Neutral Endopeptidase Expression and Distribution in Human Skin and Wounds
1999; Elsevier BV; Volume: 112; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.1999.00596.x
ISSN1523-1747
AutoresJohn E. Olerud, Marcia L. Usui, Deniz Seçkin, Diane S. Chiu, Claire L. Haycox, In-Sung Song, John C. Ansel, Nigel W. Bunnett,
Tópico(s)Biochemical and Structural Characterization
ResumoCutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds. Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds. neutral endopeptidase immunohistochemistry substance P substance P receptor human dermal fibroblasts human dermal microvascular endothelial cells methoxy-2 napthylamine The cutaneous sensory nervous system comprises a network of fine C fibers within the skin that innervate multiple cell types and plays an important role in inflammation and wound healing (Hosoi et al., 1993Hosoi J. Murphy G.F. Egan C.L. Lemer E.A. Grabbe S. Asahina A. Granstein R.D. Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide.Nature. 1993; 363: 159-163Crossref PubMed Scopus (518) Google Scholar;Ansel et al., 1996Ansel J.C. Kaynard A.H. Armstrong C.A. Olerud J. Bunnett N. Payan D. Skin–nervous system interactions.J Invest Dermatol. 1996; 106: 198-204Crossref PubMed Scopus (184) Google Scholar). These fibers convey information from the skin to the central nervous system, and thus have a sensory role. In addition, they have a local effector function by releasing neurotransmitters within the skin (Foreman and Jordan, 1984Foreman J. Jordan C. Neurogenic inflammation.Trends Pharmacol Sci. 1984; 5: 116-119Abstract Full Text PDF Scopus (89) Google Scholar). Neuropeptides, notably substance p (SP) and calcitonin gene related peptide are released from afferent fibers both centrally and peripherally (Holzer, 1988Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinin, calcitonin gene-related peptide and other neuropeptides.Neuroscience. 1988; 24: 739-768Crossref PubMed Scopus (1462) Google Scholar). SP and calcitonin gene related peptide have multiple actions within the skin that are important in inflammation and healing (Otsuka and Yoshioka, 1993Otsuka M. Yoshioka K. Neurotransmitter functions of mammalian tachykinins.Physiol Rev. 1993; 73: 229-281Crossref PubMed Scopus (1023) Google Scholar). These actions include increasing blood flow (Lembeck and Holzer, 1979Lembeck F. Holzer P. Substance P as neurogenic mediator of antidromic vasodilatation and neurogenic plasma extravasation.Naunyn-Schmeideberg's Arch Pharmacol. 1979; 310: 175-183Crossref PubMed Scopus (1006) Google Scholar), inducing plasma extravasation (Iwamoto et al., 1989Iwamoto I. Ueki I.F. Borson D.B. Nadel J.A. Neutral endopeptidase modulates tachykinin-induced increase in vascular permeability in guinea pig skin.Int Arch Allergy Appl Immunol. 1989; 88: 288-293Crossref PubMed Scopus (28) Google Scholar) and neutrophil infiltration (Öhlén et al., 1989Öhlén A. Thureson-Klein Å Lindbom L. Persson M. Hedqvist P. Substance P activates leukocytes and platelets in rabbit microvessels.Blood Vessels. 1989; 26: 84-94PubMed Google Scholar), stimulating adhesion molecule expression by endothelial cells (Matis et al., 1990Matis W.L. Lavker R.M. Murphy G.F. Substance P induces the expression of an endothelial leukocyte adhesion molecule by microvascular endothelium.J Invest Dermatol. 1990; 94: 492-495Abstract Full Text PDF PubMed Google Scholar;Quinlan et al., 1998Quinlan K.L. Song I.S. Bunnett N.W. et al.Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-I expression and function.Am J Physiol 275 (Cell Physiol). 1998; 44: C1580-C1590Google Scholar) and macrophage chemotaxis (Ruff et al., 1985Ruff M.R. Wahl S.M. Pert C.B. Substance P receptor-mediated chemotaxis of human monocytes.Peptides. 1985; 6: 107-111Crossref PubMed Scopus (168) Google Scholar), stimulating DNA synthesis by keratinocytes (Tanaka et al., 1988Tanaka T. Danno K. Ikai K. Imamura S. Effects of substance P and substance K on the growth of cultured keratinocytes.J Invest Dermatol. 1988; 90: 399-401Abstract Full Text PDF PubMed Google Scholar) and fibroblasts (Nilsson et al., 1985Nilsson J. von Euler A.M. Dalsgaard C.-J. Stimulation of connective tissue cell growth by substance P and substance K.Nature. 1985; 315: 61-63Crossref PubMed Scopus (576) Google Scholar) as well as stimulating proliferation by endothelial cells and inducing neovascularization (Ziche et al., 1990Ziche M. Morbidelli L. Pacini M. Geppetti P. Alessandri G. Maggi C.A. Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells.Microvascular Res. 1990; 40: 264-278Crossref PubMed Scopus (241) Google Scholar). These effects require local secretion and are mediated by interaction with specific G-protein coupled receptors on target cells. The biologic actions of neuropeptides are rapidly attenuated to prevent prolonged stimulation of cells in an uncontrolled manner (Bohm et al., 1997Bohm S.K. Grady E.F. Bunnett N.W. Regulatory mechanisms that modulate signaling by G-protein coupled receptors.Biochem J. 1997; 322: 1-18Crossref PubMed Scopus (451) Google Scholar). One of the earliest mechanisms of attenuation is degradation of the neuropeptide within the extracellular fluid. Neutral endopeptidase (NEP, EC 3.4 24.11), also known as enkephalinase, common acute lymphoblastic antigen (CALLA) and CD10 is a cell surface enzyme that degrades several neuropeptides (Roques et al., 1993Roques B.P. Nobel F. Dauge V. Fournié-Zaluski M.-C. Beaumont A. Neutral endopeptidase 24.11: Structure, inhibition, and experimental and clinical pharmacology.Pharmacol Rev. 1993; 45: 87-133PubMed Google Scholar). From a kinetic standpoint, SP and bradykinin are favorable substrates (Matsas et al., 1984Matsas R. Kenny J. Turner A.J. The metabolism of neuropeptides.Biochem J. 1984; 223: 433-440Crossref PubMed Scopus (303) Google Scholar), although NEP also degrades calcitonin gene related peptide (Katayama et al., 1991Katayama M. Nadel J.A. Bunnett N.W. DiMaria G.U. Haxhiu M. Borson D.B. Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase.Peptides. 1991; 12: 563-567Crossref PubMed Scopus (85) Google Scholar). Thus, NEP inactivates several proinflammatory peptides and thereby terminates inflammation. Administration of NEP inhibitors magnifies the proinflammatory effects of SP and other tachykinin in several tissues (Nadel, 1990Nadel J.A. Neutral endopeptidase modulation of neurogenic inflammation in airways.Eur Respir J. 1990; 3: 645S-651SGoogle Scholar;Roques et al., 1993Roques B.P. Nobel F. Dauge V. Fournié-Zaluski M.-C. Beaumont A. Neutral endopeptidase 24.11: Structure, inhibition, and experimental and clinical pharmacology.Pharmacol Rev. 1993; 45: 87-133PubMed Google Scholar). Furthermore, genetic deletion of NEP results in elevated plasma extravasation in multiple tissues including the skin (Lu et al., 1997Lu B. Figini M. Emanueli C. et al.The control of microvascular permeability and blood pressure by neutral endopeptidase.Nature Med. 1997; 3: 904-907Crossref PubMed Scopus (133) Google Scholar). These effects are reversed by administration of selective antagonists of SP and bradykinin receptors and thus the observed effects are mediated by diminished degradation of these peptides. Tissue responsiveness to neuropeptides depends on the presence of specific receptors and on the distribution of neuropeptide degrading enzymes such as NEP. NEP is highly expressed in the brush border of the kidney and small intestine, and in lymph nodes, although substantial NEP levels are also found in the brain, salivary glands, adrenal glands, pancreas, gut wall, nasal mucosa, and lungs (Roques et al., 1993Roques B.P. Nobel F. Dauge V. Fournié-Zaluski M.-C. Beaumont A. Neutral endopeptidase 24.11: Structure, inhibition, and experimental and clinical pharmacology.Pharmacol Rev. 1993; 45: 87-133PubMed Google Scholar;Ansel et al., 1996Ansel J.C. Kaynard A.H. Armstrong C.A. Olerud J. Bunnett N. Payan D. Skin–nervous system interactions.J Invest Dermatol. 1996; 106: 198-204Crossref PubMed Scopus (184) Google Scholar). The skin also contains neuropeptide degrading proteases, although they have not been fully characterized and localized. Frog skin secretes a 100 kDa prototypical metallopeptidase that degrades neuropeptides but which is distinct from NEP (Carvalho et al., 1992Carvalho K.M. Joudiou C. Boussetta H. Leseney A.-M. Cohen P. A peptide-hormone inactivating endopeptidase in Xenopus iaevis skin secretion.Proc Natl Acad Sci USA. 1992; 89: 84-88Crossref PubMed Scopus (27) Google Scholar). Homogenized mouse skin (Paus et al., 1994Paus R. Heinzelmann T. Schultz K.D. Furkert J. Fechner K. Czarnetzki B.M. Hair growth induction by substance P.Lab Invest. 1994; 1: 134-140Google Scholar) and guinea pig skin (Iwamoto et al., 1989Iwamoto I. Ueki I.F. Borson D.B. Nadel J.A. Neutral endopeptidase modulates tachykinin-induced increase in vascular permeability in guinea pig skin.Int Arch Allergy Appl Immunol. 1989; 88: 288-293Crossref PubMed Scopus (28) Google Scholar) have peptidase activity that can be blocked with specific inhibitors of NEP. A previous study of human skin has failed to demonstrate NEP by immunohistochemistry (IHC) in a patient with scleroderma. Cultured skin fibroblasts from the same patient, however, did express NEP measured by flow cytometry (Bou-Gharios et al., 1995Bou-Gharios G. Osman J. Atherton A. Monoghan P. Vancheeswaran R. Black C. Olsen I. Expression of ectopeptidases in scleroderma.Ann Rheum Dis. 1995; 54: 111-116Crossref PubMed Scopus (17) Google Scholar). In view of the lack of information about localization of NEP in the skin, and the established role of neuropeptides in cutaneous inflammation and in wound healing, we decided to study in detail the distribution of NEP in normal and wounded human skin. Our aims were to: (i) localize NEP in normal and wounded human skin by IHC using a specific antibody; (ii) examine expression of NEP mRNA in normal human skin, in wounds and in cells derived from the human skin; and (iii) confirm expression of enzymatically active NEP in keratinocytes. An antibody was raised in rabbits to recombinant human NEP (Khepri Pharmaceutical, San Francisco, CA) conjugated to keyhole limpet hemocyanin (KLH) (Calbiochem, La Jolla, CA). The recombinant human NEP was produced in Chinese hamster ovary (CHO) cells. This human NEP was shown to be enzymatically active using the fluorometric assay described in Materials and Methods below. The NEP (5 mg) was dissolved in 3 ml 50 mM phosphate-buffered saline (PBS), pH 7.4, containing 5 mg KLH, 30 mg carbodiimide (ICN, CA). The mixture was agitated at room temperature for 3 h and overnight at 4°C. The efficacy of conjugation, estimated by incorporation of iodinated NEP (labeled using chloramine T) was ∼30%. Three 10 wk old New Zealand rabbits were immunized at intervals of 6–8 wk by multiple intradermal injections. Emulsions were prepared of equal parts of conjugated antigen and complete Freund's adjuvant (Difco Labs, Detroit, MI). Each rabbit received 2 ml of emulsion, containing 500 μg conjugate, divided into 12–15 sites. At the first immunization, 0.5 ml Tri-immunol vaccine (Lederle Labs, Pearl River, NY) was injected intramuscularly. Useful antibodies were obtained after the second boost. All rabbits generated antibodies with titers of ∼1:10,000, as determined by ELISA. Affinity column purification was performed with recombinant NEP. Total protein from homogenized adult human skin, cultured adult human keratinocytes, and cultured fetal human keratinocytes (human keratinocytes) was extracted in urea–Tris (Fleckman et al., 1997Fleckman P. Hager B. Dale B.A. Harlequin ichthyosis keratinocytes in lifted culture differentiate poorly by morphologic and biochemical criteria.J Invest Dermatol. 1997; 109: 36-38Abstract Full Text PDF PubMed Scopus (13) Google Scholar) and centrifuged. The protein concentration of the supernatants was determined for each sample, then 15 μg of protein was loaded and separated by discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar). Purified recombinant NEP (1 μg total protein) was also fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules, CA) (Towbin et al., 1979Towbin H. Staehalin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44209) Google Scholar). The membranes were incubated with antibodies to NEP at 1:500 dilution or pan keratin AE1/AE3 at 1:200/1:1000 dilution (gift T.T. Sun) (Cooper and Sun, 1986Cooper D. Sun T.T. Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression.J Biol Chem. 1986; 261: 4646-4654Abstract Full Text PDF PubMed Google Scholar) for 1 h at room temperature. Membranes were washed and incubated with secondary antibodies. Secondary antibody for NEP was biotinylated goat anti-rabbit–horseradish peroxidase (Bio-Rad) and secondary antibody for AE1/AE3 was biotinylated goat anti-mouse–horseradish peroxidase (BioRad), each at 1:2000 dilution with a 1 h incubation. All washes used 2% dried milk, 0.1% Tween in PBS. Blots were exposed on autoradiography film then visualized using ECL detection reagents (Amersham, Arlington Heights, IL). Simplate-II (General Diagnostics Company, Organon Teknika, Durham, NC) bleeding time devices were used to create pairs of wounds on both legs of 11 normal males and three normal female volunteers 66 ± 6 y of age (mean ± SD). The Simplate-II is a spring-loaded instrument which, when activated, projects a pair of blades 5 mm in length, 1 mm in depth, and 3 mm apart. This human wound model has been described previously in detail (Olerud et al., 1995Olerud J.E. Odland G.F. Burgess E.M. Wyss C.R. Fisher L.D. Matsen F.A. 3rd. A model for the study of wounds in normal elderly adults and patients with peripheral vascular disease or diabetes mellitus.J Surg Res. 1995; 59: 349-360Abstract Full Text PDF PubMed Scopus (28) Google Scholar). Volunteers were recruited using methods approved by the University of Washington Institutional Review Board for Human Subjects and with informed consent. All subjects were screened and shown to be free of neuropathy and diabetes mellitus as previously described (Olerud et al., 1995Olerud J.E. Odland G.F. Burgess E.M. Wyss C.R. Fisher L.D. Matsen F.A. 3rd. A model for the study of wounds in normal elderly adults and patients with peripheral vascular disease or diabetes mellitus.J Surg Res. 1995; 59: 349-360Abstract Full Text PDF PubMed Scopus (28) Google Scholar). Pairs of wounds were collected with a 4 mm biopsy punch at 1 h, 6 h, 1 d, 3 d, 7 d, 14 d, and 28 d after injury. Wounds were created and collected such that each time point was represented in four or five subjects. All 14 subjects contributed unwounded control samples from both legs and one sample from thigh, sacrum, or medial arm to test for regional variation in innervation and NEP expression. Half of each biopsy was frozen in OCT (Tissue Tek Miles, Torrance, CA) for IHC studies. To examine more thoroughly NEP immunohistochemistry in hair follicles a single 6 mm biopsy was taken from the vertex scalp of a 53 y old volunteer with mild androgenetic alopecia. The cylindrical biopsy was bisected and mounted in OCT in order to obtain sections perpendicular to the epidermis (vertical sections) as well as sections parallel to the epidermis (horizontal sections). Parallel sections were taken below the dermal–epidermal junction at depths between 1 and 2 mm. A sample of human jejunum was taken after informed consent from redundant tissue excised during cancer surgery. The specimen was immediately frozen in OCT and used as a positive control for IHC studies of NEP because of the high level of NEP found in the intestinal brush boarders. Six micron frozen tissue sections of wounds and control skin washed in 0.05 M Tris-buffered saline were postfixed with 2% paraformaldehyde/Sorensons buffer for 5 min. The slides were then washed with 20 mM glycine in Tris-buffered saline to saturate any free aldehydes and incubated in H2O2/Tris-buffered saline for 30 min. Slides were incubated in 1.3% goat serum/Tris-buffered saline for 30 min and then incubated with NEP anti-serum at 1:200 dilution for 1 h at room temperature. Control slides were either incubated without primary antibody or with NEP antibody at 1:200 adsorbed with recombinant NEP at a concentration of 10 μg per ml. Biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) at 1:200 dilution for 30 min was used as secondary antibody, followed by streptavidin–biotin complex (SABC Universal Kit, Zymed Laboratories, San Francisco, CA) at 1:200 dilution for 30 min. All antibodies were diluted with 0.1% bovine serum albumin in Tris-buffered saline and all incubations were done at room temperature. Sections were then visualized for immunoreactivity using 3,3′ diaminobenzidine (Sigma, St. Louis, MO) as a chromogen (0.12% in H2O with 0.1% H2O2 and 20% 0.5 M Tris buffer for 20 min), coverslipped with glycergel (Dako, Carpenteria, CA) and photographed using a Nikon-Microphot-SA microscope using either standard image capture or differential interference contrast image capture. For studies of mRNA in normal skin and wounds a 53 y old subject allowed pairs of wounds 1, 3, and 7 d of age to be created on the leg with a Simplate-II bleeding time device. These wounds were removed with a 6 mm biopsy punch and immediately frozen in liquid nitrogen. Two biopsies of adjacent unwounded skin were likewise taken and immediately frozen in liquid nitrogen. Total RNA was extracted with Trizol (Gibco, Gaithersburg, MD) for mRNA studies. The subject met the same entry criteria as the other volunteers with regard to the absence of diabetes and neuropathy. Skin and wound extracts were studied using oligonucleotide primers synthesized (Gibco) corresponding to bp 903–921 (5′ primer, sense-ATG ACA TTG GCC CAG ATC C) and the complementary reverse sequence against base pairs 1330–1311 (3′ GCA ATC AAA TCC TCG ACC AC). The melting temperature of the primers is 46°C. RNA was extracted with Trizol (Gibco) from a pair of 6 mm punch biopsies of normal skin from the lateral leg (day 0) as well as the day 1, 3, and 7 wound specimens. Reverse transcriptase–PCR was performed using Super Script One-Step reverse transcriptase–PCR System (Gibco). Synthesis of cDNA was performed at 50°C for 30 min followed by PCR using a denaturing temperature of 94°C and an annealing temperature of 58°C and an extension temperature of 72°C for 30 cycles. Included were samples treated with RNase to control for the possibility of a PCR product generated from genomic DNA and samples were tested for consistent levels of glyceraldehyde-3-phosphate dehydrogenase expression. RNA was isolated from cultured newborn human keratinocytes using Ultraspec RNA Isolation Reagent (Biotex Labs, Alberta, Canada). Oligonucleotide primers were synthesized (Bio-Synthesis, Lewisville, TX) corresponding to base pairs 461–481 (5′ primer, sense-CCT CTA CTC AAA CTG TTA CC) and the complementary reverse sequence against base pairs 754–774 (3′ primer, anti-sense-GT CTA ACT AAG CAG TCC TTC) of NEP cDNA (Malfroy et al., 1988Malfroy B. Kuang W.-J. Seeburg P.H. Mason A.J. Schofield P.R. Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase).FEBS Lett. 1988; 229: 1206-1210Abstract Full Text PDF Scopus (162) Google Scholar). Reverse transcription was performed on keratinocyte RNA to make a complementary cDNA strand to NEP mRNA by combining 3′ primer, Maloney murine leukemia virus reverse transcriptase (MMLV RT, Gibco), 1 OX d4NTP (Pharmacia LKB Biotechnology, Piscataway, NJ), 5 × first strand buffer (Gibco), RNasin (Promega, Madison, WI), and dithiothreitol (Gibco). Reverse transcriptase mixture was incubated at 42°C for 1 h followed by heat inactivation of the MMLV RT at 65°C. PCR was performed on 0, 1, 2, and 5 μl of the preconfluent and confluent reverse transcriptase reaction products by adding equal amounts of the 3′ and 5′ primers, 1.25 × d4NTP mixture (Pharmacia LKB Biotechnology) and 1 OX PCR buffer + Mg (Boehringer Mannheim, Indianapolis, IN). Taq polymerase (Boehringer Mannheim) was added during the hot start at 85°C. The PCR reaction was carried out for 35 cycles using a denaturing temperature of 94°C, an annealing temperature of 53°C, and extension temperature of 72°C. The reverse transcriptase–PCR products from extracts of normal skin and wound as well as from keratinocytes were gel-purified (Qiagen, Valencia, CA). The products were then sequenced in both directions using a PCR sequencing reaction containing the 5′ and 3′ NEP oligonucleotide primer and Dye Terminator with AmpliTaq DNA Polymerase FS (ABI Prism, Perkin-Elmer, CT) followed by sequencing using a model 377 Fluorescence Sequencer (ABI Prism, Perkin-Elmer). Northern blot analysis was performed using total RNA extracted from 3 and 7 d wounds with Trizol (Gibco). RNA was hybridized to a NEP specific ((32P) UTP-labeled anti-sense RNA probe (Plowman et al., 1993Plowman G.D. Culouscou J.M. Whitney G.S. et al.Ligand-specific activation of HER 4/pl 80erbB4, a fourth member of the epidermal growth factor receptor family.Proc Natl Acad Sci USA. 1993; 90: 1746-1750Crossref PubMed Scopus (662) Google Scholar) transcribed from base pairs 945–1346 in the NEP cDNA sequence. Ten and 20 μg samples of total RNA were studied. Normalization of the data for variability in gel loading and transfer efficiency was accomplished by staining the RNA with 0.5M NaOAc/0.04% methylene blue. Newborn human keratinocytes (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatal. 1990; 94: 6-18Crossref PubMed Scopus (107) Google Scholar), human dermal fibroblasts (HDF) (Dale et al., 1990Dale B.A. Holbrook K.A. Fleckman P. Kimball J.R. Brumbaugh S. Sybert V.P. Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatal. 1990; 94: 6-18Crossref PubMed Scopus (107) Google Scholar), or human dermal microvascular endothelial cells (HDMEC) (Swerlick et al., 1991Swerlick R.A. Garcia-Gonzalez E. Kubota Y. Xu Y.L. Lawley T.J. Studies of the modulation of MHC antigen and cell adhesion molecule expression on human dermal microvascular endothelial cells.J Invest Dermatol. 1991; 97: 190-196Abstract Full Text PDF PubMed Google Scholar) were cultured in appropriate media to 50%, 70%, or 90% confluence, respectively, then subcultured in serum-free media overnight before mRNA isolation. Cells were harvested with or without 100 nM SP for 3 h and mRNA was isolated using a commercially available mRNA Isolation Kit (Boehringer Mannheim). Northern blot analysis was performed as previously described (Sambrook et al., 1989Sambrook J. Fritsch E.F. Matiatis T. Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). An 860 bp NEP cDNA fragment probe isolated by reverse transcriptase–PCR was radiolabeled according to the random priming method ofFeinberg and Vogelstein, 1983Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal Biochem. 1983; 132: 6-10Crossref PubMed Scopus (16456) Google Scholar, Feinberg and Vogelstein, 1984Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum.Anal Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5118) Google Scholar. The cDNA fragment used for northern blotting spanned base pairs 101–961. Identity with the published sequence for NEP cDNA (Malfroy et al., 1988Malfroy B. Kuang W.-J. Seeburg P.H. Mason A.J. Schofield P.R. Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase).FEBS Lett. 1988; 229: 1206-1210Abstract Full Text PDF Scopus (162) Google Scholar) was established by DNA sequencing. The positively charged nylon membrane blot was probed at 65°C for 3–4 h in Rapid-hyb buffer (Amersham) and washed with low stringency solution (2 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate) at room temperature then high stringency solution (0.1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate) at 65°C for 20 min prior to exposure to X-ray film at 70°C for 2–3 d. Normalization of the data for variability in gel loading and transfer efficiency was accomplished by determining the level of β-actin using the appropriate cDNA probe (Alonso et al., 1986Alonso S. Minty A. Bourlet Y. Buckingham M. Comparison of three actin-coding sequences in the mouse: evolutionary relationship between the actin genes of warm-blooded vertebrates.J Mol Evol. 1986; 23: 11-22Crossref PubMed Scopus (599) Google Scholar). Frozen keratinocytes were weighed and homogenized in 50 mM Tris (hydroxymethyl) aminomethane hydrochloride (Tris, pH 7.5) containing 1 μM pepstatin A and 1 mM phenylmethylsulfonylfluoride, and assayed for protein using Coomassie Blue. Cell membranes containing a variety of enzymes (presumably including NEP) were incubated with Glu-Ala-Ala-Phe-MNA. Enzymatic degradation of Glu-Ala-Ala-Phe-MNA results in a fluorescent product (MNA) that can be quantitated. Incubations with keratinocyte membranes were carried out both with and without the specific inhibitor of NEP (DL-thiorphan). The difference in the production of MNA, between membrane preparations with the specific inhibitors of NEP and those without, were attributed to NEP enzymatic activity. The enzymatic activity is reported in picomoles of MNA generated per hour per microgram of protein (Terashima et al., 1992Terashima H. Okamoto A. Menozzi D. Goetzl E.J. Bunnett N.W. Identification of neuropeptide-degrading enzymes in the pancreas.Peptides. 1992; 13: 741-748Crossref PubMed Scopus (20) Google Scholar). We examine
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