooth muscle where it mediates relaxation. On the other hand, activation of mAChR on fibroblasts and macrophages causes the release of PGE2, which also causes the vessel smooth muscle to relax.
animal experiments were approved by the local Animal Ethics Committee at University of Buenos Aires. The animals were subjected to ambient environmental conditions (23–25°C, 12-hour dark/light cycle) and maintained with unrestricted access to water and food. The animals were killed by cervical dislocation.
Exposure of Dental Pulp Exposure of healthy rat dental pulp of upper incisors (left and right) was performed. After incisor teeth were removed horizontally with diamond burs to expose the pulp chamber at the pulp horn, they were carefully extracted and immediately kept at room temperature in Krebs Ringer bicarbonate (KRB) solution in the presence of CO2 in oxygen until different experimental assays could be carried out. Two teeth were used to harvest pulp for each assay.
PCR Procedures NOS isoform-mRNA levels were determined by a method that involves simultaneous co-amplification of both the target cDNA and a reference template (MIMIC) with a single set of primers. MIMICs for m1, m2, m3, and m4 mAChRs and glyceraldehyde-3-phosphate dehydrogenase (g3pdh) (Table 1) were constructed using a PCR MIMIC construction kit. Each PCR MIMIC consists of a heterologous DNA fragment with 5=- and 3=-end sequences that were recognized by a pair of genespecific primers. Sizes of PCR MIMIC were distinct from those of native targets. Sequences of oligonucleotide primer pairs used for construction of MIMIC and amplification of mAChR isoforms and g3pdh mRNA were as reported previously (20). Aliquots were taken from pooled first-strand cDNA from the same group and constituted one sample for PCR. A series of 10-fold dilutions of known concentrations of the MIMIC were added to PCR amplification reactions containing the first-strand cDNA. PCR MIMIC amplification was performed in 100 L of a solution containing 1.5 mM MgCl2, 0.4 M primer, dNTPs, 2.5 U Taq DNA polymerase, and 0.056 M Taq Start antibody (Clontech Laboratories, MountainView, CA, USA). After initial denaturation at 94°C for 2 minutes, the cycle condition was 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 45 seconds for enzymatic primer extension at 72°C for 45 cycles for mAChR subtypes. The internal control was the mRNA of the housekeeping gene g3pdh. PCR amplification was performed with initial denaturation at 94°C for 2 minutes, followed by 30 cycles of amplification. Each cycle consisted of 35 seconds at 94°C, 35 seconds at 58°C, and 45 seconds at 72°C. Samples were incubated for an additional 8 minutes at 72°C before completion. PCR products were subjected to electrophoresis according to previously described procedures (19). Different mAChR subtypes and mRNA levels were normalized with the levels of g3pdh mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis. Prostaglandin E2 (PGE2) Assay Rat dental pulp (10 mg) preparations were incubated for 60 minutes in 0.50 mL of KRB gassed with 5% CO2 in oxygen at 37°C. Pilocarpine were added 30 minutes before the end of the incubation period and blockers 30 minutes before the addition of different pilocarpine concentrations. Dental pulp was then homogenized into a 1.5-mL polypropylene microcentrifuge tube. Thereafter, all procedures used were those indicated in the protocol of Prostaglandin E2 Biotrak Enzyme Immuno Assay (ELISA) System (Amersham Biosciences, Piscataway, NJ, USA). The PGE2 results were expressed as picograms/well (pg/well).
TABLE 1. Oligonucleotides of primers for PCR Gene Product m1 m2 m3 m4 g3pdh
Sense
Anti-sense
Predicted Size (bp)
5=-GGGAGC TGGCCG CCCTGC-3= 5=-GATGAA AACACA GTTTCC ACTTC-3= 5=-GTGGTG TGATGA TTGGTC TG-3= 5=-ACCCAG ACCACC AAGGAA CGGCCA-3= 5=-ACCACA GTCCAT GCCATC AC-3=
5=-GCCTTT CTTGGT GGGCCT C-3= 5=-ATGATG AAAGCC AACAGG ATAGCC-3= 5=-TCTGCC GAGGAG TTGGTG TC-3= 5=-GGTACC TCACGG TGTCTG GGAGAC-3= 5=-TCCACC CACCC TGTTGC TGTA-3=
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Figure 2. RT-PCR product of m1, m2, m3, and m4 obtained from rat dental pulp (8 teeth). Tissues were prepared as described in Materials and Methods and then mRNA isolation and DNA synthesis were extracted and determined.
Determination of Nitric Oxide Synthase (NOS) Activity NOS activity was measured in rat dental pulp by production of [U-14C]-citrulline from [U-14C]-arginine according to the procedure previously described for brain slices (21). Briefly, after 20 minutes in preincubation KRB solution, tissues were transferred to 500 mL of prewarmed KRB equilibrated with 5% CO2 in O2 in the presence of [U-14C]arginine (0.5 mCi). Drugs were added and incubated for 20 minutes under 5% CO2 in O2 at 37°C. Tissues were then homogenized with an Ultraturrax in 1 mL of medium containing 20 mM HEPES (pH 7.4), 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM leupeptin, and 0.2 mM phenylmethylsulphonyl fluoride (PMSF) at 4°C. After centrifugation at 20,000 g for 10 minutes at 4°C, supernatants were applied to 2-mL columns of Dowex AG 50 WX-8 (sodium form); [14C]-citrulline was eluted with 3 mL of water and quantified by liquid scintillation counting. Measurement of Total Labeled Inositol Phosphates (InsP) Preparations of rat dental pulp were incubated for 120 minutes in 0.5 mL of KRB gassed with 5% CO2 in O2 with 1 Ci [myo-3H]-inositol ([3H]-MI) (sp. act. 15 Ci mmol⫺1) from Dupont/New England Nuclear (Boston, MA, USA). LiCl (10 mM) was added for inositol monophosphate accumulation. Pilocarpine was added 30 minutes before the end of the incubation period and the blockers 30 minutes before the addi-
Figure 4. Correlation in the modulatory effect of pilocarpine on NOS activity and PGE2 generation. NOS was plotted as a function of PGE2 (Pearson r: 0.9912; p value: 0.0010; alpha: 0.05). Values are the mean of six experiments of each group (12 teeth). For other details see legend of Fig. 1.
tion of the agonist. Water-soluble InsP was extracted after a 120-minute incubation and the results were expressed as area per milligrams of wet weight tissue (area/mg wet weight).
Drugs Pilocarpine, AF-DX 116, pirenzepine, 4-DAMP, tropicamide, atropine, U-73122, L-arginine, NG-methyl-L-arginine acetate salt (L-NMMA), indomethacin (INDO), and prostaglandin E2 (PGE2) were purchased from Sigma Chemical Company (St. Louis, MO, USA); 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA) was obtained from Tocris Cookson (Ellisville, MO, USA). Stock solutions were freshly prepared in the corresponding buffers. Statistical Analysis Student’s t test for unpaired values was used to determine the levels of significance. Analysis of variance (ANOVA) and Student–Newman–
Figure 3. (A) Increase in NOS activity of rat dental pulp by increasing concentrations of pilocarpine (). (B) Increase in PGE2 generation of rat dental pulp by increasing concentrations of pilocarpine (). The inhibitory action of 1 ⫻ 10⫺7 M pirenzepine (), 1 ⫻ 10⫺7 M AF-DX 116 (Œ), 1 ⫻ 10⫺6 M 4-DAMP (□), and 1 ⫻ 10⫺6 M tropicamide (〫) are also shown. Values represent the mean ⫾ SEM of nine experiments in each group (18 teeth). No effects were observed with M1, M2, M3, and M4 antagonists alone at the concentration used.
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Figure 5. Effect of 1 ⫻ 10⫺6 M INDO (Œ), 1 ⫻ 10⫺5 M L-NMMA (), 1 ⫻ 10⫺6 M U-73122 (□), and 5 ⫻ 10⫺5 M OBAA (〫) on the dose–response curve of pilocarpine () upon NOS activity (A) and PGE2 generation (B). Values are mean ⫾ SEM of seven experiments in each group (14 teeth). For other details see legend of Fig. 1; p ⬍ 0.001 between pilocarpine alone vs. pilocarpine in the presence of inhibitory agents.
Keuls test were used when pairwise a multiple comparison procedure was necessary. Differences between means were considered significant if p ⬍ 0.05.
Results As can be seen in Fig. 1, using specific oligonucleotide primers, RT-PCR amplified products showed bands of the predicted size for m1, m2, m3, and m4 mAChRs detected in the tissue, indicating that all four mAChR subtypes are expressed in the rat dental pulp. Figure 2 shows that pilocarpine from 1 ⫻ 10⫺10 to 1 ⫻ 10⫺7 M induced a concentration-dependent stimulation on NOS activity (A) and PGE2 generation (B). The selective M1, M2, M3, and M4 mAChR antagonists could antagonize the increased NOS activity and PGE2 generation, shifting the dose–response curves of pilocarpine to the right (Fig. 2A and B). The orders of potency for reducing NOS activation and PGE2 generation are similar: AF-DX 116 ⬎ pirenzepine ⬎ 4-DAMP ⫽ tropicamide.
Figure 3 demonstrated that under identical experimental conditions a significant correlation (correlation coefficient ⫽ 0.05) between pilocarpine-stimulated NOS activity and PGE2 generation was found. These results indicated that M1, M2, M3, and M4 mAChR activation induces an increase in NO as a result of the increase in PGE2 generation or vice versa. To determine whether NOS activity and PGE2 generation are dependent on each other, dental pulp preparations were incubated with different inhibitors of the enzymatic pathways known to be involved in the PGE2 and NO production by activation of mAChR subtypes. Figure 4A and B show that the inhibition of: COX-1 by indomethacin (INDO) (1 ⫻ 10⫺6 M), phospholipase A2 (PLA2) by OBAA (5 ⫻ 10⫺6 M), nitric oxide synthase (NOS) by L-NMMA (1 ⫻ 10⫺5 M), and phospholipase C (PLC) by U-73122 (1 ⫻ 10⫺6 M) attenuated the stimulatory action of different pilocarpine concentrations upon both NOS activity and PGE2 generation. Moreover, 1 ⫻ 10⫺8 M exogenous PGE2 restored NOS activity inhibited by INDO (Fig. 5A). On the other hand, 1 ⫻ 10⫺5 M
Figure 6. (A) Histograms shows basal, pilocarpine (1 ⫻ 10⫺8 M) alone, pilocarpine in the presence of INDO (1 ⫻ 10⫺6 M), and pilocarpine plus INDO restored by adding PGE2 (1 ⫻ 10⫺8 M) upon rat dental pulp. (B) Histograms show basal, pilocarpine alone (1 ⫻ 10⫺8 M), pilocarpine in the presence of L-NMMA (1 ⫻ 10⫺5 M), and pilocarpine plus L-NMMA restored by adding PGE2 (1 ⫻ 10⫺8 M). Values are mean ⫾ SEM of six experiments performed by duplicate in each group (12 teeth); p ⬍ 0.001 between pilocarpine alone vs. pilocarpine ⫹ INDO or pilocarpine ⫹ L-NMMA.
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Figure 7. Proposed model for the mechanisms where pilocarpine up-regulates NO/PGE2 pathway on healthy dental pulp. Pilocarpine acting on M1/M2 and M3/M4 mAChRs activates PLC mediating production of inositol-triphosphate (IP3). IP3 triggers intracellular release of calcium (Ca⫹⫹). Free Ca⫹⫹ activates nitric oxide synthase (NOS) with an increment of nitric oxide (NO). NO in turn stimulates cyclooxygenase 1 (COX-1) with increased production of PGE2. Free Ca⫹⫹ also activates phospholipase A2 (PLA2) with increased production of PGE2. On the other hand, PGE2 activated PLC increases NOS activity using free Ca⫹⫹ as an enzyme cofactor. Gray arrows indicate the site where the inhibition agents act.
L-arginine restored PGE2 generation inhibited by L-NMMA (Fig. 5B). Also, 1 ⫻ 10⫺8 M pilocarpine increased total phosphoinositol accumulation (area/mg wet weight: basal: 52 ⫾ 5, n ⫽ 6, pilocarpine: 105 ⫾ 8, n ⫽ 6) and 1 ⫻ 10⫺7 M atropine (60 ⫾ 6, n ⫽ 5) and 1 ⫻ 10⫺6 M U-73122 (49 ⫾ 5, n ⫽ 5) inhibited its accumulation, thus confirming participation of PLC in pilocarpine effects.
Discussion The current study provides pharmacological evidence for the existence of functional mAChR subtypes in isolated rat dental pulp and indicates that both NOS activity and PGE2 generation are involved in the mAChR mediated stimulatory effect of pilocarpine. Our findings demonstrate that the pilocarpine effect is related to M1/M2 and M3/M4 subtypes, given that the specific M1, M2, M3, and M4 cholinoceptor antagonists blocked the mAChR effect. Furthermore, our current results show greater effectiveness of M1/M2 when compared to M3/M4 antagonists to inhibit the pilocarpine-dependent increases in NOS activity and PGE2 generation. 146
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The mechanism by which pilocarpine-induced stimulation of NOS activity and PGE2 generation on dental pulp appears to occur secondarily to the activation of a number of enzymatic pathways commonly associated with receptor signaling. Here we provide evidence for a functional link between NO and PGE2 generation because INDO (COX-1 inhibitor), OBAA (PLA2 inhibitor), U-73122 (PLC inhibitor), and L-NMMA (NOS inhibitor) abrogated the increase of both NOS activity and PGE2 generation by pilocarpine. The fact that addition of a low concentration of PGE2 reversed the INDO-inhibited pilocarpine-increased NOS activity indicates a direct action of this prostanoid on NOS activity. On the other hand, exogenous L-arginine was able to reverse the inhibitory action of L-NMMA on pilocarpine-induced PGE2 generation, indicating that NO subserves PGE2 generation triggered by mAChR activation. Prostaglandins and NO represent some of the most relevant local mediators that participate under basal conditions, in the modulation of many cellular functions. In vivo studies demonstrated that the NOdependent pathway plays an important role in regulating the basal vasodilator tone and blood circulation in dental pulp (22). Furthermore, the substance P released from the sensory nerve endings produces vasodilatation, thus inducing NO production from endothelial cells (23). The biosynthesis and release of NO and prostaglandins share a number of similarities. Under normal circumstances the constitutive isoforms of these enzymes (constitutive NOS and COX) are found in virtually all organs. Accumulated evidence indicates that the cross talk between NO and prostaglandins at many levels exists (24), although this point still remains to be proved in dental pulp. An important link between NOS and COX pathways has been shown through studies that raised the possibility that COX enzymes represent important endogenous “receptor” targets for modulating the multifaceted roles of NO (16). On the other hand, NO biosynthesis may also modulate arachidonic acid release and COX metabolite generation (17). The current data indicate that the activation of NOS correlated with PGE2 generation. Moreover, both NO and PGE2 interact with their own respective biosynthetic pathways by modulating molecular events underlying NOS and COX activities. Based on the reciprocal interactions between NO and PGE2 (25), we observed that PLC and PLA2 are implicated in the pilocarpine effect, as was demonstrated in intact cells and membrane preparations (25). It has been well established that activation of PLC and PLA2 constitutes an important function of G-protein– coupled receptors (26). Therefore, pilocarpine by stimulating mAChR activates PLC, which hydrolyzes PIP2 into IP3 with a subsequent increase in the level of intracellular calcium, in turn inducing the direct activation of PLA2 (27). In fact, our results show that pilocarpine is able to increase the accumulation of inositol phosphates by PLC coupled to mAChR activation. The molecular mechanism involved in both NOS and COX activation may play a crucial role in understanding the reciprocal interaction between NO and COX. NO gas directly increases COX-1 activity of microsomal seminal vesicles, as well as recombinant COX-1. The activation of COX-1 leads to a remarkable sevenfold increase in PGE2 formation (16). In this context, there is a significant body of experimental evidence that suggests that NO and/or NOS activity stimulate COX-1 activity and production of prostanoids, mainly PGE2 (28). Whether PGE2 stimulated NOS activity is an open question. Our study demonstrates a stimulatory action of PGE2 on NOS activity. The mechanism by which PGE2 stimulates NOS activity appears to involve an increase in the hydrolysis of phosphoinositides by PLC, where calcium/calmodulin could act as an enzyme cofactor as was reported from PGE2-activated NOS on the submandibulary gland (29). Our finding suggests that pilocarpine—through its mAChR subtypes—increases basal levels of NO and PGE2, modulating their own release by affecting NOS and COX activities, respectively.
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Basic Research—Biology Figure 6 shows a diagram that ties together the various systems studied and Fig. 7 proposes a model for the mechanisms where pilocarpine activating mAChR up-regulates the NOS/ PGE2 pathway in dental pulp. In conclusion, this paper indicates a modulatory role of the parasympathetic system in dental pulp. The dense parasympathetic nerve supply of dental pulp might not only exert neuronal control, but it may also modulate the large terminal arteriolar diameter (30), suggesting an important role of neuronal regulation on tone vessel (3). NO/PGE2 might represent a signaling-dependent modulation of odontoblasts, blood vessels, and parasympathetic nerve fibers in the dentin–pulp complex.
Acknowledgment We thank Mrs. Elvita Vannucchi and Fabiana Solari for outstanding technical assistance. Also, the assistance of art designer Mr. Alejandro Thornton is gratefully acknowledged.
References 1. Bletsa A, Berggreen E, Fristad I, Tenstad O, Wiig H. Cytokine signalling in rat pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-induced acute inflammation. J Physiol 2006;573:225–36. 2. Olgart L, Kostouros GD, Edwall L. Local actions of acetylcholine on vasomotor regulation in rat incisor pulp. Acta Physiol Scand 1996;158:311– 6. 3. Zhang JQ, Nagata K, Iijima T. Scanning electron microscopy and immunohistochemical observations of the vascular nerve plexuses in the dental pulp of rat incisor. Anat Rec 1998;251:214 –20. 4. Iijima T, Zhang JQ. Three-dimensional wall structure and the innervation of dental pulp blood vessels. Microsc Res Tech 2002;56:32– 41. 5. Akai M, Wakisaka S. Distribution of peptidergic nerves. In: Inoqui R, Kudo T, Olgart LM, eds. Dynamic aspects of dental pulp. Cambridge, UK: Chapman & Hall, 1990:337– 48. 6. Luthman J, Luthman G, Hökfelt T. Occurrence and distribution of different neurochemical markers in the human dental pulp. Arch Oral Biol 1992;37:193–208. 7. Kim S, Dörscher-Kim JE, Lipowsky HH. Quantitative assessment of microcirculation in the rat dental pulp in response to alpha and beta adrenergic agonists. Arch Oral Biol 1989;100:1387–95. 8. Perez Leiros C, Rosignoli F, Genaro AM, Sales ME, Sterin-Borda L, Borda E. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in the rat salivary gland. J Auton Nerv Syst 2000;79:99 –107. 9. Liaudet L, Soriano FG, Szabo C. Biology of nitric oxide signaling. Crit Care Med 2000;28:N37–N52. 10. Shouthan GJ, Szabo C. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem Pharmacol 1996;51:383–94.
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11. Genaro AM, Stranieri GM, Borda E. Involvement of the exogenous nitric oxide signaling system in bradykinin receptor activation in rat submandibular salivary gland. Arch Oral Biol 2000;45:725–9. 12. Waterhouse PJ, Whitworth JM, Nunn JH. Development of a method to detect and quantify prostaglandin E2 in pulp blood from cariously exposed, vital primary molar teeth. Int Endod J 1999;32:381–7. 13. Antila R, Pohto P. In vitro studies on the prostaglandin system in tooth pulp. Proc Finn Dent Assoc 1984;80:245–52. 14. Dewhirst FE, Goodson JM. Prostaglandin synthase inhibition by eugenol, guaiacol and other dental medicaments. J Dent Res 1974;53:199. 15. Okiji T, Morita I, Zunada I, Murota S. Involvement of arachidonic acid metabolites increases in vascular permeability in experimental dental pulp inflammation in the rat. Arch Oral Biol 1989;34:523– 8. 16. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman G. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 1993;90:7240 – 4. 17. Mollace V, Muscoli C, Masini E, Cuzzocrea S, Salvemini D. Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors. Pharmacol Rev 2005;57:217–52. 18. Chomozynski P, Saachi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Ann Biochem 1987;162:156 –9. 19. Sterin-Borda L, Ganzinelli S, Berra A, Borda E. Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels. Neuropharmacology 2003;45:260 –9. 20. Orman B, Reina S, Borda E, Sterin-Borda L. Signal transduction underlying carbachol-induced PGE2 generation and COX-1 mRNA expression of rat brain. Neuropharmacology 2005;48:757– 65. 21. Borda T, Genaro AM, Sterin-Borda L, Cremaschi G. Involvement of endogenous nitric oxide signaling system in brain muscarinic acetylcholine receptor activation. J Neural Transm 1998;105:193–204. 22. Lohinai Z, Balla I, Marczis J, Vass Z, Kovach AG. Evidence for the role of nitric oxide in the circulation of the dental pulp. J Dent Res 1995;74:1501– 6. 23. Karabucak B, Walsch H, Jou YT, Simchon S, Kim S. The role of endothelial nitric oxide in the substance P induced vasodilation in bovine dental pulp. J Endod 2005;31:733– 6. 24. Clancy R, Varenika B, Huang W, et al. Nitric oxide synthase/COX cross-talk: nitric oxide activates COX-1 but inhibits COX-2 derived prostaglandin production. J Immunol 2000;165:1582–7. 25. Wallace JL, Del Soldato P. The therapeutic potential of NO-NSAIDs. Fundam Clin Pharmacol 2003;17:11–20. 26. Chakraborti S. Phospholipase A2 isoforms: a perspective. Cell Signal 2003;15:637– 65. 27. Pochet S, Métioui M, Grosfils K, Gomez-Muñoz A, Marino A, Dehaye JP. Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells. Cell Signal 2002;15:103–13. 28. Marnett LJ, Wrigth TL, Crews BC, Tannenbaum SR, Morrow JD. Regulation of prostaglandin biosynthesis by nitric oxide by targeted deletion of iNOS. J Biol Chem 2000;5:13427–30. 29. Borda E, Heisig G, Busch L, Sterin-Borda L. Nitric oxide synthase/prostaglandin E2 cross-talk in rat submandibular gland. Prost Leuk Ess Fatty Acids 2002;67:39 – 44. 30. Kerezoudis NP, Fried K, Olgart L. Haemodynamic and inmunohistochemical studies of rat incisor pulp after denervation and subsequent re-innervation. Arch Oral Biol 1995;40:815–23.
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