Pharmacodynamics, pharmacokinetics and CYP3A4 interaction potential of the selective P2X3 receptor antagonist filapixant: A randomized multiple ascending-dose study in healthy young men.

AIMS: We report on investigations exploring the P2X3-receptor antagonist filapixant's effect on taste perception and cough-reflex sensitivity and describe its pharmacokinetics, including its CYP3A4-interaction potential. METHODS: In a randomized, placebo-controlled, double-blind study, 3 × 12 healthy men (18-45 years) were assigned (3:1) to filapixant (20, 80 or 250 mg by mouth) or placebo twice daily over 2 weeks. A single dose of midazolam (1 mg), a CYP3A4 substrate, was administered with and without filapixant. Assessments included a taste-strips test, a taste questionnaire, cough challenge with adenosine triphosphate, adverse event reports and standard safety assessments. RESULTS: Taste disturbances were observed mainly in the 250-mg group: six of nine participants (67%) in this group reported hypo- or dysgeusia in the questionnaire; eight participants (89%) reported taste-related adverse events. Five participants (56%) had a decrease in overall taste-strips-test scores ≥2 points (point estimate -1.1 points, 90% confidence interval [-3.3; 1.1]). Cough counts increased with adenosine triphosphate concentration but without major differences between treatments. Filapixant exposure increased proportionally to dose. Co-administration of filapixant had no clinically relevant effect on midazolam pharmacokinetics. Area under the concentration-time curve ratios and 90% confidence intervals were within 80-125%. No serious or severe adverse events were reported. CONCLUSIONS: Overall, filapixant was safe and well tolerated, apart from mild, transient taste disturbances. Such disturbances occurred more frequently than expected based on (in vitro) receptor-selectivity data, suggesting that other factors than P2X3:P2X2/3 selectivity might also play an important role in this context. The cough-challenge test showed no clear treatment effect. Filapixant has no clinically relevant CYP3A4 interaction potential.

Intermittent compressive force induces human mandibular-derived osteoblast differentiation via WNT/ß-catenin signaling.

Mechanical force induces an efflux of ATP that regulates osteoblast differentiation. However, the effect of mechanical force-induced ATP efflux on WNT/ß-catenin signaling remains unclarified. The aim of this study was to investigate the effect of intermittent compressive force (ICF) and ICF-induced extracellular ATP on osteoblast differentiation via WNT/ß-catenin signaling in human mandibular-derived osteoblast precursors (hMOBPs). The hMOBPs were subjected to ICF (1.5 g/cm2 , 0.3 Hz) for 20 h. To investigate the role of ATP, Apyrase (0.5 units/mL), an enzyme that hydrolyzes ATP, was added 30 min before ICF was applied. The extracellular ATP levels were measured immediately after ICF was removed. The mRNA expression of osteogenic related genes, including WNT was evaluated via quantitative real time polymerase chain reaction. In vitro mineralization was determined by Alizarin Red S staining. The localization of ß-catenin was detected using immunofluorescence staining and lentiviral-TOP-dGFP reporter assay. The results demonstrated that ICF increased ATP efflux and in vitro mineralization by hMOBPs. In addition, OSX, ALP, and WNT3A mRNA expression and ß-catenin nuclear translocation increased when ICF was applied. The upregulation of these genes was reduced by Apyrase, suggesting the role of ICF-induced ATP on osteoblast differentiation. Notably, ICF altered the mRNA expression of purinergic 2X receptors (P2XRs). A P2X1R antagonist (NF449) downregulated ICF-induced WNT3A, OSX, and ALP mRNA expression. Moreover, when 25 µM α, ß-meATP, a P2X1R agonist, was added, WNT3A, and OSX expression increased. In conclusion, our results demonstrate that ICF-induced ATP enhanced hMOBP differentiation. This enhancement was associated with WNT/ß-catenin signaling and P2X1R activation.

Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

Topical application of a P2X2/P2X3 purine receptor inhibitor suppresses the bitter taste of medicines and other taste qualities.

BACKGROUND AND PURPOSE: Many medications taste intensely bitter. The innate aversion to bitterness affects medical compliance, especially in children. There is a clear need to develop bitter blockers to suppress the bitterness of vital medications. Bitter taste is mediated by TAS2R receptors. Because different pharmaceutical compounds activate distinct sets of TAS2Rs, targeting specific receptors may only suppress bitterness for certain, but not all, bitter-tasting compounds. Alternative strategies are needed to identify universal bitter blockers that will improve the acceptance of every medication. Taste cells in the mouth transmit signals to afferent gustatory nerve fibres through the release of ATP, which activates the gustatory nerve-expressed purine receptors P2X2/P2X3. We hypothesized that blocking gustatory nerve transmission with P2X2/P2X3 inhibitors (e.g. 5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine [AF-353]) would reduce bitterness for all medications and bitter compounds. EXPERIMENTAL APPROACH: Human sensory taste testing and mouse behavioural analyses were performed to determine if oral application of AF-353 blocks perception of bitter taste and other taste qualities but not non-gustatory oral sensations (e.g. tingle). KEY RESULTS: Rinsing the mouth with AF-353 in humans or oral swabbing it in mice suppressed the bitter taste and avoidance behaviours of all compounds tested. We further showed that AF-353 suppressed other taste qualities (i.e. salt, sweet, sour and savoury) but had no effects on other oral or nasal sensations (e.g, astringency and oral tingle). CONCLUSION AND IMPLICATIONS: This is the first time a universal, reversible taste blocker in humans has been reported. Topical application of P2X2/P2X3 inhibitor to suppress bitterness may improve medical compliance.

Blocking the P2X7 receptor improves outcomes after axonal fusion.

BACKGROUND: Activation of the P2X7 receptor on peripheral neurons causes the formation of pannexin pores, which allows the influx of calcium across the cell membrane. Polyethylene glycol (PEG) and methylene blue have previously been shown to delay Wallerian degeneration if applied during microsuture repair of the severed nerve. Our hypothesis is that by modulating calcium influx via the P2X7 receptor pathway, we could improve PEG-based axonal repair. The P2X7 receptor can be stimulated or inhibited using bz adenosine triphosphate (bzATP) or brilliant blue (FCF), respectively. METHODS: A single incision rat sciatic nerve injury model was used. The defect was repaired using a previously described PEG methylene blue fusion protocol. Experimental animals were treated with 100 µL of 100 µM FCF solution (n = 8) or 100 µL of a 30 µM bzATP solution (n = 6). Control animals received no FCF, bzATP, or PEG. Compound action potentials were recorded prior to transection (baseline), immediately after repair, and 21 d postoperatively. Animals underwent behavioral testing 3, 7, 14, and 21 d postoperatively. After sacrifice, nerves were fixed, sectioned, and immunostained to allow for counting of total axons. RESULTS: Rats treated with FCF showed an improvement compared with control at all time points (n = 8) (P = 0.047, 0.044, 0.014, and 0.0059, respectively). A statistical difference was also shown between FCF and bzATP at d 7 (P < 0.05), but not shown with d 3, 14, and 21 (P > 0.05). CONCLUSIONS: Blocking the P2X7 receptor improves functional outcomes after PEG-mediated axonal fusion.

P2X7 receptor blockade reverses purinergic facilitation of neck muscle nociception in mice.

INTRODUCTION: Facilitation of neck muscle nociception mediated via purinergic signalling may play a role in the pathophysiology of tension-type headache (TTH). The present study addressed reversal of purinergic facilitation of brainstem nociception via P2X7 antagonist action in anaesthetized mice. METHODS: Following administration of α,ß-meATP (i.m. 20 µL/min, 20 µL each) into semispinal neck muscles, the impact of neck muscle nociceptive input on brainstem processing was monitored by the jaw-opening reflex in anaesthetized mice (n = 20). The hypothesized involvement of the P2X7 receptor in the α,ß-meATP effect was addressed with i.p. (systemic) and i.m. (semispinalis, 20 µL/min, 20 µL each) administration of P2X7 inhibitor A438079 during established facilitation; i.p. saline served as control. RESULTS: α,ß-meATP reliably induced jaw-opening reflex facilitation (256 ± 48% (mean ± SEM), n = 20). I.p. A438079 (150, 300 µmol/kg) completely reversed this α,ß-meATP effect dose-dependently. Neither saline nor intramuscular A438079 (100 µM) altered facilitated brainstem nociceptive processing. DISCUSSION: These data suggest that muscular structures are not directly involved in the P2X7 antagonist-mediated reversal of purinergic facilitation. Instead, involvement of neuronal structures, particularly of the central nervous system, seems more probable. The results from this animal experimental model may point to involvement of purinergic P2X7 receptors in TTH pathophysiology and may suggest potential future targets for its pharmacological treatment.

Assessment of the Effect of Pyrimethamine, a Potent Inhibitor of Multidrug and Toxin Extrusion Protein 1/2K, on the Pharmacokinetics of Gefapixant (MK-7264), a P2X3 Receptor Antagonist.

Gefapixant (MK-7264, AF-219) is a first-in-class P2X3 antagonist in development for refractory or unexplained chronic cough. Gefapixant is primarily cleared by renal excretion. To assess the importance of the multidrug and toxin extrusion protein 1 (MATE1) and MATE2K transporters in the elimination of gefapixant, a drug-drug interaction study was conducted evaluating the effect of coadministration of a single dose of pyrimethamine, a competitive inhibitor of MATE1 and MATE2K, on the single-dose pharmacokinetics of gefapixant in healthy participants. Safety and tolerability were also assessed. In this open-label, 2-period, fixed-sequence study, a 45-mg dose of gefapixant was administered to 12 participants in period 1. After a 7-day washout, a 50-mg dose of pyrimethamine was administered 3 hours before a 45-mg dose of gefapixant in period 2. Compared with the administration of gefapixant alone, concomitant dosing of gefapixant with pyrimethamine increased the total gefapixant plasma exposure (area under the plasma concentration-time curve from time 0 to infinity) by 24%, reduced gefapixant renal clearance by 30%, and increased gefapixant mean terminal half-life from 7.7 to 10.3 hours. The most frequently reported adverse events were dysgeusia, hypogeusia, and dry mouth; all adverse events were considered of mild intensity and resolved by the end of the study. These results support that MATE1 and/or MATE2K contribute to the renal clearance of gefapixant, but the effect of inhibition of these transporters on gefapixant pharmacokinetics is not considered clinically meaningful.

Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1ß (IL-1ß), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. RESULTS: Nontoxic concentrations of LL-37 (up to 10 µm) and IL-1ß significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 µm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs. CONCLUSION: These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Role of the P2X7 receptor in inflammation-mediated changes in the osteogenesis of periodontal ligament stem cells.

Accumulating evidence indicates that the pluripotency of periodontal ligament stem cells (PDLSCs) is compromised under inflammatory conditions; however, the underlying mechanisms remain largely unexplored. In this study, we hypothesize that the P2X7 receptor (P2X7R) is a key molecule linked to inflammation-associated impairment of PDLSCs. We first investigated P2X7R expression in PDLSCs under normal and inflammatory conditions and then determined the effect of a P2X7R agonist (BzATP) or antagonist (BBG) on PDLSC osteogenesis under various conditions. Gene-modified PDLSCs were used to further examine the role of P2X7R and the signaling pathway underlying P2X7R-enhanced osteogenesis. We found that inflammatory conditions decreased P2X7R expression in PDLSCs and reduced osteogenesis in these cells. In addition, activation of P2X7R by BzATP or overexpression of P2X7R via gene transduction reversed the inflammation-mediated decrease in PDLSC osteogenic differentiation. When selected osteogenesis-related signaling molecules were screened, the PI3K-AKT-mTOR pathway was identified as potentially involved in P2X7R-enhanced PDLSC osteogenesis. Our data reveal a crucial role for P2X7R in PDLSC osteogenesis under inflammatory conditions, suggesting a new therapeutic target to reverse or rescue inflammation-mediated changes in PDLSCs for future mainstream therapeutic uses.

Effect of A-317491 delivered by glycolipid-like polymer micelles on endometriosis pain.

Endometriosis is a common gynecological disease with a lack of effective clinical treatment. Current therapy often results in endometriosis pain recurrence and serious side effects. P2X3 receptor, an adenosine triphosphate (ATP)-gated ion channel, might be implicated in endometriosis pain. In this study, chitosan oligosaccharide-g-stearic acid (CSOSA) polymer micelles-coated nanostructured lipid carriers (NLCs) were developed as a novel delivery system for A-317491, a selective P2X3 receptor antagonist for endometriosis pain therapy. A-317491-loaded NLC (NLC/A-317491) could be coated by CSOSA micelles to form CSOSA/NLC/A-317491 nanoparticles. Pheochromocytoma PC12 cells, which highly expressed P2X3 receptors, were used as a cell model, and the CSOSA/NLC/A-317491 partly blocked the Ca2+ influx induced by ATP stimulation. In nude mouse and rat endometriotic models, CSOSA/NLC could accumulate into endometriotic lesions after vein injection. In endometriotic rats, CSOSA/NLC/A-317491 reversed mechanical and heat hyperalgesia with long-term efficacy, which might be attributed to the massive CSOSA/NLC/A-317491 distribution in the endometriotic lesions. In conclusion, A-317491 delivered by CSOSA/NLC nanoparticles attenuated endometriosis pain in rats, and CSOSA/NLC/A-317491 could be used as an effective treatment strategy for P2X3-targeted therapy in endometriosis pain.

[Trigeminal purinergic P2X4 receptor involved in experimental occlusal interference-induced hyperalgesia in rat masseter muscle].

OBJECTIVE: To explore the expression of purinergic p2X4 receptor (P2X4R) in trigeminal ganglion of rats after occlusal interference. Investigation of peripheral receptor mechanism of occlusal interference-induced masticatory muscle pain will aid the development of drug intervention against this condition. METHODS: Experimental occlusal interference was established by application of 0.4 mm metal crown to the upper right first molar of male Sprague-Dawley rats. Real-time PCR assay was used to investigate P2X4R mRNA level in trigeminal ganglion in rats with occlusal interference for 3, 7, 10 and 14 days and in control rats without occlusal interference (n=5 in each). Retrograde labelling combining immunofluorescence was performed to evaluate the percentage of P2X4R-positive cells in masseter afferent neurons (n=5 in each group). Graded concentrations of P2XR antagonist TNP-ATP (0.1, 10, 125, 250, 500 µmol/L) or saline (n=5 in each group) was administrated in right masseter and the mechanical sensitivity of bilateral masseters was measured before occlusal interference application, before the injection, and 30 min as well as 60 min after the injection. RESULTS: Compared with control rats (P2X4R mRNA: right side: 1.00±0.26, left side: 0.94± 0.21; percentage of P2X4R-positive masseter afferents: right side: [64.3±6.3]%, left side: [67.7±5.8]%), the level of P2X4R mRNA in bilateral trigeminal ganglia (right side: 5.98±3.56; left side: 5.06±2.88) of rats with occlusal interference for 7 days up-regulated (P<0.01) and the percentage of P2X4R-positive masseter afferent neurons(right side: [81.7±1.5]%; left side: [82.9±2.3]%) increased (P<0.05). Local administration of 10, 125, 250, 500 µmol/L TNP-ATP increased the mechanical withdrawal threshold in masseter 30 min after injection, compared with those before injection (P<0.05). CONCLUSIONS: Increased expression of trigeminal P2X4R involves in the development of occlusal interference-induced masseter hyperalgesia.

Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat.

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine.

Upregulation of the Purinergic Receptor Subtype P2X3 in the Trigeminal Ganglion Is Involved in Orofacial Pain Induced by Occlusal Interference in Rats.

AIMS: To evaluate whether the purinergic receptor subtype P2X3 (P2X3R) in trigeminal ganglion (TG) neurons is involved in hyperalgesia of the temporomandibular joints (TMJs) and masseter muscles associated with placement of an occlusal interference. METHODS: Forty-five rats were randomized into five groups (ie, for days 1, 3, 7, 14, or 28; nine rats per group). Six rats from each group were chosen to receive the occlusal interference, and the remaining three rats were sham-treated controls. On days 1, 3, 7, 14, and 28 after placement of the occlusal interference, the mechanical pain threshold (MPT) to stimulation of the TMJs or masseter muscles was examined using von Frey filaments. Seven days after the occlusal interference placement, changes in MPT were tested after administration of the P2X3R antagonist A-317491 into the TMJs and masseter muscles (60 µg/site) in six rats. The expression of P2X3R in the TGs was investigated by immunohistochemistry and quantitative polymerase chain reaction (qPCR). Retrograde tracing was combined with immunofluorescence to identify TMJ and masseter muscle afferent neurons in the TGs of six premature rats. RESULTS: The TMJ and masseter muscle MPTs were decreased after placement of the occlusal interference, and the P2X3R antagonist reversed the mechanical hyperalgesia that was caused by the occlusal interference placement. The frequency of P2X3R-immunoreactive cells increased in small-sized neurons in the TG after occlusal interference. By contrast, there was no increase in medium-sized TG neurons. P2X3R mRNA increased on day 3. Retrograde tracing indicated that the TMJ and masseter muscle afferent neurons in the TG expressed P2X3R. CONCLUSION: Upregulated P2X3R expression in the TG may contribute to orofacial pain development induced by an occlusal interference. P2X3R may be a therapeutic target for chronic TMJ or masseter muscle pain.

Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor.

The P2X receptors are seven-transmembrane domain G protein-coupled receptors and the 7 subtypes of P2X receptors identified in humans, and named P2X1 to P2X7, are channel receptors whose endogenous ligand is ATP. New antagonists of the P2X7 receptor were developed, since this purinergic receptor was highlighted to be involved in many diseases such as different types of pain, cancer, ischemia, neurodegenerative diseases (including Parkinson's and Alzheimer's diseases) characterized by inflammatory processes. With the aim of evaluate the impact of chirality on the pharmacological activity of a new P2X7R antagonist, a semi-preparative method was developed in supercritical fluid chromatography (SFC). Among four polysaccharide based chiral stationary phases: Chiralcel OD-H and OJ-H and Chiralpak AS-H and AD-H, the last one namely amylose tris (3,5-dimethylphenylcarbamate) with a mobile phase consisted of carbon dioxide-ethanol (80:20, v/v), led to the successful separation of the enantiomers in short run time and with good resolution. Limits of detection and quantification were calculated and were found equal for compound 1, to 1.37 µM and 4.57 µM respectively, for peak 1 and were equal to 1.60 µM and 5.30 µM respectively, for peak 2 at λ=210 nm. Before carrying out the pharmacological evaluation of each enantiomer, two complementary methodologies, e.g. liquid chromatography and capillary electrophoresis were performed in parallel to improve the limits of detection and quantification to assess the enantiomeric purity. HPLC using a Chiralpak AD stationary phase led to four times lower limits of detection and quantification with regard to SFC. In the same time, capillary electrophoresis involving dual cyclodextrins system constituted of a SBE-ß-CD and a MM-ß-CD mixture enhanced the signal-to-noise ratio and led to similar limits of detection and quantification with regard to SFC. No trace of the other enantiomer was found in the isolated one. Biological activities of individual enantiomers were then evaluated and revealed no cytotoxicity against cell lines and a significant difference in terms of their IC50 values with respect to the investigated racemate (6.43 µM): 3.49 µM for the (R)-enantiomer and >10(-4)µM for the (S)-enantiomer, for compound 1, showing that, this antagonist activity is stereospecific.

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

AIM: The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. METHODS: Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. RESULTS: Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. CONCLUSION: Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement.

Role of endothelial cells in antihyperalgesia induced by a triptan and ß-blocker.

While blood vessels have long been implicated in diverse pain syndromes (e.g., migraine headache, angina pectoris, vasculitis, and Raynaud's syndrome), underlying mechanisms remain to be elucidated. Recent evidence supports a contribution of the vascular endothelium in endothelin-1-induced hyperalgesia, and its enhancement by repeated mechanical stimulation; a phenomenon referred to as stimulus-induced enhancement of (endothelin) hyperalgesia (SIEH). SIEH is thought to be mediated by release of ATP from endothelial cells, to act on P2X3 receptors on nociceptors. In the present study we evaluated the ability of another vasoactive hyperalgesic agent, epinephrine, to induce endothelial cell-dependent hyperalgesia and SIEH. We found that epinephrine also produces hyperalgesia and SIEH. Both P2X3 receptor antagonists, A317491 and octoxynol-9, which attenuate endothelial cell function, eliminated SIEH without affecting epinephrine hyperalgesia. We further evaluated the hypothesis that members of two important classes of drugs used to treat migraine headache, whose receptors are present in endothelial cells - the triptans and ß blockers - have a vascular component to their anti-hyperalgesic action. For this, we tested the effect of ICI-118,551, a ß2-adrenergic receptor antagonist and sumatriptan, an agonist at 5-HT1B and 5-HT1D receptors, on nociceptive effects of endothelin and epinephrine. ICI-118,551 inhibited endothelin SIEH, and attenuated epinephrine hyperalgesia and SIEH. Sumatriptan inhibited epinephrine SIEH and inhibited endothelin hyperalgesia and SIEH, while having no effect on epinephrine hyperalgesia or the hyperalgesia induced by a prototypical direct-acting inflammatory mediator, prostaglandin E2. These results support the suggestion that triptans and ß-blockers interact with the endothelial cell component of the blood vessel to produce anti-hyperalgesia.

Sensory dysfunction of bladder mucosa and bladder oversensitivity in a rat model of metabolic syndrome.

PURPOSE: To study the role of sensory dysfunction of bladder mucosa in bladder oversensitivity of rats with metabolic syndrome. MATERIALS AND METHODS: Female Wistar rats were fed a fructose-rich diet (60%) or a normal diet for 3 months. Based on cystometry, the fructose-fed rats (FFRs) were divided into a group with normal detrusor function or detrusor overactivity (DO). Acidic adenosine triphosphate (ATP) solution (5mM, pH 3.3) was used to elicit reflex micturition. Cystometric parameters were evaluated before and after drug administration. Functional proteins of the bladder mucosa were assessed by western blotting. RESULTS: Compared to the controls, intravesical acidic ATP solution instillation induced a significant increase in provoked phasic contractions in both FFR groups and a significant decrease in the mean functional bladder capacity of group DO. Pretreatment with capsaicin for C-fiber desentization, intravesical liposome for mucosal protection, or intravenous pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid for antagonized purinergic receptors can interfere with the urodynamic effects of intravesical ATP in FFRs and controls. Over-expression of TRPV1, P2X(3), and iNOS proteins, and down-regulation of eNOS proteins were observed in the bladder mucosa of both fructose-fed groups. CONCLUSIONS: Alterations of sensory receptors and enzymes in the bladder mucosa, including over-expression of TRPV1, P2X(3), and iNOS proteins, can precipitate the emergence of bladder phasic contractions and oversensitivity through the activation of C-afferents during acidic ATP solution stimulation in FFRs. The down-regulation of eNOS protein in the bladder mucosa of FFRs may lead to a failure to suppress bladder oversensitivity and phasic contractions. Sensory dysfunction of bladder mucosa and DO causing by metabolic syndrome are easier to elicit bladder oversensitivity to certain urothelium stimuli.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: a microdialysis study.

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.