RESUMEN
The endocrine body rhythms including the hypothalamic-pituitary-thyroid axis seem to be regulated by the circadian timing system, and daily rhythmicity of circulating thyroid-stimulating hormone (TSH) is well established. The circadian rhythms are generated by endogenous clocks in the central brain oscillator located in the hypothalamic suprachiasmatic nucleus (SCN) as well as multiple peripheral clocks, but information on the existence and function of a thyroid clock is limited. The molecular machinery in all clock cells is composed of a number of clock genes and their gene products are connected by autoregulatory feedback loops. Here, we provide evidence for a thyroid clock in the rat by demonstrating 24-h antiphase oscillations for the mRNA of the canonical clock genes Per1 and Bmal1, which was unaffected by hypophysectomy. By immunostaining, we supported the existence of a core oscillator in the individual thyroid cells by demonstrating a daily cytoplasmatic-nuclear shuttling of PER1 protein. In normal rats, we found a significant daily rhythmicity in the circulating thyroid hormones preceded by a peak in TSH. In hypophysectomised rats, although the thyroid clock was not affected, the oscillations in circulating thyroid hormones were abolished and the levels were markedly lowered. No daily oscillations in the expression of TSH receptor mRNA were observed in neither control rats nor hypophysectomised rats. Our findings indicate that the daily rhythm of thyroid hormone secretion is governed by SCN signalling via the rhythmic TSH secretion rather than by the local thyroid clock, which was still ticking after hypophysectomy.
Asunto(s)
Relojes Biológicos/fisiología , Hipofisectomía/métodos , Glándula Tiroides/fisiología , Tiroxina/fisiología , Triyodotironina/fisiología , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Femenino , Regulación de la Expresión Génica/fisiología , Masculino , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
BACKGROUND: Ablative fractional laser (AFL) generates microchannels in skin surrounded by a zone of thermally altered tissue, termed the coagulation zone (CZ). The thickness of CZ varies according to applied wavelength and laser settings. It is well-known that AFL channels facilitate uptake of topically applied compounds, but the importance of CZ is unknown. METHODS: Franz Cells were used to investigate skin uptake and permeation of fluorescent labeled polyethylene glycols (PEGs) with mean molecular weights (MW) of 350, 1,000, and 5,000 Da. Microchannels with CZ thicknesses ranging from 0 to 80 µm were generated from micro-needles (0 µm, CZ-0), and AFL (10,600 nm) applied to -80°C deep frozen skin (20 µm, CZ-20) and skin equilibrated to room temperature (80 µm, CZ-80). Channels penetrated into similar mid-dermal skin depths of 600-700 µm, and number of channels per skin area was similar. At 4 hours incubation, skin uptake of PEGs into CZ and dermis was evaluated by fluorescence microscopy at specific skin depths of 150, 400, and 1,000 µm and the transcutaneous permeation was quantified by fluorescence of receptor fluids. RESULTS: Overall, the highest uptake of PEGs was reached through microchannels surrounded by CZ compared to channels with no CZ (CZ-20 and CZ-80>CZ-0).The thickness of CZ affected PEG distribution in skin. A thin CZ-20 favored significantly higher mean fluorescence intensities inside CZ areas compared to CZ-80 (PEG 350, 1,000, and 5,000; P < 0.001). In dermis, the uptake through CZ-20 channels was significantly higher than through CZ-80 and CZ-0 at all skin depths (PEG 350, 1,000 and 5,000, 150-1,000 µm; P < 0.001). Correspondingly, transcutaneous permeation of PEG 350 was highest in CZ-20 compared to CZ-80 and CZ-0 samples (P < 0.001). Permeation of larger molecules (PEG 1,000 and PEG 5,000) was generally low. CONCLUSION: Uptake of topical compounds is higher through microchannels surrounded by a CZ than without a CZ. Moreover, CZ thickness influences PEG distribution, with highest PEG uptake achieved from microchannels surrounded by a thin CZ. Lasers Surg. Med. 49:582-591, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Coagulación Sanguínea , Fármacos Dermatológicos/farmacocinética , Sistemas de Liberación de Medicamentos , Polietilenglicoles/farmacocinética , Piel/metabolismo , Administración Cutánea , Animales , Células Cultivadas , Fármacos Dermatológicos/administración & dosificación , Femenino , Microscopía Fluorescente , Polietilenglicoles/administración & dosificación , Distribución Aleatoria , Piel/diagnóstico por imagen , PorcinosRESUMEN
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
Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). The ipRGCs regulate other nonimage-forming visual functions such as the pupillary light reflex, masking behavior, and light-induced melatonin suppression. To evaluate whether PACAP-immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin-expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer cholera toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be costored in 99% of melanopsin-expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP-immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey, supporting previous retrograde tracer studies demonstrating that melanopsin-containing retinal projections reach areas in the primate brain involved in both image- and nonimage-forming visual processing.
Asunto(s)
Encéfalo/anatomía & histología , Macaca/anatomía & histología , Células Ganglionares de la Retina/citología , Vías Visuales/anatomía & histología , Animales , Encéfalo/metabolismo , Toxina del Cólera , Inmunohistoquímica , Macaca/metabolismo , Masculino , Microscopía Confocal , Técnicas de Trazados de Vías Neuroanatómicas , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Vías Visuales/metabolismoRESUMEN
Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.
Asunto(s)
Globinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Opsinas de Bastones/metabolismo , Núcleo Supraquiasmático/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Globinas/biosíntesis , Globinas/genética , Masculino , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuroglobina , Oxígeno/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismoRESUMEN
Neuroglobin (Ngb) is a myoglobin-like (Mb) heme-globin, belonging the globin family located only in neuronal tissue of the central nervous system. Ngb has been shown to be upregulated in and to protect neurons from hypoxic and ischemic injury, but the function of Ngb-in particular how Ngb may protect neurons-remains largely elusive. We have previously described the localization of Ngb in the rat brain and found it to be expressed in areas primarily involved in sleep/wake, circadian, and food regulation. The present study was undertaken, using immunohistochemistry, to characterize the localization, colocalization, innervation, and response to light of Ngb-immunoreactive (IR) cells in the rat suprachiasmatic nucleus (SCN). Our results demonstrate that the majority of Ngb-expressing neurons in the SCN belong to a cell group not previously characterized by neurotransmitter content; only a small portion was found to co-store GRP in the ventral SCN. Furthermore, some Ngb-containing neurons were responsive to light stimulation at late night evaluated by the induction of cFOS and only a few cells were found to express the core clock gene PER1 during the 24-hour light/dark cycle. The Ngb-containing cells received input from neuropeptide Y (NPY)-containing nerve fibers of the geniticulo-hypothalamic tract (GHT), whereas no direct input from the eye or the midbrain raphe system was demonstrated. The results indicate that the Ngb could be involved in both photic and nonphotic entrainment via input from the GHT.
Asunto(s)
Globinas/metabolismo , Luz , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Péptido Liberador de Gastrina/metabolismo , Humanos , Masculino , Vías Nerviosas/anatomía & histología , Neuroglobina , Neuronas/citología , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Wistar , Núcleo Supraquiasmático/citología , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus (SCN), and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. Rhythmic expression of clock genes in the adrenal glands has previously been reported. Since the central clock in the SCN communicates with the adrenal glands via circadian release of adrenocorticotrophic hormone, we quantified the mRNAs for the canonical clock genes, Per1, Per2 and Bmal1 in the adrenal glands by real-time reverse transcription-polymerase chain reaction during a 24-h-cycle in normal and hypophysectomised rats. The mRNAs for all the three clock genes disclosed rhythmic oscillations with a period of 24 h and the phase did not differ between the hypophysectomised and intact rats. The expression pattern of Per1 and Bmal1 was in antiphase in both groups of animals. In situ hybridisation histochemistry using antisense RNA probes demonstrated that, at times of peak expression, mRNAs for all the three clock genes were expressed in the adrenal cortex with a particularly strong labelling in the zona reticularis. In accordance with the mRNA localisation, immunostaining for PER1 protein was visualised in cells of the adrenal cortex, being most intense in the inner zone. The immunostaining also demonstrated a translocation of PER1 protein from the cytoplasm to the nucleus during the daily cycle, supporting the existence of a core oscillator in the individual adrenal gland cells. Our findings support the existence of a circadian core oscillator in cells of the rat adrenal cortex and indicate that the activity of the oscillator is independent of SCN signalling via the pituitary gland. The adrenal cortical clock could be involved in rhythmic transcriptional activation of genes associated with hormonal biosynthesis, involved in gating of the response of the adrenal cortex to external cues or involved in apoptosis of adrenal cortical cells.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Ritmo Circadiano/genética , Hipofisectomía , Proteínas Nucleares/genética , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano/fisiología , Femenino , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fotoperiodo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Circadian rhythms are entrained daily by environmental photic and non-photic cues. The present review describes the anatomy and functional characteristics of the three major input pathways to the circadian clock mediating entrainment: the retino-hypothalamic tract, the geniculo-hypothalamic tract and the midbrain raphe projection.
Asunto(s)
Vías Aferentes/fisiología , Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Núcleo Supraquiasmático/fisiología , Vías Aferentes/citología , Animales , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Ácido Glutámico/metabolismo , Humanos , Proteínas Circadianas Period , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Núcleo Supraquiasmático/citologíaRESUMEN
Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.
Asunto(s)
Ritmo Circadiano/efectos de los fármacos , Hipotálamo/fisiología , Luz , Neuropéptidos/farmacología , Neuropéptidos/fisiología , Fragmentos de Péptidos/farmacología , Retina/fisiología , Núcleo Supraquiasmático/efectos de los fármacos , Animales , Anticuerpos , Conducta Animal , Ritmo Circadiano/fisiología , Cricetinae , Inmunohistoquímica , Masculino , Mesocricetus , Vías Nerviosas/química , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Neuropéptidos/administración & dosificación , Neuropéptidos/análisis , Neuropéptidos/inmunología , Neurotransmisores/análisis , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Retina/química , Opsinas de Bastones/efectos de los fármacos , Opsinas de Bastones/fisiología , Carrera , Transmisión Sináptica/efectos de los fármacosRESUMEN
Circadian rhythms of physiology and behaviour generated by the brain's biological clock located in the suprachiasmatic nucleus are entrained by light via the retinohypothalamic tract. Two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), found in this monosynaptic pathway mediate the effects of light to the clock. It is well known that not only light entrains the clock. Nonphotic cues mediated by neurotransmitters such as serotonin reaching the suprachiasmatic nucleus from the midbrain raphe nucleus modulate light-induced phase shifts at night. Two clock genes, per1 and per2, have been attributed a role in light-induced phase shift. In the present study, using an in vitro brain slice model and quantitative in situ hybridization for per1 and per2, we have shown that serotonin induces per1 gene expression at late subjective night but not at early night. Furthermore, serotonin application before glutamate or PACAP blocked glutamate-induced per1 expression at early night and per2 gene expression at late night. In contrast, serotonin did not influence PACAP-induced per gene expression at late night. Triple antigen immunohistochemistry and confocal microscopy supported both a pre- and post-synaptic interaction of retinohypothalamic tract (PACAP-immunoreactive) and serotonin projections on vasoactive intestinal peptide- and gastrin-releasing peptide-containing cell bodies in the ventro-lateral suprachiasmatic nucleus. Our findings suggest that the per genes could be the molecular target for the modulatory effects of serotonin on light signalling to the clock.
Asunto(s)
Ácido Glutámico/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Proteínas Nucleares/metabolismo , Serotonina/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Proteínas de Ciclo Celular , Oscuridad , Péptido Liberador de Gastrina/metabolismo , Regulación de la Expresión Génica , Ácido Glutámico/administración & dosificación , Inmunohistoquímica , Hibridación in Situ , Luz , Masculino , Microscopía Confocal , Neuropéptidos/administración & dosificación , Neurotransmisores/administración & dosificación , Proteínas Circadianas Period , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Factores de Transcripción , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.
Asunto(s)
Genes fos/genética , Genes fos/efectos de la radiación , Hipotálamo/fisiología , Neuropéptidos/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de la radiación , Animales , Enucleación del Ojo , Técnica del Anticuerpo Fluorescente , Herpesvirus Suido 1 , Inmunohistoquímica , Luz , Masculino , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Glutamato de Sodio/farmacología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efectos de la radiación , Vías Visuales/citología , Vías Visuales/metabolismo , Vías Visuales/efectos de la radiaciónRESUMEN
UNLABELLED: The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. CONCLUSION: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.
Asunto(s)
Motilidad Gastrointestinal , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Fragmentos de Péptidos/farmacología , Antro Pilórico/inervación , Animales , Técnicas de Cultivo , Estimulación Eléctrica , Péptido Liberador de Gastrina/metabolismo , Gastrinas/metabolismo , Motilidad Gastrointestinal/efectos de los fármacos , Inmunohistoquímica , Plexo Mientérico/metabolismo , Neuropéptidos/inmunología , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Antro Pilórico/metabolismo , Antro Pilórico/fisiología , ARN Mensajero/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/antagonistas & inhibidores , Receptores de la Hormona Hipofisaria/biosíntesis , Receptores de la Hormona Hipofisaria/genética , Somatostatina/metabolismo , Sustancia P/metabolismo , Porcinos , Nervio Vago/fisiología , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1(-)/-) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1(-)/- mice. However, in contrast to the phase advance seen in wild-type mice, PAC1(-)/- mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.
Asunto(s)
Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Receptores de la Hormona Hipofisaria/deficiencia , Ciclos de Actividad/fisiología , Ciclos de Actividad/efectos de la radiación , Animales , Proteínas de Ciclo Celular , Ritmo Circadiano/efectos de la radiación , Cruzamientos Genéticos , Oscuridad , Regulación de la Expresión Génica/efectos de la radiación , Inmunohistoquímica , Luz , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Actividad Motora/genética , Actividad Motora/efectos de la radiación , Neuropéptidos/metabolismo , Proteínas Nucleares/genética , Proteínas Circadianas Period , Estimulación Luminosa , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/genética , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiación , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Factores de TranscripciónRESUMEN
The suprachiasmatic nucleus generates circadian rhythms which are synchronized to the environmental light-dark cycle via the retinohypothalamic tract. Pituitary adenylate cyclase-activating polypeptide and glutamate, two neurotransmitters co-stored in the retinohypothalamic tract of the rat, are able to phase shift the endogenous rhythm similar to light. The "clock genes" period1 (per1) and per2, which show circadian oscillation within the suprachiasmatic nucleus, have been attributed a role in light-induced resetting of the mammalian circadian clock due to rapid induction of the period (per) genes after light stimulation at night. Using a rat in vitro brain slice model, we demonstrate by quantitative in situ hybridization histochemistry that the diurnal alteration in expression of both per genes in the suprachiasmatic nucleus was retained in vitro. In the model, we examined the effects of pituitary adenylate cyclase-activating polypeptide and glutamate alone and in combination on per1 and per2 gene expression at late subjective night (circadian time 19). Glutamate administration (10(-3)M) induced both per1 and per2 gene expression in the suprachiasmatic nucleus of the brain slice within 1h. The per gene responses were similar to the induction of gene expression observed after light stimulation in vivo at late night. Pituitary adenylate cyclase-activating polypeptide (10(-6)M) administered alone had no effect on the per gene expression, but when pituitary adenylate cyclase-activating polypeptide in micromolar concentration was applied before glutamate, the neuropeptide blocked the glutamate-induced per1 and per2 gene expression in the suprachiasmatic nucleus. In contrast to the lack of effect of pituitary adenylate cyclase-activating polypeptide itself in micromolar concentration, pituitary adenylate cyclase-activating polypeptide (10(-9)M) induced both per1 and per2 gene expression, an effect which was not augmented by co-application of glutamate. Our results provide the molecular substrate for the previous electrophysiological findings that pituitary adenylate cyclase-activating polypeptide in high concentration is able to block glutamate-induced phase advance at late night, and that the peptide in low concentration can induce a phase advance similar to light and glutamate.
Asunto(s)
Ritmo Circadiano/fisiología , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Proteínas Nucleares/genética , Núcleo Supraquiasmático/fisiología , Animales , Proteínas de Ciclo Celular , Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Técnicas In Vitro , Masculino , Proteínas Circadianas Period , Estimulación Luminosa , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/análisis , Ratas , Ratas Wistar , Núcleo Supraquiasmático/efectos de los fármacos , Factores de Transcripción , Vías Visuales/efectos de los fármacos , Vías Visuales/fisiologíaRESUMEN
The concentration of pituitary adenylyl cyclase-activating polypeptide [PACAP-(1-38)] in porcine adrenal glands amounted to 14 +/- 3 pmol/g tissue. PACAP immunoreactive (PACAP-IR) fibers innervated adrenal chromaffin cells (often co-localized with choline acetyltransferase). Subcapsular fibers traversed the cortex-innervating endocrine cells and blood vessels [some co-storing mainly calcitonin gene-related peptide but also vasoactive intestinal polypeptide (VIP)]. PACAP-IR fibers were demonstrated in the splanchnic nerves, whereas IR adrenal nerve cell bodies were absent. In isolated, vascularly perfused adrenal gland, splanchnic nerve stimulation (16 Hz) and capsaicin (10(-5) M) increased PACAP-(1-38) release (1.6-fold and 6-fold respectively, P = 0.02). PACAP-(1-38) dose-dependently stimulated cortisol (2 x 10(-10) M; 24-fold increase, P = 0.02) and chromogranin A fragment (2 x 10(-9) M; 15-fold increase, P = 0.05) secretion. Both were strongly inhibited by the PAC(1)/VPAC(2) receptor antagonist PACAP-(6-38) (10(-7) M). PACAP-(6-38) also inhibited splanchnic nerve (10 Hz)-induced cortisol secretion but lacked any effect on splanchnic nerve-induced pancreastatin secretion. PACAP-(1-38) (2 x 10(-10) M) decreased vascular resistance from 5.5 +/- 0.6 to 4.6 +/- 0.4 mmHg. min. ml(-1). PACAP-(6-38) had no effect on this response. We conclude that PACAP-(1-38) may play a role in splanchnic nerve-induced adrenal secretion and in afferent reflex pathways.
Asunto(s)
Glándulas Suprarrenales/química , Glándulas Suprarrenales/inervación , Fibras Nerviosas/química , Neuropéptidos/análisis , Fragmentos de Péptidos/análisis , Animales , Capsaicina/farmacología , Cromatografía Líquida de Alta Presión , Cromogranina A , Relación Dosis-Respuesta a Droga , Epinefrina/metabolismo , Expresión Génica/fisiología , Hidrocortisona/metabolismo , Inmunohistoquímica , Hibridación in Situ , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Norepinefrina/metabolismo , Hormonas Pancreáticas/análisis , Hormonas Pancreáticas/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/análisis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/genética , Nervios Esplácnicos/química , Nervios Esplácnicos/citología , Nervios Esplácnicos/metabolismo , Porcinos , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The two structurally related peptides, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), are present in cerebral vascular nerve fibers. Biologic actions of VIP are exerted through two receptors, VPAC1 and VPAC2, having similar binding affinity for both VIP and PACAP. In the current study, the authors have developed a specific antibody against the rVPAC1 receptor to examine the localization of rVPAC1 immunoreactivity in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence confocal microscopy. Specificity of the antiserum was ensured by immunoblotting and immunocytochemistry of cells transfected with cDNA encoding the different PACAP-VIP receptor subtypes. The rVPAC1 receptor immunoreactivity was localized to the plasmalemma of circularly orientated smooth muscle cells on superficial cerebral arteries and arterioles taken from the basal surface of the brain. By double immunostaining VIP immunoreactive nerve fibers and, to a lesser extent, those containing PACAP were shown to have intimate contact with the receptor protein. Vasoactive intestinal polypeptide and PACAP containing cerebrovascular nerve fibers were found in separate nerve populations with different distribution pattern and density. In brain sections processes of cortical VIP-, but not PACAP-, containing neurons seemed to innervate the rVPAC1 receptor of pial arterioles on the brain surface. The current findings provide the neuroanatomical substrate for a role of VIP and maybe PACAP in the regulation of cerebral blood flow.
Asunto(s)
Circulación Cerebrovascular/fisiología , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Animales , Arterias/inervación , Arterias/metabolismo , Arteriolas/inervación , Arteriolas/metabolismo , Células CHO/metabolismo , Cricetinae , Immunoblotting , Inmunohistoquímica , Masculino , Fibras Nerviosas/metabolismo , Neuropéptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Distribución Tisular , Transfección , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
We examined the effects of kainic acid (KA)-induced seizure on the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in the paraventricular nucleus (PVN) of rats using in situ hybridization histochemistry. Subcutaneous administration of KA (12 mg/kg) in adult male Sprague-Dawley rats caused a progressive development of seizure behavior. An induction of the PACAP gene expression in the medial parvocellular part of the PVN (mpPVN) was observed 3, 6, 12, 24 and 48 h after subcutaneous administration of KA. From a nearly undetectable level, PACAP gene expression increased in the mpPVN and reached maximum 12 h after subcutaneous administration of KA. PACAP gene expression returned to near basal level 48 h after stimulation with KA. Using a specific monoclonal PACAP antibody, PACAP immunoreactivity (-IR) gradually increased during the following 24 h after KA administration. In controls, PACAP-IR was located exclusively in nerve fibers of the mpPVN, whereas KA administration induced PACAP-IR in cell bodies of the mpPVN, and a dense accumulation of PACAP-IR nerve fibers in the external zone of the median eminence was observed. Induction of the PACAP gene expression following KA-induced seizure was significantly reduced by pretreatment with diazepam or MK-801 (nonselective N-methly-D-aspartate receptor antagonist). These results suggest that PACAP in the hypothalamo-adenohypophysial system may have a hypophysiotropic role during KA-induced seizure.
Asunto(s)
Agonistas de Aminoácidos Excitadores , Regulación de la Expresión Génica , Ácido Kaínico , Neuropéptidos/genética , Núcleo Hipotalámico Paraventricular/metabolismo , ARN Mensajero/metabolismo , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Animales , Anticonvulsivantes/farmacología , Diazepam/farmacología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Masculino , Núcleo Hipotalámico Paraventricular/fisiopatología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Sprague-Dawley , Convulsiones/genéticaRESUMEN
We have characterised the innervation pattern and intracellular Ca2+-signalling in labial salivary glands (LSG) of 16 patients with primary Sjögren's syndrome (pSS) and 27 healthy controls. Numerous immunoreactive nerve fibers (IRF) containing vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were found around acini, ducts and blood vessels. Substance P (SP)-, neuropeptide Y-, tyrosine hydroxylase- and nitric oxide synthase-IRF were mainly surrounding ducts and blood vessels. The majority of pSS patients had inflamed LSG and the presence of focal lymphocytic infiltrates (FI) were more frequent and pronounced as compared with healthy controls. In areas with normal or diffusely inflamed LSG tissue, pSS patients demonstrated the same distribution of IRF as healthy controls with similar histology. However, IRF were absent in central areas of FI both in pSS and age-matched healthy controls. Although all pSS patients had hyposalivation, stimulation with acetylcholine, norepinephrine, phenylephrine, isoproterenol, VIP, PACAP, SP, adenosine 5'-triphosphate and uridine 5'-triphosphate induced the same increase in the intracellular free Ca2+ concentration in LSG acini from both pSS patients and healthy controls, indicating the presence of functional receptor systems in vitro.
Asunto(s)
Señalización del Calcio/fisiología , Glándulas Salivales Menores/inervación , Glándulas Salivales Menores/metabolismo , Síndrome de Sjögren/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Calcio/agonistas , Calcio/análisis , Estudios de Casos y Controles , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Labio , Masculino , Persona de Mediana Edad , Neurotransmisores/agonistas , Neurotransmisores/análisis , Análisis de RegresiónRESUMEN
The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway plays a role in adjusting circadian timing to the light/dark environment. Two transmitters, glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) having phase shifting capacity during the night and day, respectively, are located in the RHT. Using double staining immunohistochemistry at the light and electron microscopic level, we showed that PACAP was co-stored with glutamate in a subset of retinal ganglion cells and in nerve terminals in the retino-recipient area of the SCN. These findings provide an anatomical basis for the recent demonstration of the interaction between these two transmitters on the SCN phase response at night.
Asunto(s)
Ácido Glutámico/metabolismo , Hipotálamo/metabolismo , Neuropéptidos/metabolismo , Retina/metabolismo , Animales , Hipotálamo/citología , Hipotálamo/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Electrónica , Neuronas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Wistar , Retina/citología , Retina/ultraestructuraRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.