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Expression of Neuropeptide Galanin and Galanin Receptors in Human Skin

2004; Elsevier BV; Volume: 122; Issue: 4 Linguagem: Inglês

10.1111/j.0022-202x.2004.22418.x

1523-1747

Barbara Kofler, Alexandra Berger, Radmila Santic, Kerstin Moritz, Dietmar Almer, Camilla Tuechler, Roland Lang, Michael Emberger, Alfred Klausegger, Wolfgang Sperl, Johann Bauer,

Hypothalamic control of reproductive hormones

alpha-melanocyte stimulating hormone galanin GAL-like immunoreactivity phosphate-buffered saline To the Editor: The neural system in human skin contributes to the regulation of body homeostasis by transporting afferent stimuli to the central nervous system. Efferent activities like the modulation of inflammatory reactions, which are partially mediated by neuropeptides released from neurons in the skin, have also been detected (Scholzen et al., 1998Scholzen T. Armstrong C.A. Bunnett N.W. Luger T.A. Olerud J.E. Ansel J.C. Neuropeptides in the skin: Interactions between the neuroendocrine and the skin immune systems.Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (366) Google Scholar). One of these neuropeptides is galanin (GAL), a 29–30 amino-acid peptide (Tatemoto et al., 1983Tatemoto K. Rokaeus A. Jornvall H. McDonald T.J. Mutt V. Galanin—A novel biologically active peptide from porcine intestine.FEBS Lett. 1983; 164: 124-128Abstract Full Text PDF PubMed Scopus (1357) Google Scholar), which has been shown to have a widespread distribution in the central and peripheral nervous systems of many mammalian species (Kordower et al., 1992Kordower J.H. Le H.K. Mufson E.J. Galanin immunoreactivity in the primate central nervous system.J Comp Neurol. 1992; 319: 479-500Crossref PubMed Scopus (150) Google Scholar). In human skin,Johansson et al., 1988Johansson O. Vaalasti A. Tainio H. Ljungberg A. Immunohistochemical evidence of galanin in sensory nerves of human digital skin.Acta Physiol Scand. 1988; 132: 261-263Crossref PubMed Scopus (32) Google Scholar reported GAL-like immunoreactivity (GAL-LI) in nerve terminals and fibers of the dermis and sometimes in the basal layer of the epidermis. Furthermore, the nerves beneath the epidermis, around hair follicles, Meissner's corpuscles, and sweat glands were found to be immunoreactive for GAL (Tainio et al., 1987Tainio H. Vaalasti A. Rechardt L. The distribution of substance P-, CGRP-, galanin- and ANP-like immunoreactive nerves in human sweat glands.Histochem J. 1987; 19: 375-380Crossref PubMed Scopus (83) Google Scholar;Johansson et al., 1999Johansson O. Fantini F. Hu H. Neuronal structural proteins, transmitters, transmitter enzymes and neuropeptides in human Meissner's corpuscles: A reappraisal using immunohistochemistry.Arch Dermatol Res. 1999; 291: 419-424Crossref PubMed Scopus (32) Google Scholar).Pincelli et al., 1990Pincelli C. Fantini F. Massimi P. Girolomoni G. Seidenari S. Giannetti A. Neuropeptides in skin from patients with atopic dermatitis: An immunohistochemical study.Br J Dermatol. 1990; 122: 745-750Crossref PubMed Scopus (148) Google Scholar noted a diffuse intracellular staining in the epidermis of human skin, which they regarded as probably due to a cross-reactivity of the antibody used. A first hint for extra-neuronal expression of GAL in the skin was provided byJi et al., 1995Ji R.R. Zhang X. Zhang Q. Dagerlind A. Nilsson S. Wiesenfeld-Hallin Z. Hokfelt T. Central and peripheral expression of galanin in response to inflammation.Neuroscience. 1995; 68: 563-576Crossref PubMed Scopus (103) Google Scholar, who found GAL-LI and GAL mRNA in the dermis and epithelium of the rat hindpaw. Recently, the expression of GAL in the epithelium of the rat molar gingiva was reported (Baumann et al., 2003Baumann M.A. Korkmaz Y. Bloch W. Schmidt A. Addicks K. Schroder H. Localization of the neuropeptide galanin in nerve fibers and epithelial keratinocytes of the rat molar gingiva.Eur J Oral Sci. 2003; 111: 175-178Crossref PubMed Scopus (11) Google Scholar). GAL exerts its diverse actions via GAL receptors, which belong to the superfamily of G-protein coupled transmembrane receptors. Three GAL receptor subtypes (GalR1-3) have been cloned and pharmacologically characterized to date (Branchek et al., 2000Branchek T.A. Smith K.E. Gerald C. Walker M.W. Galanin receptor subtypes.Trends Pharmacol Sci. 2000; 21: 109-117Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Although GAL receptors are the key to the functional significance of GAL action, to our knowledge, their expression in human skin has not been studied to date. We analyzed GAL-LI in human skin biopsies of different anatomical sites, including the inner aspect of the upper arm, temple, ear, thigh, and foreskin by immunohistochemistry. GAL-LI was detected in the follicular and interfollicular epidermis with no apparent differences between basal and suprabasal layers and anatomical sites Figure 1a, c. In our study, the specificity of the GAL-LI in the epidermis was shown by absorption of the antibody with 1 μM GAL peptide, which abolished the staining Figure 1b, d. The dermis of foreskin displayed GAL-LI-positive nerve fiber bundles (Figure 1e, Table I). Arteries, small pre-capillary arterioles, and capillaries in the foreskin were also GAL-LI-positive (Figure 1e, Table I). Smooth muscle cells of the lamina muscularis of the vessels are most likely creating this signal. GAL-LI was also observed in ductal cells of eccrine sweat glands but not in the cells of the secretory coil Figure 1g. In sebaceous glands, no specific GAL-LI could be detected.Table IExpression of GAL-LI and GAL binding sites in the human skinGAL-LIaFor GAL-LI also references to previous studies are given.[125I]-GAL bindingbGAL binding sites were determined by receptor autoradiography as described in Figure 2.Interfollicular epidermis++-Follicular epidermis++-Fibroblasts--Dermal arteries/arterioles+ (smooth muscle cells)+ (most likely innervating nervescCurrently, no distinction can be made if this labeling is due to nerves innervating these structures or due to receptors on these cells. ++, strong staining; +, moderate staining; -, no staining; n.d., not determined.)Dermal venules--Sweat glands++ (ductal cells)++ (most likely innervating nervescCurrently, no distinction can be made if this labeling is due to nerves innervating these structures or due to receptors on these cells. ++, strong staining; +, moderate staining; -, no staining; n.d., not determined.)+ (innervating nerves; this study andTainio et al., 1987Tainio H. Vaalasti A. Rechardt L. The distribution of substance P-, CGRP-, galanin- and ANP-like immunoreactive nerves in human sweat glands.Histochem J. 1987; 19: 375-380Crossref PubMed Scopus (83) Google Scholar)Nerve fibers+ (this study andJohansson et al., 1988Johansson O. Vaalasti A. Tainio H. Ljungberg A. Immunohistochemical evidence of galanin in sensory nerves of human digital skin.Acta Physiol Scand. 1988; 132: 261-263Crossref PubMed Scopus (32) Google Scholar,Johansson et al., 1999Johansson O. Fantini F. Hu H. Neuronal structural proteins, transmitters, transmitter enzymes and neuropeptides in human Meissner's corpuscles: A reappraisal using immunohistochemistry.Arch Dermatol Res. 1999; 291: 419-424Crossref PubMed Scopus (32) Google Scholar;Pincelli et al., 1990Pincelli C. Fantini F. Massimi P. Girolomoni G. Seidenari S. Giannetti A. Neuropeptides in skin from patients with atopic dermatitis: An immunohistochemical study.Br J Dermatol. 1990; 122: 745-750Crossref PubMed Scopus (148) Google Scholar)+Sebaceous glands--Merkel cells+ (Fantini and Johansson, 1995Fantini F. Johansson O. Neurochemical markers in human cutaneous Merkel cells. An immunohistochemical investigation.Exp Dermatol. 1995; 4: 365-371Crossref PubMed Scopus (59) Google Scholar)n.d.a For GAL-LI also references to previous studies are given.b GAL binding sites were determined by receptor autoradiography as described in Figure 2.c Currently, no distinction can be made if this labeling is due to nerves innervating these structures or due to receptors on these cells. ++, strong staining; +, moderate staining; -, no staining; n.d., not determined. Open table in a new tab In order to determine if GAL is a secretory product of human keratinocytes, cell culture supernatants of primary cultured keratinocytes from neonatal foreskins were analyzed for the presence of GAL-LI. GAL peptide elution, and radioimmunoassays were performed according toBerger et al., 2002Berger A. Tuechler C. Almer D. et al.Elevated expression of galanin receptors in childhood neuroblastic tumors.Neuroendocrinology. 2002; 75: 130-138Crossref PubMed Scopus (27) Google Scholar (detection limit: 0.2 fmol GAL/1 × 106 cells per h). GAL was secreted into the supernatant of primary cultured keratinocytes at a rate of 0.84±0.33 fmol/1 × 106 cells per h (n=5), which leads to a steady-state level of 5.0±2.1 fmol per mL GAL in the cell culture supernatant after 18 h. This amount is comparable to the GAL concentration found in the serum of healthy humans (8 fmol per mL;Baranowska et al., 1997Baranowska B. Wasilewska-Dziubinska E. Radzikowska M. Plonowski A. Roguski K. Neuropeptide Y, galanin, and leptin release in obese women and in women with anorexia nervosa.Metabolism. 1997; 46: 1384-1389Abstract Full Text PDF PubMed Scopus (107) Google Scholar). [125I]-GAL receptor autoradiography was used to detect GAL binding sites in different anatomical sites. In contrast to GAL, no specific GAL binding sites were present in the human epidermis of all sites Figure 2a, b. GAL binding sites could mainly be detected around eccrine sweat glands Figure 2e, f. However, by the use of this method it cannot be distinguished if the receptors are expressed on myoepithelial cells or on surrounding nerve fibers. To a lesser extent, GAL binding was found on nerve fibers and in the vicinity of arteries and arterioles of foreskin (Figure 2c, d; Table I). In this study, we show that GAL and GAL receptors are expressed in human skin in a neuronal and extra-neuronal position Table I. This finding further strengthens the notion that neuropeptides can also be expressed outside of the nervous system to exert multiple biological functions, including mitogenic and immunomodulatory effects. The neuropeptide, which has been shown to be paradigmatic for this dichotomy in human skin, is alpha-melanocyte stimulating hormone (α-MSH), a proopiomelanocortin-derived peptide. α-MSH exerts its effects via binding to melanocortin receptors 1–5, being expressed on various tissues including melanocytes, keratinocytes, fibroblasts, and endothelial cells (for a review, seeScholzen et al., 1998Scholzen T. Armstrong C.A. Bunnett N.W. Luger T.A. Olerud J.E. Ansel J.C. Neuropeptides in the skin: Interactions between the neuroendocrine and the skin immune systems.Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (366) Google Scholar). In contrast, we could not find GAL binding sites on keratinocytes and endothelial membrane preparations, although GAL receptors have been reported to be expressed in other epithelia, e.g. the human intestinal epithelium (Engelis et al., 1998Engelis A. Mozgis D.Z. Pine K.A. Shine J. Iismaa T.P. Pilmane M. Expression of galanin and the GALR1 galanin receptor subtype in the colon of children with paradoxical fecal incontinence.Ann NY Acad Sci. 1998; 863: 425-429Crossref PubMed Scopus (1) Google Scholar;Berger et al., 2003Berger A. Kofler B. Santic R. Zipperer E. Sperl W. Hauser-Kronberger C. 125I-labeled galanin binding sites in congenital innervation defects of the distal colon.Acta Neuropathol (Berl). 2003; 105: 43-48PubMed Google Scholar). Most likely GAL being expressed and secreted by keratinocytes exerts its actions on the skin nervous system, by diffusion into the dermis or via the vascular system. A possible role of GAL in the regulation of vasomotoric action is suggested bySantha et al., 1998Santha P. Pierau F.K. Jancso G. Evidence for an inhibition by endogenous galanin of neurogenic cutaneous vasodilatation in the pigeon.Neurosci Lett. 1998; 243: 101-104Crossref PubMed Scopus (19) Google Scholar,Santha et al., 1999Santha P. Pierau F.K. Jancso G. Inhibitory modulation of cutaneous vascular responses by endogenous galanin in the pigeon.Neurosci Lett. 1999; 273: 64-66Crossref PubMed Scopus (12) Google Scholar. Expression of GAL on smooth muscle cells of the lamina muscularis of small vessels and GAL receptors in nervous tissue surrounding small arterioles supports the idea of a modulatory action in vasodilatation and therefore neurogenic inflammation. GAL-LI surrounding sweat glands were consistent with GAL-positive nerve fibers innervating the glands as reported previously (Tainio et al., 1987Tainio H. Vaalasti A. Rechardt L. The distribution of substance P-, CGRP-, galanin- and ANP-like immunoreactive nerves in human sweat glands.Histochem J. 1987; 19: 375-380Crossref PubMed Scopus (83) Google Scholar). Intriguingly, GAL-LI was found to be restricted to ductal cells. Other neuropeptides found in sweat glands were mainly found to be localized in the secretory coil (Zancanaro et al., 1999Zancanaro C. Merigo F. Crescimanno C. Orlandini S. Osculati A. Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands.J Anat. 1999; 194: 433-444Crossref PubMed Google Scholar) and are therefore regarded to be secreted into the lumen. Being located in the ductal cells GAL could function in a regulatory feedback loop upon secretion of sweat by targeting of GAL receptors and consecutively activation/inactivation of sweat gland function. Taken together, these studies suggest a possible role of GAL in skin physiology. These studies were supported by a Grant of the Austrian Science Foundation (P14906), the Vereinigung zur Förderung Pädiatrischer Forschung und Fortbildung Salzburg, and the Medizinische Forschungsgesellschaft Salzburg. We thank Peter Petzelbauer (Department of Dermatology, University of Vienna) for providing samples of cultured human dermal microvascular endothelial cells.