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• W2069484757 abstract "The cutaneous vasculature plays a key role in the pathophysiology of inflammatory skin diseases. The vascular activity is under the control of the peripheral nervous system that includes locally released neuropeptides. Recently, we detected receptors for the neuropeptide galanin in association with dermal blood vessels, suggesting a role of the galanin-peptide-family in the regulation of the cutaneous microvasculature. Therefore, we have investigated galanin and galanin-like peptide (GALP), a neuropeptide previously only considered to be involved in metabolism and reproduction in the central nervous system, for vaso-modulatory activity in the murine skin in vivo. Picomole amounts of intradermally injected galanin and GALP decreased cutaneous blood flow and inhibited inflammatory edema formation. Both the full-length GALP (1–60) and the putative smaller proteolytic fragment GALP (3–32) showed similar effects. These activities are most likely mediated by galanin receptors galanin receptor subtype 2 (GalR2) and/or galanin receptor subtype 3 (GalR3), because reverse transcription-PCR analysis of murine skin revealed messenger RNA (mRNA) expression of GalR2 and GalR3 but not of galanin receptor subtype 1. The lack of galanin receptor mRNAs in endothelial and smooth muscle cells indicates a neuronal localization of these receptors around the vessels. These results indicate functional activity of GALP in the periphery in vivo and suggest a potential role as an inflammatory modulator. The cutaneous vasculature plays a key role in the pathophysiology of inflammatory skin diseases. The vascular activity is under the control of the peripheral nervous system that includes locally released neuropeptides. Recently, we detected receptors for the neuropeptide galanin in association with dermal blood vessels, suggesting a role of the galanin-peptide-family in the regulation of the cutaneous microvasculature. Therefore, we have investigated galanin and galanin-like peptide (GALP), a neuropeptide previously only considered to be involved in metabolism and reproduction in the central nervous system, for vaso-modulatory activity in the murine skin in vivo. Picomole amounts of intradermally injected galanin and GALP decreased cutaneous blood flow and inhibited inflammatory edema formation. Both the full-length GALP (1–60) and the putative smaller proteolytic fragment GALP (3–32) showed similar effects. These activities are most likely mediated by galanin receptors galanin receptor subtype 2 (GalR2) and/or galanin receptor subtype 3 (GalR3), because reverse transcription-PCR analysis of murine skin revealed messenger RNA (mRNA) expression of GalR2 and GalR3 but not of galanin receptor subtype 1. The lack of galanin receptor mRNAs in endothelial and smooth muscle cells indicates a neuronal localization of these receptors around the vessels. These results indicate functional activity of GALP in the periphery in vivo and suggest a potential role as an inflammatory modulator. calcitonin gene-related peptide galanin-like peptide galanin receptor subtype 2 galanin receptor subtype 3 intradermally messenger RNA reverse transcription; substance P The skin has an essential protective function in responding to challenges from the environment. Cutaneous nerves transmit nociceptive information to the central nervous system and release biologically active neuropeptides (McDonald et al., 1996McDonald D.M. Bowden J.J. Baluk P. Bunnett N.W. Neurogenic inflammation. A model for studying efferent actions of sensory nerves.Adv Exp Med Biol. 1996; 410: 453-462Crossref PubMed Scopus (84) Google Scholar). The efferent activities of sensory nerves, mediated via neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), may contribute to the physiological and pathophysiological modulation of skin responses (Hokfelt and Ljungdahl, 1971Hokfelt T. Ljungdahl A. Light and electron microscopic autoradiography on spinal cord slices after incubation with labeled glycine.Brain Res. 1971; 32: 189-194Crossref PubMed Scopus (86) Google Scholar; Brain, 1997Brain S.D. Sensory neuropeptides: their role in inflammation and wound healing.Immunopharmacology. 1997; 37: 133-152Crossref PubMed Scopus (194) Google Scholar). Galanin is a 29 (30 human) amino-acid peptide involved in a variety of peripheral and central physiological and pathophysiological processes (Bartfai et al., 1993Bartfai T. Hokfelt T. Langel U. Galanin – a neuroendocrine peptide.Crit Rev Neurobiol. 1993; 7: 229-274PubMed Google Scholar), including secretion of hormones (McDonald et al., 1985McDonald T.J. Dupre J. Tatemoto K. Greenberg G.R. Radziuk J. Mutt V. Galanin inhibits insulin secretion and induces hyperglycemia in dogs.Diabetes. 1985; 34: 192-196Crossref PubMed Google Scholar; Bauer et al., 1986Bauer F.E. Ginsberg L. Venetikou M. MacKay D.J. Burrin J.M. Bloom S.R. Growth hormone release in man induced by galanin, a new hypothalamic peptide.Lancet. 1986; 2: 192-195Abstract PubMed Scopus (190) Google Scholar), inhibition of cardiac vagal action (Ulman et al., 1994Ulman L.G. Potter E.K. McCloskey D.I. Functional effects of a family of galanin antagonists on the cardiovascular system in anaesthetised cats.Regul Peptide. 1994; 51: 17-23Crossref PubMed Scopus (13) Google Scholar), mitogenic properties (Sethi et al., 1992Sethi T. Langdon S. Smyth J. Rozengurt E. Growth of small cell lung cancer cells: stimulation by multiple neuropeptides and inhibition by broad spectrum antagonists in vitro and in vivo.Cancer Res. 1992; 52: 2737-2742PubMed Google Scholar; Wynick et al., 1993Wynick D. Hammond P.J. Akinsanya K.O. Bloom S.R. Galanin regulates basal and oestrogen-stimulated lactotroph function.Nature. 1993; 364: 529-532Crossref PubMed Scopus (112) Google Scholar), and analgesic effects in response to nerve injury (Wiesenfeld-Hallin et al., 1992Wiesenfeld-Hallin Z. Xu X.J. Langel U. Bedecs K. Hokfelt T. Bartfai T. Galanin-mediated control of pain: enhanced role after nerve injury.Proc Natl Acad Sci USA. 1992; 89: 3334-3337Crossref PubMed Scopus (197) Google Scholar). A second peptide of the galanin family, galanin-like peptide (GALP) shares the amino acids at position 9–21 with the first 13 amino acids of galanin, which are required to activate galanin receptors. The sequence of GALP (1–60) contains a potential proteolytic cleavage site between two basic amino acids at position 33, which might lead to shorter C-terminally amidated peptides. In addition, the first two amino acids of human GALP could be potentially removed by dipeptidyl dipeptidase IV (Lang et al., 2005Lang R. Berger A. Santic R. Geisberger R. Hermann A. Herzog H. et al.Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists.Neuropeptides. 2005; 39: 179-184Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Like galanin, GALP has potent species-specific and time-dependent effects in the central nervous system and has been suggested to constitute a link between metabolism and reproduction (Gottsch et al., 2004Gottsch M.L. Clifton D.K. Steiner R.A. Galanin-like peptide as a link in the integration of metabolism and reproduction.Trends Endocrinol Metab. 2004; 15: 215-221Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To our knowledge, GALP has not been shown to possess biological activity in the periphery. The effects of galanin and GALP are mediated by G-protein-coupled receptor subtypes, which are uniquely distributed throughout the central nervous system and periphery (Burgevin et al., 1995Burgevin M.C. Loquet I. Quarteronet D. Habert-Ortoli E. Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor.J Mol Neurosci. 1995; 6: 33-41Crossref PubMed Scopus (168) Google Scholar; Fathi et al., 1998Fathi Z. Battaglino P.M. Iben L.G. Li H. Baker E. Zhang D. et al.Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.Brain Res Mol Brain Res. 1998; 58: 156-169Crossref PubMed Scopus (72) Google Scholar; Kolakowski et al., 1998Kolakowski Jr, L.F. O’Neill G.P. Howard A.D. Broussard S.R. Sullivan S.R. Feighner S.D. et al.Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.J Neurochem. 1998; 71: 2239-2251Crossref PubMed Scopus (155) Google Scholar). Galanin has high affinity for all three galanin receptor subtypes, whereas GALP (1–60) displays high affinity only for galanin receptor subtype 2 (GalR2) and galanin receptor subtype 3 (GalR3) (Ohtaki et al., 1999Ohtaki T. Kumano S. Ishibashi Y. Ogi K. Matsui H. Harada M. et al.Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus.J Biol Chem. 1999; 274: 37041-37045Crossref PubMed Scopus (237) Google Scholar; Berger et al., 2004Berger A. Lang R. Moritz K. Santic R. Hermann A. Sperl W. et al.Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.Endocrinology. 2004; 145: 500-507Crossref PubMed Scopus (55) Google Scholar). Recently, it was shown that GALP (3–32) is a more potent agonist on the GalR2 than the full-length peptide in neuroblastoma cells expressing the GalR2 (Lang et al., 2005Lang R. Berger A. Santic R. Geisberger R. Hermann A. Herzog H. et al.Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists.Neuropeptides. 2005; 39: 179-184Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The upregulation of galanin and galanin-binding sites in rat skin upon inflammation indicates a role of the galanin system in the plasticity of the cutaneous microvasculature (Ji et al., 1995Ji R.R. Zhang X. Zhang Q. Dagerlind A. Nilsson S. Wiesenfeld-Hallin Z. et al.Central and peripheral expression of galanin in response to inflammation.Neuroscience. 1995; 68: 563-576Crossref PubMed Scopus (102) Google Scholar). Accordingly, galanin inhibits plasma extravasation induced by antidromic C-fiber stimulation in the rat hind paw (Xu et al., 1991Xu X.J. Hao J.X. Wiesenfeld-Hallin Z. Hakanson R. Folkers K. Hokfelt T. Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hind paw.Neuroscience. 1991; 42: 731-737Crossref PubMed Scopus (69) Google Scholar) and histamine-induced plasma extravasation in the pigeon skin (Jancso et al., 2000Jancso G. Santha P. Horvath V. Pierau F. Inhibitory neurogenic modulation of histamine-induced cutaneous plasma extravasation in the pigeon.Regul Peptide. 2000; 95: 75-80Crossref PubMed Scopus (26) Google Scholar). Furthermore, galanin-overexpressing mice showed a significant decrease in plasma extravasation upon activation of neurogenic inflammation following mustard oil treatment (Holmberg et al., 2005Holmberg K. Kuteeva E. Brumovsky P. Kahl U. Karlstrom H. Lucas G.A. et al.Generation and phenotypic characterization of a galanin overexpressing mouse.Neuroscience. 2005; 133: 59-77Crossref PubMed Scopus (49) Google Scholar). Consistent with these vascular effects, we were able to demonstrate that galanin-binding sites are present around blood vessels (Kofler et al., 2004Kofler B. Berger A. Santic R. Moritz K. Almer D. Tuechler C. et al.Expression of neuropeptide galanin and galanin receptors in human skin.J Invest Dermatol. 2004; 122: 1050-1053Crossref PubMed Scopus (45) Google Scholar). Although there is evidence for a role of galanin in modulating inflammatory edema in the periphery, the possible functional significance of GALP in the periphery has not been investigated. Moreover, the mechanism of edema formation and the galanin receptor subtype involved, are so far unclear. The purpose of this study was to analyze the effects of galanin and GALP on cutaneous microvasculature in mice. We have examined the ability of galanin and GALP to modulate inflammatory edema formation and blood flow in the murine skin in vivo. We have also examined the skin for the presence of galanin receptors, that both galanin and GALP can mediate their effects through. Plasma extravasation was induced in skin by the coinjection of the potent mediator of increased microvascular permeability SP and the vasodilator CGRP, which act in a synergistic manner to induce an acute inflammatory edema in skin (Brain and Williams, 1985Brain S.D. Williams T.J. Inflammatory oedema induced by synergism between calcitonin gene-related peptide (CGRP) and mediators of increased vascular permeability.Br J Pharmacol. 1985; 86: 855-860Crossref PubMed Scopus (363) Google Scholar; Cao et al., 1999Cao T. Gerard N.P. Brain S.D. Use of NK(1) knockout mice to analyze substance P-induced edema formation.Am J Physiol. 1999; 277: 476-481PubMed Google Scholar) (Figure 1a). The β-adrenergic anti-permeability agent salbutamol (Teixeira et al., 1995Teixeira M.M. Williams T.J. Hellewell P.G. Anti-inflammatory effects of a short-acting and a long-acting beta 2-adrenoceptor agonist in guinea pig skin.Eur J Pharmacol. 1995; 272: 185-193Crossref PubMed Scopus (21) Google Scholar) was used (Figure 1a), and the potent vasoconstrictor endothelin (Brain et al., 1989Brain S.D. Crossman D.C. Buckley T.L. Williams T.J. Endothelin-1: demonstration of potent effects on the microcirculation of humans and other species.J Cardiovasc Pharmacol. 1989; 13: 147-149Crossref PubMed Scopus (75) Google Scholar) as positive controls for the assay. It can be seen that galanin, at a 1 pmol dose, acted in a similar manner to endothelin to abolish this edema. The effect of the full-length mature GALP (1–60), the truncated peptides (1–32) and (3–32), as well as the peptide fragment GALP (19–37), which lacks the galanin receptor-binding domain, were also examined at a dose of 10 pmol/site for their ability to modulate edema formation (Figure 1a). Although GALP (1–60) and GALP (3–32) demonstrated an inhibitory effect, GALP (1–32) exhibited a lesser but albeit significant inhibitory response and GALP (19–37) was inactive (Figure 1a). Further studies were then carried out to learn the dose-dependent activities. Results for galanin are shown in Figure 2, where galanin (0.1–10 pmol) acted in a dose-dependent manner to inhibit edema formation, although higher doses had less effect, giving a bell-shaped dose–response curve. The ability of galanin to inhibit non-neurogenic edema formation was investigated through the study of histamine coinjected with CGRP-induced responses. Figure 1b shows that galanin also inhibited histamine- and CGRP-induced edema formation. The dose-related effect of the two GALP peptides that showed potent anti-edema activity, GALP (1–60) (1–100 pmol) and GALP (3–32) (1–100 pmol) is shown in Figure 3. Again bell-shaped dose–response curves were observed. The anti-edema effect of GALP (3–32) was confirmed using a second technique (Evans Blue) to assess plasma extravasation (Figure 3c). There are two obvious mechanisms via galanin peptides could be acting to modulate inflammatory edema formation. Either by inhibition of microvascular permeability (as salbutamol) or as an indirect mechanism through reduction of skin blood flow (as endothelin). To investigate possible effects on blood flow, a 99 m technetium clearance technique was utilized. Injection of galanin, GALP (1–60), and GALP (3–32) all induced dose-dependent reductions in inhibiting cutaneous blood flow (Figure 4; data for GALP 1–60 not shown). Figure 5 shows the expected lack of effect of salbutamol, and the potent constrictor effect of endothelin-1, and again GALP (1–32) was not effective (Figure 5).Figure 5Effect of galanin and GALP peptides on blood flow in cutaneous dorsal microvasculature. The responses of salbutamol and ET-1 as negative and positive controls, respectively, are shown alongside responses of the most potent concentration (according to Figure 4) of galanin and GALP peptides (GALP (1–60), GALP (1–32), and GALP (3–32)). Responses are shown as decrease in % clearance (mean±SEM) compared with vehicle (Tyrode-injected) skin (n=8). Results that are significantly different from clearance at Tyrode-injected sites are shown (**P<0.01).View Large Image Figure ViewerDownload (PPT) Reverse transcription (RT)-PCR analysis of dorsal murine skin messenger RNA (mRNA) revealed no expression of galanin receptor subtype 1 (Figure 6). GalR2 was consistently expressed at substantial levels (Figure 6). GalR3 was detected at low levels of mRNA expression (Figure 6). To determine which cell types are expressing galanin receptor mRNA in the microcvasulature, endothelial and smooth muscle cells were separately used for galanin receptor-specific RT-PCR analysis. Neither primary human endothelial cells (human dermal microvascular endothelial cell) nor smooth muscle cells (human umbilical artery smooth muscle cell) showed an expression of galanin receptor subtype 1, GalR2, or GalR3 mRNA (data not shown). This finding was also supported by a receptor membrane-binding study. Both cell types were below the detection limit, which was defined as ≤0.006 pmol bound galanin/mg membran protein. As a control for the assay, SH-SY5Y/GalR2 were used with a binding capacity of 3.2±0.3 pmol bound galanin/mg membrane protein (data not shown). This study demonstrates the potent ability of galanin and GALP to inhibit inflammatory edema, which is most likely secondary to vasoconstricton and inhibition of blood flow in the cutaneous microvasculature. Thus, we show that either the full-length peptide GALP (1–60) or GALP (3–32) is able to exert vasoactive effects in the cutaneous microcirculation. There is evidence that galanin inhibits plasma extravasation in experimental models of neurogenic inflammation induced either chemically or by C-fiber stimulation (Xu et al., 1991Xu X.J. Hao J.X. Wiesenfeld-Hallin Z. Hakanson R. Folkers K. Hokfelt T. Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hind paw.Neuroscience. 1991; 42: 731-737Crossref PubMed Scopus (69) Google Scholar; Green et al., 1992Green P.G. Basbaum A.I. Levine J.D. Sensory neuropeptide interactions in the production of plasma extravasation in the rat.Neuroscience. 1992; 50: 745-749Crossref PubMed Scopus (88) Google Scholar; Holmberg et al., 2005Holmberg K. Kuteeva E. Brumovsky P. Kahl U. Karlstrom H. Lucas G.A. et al.Generation and phenotypic characterization of a galanin overexpressing mouse.Neuroscience. 2005; 133: 59-77Crossref PubMed Scopus (49) Google Scholar), and that SP-induced plasma extravasation was inhibited by galanin in the rat hind paw (Xu et al., 1991Xu X.J. Hao J.X. Wiesenfeld-Hallin Z. Hakanson R. Folkers K. Hokfelt T. Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hind paw.Neuroscience. 1991; 42: 731-737Crossref PubMed Scopus (69) Google Scholar). Galanin-induced inhibition of plasma extravasation could be mediated by presynaptic or postsynaptic effects. Previously, galanin has been suggested to modulate neurogenic edema formation via a presynaptic mechanism by inhibiting release of SP and CGRP (Holmberg et al., 2005Holmberg K. Kuteeva E. Brumovsky P. Kahl U. Karlstrom H. Lucas G.A. et al.Generation and phenotypic characterization of a galanin overexpressing mouse.Neuroscience. 2005; 133: 59-77Crossref PubMed Scopus (49) Google Scholar). The experiments presented in our work utilizing an experimental design with coinjection of SP and CGRP with galanin or GALP indicate that the effect of galanin on plasma extravasation is primarily a postsynaptic effect in our system. Possible downstream postsynaptic consequences could be an interaction with the signaling of SP and CGRP, or a direct vasoconstrictor activity of galanin. A first indication of an involvement of galanin in the regulation of skin blood flow came from the observation that mustard oil-mediated vasodilatation in pigeon skin was potentiated by a galanin antagonist (Santha et al., 1999Santha P. Pierau F.K. Jancso G. Galanin mediated inhibitory nervous modulation of cutaneous vascular reactions.Acta Physiol Hung. 1999; 86: 279-285PubMed Google Scholar). The vasoconstrictor potential of galanin was confirmed in the hamster cheek pouch, measured by the change of arteriolar diameter (Dagar et al., 2003Dagar S. Onyuksel H. Akhter S. Krishnadas A. Rubinstein I. Human galanin expresses amphipathic properties that modulate its vasoreactivity in vivo.Peptides. 2003; 24: 1373-1380Crossref PubMed Scopus (11) Google Scholar). This group found a similar potent vasoconstrictor effect of human galanin when administered alone as presented in this study, but also importantly that micelle formation can amplify its vasoactive effects in vivo. In our studies, a direct vasoactive effect was supported by a series of experiments with histamine. Histamine, a potent mediator of increased microvascular permeability, was co-injected with vasodilator CGRP to induce edema formation. This edema formation was inhibited by galanin coinjection. In further support of our findings, Holmberg et al., 2005Holmberg K. Kuteeva E. Brumovsky P. Kahl U. Karlstrom H. Lucas G.A. et al.Generation and phenotypic characterization of a galanin overexpressing mouse.Neuroscience. 2005; 133: 59-77Crossref PubMed Scopus (49) Google Scholar have reported that galanin-overexpressing mice show reduced edema formation following chemical stimulation of neurogenic inflammation in the mouse paw, although they did not measure blood flow changes. To further clarify the vasoconstrictive potency of galanin and GALP, skin blood flow was measured using a 99 m technetium clearance assay, which was initially developed in order to assess the vasoconstrictor potential of the neuropeptide Y (Chu et al., 2003Chu D.Q. Cox H.M. Costa S.K. Herzog H. Brain S.D. The ability of neuropeptide Y to mediate responses in the murine cutaneous microvasculature: an analysis of the contribution of Y1 and Y2 receptors.Br J Pharmacol. 2003; 140: 422-430Crossref PubMed Scopus (18) Google Scholar). In this assay, we show that galanin, GALP (1–60), and GALP (3–32) decreased skin blood flow and inflammatory edema, whereas salbutamol, a β2-adrenergic receptor agonist which decreases blood vessel leakiness (Kwan et al., 2001Kwan M.L. Gomez A.D. Baluk P. Hashizume H. McDonald D.M. Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P.Am J Physiol Lung Cell Mol Physiol. 2001; 280: 286-297PubMed Google Scholar), was only active in the edema model. This suggests that the primary activity of galanin, GALP (1–60), and GALP (3–32) is that of cutaneous vasoconstriction. However, we cannot rule out the possibility of a concomitant permeability decrease. Pharmacologically, the galanin and the GALP effects showed bell-shaped dose–response curves in the plasma extravasation and vasoconstriction assays. It is possible that whereas at lower doses galanin is decreasing in blood flow, at higher doses other mechanisms mediating direct effects in promoting vascular permeability become active. We have recently shown the presence of galanin-binding sites in human skin. They were detected in a high density around small vessels, indicating that galanin receptors located around these vessels mediate the vascular activity of galanin (Kofler et al., 2004Kofler B. Berger A. Santic R. Moritz K. Almer D. Tuechler C. et al.Expression of neuropeptide galanin and galanin receptors in human skin.J Invest Dermatol. 2004; 122: 1050-1053Crossref PubMed Scopus (45) Google Scholar). The type of galanin receptor could not be specified by the galanin receptor autoradiography used. In this study, we demonstrate that dermal endothelial and smooth muscle cells do not express galanin receptors. These observations suggest that galanin receptors around blood vessels in the human dermal microvasculature are most likely located on neurons surrounding the vessels, indicating that the effect on the dermal microvasculature is indirect owing to mediators secondarily released by other cells (e.g. nerve cells). However, we cannot exclude that species- (human/mouse) and tissue-specific (skin/umbilical cord) differences of galanin receptor location exist. We show that the potency order of galanin peptide fragments on edema formation was as follows: galanin <GALP (3–32)<GALP (1–60)<GALP (1–32), and this is in accordance with the affinities for the human GalR2 receptor reported previously (Lang et al., 2005Lang R. Berger A. Santic R. Geisberger R. Hermann A. Herzog H. et al.Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists.Neuropeptides. 2005; 39: 179-184Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Moreover, the substantial expression of GalR2 in the murine skin further implies a more likely GalR2 than GalR3-mediated mechanism. In other model systems, GalR2 is upregulated following inflammation whereas galanin receptor subtype 1 is downregulated, suggesting that under inflammatory conditions, there may be preferential activity of the GalR2 receptor (Xu et al., 1996Xu Z.Q. Shi T.J. Landry M. Hokfelt T. Evidence for galanin receptors in primary sensory neurones and effect of axotomy and inflammation.Neuroreport. 1996; 8: 237-242Crossref PubMed Scopus (87) Google Scholar). We demonstrate here, that active members of the galanin family of peptides possess activities that are in the region of 10- to 100-fold more potent than those of the established potent constrictor agent neuropeptide Y (Chu et al., 2003Chu D.Q. Cox H.M. Costa S.K. Herzog H. Brain S.D. The ability of neuropeptide Y to mediate responses in the murine cutaneous microvasculature: an analysis of the contribution of Y1 and Y2 receptors.Br J Pharmacol. 2003; 140: 422-430Crossref PubMed Scopus (18) Google Scholar). Thus, our findings suggest a far-reaching influence in the pathophysiology of inflammatory skin disorders and should prompt experiments evaluating the therapeutic potential of galanin and GALP in the skin. Normal female CD-1 mice (22–27 g, 8–12 weeks) were obtained from Charles River, UK. All mice were maintained on normal diet, with free access to food and water, in a climatically controlled environment. Animals were anesthetized with urethane (25% w v−1; 2.5 g kg−1 intraperitoneally) and the dorsal skin was shaved. Injection sites were chosen according to a randomized site pattern on the dorsal skin of the anesthetized mouse. Experiments involving mice were conducted under the Animals (Scientific Procedures) Act, 1986. Primary human dermal microvascular endothelial cells were isolated from normal adult foreskin and cultured as described previously by Nguyen et al., 2002Nguyen V.A. Ebner S. Furhapter C. Romani N. Kolle D. Fritsch P. et al.Adhesion of dendritic cells derived from CD34+ progenitors to resting human dermal microvascular endothelial cells is down-regulated upon maturation and partially depends on CD11a-CD18, CD11b-CD18 and CD36.Eur J Immunol. 2002; 32: 3638-3650Crossref PubMed Scopus (21) Google Scholar. Primary human umbilical artery smooth muscle cells were purchased from Promocell (Heidelberg, Germany) and cultured in smooth muscle basal medium 2 (Promocell, Heidelberg, Germany) at 37°C in a humified atmosphere of 5% CO2. Stable transfected SH-SY5Y neuroblastoma cells with the human galanin receptor GalR2 (SH-SY5Y/GalR2) were cultivated as recently described (Berger et al., 2004Berger A. Lang R. Moritz K. Santic R. Hermann A. Sperl W. et al.Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.Endocrinology. 2004; 145: 500-507Crossref PubMed Scopus (55) Google Scholar). As the galanin receptor expression is under control of a tetracycline-regulated expression system (T-Rex System, Invitrogen Corporation, CA), receptor expression was induced overnight with 1 μg/ml tetracycline. SP, CGRP, endothelin-1, and salbutamol were from Sigma (Poole, UK), as well as all others agents unless specified. Galanin (rat) and GALP (1–60) (human) were purchased from Bachem (Bubendorf, Switzerland). GALP (1–32)-amide (human) GALP (3–32)-amide (human), GALP (19–37) (human) were custom synthesized by NeoMPS Inc. (Strasbourg, France). All peptides were dissolved in distilled water. The stock solutions (10 nM) were stored at −20°C and further diluted in Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 11.9 mM NaHCO3, and 5.6 mM glucose) just prior use. Plasma extravasation was used as an index of inflammatory edema formation and measured as described previously (Cao et al., 1999Cao T. Gerard N.P. Brain S.D. Use of NK(1) knockout mice to analyze substance P-induced edema formation.Am J Physiol. 1999; 277: 476-481PubMed Google Scholar). Briefly, test agents (Galanin, GALP (1–60), GALP (1–32), GALP (3–32), GALP (19–37), endothelin-1, and salbutamol) were diluted in Tyrode's solution and stored on ice. 125I-BSA (45 kBq in 100 μl of saline) was administered intravenously into the tail vein, and 5 minutes later test agents (50 μl/site) were injected intradermally (i.d.). Plasma extravasation was allowed for 30 minutes and then a blood sample (0.5 ml) in a heparine-coated syringe was obtained via cardiac puncture and centrifuged at 10,000 g for 4 minutes to obtain plasma. The mice were then killed, the dorsal skin was removed, and the injected sites punched out (8 mm). The amount of plasma extravasated (μl g−1 tissue) was calculated by comparing the amount of radioactivity in each skin site with that in 100 μl plasma from the same animal. In a separate set of experiments, edema formation was observed by the extravascular accumulation of intravenously injected Evans Blue (0.1 ml of 2.5% w v−1 in saline) that binds to endogenous plasma albumin. After 5 minutes, test agents (50 μl in Tyrode solution) were injected i.d. Animals were left for 30 minutes to allow plasma extravasation to occur. Animals were then killed by cervical dislocation. Dorsal skin was removed and photographed to identify sites of plasma extravasation. Blood flow changes were measured using a 99 m technetium clearance technique (Chu et al., 2003Chu D.Q. Cox H.M. Costa S.K. Herzog H. Brain S.D. The ability of neuropeptide Y to mediate responses in the murine cutaneous microvasculature: an analysis of the contribution of Y1 and Y2 receptors.Br J Pharmacol. 2003; 140: 422-430Crossref PubMed Scopus (18) Google Scholar). Briefly, test agents (Galanin, GALP (1–60), GALP (1–32), GALP (3–32), endothelin-1, and salbutamol) were made up in Tyrode's solution and an equal amount of 99 m technetium (aprox. 200 kBq) was added to all samples, and kept on ice until use. Test agents (50 μl/site) were injected i.d., with an identical amount placed into a vial for measurement of the total radioactivity. The mouse was killed after a clearance period (15 minutes) and the skin was removed and the injected sites punched out for measurement of the remaining radioactivity. Initially, the amount of 99 m technetium cleared from each injection-site was calculated by comparing counts in skin with counts in the respective paired aliquot of total radioactivity. From this, the clearance at test agent-injected sites was then calculated by comparing with the Tyrode-value (which was normalized to 100 for each experiment), and expressed as % change in clearance compared to Tyrode, with positive numbers indicating a decreased blood flow. Membrane preparation and radioligand-binding assay were performed as described previously (Berger et al., 2004Berger A. Lang R. Moritz K. Santic R. Hermann A. Sperl W. et al.Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.Endocrinology. 2004; 145: 500-507Crossref PubMed Scopus (55) Google Scholar). Radio-labeled galanin binding to membrane preparation (15 μg) was carried out in duplicates in a total volume of 120 μl of binding buffer containing 50 pM [125I]galanin (2.000 Ci/mmol; Amersham Pharmacia Biotech (Little Chalfont, UK)) and non-specific binding was determined in the presence of 1 μM human galanin. Murine skin tissues where homogenized within 10 minutes after withdrawal of a freshly killed animal in Tri Reagent (Molecular Research Center Inc., Cincinnati, OH), using an Ultra-Turrax T25 (IKA Werke GmbH & Co., KG, Germany). Total RNA from tissues and cell lines was isolated according to the instructions of the manufacturer. RNA (1 μg) was reverse transcribed using 200 U SUPERSCRIPT II™ reverse transcriptase (Life Technologies Inc., Gaithersburg, MD). PCR amplification using 100 ng of cDNA was performed using Thermo Start polymerase (ABgene, Surrey, UK) in the presence of 10 pmol of each primer. Primers and PCR conditions are listed as Supplementary Material in Table S1. The PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide. Download .pdf (.02 MB) Help with pdf files Supplementary material in Table S1 Results for functional studies are shown mainly as mean±SEM. Statistical analyses were performed on original data by one-way analysis of variance, followed by Dunnett's or Bonferroni multiple comparison test. P<0.05 was considered as significant. N represents the number of animals. The authors state no conflict of interest. This study was supported by a grant of the Austrian Science Foundation (P14906), the Salzburg “Auslandsstipendim für kurzfristige wissenschaftliche Arbeiten im Ausland,” and the British Heart Foundation and the BBSRC. We thank the Department of Nuclear Medicine, Guy's Hospital, London, UK for 99 m technetium. Primary human dermal endothelia cells (HDMEC) were kindly provided by Dr Sepp Norbert, Department of Dermatology, University of Innsbruck, Austria. Table S1. Primers and PCR conditions." @default.
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