RESUMO
Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (â¼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.
Assuntos
Comportamento Animal/fisiologia , Ritmo Circadiano/fisiologia , Periodicidade , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas Mutantes Quiméricas/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Núcleo Supraquiasmático/fisiologiaRESUMO
The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24â¯h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24â¯h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24â¯h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Pressão Intraocular/fisiologia , Luz , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Receptores Tipo II de Peptídeo Intestinal Vasoativo/deficiência , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Tonometria OcularRESUMO
The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices.
Assuntos
Dendritos , Extinção Psicológica/fisiologia , Medo/fisiologia , Córtex Pré-Frontal/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Animais , Condicionamento Clássico , Sinais (Psicologia) , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Pré-Frontal/citologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genéticaRESUMO
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.
Assuntos
Glândulas Écrinas/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Suor/metabolismo , Adulto , Animais , Feminino , Pé , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , RNA Mensageiro/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/fisiologiaRESUMO
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.
Assuntos
Macrófagos/efeitos dos fármacos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Artrite Reumatoide/patologia , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunofenotipagem , Inflamação , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , RNA Mensageiro/biossíntese , Receptores Tipo II de Peptídeo Intestinal Vasoativo/biossíntese , Receptores Tipo II de Peptídeo Intestinal Vasoativo/efeitos dos fármacos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/biossíntese , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/efeitos dos fármacos , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Líquido Sinovial/citologia , Regulação para CimaRESUMO
To serve as a robust internal circadian clock, the cell-autonomous molecular and electrophysiological activities of the individual neurons of the mammalian suprachiasmatic nucleus (SCN) are coordinated in time and neuroanatomical space. Although the contributions of the chemical and electrical interconnections between neurons are essential to this circuit-level orchestration, the features upon which they operate to confer robustness to the ensemble signal are not known. To address this, we applied several methods to deconstruct the interactions between the spatial and temporal organisation of circadian oscillations in organotypic slices from mice with circadian abnormalities. We studied the SCN of mice lacking Cryptochrome genes (Cry1 and Cry2), which are essential for cell-autonomous oscillation, and the SCN of mice lacking the vasoactive intestinal peptide receptor 2 (VPAC2-null), which is necessary for circuit-level integration, in order to map biological mechanisms to the revealed oscillatory features. The SCN of wild-type mice showed a strong link between the temporal rhythm of the bioluminescence profiles of PER2::LUC and regularly repeated spatially organised oscillation. The Cry-null SCN had stable spatial organisation but lacked temporal organisation, whereas in VPAC2-null SCN some specimens exhibited temporal organisation in the absence of spatial organisation. The results indicated that spatial and temporal organisation were separable, that they may have different mechanistic origins (cell-autonomous vs. interneuronal signaling) and that both were necessary to maintain robust and organised circadian rhythms throughout the SCN. This study therefore provided evidence that the coherent emergent properties of the neuronal circuitry, revealed in the spatially organised clusters, were essential to the pacemaking function of the SCN.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Criptocromos/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rede Nervosa/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genéticaRESUMO
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.
Assuntos
Lagartos/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Testículo/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Hibridização In Situ , Células Intersticiais do Testículo/química , Células Intersticiais do Testículo/fisiologia , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Peptídeo Intestinal Vasoativo/análise , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/análise , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/análise , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/fisiologia , Alinhamento de Sequência , Testículo/química , Peptídeo Intestinal Vasoativo/análise , Peptídeo Intestinal Vasoativo/genéticaRESUMO
PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 µM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 µM) but not the MAPKK inhibitor (PD98059, 50 µM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.
Assuntos
Proteínas da Mielina/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Células de Schwann/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Células de Schwann/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Vasoactive intestinal polypeptide (VIP) signaling is critical for circadian rhythms. For example, the expression of VIP and its main receptor, VPAC2R, is necessary for maintaining synchronous daily rhythms among neurons in the suprachiasmatic nucleus (SCN), a master circadian pacemaker in animals. Where and when VPAC2R protein is expressed in the SCN and other brain areas has not been examined. Using immunohistochemistry, we characterized a new antibody and found that VPAC2R was highly enriched in the SCN and detectable at low levels in many brain areas. Within the SCN, VPAC2R was circadian, peaking in the subjective morning, and abundantly expressed from the rostral to caudal margins with more in the dorsomedial than ventrolateral area. VPAC2R was found in nearly all SCN cells including neurons expressing either VIP or vasopressin (AVP). SCN neurons mainly expressed VPAC2R in their somata and dendrites, not axons. Finally, constant light increased VIP and AVP expression, but not VPAC2R. We conclude that the circadian clock, not the ambient light level, regulates VPAC2R protein localization. These results are consistent with VPAC2R playing a role in VIP signaling at all times of day, broadly throughout the brain and in all SCN cells.
Assuntos
Neurônios/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/química , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Núcleo Supraquiasmático/química , Núcleo Supraquiasmático/metabolismo , Animais , Células Cultivadas , Ritmo Circadiano/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/fisiologiaRESUMO
The circadian time-keeping system consists of clocks in the suprachiasmatic nucleus (SCN) and in peripheral organs including an adrenal clock linked to the rhythmic corticosteroid production by regulating steroidogenic acute regulatory protein (StAR). Clock cells contain an autonomous molecular oscillator based on a group of clock genes and their protein products. Mice lacking the VPAC2 receptor display disrupted circadian rhythm of physiology and behaviour, and therefore, we using real-time RT-PCR quantified (1) the mRNAs for the clock genes Per1 and Bmal1 in the adrenal gland and SCN, (2) the adrenal Star mRNA and (3) the serum corticosterone concentration both during a light/dark (L/D) cycle and at constant darkness in wild type (WT) and VPAC2 receptor-deficient mice (VPAC2-KO). We also examined if PER1 and StAR were co-localised in the adrenal steroidogenic cells. Per1 and Bmal1 mRNA showed a 24-h rhythmic expression in the adrenal of WT mice under L/D and dark conditions. During a L/D cycle, the adrenal clock gene rhythm in VPAC2-KO mice was phase-advanced by approximately 6 h compared to WT mice and became arrhythmic in constant darkness. A significant 24-h rhythmic variation in the adrenal Star mRNA expression and circulating corticosterone concentration was similarly phase-advanced during the L/D cycle. The loss of adrenal clock gene rhythm in the VPAC2 receptor knockout mice after transfer into constant darkness was accompanied by disappearance of rhythmicity in Star mRNA expression and serum corticosterone concentration. Double immunohistochemistry showed that the PER1 protein and StAR were co-localised in the same steroidogenic cells. Circulating corticosterone plays a role in the circadian timing system and the misaligned corticosterone rhythm in the VPAC2 receptor knockout mice could be involved in their abnormal rhythms of physiology.
Assuntos
Fatores de Transcrição ARNTL/genética , Córtex Suprarrenal/metabolismo , Ritmo Circadiano/fisiologia , Corticosterona/sangue , Regulação da Expressão Gênica/fisiologia , Proteínas Circadianas Period/genética , Fosfoproteínas/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/deficiência , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição ARNTL/biossíntese , Medula Suprarrenal/metabolismo , Animais , Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , Corticosterona/metabolismo , Escuridão , Regulação da Expressão Gênica/efeitos da radiação , Hibridização In Situ , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Circadianas Period/biossíntese , Fosfoproteínas/biossíntese , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas able to grow under conditions of metabolic stress caused by insufficient nutrients or oxygen. Both pituitary adenylate cyclase-activating polypeptide (PACAP) and activity-dependent neuroprotective protein (ADNP) have glioprotective potential. However, whether PACAP/ADNP signaling is involved in the resistance to cell death in MPNST cells remains to be clarified. Here, we investigated the involvement of this signaling system in the survival response of MPNST cells against hydrogen peroxide (H(2)O(2))-evoked death both in the presence of normal serum (NS) and in serum-starved (SS) cells. Results showed that ADNP levels increased time-dependently (6-48 h) in SS cells. Treatment with PACAP38 (10(-9) to 10(-5) M) dose-dependently increased ADNP levels in NS but not in SS cells. PAC(1)/VPAC receptor antagonists completely suppressed PACAP-stimulated ADNP increase and partially reduced ADNP expression in SS cells. NS-cultured cells exposed to H(2)O(2) showed significantly reduced cell viability (~50 %), increased p53 and caspase-3, and DNA fragmentation, without affecting ADNP expression. Serum starvation significantly reduced H(2)O(2)-induced detrimental effects in MPNST cells, which were not further ameliorated by PACAP38. Altogether, these finding provide evidence for the involvement of an endogenous PACAP-mediated ADNP signaling system that increases MPNST cell resistance to H(2)O(2)-induced death upon serum starvation.
Assuntos
Proteínas de Neoplasias/fisiologia , Neoplasias de Bainha Neural/patologia , Proteínas do Tecido Nervoso/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Meios de Cultura Livres de Soro/farmacologia , Replicação do DNA , DNA de Neoplasias/análise , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
OBJECTIVE: The liver is innervated by the vagus nerve. Its efferent neurotransmitters acetylcholine (ACh) and vasoactive intestinal peptide (VIP) are both well-known vasodilators. A study was undertaken to determine whether electrical vagus nerve stimulation (STIM) influences portal vein pressure. METHODS: The left vagus nerve upstream of the hepatic branch was stimulated at 5 Hz (ACh release) and 10 Hz (VIP release) in normal and cirrhotic rats. RESULTS: STIM at both frequencies decreased portal pressure in normal rats while, in cirrhotic rats, only 10 Hz STIM resulted in long-lasting reduction of portal pressure. Hepatic branch vagotomy prevented the STIM-induced decrease in pressure, proving that the effect is a direct hepatic effect. Deafferentation of the left vagus nerve by pretreatment with capsaicin did not change the effect of STIM, showing that the vagus efferents and not the afferents are responsible for the decrease in portal pressure. Injecting microspheres before and after STIM showed that STIM did not lead to redistribution of systemic blood flow but decreased portal pressure by lowering intrahepatic resistance. Using in situ liver perfusion to evaluate the intrahepatic effect of ACh and VIP, both neurotransmitters significantly decreased the perfusion pressure in normal rats. VIP also decreased portal pressure in cirrhotic rats, confirming the results of STIM. This VIP-induced decrease in pressure could be prevented by a VIP receptor 2 antagonist. L-NAME did not inhibit the VIP effect in cirrhotic rats, indicating that VIP does not act via nitric oxide. CONCLUSION: High-frequency electrical vagus stimulation improves portal hypertension in cirrhotic rats, most likely through release of VIP, binding to VIP receptor 2. As the technology is already in use for other applications, vagus nerve stimulation might be an important new strategy in the treatment of portal hypertension.
Assuntos
Hipertensão Portal/terapia , Cirrose Hepática Experimental/complicações , Estimulação do Nervo Vago/métodos , Acetilcolina/administração & dosagem , Acetilcolina/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Hipertensão Portal/etiologia , Hipertensão Portal/fisiopatologia , Circulação Hepática/fisiologia , Cirrose Hepática Experimental/fisiopatologia , Masculino , Microcirculação/fisiologia , Microesferas , Pressão na Veia Porta/efeitos dos fármacos , Pressão na Veia Porta/fisiologia , Ratos , Ratos Wistar , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Transdução de Sinais/fisiologia , Nervo Vago/fisiopatologia , Peptídeo Intestinal Vasoativo/administração & dosagem , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologiaRESUMO
Excitotoxicity is a key molecular mechanism of perinatal brain damage and is associated with cerebral palsy and long term cognitive deficits. VIP induces a potent neuroprotection against perinatal excitotoxic white matter damage. VIP does not prevent the initial appearance of white matter lesion but promotes a secondary repair with axonal regrowth. This plasticity mechanism involves an atypical VPAC2 receptor and BDNF production. Stable VIP agonists mimic VIP effects when given systemically and exhibit a large therapeutic window. Unraveling cellular and molecular targets of VIP effects against perinatal white matter lesions could provide a more general rationale to understand the neuroprotection of the developing white matter against excitotoxic insults.
Assuntos
Encéfalo/embriologia , Paralisia Cerebral/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Peptídeo Intestinal Vasoativo/fisiologia , Peptídeo Intestinal Vasoativo/uso terapêutico , Animais , Encéfalo/metabolismo , Paralisia Cerebral/prevenção & controle , Humanos , Plasticidade Neuronal/efeitos dos fármacos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Transdução de Sinais/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/análogos & derivados , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Vasoactive intestinal peptide (VIP) facilitates a "pro-allergy" phenotype when signaling through its G protein-coupled receptor, VPAC(2). We have shown that VPAC(2) knock-out (KO) mice developed an allergic phenotype marked by eosinophilia and elevated serum IgE. Therefore, we hypothesized that the humoral response to allergen challenge in these mice was T(H)2 dominant similar to wild-type (WT) C57BL/6 mice. Antibody responses in WT and KO mice were measured after Aspergillus fumigatus conidia inhalation. In contrast to previous reports, basal levels of serum IgG(2a) and IgA were significantly higher in naïve VPAC(2) KO animals. Antibody availability in the serum as well as the bronchoalveolar lavage fluid after fungal challenge was dominated by the pro-inflammatory isotype IgG(2a) and the mucosal isotype, IgA. IgA localizing cells dominated in the peribronchovascular areas of allergic KO mice while IgE immune complexes were found in WT allergic lungs. This research shows for the first time that VPAC(2) has a significant effect on antibody regulation, in the context of allergy.
Assuntos
Formação de Anticorpos , Aspergillus fumigatus/imunologia , Hipersensibilidade Imediata/imunologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Animais , Formação de Anticorpos/genética , Hipersensibilidade Imediata/genética , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/análise , Peptídeo Intestinal Vasoativo/imunologiaRESUMO
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.
Assuntos
Aorta/metabolismo , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Artéria Pulmonar/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Aorta/citologia , Western Blotting , Células Cultivadas , AMP Cíclico/metabolismo , Expressão Gênica , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Imuno-Histoquímica , Masculino , Fragmentos de Peptídeos/farmacologia , Artéria Pulmonar/citologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vasoactive intestinal peptide (VIP) is implicated in cutaneous active vasodilation in humans. VIP and the closely related pituitary adenylate cyclase activating peptide (PACAP) act through several receptor types: VIP through VPAC1 and VPAC2 receptors and PACAP through VPAC1, VPAC2, and PAC1 receptors. We examined participation of VPAC2 and/or PAC1 receptors in cutaneous vasodilation during heat stress by testing the effects of their specific blockade with PACAP6-38. PACAP6-38 dissolved in Ringer's was administered by intradermal microdialysis at one forearm site while a control site received Ringer's solution. Skin blood flow was monitored by laser-Doppler flowmetry (LDF). Blood pressure was monitored noninvasively and cutaneous vascular conductance (CVC) calculated. A 5- to 10-min baseline period was followed by approximately 70 min of PACAP6-38 (100 microM) perfusion at one site in normothermia and a 3-min period of body cooling. Whole body heating was then performed to engage cutaneous active vasodilation and was maintained until CVC had plateaued at an elevated level at all sites for 5-10 min. Finally, 58 mM sodium nitroprusside was perfused through both microdialysis sites to effect maximal vasodilation. No CVC differences were found between control and PACAP6-38-treated sites during normothermia (19 +/- 3%max untreated vs. 20 +/- 3%max, PACAP6-38 treated; P > 0.05 between sites) or cold stress (11 +/- 2%max untreated vs. 10 +/- 2%max, PACAP6-38 treated, P > 0.05 between sites). PACAP6-38 attenuated the increase in CVC during whole body heating when compared with untreated sites (59 +/- 3%max untreated vs. 46 +/- 3%max, PACAP6-38 treated, P < 0.05). We conclude that VPAC2 and/or PAC1 receptor activation is involved in cutaneous active vasodilation in humans.
Assuntos
Resposta ao Choque Térmico/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Pele/irrigação sanguínea , Vasodilatação/fisiologia , Adulto , Regulação da Temperatura Corporal/fisiologia , Feminino , Antebraço , Temperatura Alta , Humanos , Masculino , Nitroprussiato/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Pele/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Peptídeo Intestinal Vasoativo/fisiologia , Vasodilatadores/farmacologiaRESUMO
Allergic asthma is a T(H)2-mediated disease marked by airway inflammation, increased mucus production, and elevated serum IgE in response to allergen provocation. Among its ascribed functions, the neuropeptide vasoactive intestinal peptide (VIP) is believed to promote a T(H)2 phenotype when signaling through its VPAC(2) receptor. In this study, we assessed the requirement for the VIP/VPAC(2) axis in initiating the allergic pulmonary phenotype in a murine model of fungal allergic asthma. C57BL/6 wild-type (WT) and VPAC(2) knock-out (KO) mice were sensitized with Aspergillus fumigatus antigen and challenged with an aerosol of live conidia to induce allergic airways disease. WT and KO mice exhibited similar peribronchovascular inflammation, increased number of goblet cells, and elevated serum IgE. However, the absence of VPAC(2) receptor resulted in a marked enhancement of MUC5AC mRNA with an associated increase in goblet cells and a reduction in eosinophils in the airway lumen at day 3 when VIP mRNA was undetectable in the KO lung. Sustained elevation of serum IgE was noted in KO mice at day 14, while the level in WT mice declined at this time point. These data suggest that the absence of VPAC(2) does not protect mice from developing the signs and symptoms of allergic asthma.
Assuntos
Asma/imunologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/deficiência , Animais , Antígenos de Fungos/imunologia , Aspergillus/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Células Caliciformes/patologia , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-5AC/biossíntese , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
Among several factors known to modulate embryo implantation and survival, uterine quiescence and neovascularization, maternal immunotolerance through the Th1/Th2 cytokine balance towards a Th2 profile, local regulatory T-cell (Treg) activation, and high levels of progesterone were assigned a prominent role. Vasoactive intestinal peptide (VIP) is a neuroimmunopeptide that has anti-inflammatory effects, promotes Th2 cytokines and CD4(+)CD25(+)FOXP3(+) Treg activation, and stimulates exocrine secretion, smooth muscle relaxation, and vasodilatation favoring uterus quiescence. The goal of the present work was to explore the participation of VIP in the implantation sites of normal and pregnant prediabetic nonobese diabetic (NOD) females, a mouse strain that spontaneously develops an autoimmune exocrinopathy similar to Sjögren's syndrome. Our results indicate a reduction in litter size from the third parturition onwards in the NOD female lifespan with increased resorption rates. Progesterone systemic levels were significantly decreased in pregnant NOD mice compared with BALB/c mice, although the allogeneic response to progesterone by spleen cells was not impaired. VIP receptors, Vipr1 and Vipr2 (Vpac1 and Vpac2), were expressed at the implantation sites and VIP induced leukemia inhibitory factor (LIF) and Treg marker expression in both strains; however, a reduced Vip expression was found in NOD implantation sites. We conclude that the reduced birth rate at 16-week-old NOD mice with a Th1 systemic cytokine profile involves resorption processes with a lower expression of VIP at the sites of implantation, which acts as a local inducer of pro-implantatory LIF and Treg activation.
Assuntos
Implantação do Embrião/imunologia , Fatores Imunológicos/fisiologia , Estado Pré-Diabético , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Diabetes Gestacional/genética , Diabetes Gestacional/imunologia , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patologia , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/genética , Perda do Embrião/genética , Perda do Embrião/imunologia , Perda do Embrião/metabolismo , Perda do Embrião/patologia , Feminino , Fatores Imunológicos/farmacologia , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Estado Pré-Diabético/genética , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/patologia , Gravidez , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The hypothalamic suprachiasmatic nucleus (SCN) harbors the master circadian pacemaker. SCN neurons produce the amino acid gamma-aminobutyric acid (GABA) and several peptide molecules for coordination and communication of their circadian rhythms. A subpopulation of SCN cells synthesizes vasoactive intestinal polypeptide (VIP) and provides a dense innervation of the subparaventricular zone (SPZ), an important CNS target of the circadian pacemaker. In this study, using patch-clamp recording techniques and rat brain slice preparations, the contribution of VIP to SCN efferent signaling to SPZ was evaluated by examining membrane responses of SPZ neurons to exogenous VIP receptor ligands. In approximately 50% of the SPZ neurons receiving monosynaptic GABAA receptor-mediated inputs from SCN, bath-applied VIP (0.5-1 microM) resulted in a membrane depolarization caused by tetrodotoxin-resistant inward currents reversing at approximately -23 mV. These data suggest the existence of postsynaptic receptors that activate a nonselective cationic conductance. In addition, a subset of SPZ neurons showed an increase in the amplitude of SCN-evoked GABAergic inhibitory postsynaptic currents (IPSCs) and a decrease in their paired-pulse ratios. This, together with an increase in frequency of spontaneous and miniature IPSCs, implies the presence of presynaptic receptors that facilitate GABA release from SCN and possibly other synaptic terminals. The effects occurred in separate neurons and could be mimicked by the selective VPAC2 receptor agonist BAY 55-9837 (0.2-0.5 microM) and partially blocked by the VIP receptor antagonist VIP(6-28) (5 microM). The results indicate that VIP acts via both post- and presynaptic VPAC2 receptors to differentially modulate SCN GABAergic signaling to distinct subpopulations of SPZ neurons.