P2X Purinergic Receptor Antagonist Accelerates SkinBarrier Repair and Prevents Epidermal Hyperplasia Inducedby Skin Barrier Disruption
2002; Elsevier BV; Volume: 119; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2002.19505.x
ISSN1523-1747
AutoresMitsuhiro Denda, Kaori Inoue, Shigeyoshi Fuziwara, Sumiko Denda,
Tópico(s)Ocular Surface and Contact Lens
ResumoThe effects of ATP receptor agonists/antagonists on skin barrier recovery rate were evaluated in hairless mice. Topical application of ATP and α,β-methylene ATP (agonist of P2X receptor) delayed barrier recovery. Topical application of suramin (nonspecific ATP receptor antagonist), pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (P2X receptor antagonist), and 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) (P2X1, P2X3, P2X2/3 antagonist) after barrier disruption accelerated the barrier repair. The P2Y type receptor antagonist Reactive Blue 2 did not affect the barrier repair process. Moreover, topical application of TNP-ATP prevented epidermal hyperplasia induced by barrier insult under low environmental humidity. ATP was secreted immediately after tape stripping on skin in organ culture. α,β-Methylene ATP increased intercellular calcium in cultured keratinocytes and the increase was blocked by TNP-ATP. Both reverse transcription polymerase chain reaction assay and immunohistochemical study showed the existence of protein that had a structure similar to P2X3 on hairless mouse epidermis. These results suggest that cutaneous barrier homeostasis can be regulated by cation flux through a P2X3-like ATP receptor. The effects of ATP receptor agonists/antagonists on skin barrier recovery rate were evaluated in hairless mice. Topical application of ATP and α,β-methylene ATP (agonist of P2X receptor) delayed barrier recovery. Topical application of suramin (nonspecific ATP receptor antagonist), pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (P2X receptor antagonist), and 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) (P2X1, P2X3, P2X2/3 antagonist) after barrier disruption accelerated the barrier repair. The P2Y type receptor antagonist Reactive Blue 2 did not affect the barrier repair process. Moreover, topical application of TNP-ATP prevented epidermal hyperplasia induced by barrier insult under low environmental humidity. ATP was secreted immediately after tape stripping on skin in organ culture. α,β-Methylene ATP increased intercellular calcium in cultured keratinocytes and the increase was blocked by TNP-ATP. Both reverse transcription polymerase chain reaction assay and immunohistochemical study showed the existence of protein that had a structure similar to P2X3 on hairless mouse epidermis. These results suggest that cutaneous barrier homeostasis can be regulated by cation flux through a P2X3-like ATP receptor. pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid transepidermal water loss 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate The most important role of the skin for terrestrial animals is protecting water-rich internal organs from environmental dryness. Stratum corneum plays a crucial role as the water-impermeable barrier. The stratum corneum is composed of two components, i.e., protein-rich nonviable cells and intercellular lipid domains (Elias and Feingold, 2001Elias P.M. Feingold K.R. Coordinate regulation of epidermal differentiation and barrier homeostasis.Skin Pharmacol Appl Skin Physiol. 2001; 14: S28-S34Crossref PubMed Scopus (77) Google Scholar). When the barrier function is damaged by a surfactant, organic solvent, or tape stripping, a series of homeostatic systems is accelerated and the barrier function recovers to its original level (Elias and Feingold, 2001Elias P.M. Feingold K.R. Coordinate regulation of epidermal differentiation and barrier homeostasis.Skin Pharmacol Appl Skin Physiol. 2001; 14: S28-S34Crossref PubMed Scopus (77) Google Scholar). At the first stage of the barrier repair process, exocytosis of lipid-containing granules, lamellar bodies, is accelerated and the inside lipid is secreted into the intercellular domain between the stratum granulosum and stratum corneum and forms a water-impermeable membrane (Elias and Feingold, 2001Elias P.M. Feingold K.R. Coordinate regulation of epidermal differentiation and barrier homeostasis.Skin Pharmacol Appl Skin Physiol. 2001; 14: S28-S34Crossref PubMed Scopus (77) Google Scholar). Previous studies suggested that ion gradation in the epidermis is strongly associated with the barrier repair system (Lee et al., 1992Lee S.H. Elias P.M. Proksch E. Menon G.K. Man M.Q. Feingold K.R. Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.J Clin Invest. 1992; 89: 530-538Crossref PubMed Scopus (177) Google Scholar; Menon et al., 1994Menon K. Price L.F. Bommannan B. Elias P.M. Feingold K.R. Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion.J Invest Dermatol. 1994; 102: 789-795Abstract Full Text PDF PubMed Google Scholar; Denda et al., 2000Denda M. Hosoi J. Ashida Y. Visual imaging of ion distribution in human epidermis.Biochem Biophys Res Commun. 2000; 272: 134-137Crossref PubMed Scopus (84) Google Scholar). When the calcium concentration in the upper epidermis increases by sonophoresis, lamellar body secretion is prevented (Menon et al., 1994Menon K. Price L.F. Bommannan B. Elias P.M. Feingold K.R. Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion.J Invest Dermatol. 1994; 102: 789-795Abstract Full Text PDF PubMed Google Scholar). Lee et al demonstrated that the Ca2+ channel blocker verapamil prevented the delay of the barrier repair induced by increased extracellular calcium concentration (Lee et al., 1994Lee S.H. Elias P.M. Feingold K.R. Mauro T. A role for ions in barrier recovery after acute perturbation.J Invest Dermatol. 1994; 102: 976-979Abstract Full Text PDF PubMed Google Scholar). These results suggest that calcium flux into keratinocytes perturbs the lamellar body secretion and the barrier repair and that blocking the calcium flux prevents the delay of the barrier recovery. The type of channel related to skin barrier homeostasis has not been identified yet. Various types of ligand-gated cation channels ordinarily found in neurons are also found in epithelial cells including keratinocytes (Ndoye et al., 1998Ndoye A. Buchli R. Greenberg B. et al.Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis.J Invest Dermatol. 1998; 111: 410-416Crossref PubMed Scopus (108) Google Scholar; Genever et al., 1999Genever P.G. Maxfield S.J. Kennovin G.D. Maltman J. Bowgen C.J. Raxworthy M.J. Skerry T.M. Evidence for a novel glutamate-mediated signaling pathway in keratinocytes.J Invest Dermatol. 1999; 112: 337-342Crossref PubMed Scopus (91) Google Scholar; Stoebner et al., 1999Stoebner P.E. Carayon P. Penarier G. et al.The expression of peripheral benzodiapine receptors in human skin the relationship with epidermal cell differentiation.Br J Dermatol. 1999; 140: 1010-1016Crossref PubMed Scopus (43) Google Scholar; Denda et al., 2001Denda M. Fuziwara S. Inoue K. Denda S. Akamatsu H. Tomitaka A. Matsunaga K. Immunoreactivity of VR1 on epidermal keratinocyte of human skin.Biochem Biophys Res Commun. 2001; 285: 1250-1252Crossref PubMed Scopus (181) Google Scholar). Several reports demonstrated that epithelial cells secrete ATP under stress (Ferguson et al., 1997Ferguson D.R. Kennedy I. Burton T.J. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes – a possible sensory mechanism?.J Physiol. 1997; 505: 503-511Crossref PubMed Scopus (517) Google Scholar; Homolya et al., 2000Homolya L. Steinberg T.H. Boucher R.C. Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia.J Cell Biol. 2000; 150: 1349-1359Crossref PubMed Scopus (216) Google Scholar). Moreover, ATP affects keratinocyte calcium flux and its terminal differentiation (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar). Enomoto et al demonstrated that a calcium wave was induced in cultured epithelial cells by mechanical stress and ATP was the mediator of calcium movement (Enomoto et al., 1994Enomoto K. Furuya K. Yamagishi S. Oka T. Maeno T. The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells.Pflugers Arch. 1994; 427: 533-542Crossref PubMed Scopus (94) Google Scholar). Thus, we hypothesized that the ATP-gated cation channel plays an important role in skin barrier homeostasis. In this study, we examined the effects of ATP receptor (purinergic receptor) agonists and antagonists on the barrier repair process after barrier disruption. We also showed the effects of these reagents on the calcium concentration in cultured keratinocytes. All experiments were performed on 7–10-wk-old male hairless mice (HR-1, Hoshino, Japan). All procedures of the measurement of skin barrier function, disruption of the barrier, and application of test sample were carried out under anesthesia. All experiments were approved by the Animal Research Committee of the Shiseido Research Center in accordance with the National Research Council Guide (National Research Council (NRC):, 1996National Research Council (NRC) National Research Council Guide. National Academy Press, Washington1996Google Scholar). ATP was purchased from Wako (Wako, Osaka, Japan). α,β-methylene ATP, suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) were purchased from Sigma (Sigma, MO). Normal human keratinocytes (neonatal skin) were purchased from BioWhittaker (Walkersville, MD). Permeability barrier function was evaluated by measurement of transepidermal water loss (TEWL) with an electric water analyzer (Meeco, Warrington, PA), as described previously (Denda et al., 1998aDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (186) Google Scholar). For barrier recovery experiments, both sides of flank skin were treated with repeated tape stripping until the TEWL reached 7–10 mg per cm2 per h, as described previously (Denda et al., 1998aDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (186) Google Scholar). Immediately after barrier disruption, 100 μl of an aqueous solution containing 1 mM reagent or water alone (control) was applied to the treated area (2×3 cm2). We did not apply the same reagent to both sides of the flank. The areas were covered by plastic membrane for 15 min and the membrane was removed. Two points on one side of the flank were measured and four to 12 mice were used to evaluate the effects of each treatment. TEWL was then measured over the same sites at 1, 3, and 6 h after barrier disruption. All of the barrier disruption was done between 7:00 a.m. and 8:00 a.m. and then evaluation of the barrier recovery rate was carried out to avoid the deviation of the results by circadian rhythm (Denda and Tsuchiya, 2000Denda M. Tsuchiya T. Barrier recovery rate varies time-dependently in human skin.Br J Dermatol. 2000; 142: 881-884Crossref PubMed Scopus (68) Google Scholar). The barrier recovery results are expressed as percent of recovery, because of variations from day to day in the extent of barrier disruption. In each animal, the percentage of recovery was calculated by the following formula: (TEWL immediately after barrier disruption –TEWL at indicated time point)/(TEWL immediately after barrier disruption –baseline TEWL)×100%. Animals were kept separately in 7.2 l cages in which the relative humidity was maintained at less than 10% with dry air as described previously (Denda et al., 1998aDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (186) Google Scholar). The temperature was the same in all cases (22–25°C), and fresh air was circulated 100 times per hour. Animals were kept out of the direct stream of air. During the experiments, the animal's behavior was not restricted. The level of NH3 was always below 1 ppm. Animals were first kept in the dry condition for 48 h and then both sides of the flank skin were treated with acetone-soaked cotton balls, as described previously (Denda et al., 1998aDenda M. Sato J. Masuda Y. et al.Exposure to a dry environment enhances epidermal permeability barrier function.J Invest Dermatol. 1998; 111: 858-863Crossref PubMed Scopus (186) Google Scholar). The procedure was terminated when the TEWL reached 2.5–3.5 mg per cm2 per h. Immediately after the barrier disruption, 100 μl of TNP-ATP aqueous solution (100 nM) was applied on one side of the treated area. Water was applied on the other side. Then the animals were again kept in a dry condition for 48 h. After the experiments, animals were euthanized with diethylether inhalation and skin samples were taken from the treated area. One hour before the euthanization, 20 μl per g body weight bromodeoxyuridine (BrdU) 10 mM solution was injected intraperitoneally. Untreated control mice were also treated with BrdU at the same time. After fixation with 4% paraformaldehyde, full thickness skin samples were embedded in paraffin, sectioned (4 μm), and processed for hematoxylin and eosin staining. Five areas were selected at random from each section; the thickness of the epidermis was measured with an optical micrometer, and the mean value was calculated. For the assessment of DNA synthesis, the sections were immunostained with anti-BrdU antibodies. Five areas were selected at random from each section; the number of immunostained cells per millimeter of epidermis was counted and the mean value was calculated. Measurements were carried out in an observer-blinded fashion. We quantified ATP secretion from skin organ culture by a method adopted from previous reports (Wood et al., 1996Wood L.C. Elias P.M. Calhoun C. Feingold K.R. Barrier disruption stimulates interleukin-1 alpha expression and release from a preformed pool in murine epidermis.J Invest Dermatol. 1996; 106: 397-403Crossref PubMed Scopus (231) Google Scholar; Ashida et al., 2001aAshida Y. Denda M. Hirao T. Histamine H1 and H2 receptor antagonists accelerate skin barrier repair and prevent epidermal hyperplasia induced by barrier disruption in a dry environment.J Invest Dermatol. 2001; 116: 261-265Crossref PubMed Scopus (57) Google Scholar). Skin samples were taken from both flanks immediately. Subcutaneous fat was removed with a scalpel and the skin samples were cut into exact squares (1.5±1.5 cm2). The two pieces of skin from the two flanks were placed, epidermis side upwards, in separate culture dishes kept in an ice–water bath, and one of them was tape stripped four times. The other piece of skin was not treated. One milliliter of chilled buffered saline solution containing 150 mM NaCl, 10 mM glucose, 25 mM HEPES, 5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, and 1.8 mM CaCl2, adjusted to pH 7.4 with NaOH, was added to both dishes, which were incubated on ice for 10 min, and 100 μl aliquots of the solution were removed; the ATP was immediately quantified with an ATP determination kit (A-6608, Molecular Probes, OR) according to the manufacturer's instructions. Evaluation of [Ca2+]i in cultured human keratinocytes was carried out by two methods. First, changes in [Ca2+]i in a single cell were measured with fura-2 as described by Grynkiewicz et al., 1985Grynkiewicz G. Poenie M. Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties.J Biol Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar with minor modifications (Koizumi and Inoue, 1997Koizumi S. Inoue K. Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurons.Br J Pharmacol. 1997; 122: 51-58Crossref PubMed Scopus (69) Google Scholar). First, we incubated cells in low calcium medium (0.1 mM calcium, Defined Keratinocyte SFM, Gibco BRL, Grand Island, NY) for at least 5 d and used within 10 d. At the measurements, the keratinocytes showed 80%–100% confluency. We calibrated the calcium concentration and the parameter was the absolute value of the calcium. All of the in vitro cell culture measurements were carried out using human keratinocytes. Briefly, the culture medium was replaced with buffered saline solution containing 150 mM NaCl, 10 mM glucose, 25 mM HEPES, 5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, and 1.8 mM CaCl2, adjusted to pH 7.4 with NaOH. Then, after adding fura-2 acetoxymethylester (fura-2AM, Molecular Probes, Eugene, OR) to a final concentration of 5 μM, cells were incubated for 45 min at room temperature (21°C–23°C). The cells were washed in saline and then incubated a further 15 min to allow de-esterification of the loaded fura-2AM. The coverslip was mounted on a fluorescence microscope (IX70, TS Olympus, Tokyo, Japan) equipped with a 75 W xenon lamp and band-pass filters of 340 nm and 380 nm wavelengths. Measurements were carried out at room temperature. Imaging data, recorded by a high-sensitive silicon intensifier target camera (C4742, Hamamatsu Photonics, Hamamatsu, Japan), were regulated with a Ca2+ analyzing system (AQUA/RATI01, Hamamatsu Photonics). We also evaluated [Ca2+]i in cultured keratinocytes using fluo-3AM by the method reported by Watson et al., 1998Watson J. Brough S. Coldwell M.C. et al.Functional effects of the muscaric receptor agonist, xanomeline, at 5-HT1 and 5HT2 receptors.Br J Pharmacol. 1998; 125: 1413-1420Crossref PubMed Scopus (45) Google Scholar. Cultured human keratinocytes were plated onto 96-well plates at a density of 65,000 cells per well in the buffered saline solution we described above. Fluo-3AM (final 4 μM) was added and incubated at 37°C for 90 min. After the cells were washed with the saline solution, the plate was placed into a Fluometric Imaging Plate Reader (Fluoskan, Ascent FL Labsystems, Beverly, MA) to monitor cell fluorescence. We used four mice for the assay. Epidermis of the skin tissue was removed by incubation in a 10 mM ethylenediamine tetraacetic acid (EDTA) phosphate-buffered saline (PBS) solution at 37°C for 30 min and total RNA was isolated by ISOGEN (Wako, Osaka, Japan), containing phenol and guanidine thiocyanate, according to the manufacturer's instructions. Briefly, the keratinocytes were homogenized with ISOGEN, and then chloroform was added. After centrifugation, the aqueous phase that contained RNA was isolated and the RNA was precipitated with 10 mM ammonium acetate and ethanol. The resulting pellet was suspended in 10 μl of water, and 2 μl was analyzed by PCR. For P2X1 analysis, primer 1 (5′-GAATGGCACAAACCGTCGTCACCTCTTCAA 1061–1190) and primer 2 (5′-GCACGAAGCTAGGGTACTGGTGTGTG-AGGA 1593–1622) corresponding to the mouse nucleotides were used (Liang et al., 2001Liang S.X. Jenkins N.A. Gilbert D.J. Copeland N.G. Phillips W.D. Structure and chromosome localization of the mouse P2X1 purinoceptor gene.Cytogenet Cell Genet. 2001; 92: 333-336Crossref PubMed Google Scholar). For P2X3 analysis, primer 1 (5′-TGGAGTTCTGG-GCATTAAGATCGG 879–902) and primer 2 (5′-TTAGTGACCAATA-GAATAGGCCC 1343–1365) corresponding to the mouse nucleotides were used (Souslova et al., 1997Souslova V. Ravenall S. Fox M. Wells D. Wood J.N. Akopian A.N. Structure and chromosomal mapping of mouse P2X3 gene.Gene. 1997; 195: 101-111Crossref PubMed Scopus (18) Google Scholar). Polyclonal antiserum and blocking peptide of P2X3 were purchased from Neuromics (Minneapolis, MN). The immunogen sequence was VEKQSTDSGAYSIGH, which is ordinarily found in rat P2X3, and the host was a guinea pig. A totally identical amino acid sequence has been reported in mouse P2X3 protein (Souslova et al., 1997Souslova V. Ravenall S. Fox M. Wells D. Wood J.N. Akopian A.N. Structure and chromosomal mapping of mouse P2X3 gene.Gene. 1997; 195: 101-111Crossref PubMed Scopus (18) Google Scholar). The antiserum was diluted 500: 1 with blocking solution, i.e., 3% bovine albumin PBS solution including 10% heat-inactivated goat serum and 0.4% Triton X-100. Fluorescence secondary antibody was purchased from Molecular Probes (Alexa Fluor 488, antiguinea pig IgG conjugate). The secondary antibody was also diluted 500: 1 with the blocking solution. The blocking peptides were reconstituted with 200 μl of PBS solution. For blocking the antibody/antigen binding, 10 μM of the peptide at the final concentration was used as suggested by the manufacturer. A 5 μm frozen skin section was fixed with -20°C methanol for 10 min and soaked in PBS solution. Then the section was blocked with blocking solution for 1 h at room temperature. The section was covered with diluted antiserum solution and kept at 4°C overnight. The section was washed with PBS solution containing 0.05% Tween 20 for 15 min three times and covered with the secondary fluorescent antibody solution for 1 h at room temperature. Then the section was washed with PBS solution including 0.05% Tween 20 for 15 min three times, and mounted with Vectashield (Vectashield with DAPI, Vector Laboratories, Burlingame, CA). The section was observed and photographs were taken within 6 h. The results are expressed as the mean±SD. Statistical differences between two groups were determined by a two-tailed Student's t test. In the case of more than two groups, differences were determined by ANOVA test (Fisher's protected least significant difference). Figure 1 shows the effects of topical application of ATP receptor antagonists on skin barrier recovery after tape stripping. The non-specific ATP receptor antagonist suramin (1 mM), the P2X receptor antagonist PPADS (1 mM), and the P2X1, P2X3, P2X2/3 receptor antagonist TNP-ATP (100 nM) (Virginio et al., 1998Virginio C. Robertson G. Surprenant A. North R.A. Trinitrophenyl-substituted nucleotides are potent antagonists selective for P2X1, P2X3 and heteromeric P2X2/3 receptors.Mol Pharmacol. 1998; 53: 673-696Google Scholar) accelerate the barrier recovery, 1, 3, and 6 h after barrier disruption. On the other hand, P2Y receptor antagonist Reactive Blue 2 (1 mM) did not affect the barrier recovery rate. Figure 2 shows the effects of topical application of ATP receptor agonists on skin barrier recovery after tape stripping. ATP delayed the barrier repair. α,β-methylene ATP, an agonist of the P2X receptor (Burnstock and Williams, 2000Burnstock G. Williams M. P2 purinergic receptors modulation of cell function and therapeutic potential.J Pharmacol Exp Therap. 2000; 295: 862-869PubMed Google Scholar), also delayed the barrier recovery. When we applied 1 mM α,β-methylene ATP and 100 nM TNP-ATP together, the barrier recovery was slightly, but statistically significantly, accelerated. These results suggest that P2X1, P2X3, or P2X2/3-like receptors are associated with the skin barrier repair process.Figure 2The effects of topical application of ATP receptor agonists on skin barrier recovery after tape stripping. Topical application of ATP (1 mM) delayed the barrier repair. Topical application of α,β-methylene ATP (1 mM), which is an agonist of P2X, also delayed the barrier recovery. When we applied 1 mM α,β-methylene ATP and 100 nM TNP-ATP together, the barrier recovery was slightly, but significantly, accelerated. The same tendency was seen 1, 3, and 6 h after the barrier disruption. Two points were measured in one flank skin and four to eight animals were used for each treatment. F=184.39, p<0.0001.View Large Image Figure ViewerDownload (PPT) Figure 3 shows the effect of topical application of TNP-ATP on epidermal hyperplasia induced by acetone treatment under dry conditions. As previously demonstrated (Denda et al., 1998bDenda M. Sato J. Tsuchiya T. Elias P.M. Feingold K.R. Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruptionimplication for seasonal exacerbations of inflammatory dermatoses.J Invest Dermatol. 1998; 111: 873-878Crossref PubMed Scopus (207) Google Scholar), the barrier disruption induced approximately an 80% increase of epidermal thickness to the original level, i.e., obvious epidermal hyperplasia on the water-treated control under dry conditions (Figure 3A). The epidermal hyperplasia was partially and significantly prevented by the topical application of 100 nM TNP-ATP (Figure 3A). The results of evaluation of epidermal DNA synthesis in the same experiments are shown in Figure 3(B). The levels of BrdU-positive cells in epidermis showed good agreement with the epidermal thickness presented in Figure 3(A). In Figure 3(C), (D), (E), a representative section of untreated skin is shown. The treatment with TNP-ATP significantly reduced the increase in epidermal thickness and DNA synthesis on the epidermal basal layer induced by acetone treatment under a low environmental humidity. These results suggest that P2X1, P2X3, or P2X2/3-like receptor is also associated with an epidermal proliferative response induced by barrier disruption. The results of Figure 1 suggest an increase of endogenous ATP in the epidermis immediately after barrier disruption. We used seven animals for the measurement. Figure 4 shows the amount of ATP that was released from skin in organ culture with or without barrier disruption by tape stripping. The barrier disruption significantly increased the secretion of ATP from the skin tissue within 15 min after the barrier disruption. Intracellular calcium concentration in a single cell using fura-2AM was increased when we added 10 μM α,β-methylene ATP (Figure 5A ). We observed four pools for each treatment. In each field of microscopic vision, we could observe approximately 50–60 cells together. Figure 5(A) shows the representative results of a single cell. The parameter was the ratio of the emission intensity 340 nm to 380 nm. The increase induced by α,β-methylene ATP was blocked by 100 nM TNP-ATP (Figure 5A). An increase of [Ca2+]i by α,β-methylene ATP was observed in more than 60% of cells among a total of 200 cells we observed and application of TNP-ATP completely blocked the increase. Representative patterns are shown in Figure 5(A). We measured seven pools for control, five pools for α,β-methylene ATP, and four pools for α,β-methylene ATP+TNP-ATP. A significant increase of [Ca2+]i on cultured keratinocytes (65,000 cells per pool) was also observed by α,β-methylene ATP using fluo-3AM (Figure 5B) and the increase was blocked by 100 nM TNP-ATP (Figure 5B). These results suggest the presence of functional P2X1, P2X3, or P2X2/3-like receptors on epidermal keratinocytes. RT-PCR analysis of P2X3 (Figure 6A ) on the total RNA from mouse epidermis showed a positive band at the expected sizes. The same band was observed on the RNA from the brain of the mice (data not shown). If genomic DNA were contaminated in the RNA from epidermis, a much longer sequence, approximately 23,000 bp, which includes four introns, would be amplified because the primers covered the DNA sequence from exon 8 to exon 12. Thus, the result probably showed the amplification of mRNA of P2X3. RT-PCR analysis of P2X1 on total RNA from mouse epidermis did not show the bands even when the expected band was observed on RNA from the brain of mouse (data not shown). Immunoreactivity against P2X3 antiserum was observed in the whole living layer of epidermis (between arrows, Figure 6B) and it was blocked by the blocking peptide (Figure 6D). To confirm the reproducibility of the result, we observed four different sections and found the same tendency in all of them. In Figure 6(C), the Nomarski microscopic image of the same section with DAPI staining is shown. Of note, immunoreactivity against P2X3 antiserum did not appear on the stratum corneum (*, Figure 6C). A Nomarski microscopic image of the same section of Figure 6(D) is shown in Figure 6(E). Previously, Guo et al., 1999Guo A. Vulchanova L. Wang X.L. Elde R. Immunocytochemical localization of the vanilloid receptor 1 (VR1) relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites.Eur J Neuroscience. 1999; 11: 946-958Crossref PubMed Scopus (752) Google Scholar demonstrated the immunoreactivity of nerve fibers in the epidermis against P2X3 antiserum, but they did not show the immunoreactivity of keratinocytes. They used thick sections (40 μm) incubated with antiserum for 48 h and observed the samples with a confocal microscope to see the longitudinal image of the fibers. In this study, we used thin sections (5 μm) with a shorter period of incubation (16 h) and observed the sections with a usual microscope. In the thin sections, one could only see a cross-section of nerve fibers and it might be too small to observe. We could not get a clear image of the keratinocyte membrane when we observed such thick sections (15–20 μm, data not shown). This might be why we could observe the immunoreactivity against P2X3 antiserum on keratinocytes. Two distinct ATP receptors exist in nerve systems (Burnstock and Williams, 2000Burnstock G. Williams M. P2 purinergic receptors modulation of cell function and therapeutic potential.J Pharmacol Exp Therap. 2000; 295: 862-869PubMed Google Scholar). One is the P2X family, which is a ligand-gated ion channel, and the other family, P2Y, is a metabotroipic, heptahelical G-protein coupled receptor. The P2Xn subclass is subclassified into P2X1 to P2X7. P2X2 is inactive against α,β-methylene ATP (Burnstock and Williams, 2000Burnstock G. Williams M. P2 purinergic receptors modulation of cell function and therapeutic potential.J Pharmacol Exp Therap. 200
Referência(s)