RESUMEN
Diabetic keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by resiniferatoxin severely impaired corneal wound healing and markedly upregulated proinflammatory gene expression. Exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) partially reversed resiniferatoxin's effects, with VIP specifically inducing interleukin-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIP type 1 receptor (VIPR1) expression in normal (NL) corneas, but not corneas from mice with diabetes mellitus (DM). Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses, while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (Shh) in a VIP-dependent manner. Downregulating Shh expression in NL corneas decreased while exogenous Shh in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of Shh signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in an Shh-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.
Asunto(s)
Enfermedades de la Córnea/fisiopatología , Diabetes Mellitus Experimental/complicaciones , Epitelio Corneal/fisiopatología , Proteínas Hedgehog/fisiología , Regeneración Nerviosa/fisiología , Péptido Intestinal Vasoactivo/fisiología , Cicatrización de Heridas/fisiología , Animales , Citocinas/análisis , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. RESULTS: Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. CONCLUSIONS: This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders.
Asunto(s)
Glándulas Ecrinas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Sudor/metabolismo , Adulto , Animales , Femenino , Pie , Humanos , Masculino , Ratones Endogámicos C57BL , Fibras Nerviosas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , ARN Mensajero/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiologíaRESUMEN
This study tested the hypothesis that vasoactive intestinal peptide (VIP) is able to modify the macrophage inflammatory profile, thus supporting its therapeutic role in autoimmune diseases. Macrophages are innate immune cells that display a variety of functions and inflammatory profiles in response to the environment that critically controls their polarization. Deregulation between the pro- and anti-inflammatory phenotypes has been involved in different pathologies. Rheumatoid arthritis (RA) is an autoimmune disease, in which macrophages are considered central effectors of synovial inflammation, displaying a proinflammatory profile. VIP is a pleiotropic neuropeptide with proven anti-inflammatory actions. As modulation of the macrophage phenotype has been implicated in the resolution of inflammatory diseases, we evaluated whether VIP is able to modulate human macrophage polarization. In vitro-polarized macrophages by GM-CSF (GM-MØ), with a proinflammatory profile, expressed higher levels of VIP receptors, vasoactive intestinal polypeptide receptors 1 and 2 (VPAC1 and VPAC2, respectively), than macrophages polarized by M-CSF (M-MØ) with anti-inflammatory activities. RA synovial macrophages, according to their GM-CSF-like polarization state, expressed both VPAC1 and VPAC2. In vitro-generated GM-MØ exposed to VIP exhibited an up-regulation of M-MØ gene marker expression, whereas their proinflammatory cytokine profile was reduced in favor of an anti-inflammatory function. Likewise, in GM-MØ, generated in the presence of VIP, VIP somehow changes the macrophages physiology profile to a less-damaging phenotype. Therefore, these results add new value to VIP as an immunomodulatory agent on inflammatory diseases.
Asunto(s)
Macrófagos/efectos de los fármacos , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Péptido Intestinal Vasoactivo/farmacología , Artritis Reumatoide/patología , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunofenotipificación , Inflamación , Activación de Macrófagos/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/metabolismo , ARN Mensajero/biosíntesis , Receptores de Tipo II del Péptido Intestinal Vasoactivo/biosíntesis , Receptores de Tipo II del Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/biosíntesis , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Líquido Sinovial/citología , Regulación hacia ArribaRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on multiple processes of glucose and energy metabolism. PACAP potentiates insulin action in adipocytes and insulin release from pancreatic ß-cells, thereby enhancing glucose tolerance. Contrary to these effects at organ levels, PACAP null mice exhibit hypersensitivity to insulin. However, this apparent discrepancy remains to be solved. We aimed to clarify the mechanism underlying the antidiabetic phenotype of PACAP null mice. Feeding with high-fat diet (HFD) impaired insulin sensitivity and glucose tolerance in wild type mice, whereas these changes were prevented in PACAP null mice. HFD also impaired insulin-induced Akt phosphorylation in the liver in wild type mice, but not in PACAP null mice. Using GeneFishing method, HFD increased the leukocyte common antigen-related (LAR) protein tyrosine phosphatase in the liver in wild type mice. Silencing of LAR restored the insulin signaling in the liver of HFD mice. Moreover, the increased LAR expression by HFD was prevented in PACAP null mice. HFD increased the expression of VPAC1 receptor (VPAC1-R), one of three PACAP receptors, in the liver of wild type mice. These data indicate that PACAP-VPAC1-R signaling induces LAR expression and insulin resistance in the liver of HFD mice. Antagonism of VPAC1-R may prevent progression of HFD-induced insulin resistance in the liver, providing a novel antidiabetic strategy.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Resistencia a la Insulina , Hígado/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Animales , Expresión Génica/efectos de los fármacos , Hipoglucemiantes , Hígado/química , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/análisis , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genéticaRESUMEN
Using molecular, biochemical, and cytological tools, we studied the nucleotide and the deduced amino acid sequence of PHI/VIP and the distribution of VIP/VPAC receptor system in the testis of the Italian wall lizard Podarcis sicula to evaluate the involvement of such a neuropeptide in the spermatogenesis control. We demonstrated that (1) Podarcis sicula VIP had a high identity with other vertebrate VIP sequences, (2) differently from mammals, VIP was synthesized directly in the testis, and (3) VIP and its receptor VPAC2 were widely distributed in germ and somatic cells, while the VPAC1 R had a distribution limited to Leydig cells. Our results demonstrated that in Podarcis sicula the VIP sequence is highly preserved and that this neuropeptide is involved in lizard spermatogenesis and steroidogenesis.
Asunto(s)
Lagartos/fisiología , Receptores de Péptido Intestinal Vasoactivo/fisiología , Testículo/fisiología , Péptido Intestinal Vasoactivo/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Hibridación in Situ , Células Intersticiales del Testículo/química , Células Intersticiales del Testículo/fisiología , Masculino , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Péptido Intestinal Vasoactivo/análisis , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/análisis , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/análisis , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Alineación de Secuencia , Testículo/química , Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/genéticaRESUMEN
BACKGROUND: Vasoactive intestinal peptide (VIP) has a number of important effects in intestinal physiology and pathology, including in ulcerative colitis (UC). The expression patterns of the predominant VIP receptor in the mucosa (the VPAC1 receptor) are unknown for the mucosa in UC. It is assumed that the sources of VIP in the intestine are the innervation and the inflammatory cells. AIMS: The VIP and VPAC1 receptor expression patterns in the epithelial layer of UC and non-UC patients were examined in the present study. The influence of marked inflammation of the mucosa was evaluated. METHODS: Specimens of the human colon, including the colon of UC patients, were examined concerning expressions of VIP and VPAC1 receptor, focusing on the epithelial layer. Immunohistochemistry and in situ hybridization were utilized. RESULTS: There were VIP mRNA reactions and also marked VPAC1 receptor immunoreactions in the normal and slightly/moderately affected epithelium. VIP mRNA reactions were not detected and VPAC1 immunoreactions were minimal in response to marked mucosal derangement. CONCLUSIONS: The findings suggest that there is a local production of VIP in the epithelial cells in normal and slightly/moderately inflamed mucosa but not in severely inflamed mucosa. Furthermore, a marked downregulation in VPAC1 receptor expressions occurs in the epithelium in severe UC. Based on the knowledge that VIP can have trophic, healing and anti-inflammatory effects, it is likely that the decrease in VIP mRNA and VPAC1 receptor reactions seen in severely affected mucosa in UC may be associated with adverse effects on intestinal function.
Asunto(s)
Colitis Ulcerosa/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Péptido Intestinal Vasoactivo/metabolismo , Adulto , Colitis Ulcerosa/fisiopatología , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Mucosa Intestinal/metabolismo , Masculino , Persona de Mediana Edad , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismoRESUMEN
OBJECTIVES: These studies examined the effect of homozygous deletion of vasoactive intestinal peptide receptor type 1 (VPAC1) on development and function of intestines and pancreas. METHODS: Genetically engineered VPAC1-null mutant mice were monitored for growth, development, and glucose homeostasis. Expression of VPAC1 was examined during embryonic development using VPAC1 promoter-driven ß-galactosidase transgenic mice. RESULTS: Homozygous deletion of VPAC1 resulted in fetal, neonatal, and postweaning death owing to failure to thrive, intestinal obstruction, and hypoglycemia. Histological findings demonstrated disorganized hyperproliferation of intestinal epithelial cells with mucus deposition and bowel wall thickening. The pancreas demonstrated small dysmorphic islets of Langerhans containing α, ß, and δ cells. Expression of a VPAC1 promoter-driven transgene was observed in E12.5 and E14.5 intestinal epithelial and pancreatic endocrine cells. Vasoactive intestinal peptide receptor type 1-null mutant animals had lower baseline blood glucose levels compared to both heterozygous and wild-type littermates. Vasoactive intestinal peptide receptor type 1-deficient mice responded to oral glucose challenge with normal rise in blood glucose followed by rapid hypoglycemia and failure to restore baseline glucose levels. Insulin challenge resulted in profound hypoglycemia and inadequate glucose homeostasis in VPAC1-null mutant animals. CONCLUSIONS: These observations support a role for VPAC1 during embryonic and neonatal development of intestines and endocrine pancreas.
Asunto(s)
Intestinos/embriología , Intestinos/fisiopatología , Páncreas/embriología , Páncreas/fisiopatología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/deficiencia , Animales , Secuencia de Bases , Glucemia/metabolismo , Cartilla de ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Prueba de Tolerancia a la Glucosa , Heterocigoto , Homocigoto , Intestinos/patología , Islotes Pancreáticos/embriología , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/patología , Embarazo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiologíaRESUMEN
The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias de la Próstata/inducido químicamente , Péptido Intestinal Vasoactivo/toxicidad , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/análisis , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Neoplasias de la Próstata/patología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiologíaRESUMEN
Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.
Asunto(s)
Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/antagonistas & inhibidores , Trombocitopenia/terapia , Trombopoyesis/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Trasplante de Médula Ósea , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Megacariocitos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/fisiopatología , Púrpura Trombocitopénica Idiopática/terapia , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Transducción de Señal , Trombocitopenia/fisiopatología , Irradiación Corporal TotalRESUMEN
Recent studies have suggested the potential use of vasoactive intestinal peptide (VIP) in the treatment of pulmonary arterial hypertension (PAH). An understanding of the mechanism of action of VIP is important for the development of new therapies for PAH. The biological effects of VIP are mediated by two type II guanine nucleotide binding protein (G-protein)-coupled receptors VIP/PACAP (pituitary adenylate cyclase activating peptide) receptor type1 (VPAC1) and VIP/PACAP receptor type 2 (VPAC2). In the present study, the distribution and role of these receptors were investigated and compared in cultured smooth muscle cells from rat aorta and pulmonary artery, as well as in fixed tissue sections of the aorta and pulmonary artery. Western blot analysis, RT-PCR and immunohistochemistry showed the expression of both VIP receptors in tissue sections of the aorta and pulmonary artery as well as in cultured smooth muscle cells from these vessels. The application of a specific antagonist of VPAC1 resulted in a small release from VIP induced inhibition of cell proliferation. In contrast (VIP 6-28; 300nM) which is an antagonist against both receptors resulted in a significant restoration of proliferation. The expression of cAMP was reduced in the presence of VIP 6-28 and slightly decreased by VPAC1 antagonist. These findings suggest a dual role for VPAC1 and VPAC2 receptors in mediating the antiproliferative effects of VIP with VPAC2 appearing to play a more dominant role.
Asunto(s)
Aorta/metabolismo , Proliferación Celular/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Arteria Pulmonar/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Péptido Intestinal Vasoactivo/fisiología , Animales , Aorta/citología , Western Blotting , Células Cultivadas , AMP Cíclico/metabolismo , Expresión Génica , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/fisiopatología , Inmunohistoquímica , Masculino , Fragmentos de Péptidos/farmacología , Arteria Pulmonar/citología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Tipo II del Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Megakaryopoiesis and platelet production are very complex processes, orchestrated by different growth factors, cytokines and transcription factors. It's only recently that a role was assigned for the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasoactive intestinal peptide (VIP) via their common Gs-coupled receptor VPAC1 in this process. The basis for this idea originated from studies in two related patients with a partial trisomy 18p11 and therefore carrying 3 copies of the PACAP gene and elevated PACAP concentrations in their plasma which resulted in a bleeding tendency with thrombopathy and a mild thrombocytopenia. This platelet functional and formation defect could be phenocopied in transgenic megakaryocyte specific PACAP-overexpressing mice. The addition of PACAP or VIP to hematopoietic stem cells resulted in an decreased megakaryocyte maturation and DNA polyploidization. In contrast, mice subcutaneously injected with inhibitory anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies presented with increased platelet numbers. This last concept was the basis for the development of these antibodies for the treatment of different types of thrombocytopenia as the therapeutic value for these antibodies was established in mice with a low platelet count due to chemotherapy, anti-platelet antibodies, a congenital factor of after bone marrow transplantation. For all models, the addition of 23A11 or PP1A4 resulted in an increased platelet recovery compared to the control antibody. Further studies are needed to identify the downstream signal transduction components after VPAC1 stimulation in megakaryocytes and platelets.
Asunto(s)
Plaquetas/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Animales , Plaquetas/efectos de los fármacos , Humanos , Megacariocitos , Ratones , Ratones Transgénicos , Neuropéptidos/genética , Neuropéptidos/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Recuento de Plaquetas , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Transducción de Señal , TrisomíaRESUMEN
Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC(1), VPAC(2) and PAC(1)R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.
Asunto(s)
Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , AMP Cíclico/metabolismo , Ligandos , Ratones , Ratones Transgénicos , Especificidad de Órganos , Fosfolipasa D/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/agonistas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/agonistas , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/agonistas , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Transducción de Señal , Péptido Intestinal Vasoactivo/fisiologíaAsunto(s)
Páncreas/irrigación sanguínea , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Animales , Atropina/farmacología , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Velocidad del Flujo Sanguíneo/fisiología , Masculino , Páncreas/efectos de los fármacos , Páncreas/fisiología , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Tipo II del Péptido Intestinal Vasoactivo/agonistas , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/agonistas , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Vasoactive intestinal peptide receptor-1 (VPAC-1) is an anti-proliferative, G-protein coupled receptor that is highly expressed on naïve T cells, and has been reported to be downregulated upon T cell activation. The T cell signaling molecules involved in mediating low VPAC-1 levels have not been identified. Therefore, to gain a greater understanding into this regulation, this study investigated the signaling pathways that regulate (VPAC-1) in murine, primary CD4 T cells. To this end, murine, splenic CD4 T cells were pretreated separately with 10 different pharmacological inhibitors and incubated +/- anti-CD3 for 24h. Total RNA was isolated, and VPAC-1 mRNA levels were measured by qPCR. Our results support that JNK kinases, downstream from the protein kinase, Zap70, are involved in suppressive regulation of VPAC-1 steady-state mRNA levels after anti-CD3 treatment. In contrast, inhibitors against PKC, ERK, p38, Zap70 and Rac1 supported a stimulatory influence in VPAC-1 regulation in the absence of T cell signaling. By studying the signaling pathways that regulate VPAC-1 in T cells, we can gain greater insight into the role of this anti-inflammatory receptor in autoimmunity and infectious diseases.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Transducción de Señal/fisiología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/metabolismo , Estaurosporina/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors PAC1-R, VPAC1-R, and VPAC2-R play a role in various physiological processes, including proopiomelanocortin (POMC) and brain-derived neurotrophic factor (BDNF) gene expression. We have previously found that PACAP stimulates POMC gene expression, POMC biosynthesis, and alpha-MSH secretion in the melanotrope cell of the amphibian Xenopus laevis. This cell hormonally controls the process of skin color adaptation to background illumination. Here, we have tested the hypothesis that PACAP is involved in the regulation of Xenopus melanotrope cell activity during background adaptation and that part of this regulation is through the control of the expression of autocrine acting BDNF. Using quantitative RT-PCR, we have identified the Xenopus PACAP receptor, VPAC1-R, and show that this receptor in the melanotrope cell is under strong control of the background light condition, whereas expression of PAC1-R was absent from these cells. Moreover, we reveal by quantitative immunocytochemistry that the neural pituitary lobe of white-background adapted frogs possesses a much higher PACAP content than the neural lobe of black-background adapted frogs, providing evidence that PACAP produced in the hypothalamic magnocellular nucleus plays an important role in regulating the activity of Xenopus melanotrope cells during background adaptation. Finally, an in vitro study demonstrates that PACAP stimulates the expression of BDNF transcript IV.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Exones , Melanotrofos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Xenopus laevis/genética , Animales , Encéfalo/metabolismo , Clonación Molecular , Regulación de la Expresión Génica/efectos de los fármacos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , ARN Mensajero/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Distribución TisularRESUMEN
Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34(+) cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34(+) cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.
Asunto(s)
Megacariocitos/citología , Megacariocitos/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Animales , Animales Modificados Genéticamente , Antígenos CD/análisis , Antígenos CD34/análisis , Línea Celular Tumoral , Cromosomas Humanos Par 18 , AMP Cíclico/fisiología , Humanos , Ratones , Ratones Transgénicos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Conejos , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Trombocitopenia/genética , Trisomía/genéticaRESUMEN
Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VPAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the alpha-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPAC1 receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr11 ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of [11Glyc]VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg12-Leu13 peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.
Asunto(s)
Glicosilación , Péptido Intestinal Vasoactivo/farmacología , Unión Proteica , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Relación Estructura-Actividad , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/química , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
AIM: To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R)and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. METHODS: The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone,8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The VPCAP2-R mRNA expression level in the control group (1.09+/-0.58) was lower than that in the gallbladder polyp group (1.64+/-0.56) and the gallstone group (1.55+/-0.45) (P<0.05) while the VPCAP1-R mRNA expression level in the control group (1.15+/-0.23) was not apparently different from that in the gallbladder polyp group (1.28+/-0.56) and the gallstone group (1.27+/-0.38). CONCLUSION: The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps.
Asunto(s)
Enfermedades de la Vesícula Biliar/genética , Vesícula Biliar/química , Cálculos Biliares/genética , Pólipos/genética , ARN Mensajero/análisis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Adulto , Femenino , Enfermedades de la Vesícula Biliar/metabolismo , Cálculos Biliares/química , Cálculos Biliares/etiología , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Pólipos/química , Pólipos/etiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Osteoarthritis (OA) is a debilitating disease in which primarily weight-bearing joints undergo progressive degeneration. Despite the widespread prevalence of OA in the adult population, very little is known about the factors responsible for the generation and maintenance of OA pain. Vasoactive intestinal peptide (VIP) was identified in the synovial fluid of arthritis patients nearly 20 years ago and the aim of this study was to examine whether VIP could be involved in the generation of OA pain. Hindlimb weight bearing was used as a measure of joint pain, while von Frey hair algesiometry applied to the plantar surface of the ipsilateral hindpaw tested for secondary mechanical hyperalgesia. Intra-articular injection of VIP into normal rat knee joints caused a significant shift in weight bearing in favour of the contralateral non-injected hindlimb as well as causing a reduction in ipsilateral paw withdrawal threshold. These pain responses were blocked by co-administration of the VPAC receptor antagonist VIP6-28. Induction of OA by intra-articular sodium monoiodoacetate injection resulted in a reduction in weight bearing on the affected leg, but no evidence of secondary hyperalgesia in the paw. Treatment of OA knees with a single injection of VIP6-28 diminished hindlimb incapacitance while increasing paw withdrawal threshold. This study showed for the first time that peripheral application of VIP causes increased knee joint allodynia and secondary hyperalgesia. Furthermore, antagonists that inhibit VIP activity may prove beneficial in the alleviation of OA pain.
Asunto(s)
Osteoartritis de la Rodilla/fisiopatología , Dolor/etiología , Péptido Intestinal Vasoactivo/fisiología , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Miembro Posterior , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Inyecciones Intraarticulares , Yodoacetatos/administración & dosificación , Yodoacetatos/toxicidad , Masculino , Dolor/fisiopatología , Umbral del Dolor/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Ratas , Ratas Wistar , Receptores de Tipo II del Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/antagonistas & inhibidores , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología , Rodilla de Cuadrúpedos , Estrés Mecánico , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/farmacología , Péptido Intestinal Vasoactivo/uso terapéutico , Péptido Intestinal Vasoactivo/toxicidad , Soporte de PesoRESUMEN
Vasoactive intestinal peptide (VIP) is a vasodilator peptide present in cerebrovascular nerves. Vasoactive intestinal peptide can activate VPAC1, VPAC2 and the NPR-C receptor. This study sought to determine the receptors involved in VIP-induced vasodilation of porcine basilar arteries. Porcine basilar arteries contained the messenger ribonucleic acid of all three receptors. Immunocytochemical analysis of porcine basilar arteries revealed that the VPAC1 receptor is expressed on the endothelium, VPAC2 on the outer layers of the media and the NPR-C receptor throughout the artery, including nerves. Vasodilator responses to all receptor agonists showed that the receptors are functional. The vasodilator response to the VPAC1 receptor agonist was inhibited by L-NAME and abolished by endothelial denudation. Vasodilation induced by Ro-25-1553, the VPAC2 agonist, was unaffected by NOS inhibition or removal of the endothelium. Activation of the NPR-C receptor produced a vasodilation, which was susceptible to NOS inhibition and independent of endothelium. The vasodilator response to electrical stimulation at 20 Hz was attenuated by PG-99-465, the VPAC2 antagonist. This study shows that all known VIP receptors are involved in VIP-mediated vasodilation of porcine basilar arteries. The VPAC1 receptor is located on the endothelium and elicits vasodilation by generating nitric oxide (NO). The VPAC2 receptor is mainly expressed in the outer layers of the smooth muscle and induces vasodilation independently of NO in response to VIP released from intramural nerves. The NPR-C receptor produces NO-dependent vasodilation independently of the endothelium by stimulation of nNOS in intramural nerves.