RESUMEN
Phoenixin (PNX) is a 14-amino acid amidated peptide (PNX-14) or an N-terminal extended 20-residue amidated peptide (PNX-20) recently identified in neural and non-neural tissue. Mass spectrometry analysis identified a major peak corresponding to PNX-14, with negligible PNX-20, in mouse spinal cord extracts. Using a previously characterized antiserum that recognized both PNX-14 and PNX-20, PNX-immunoreactivity (irPNX) was detected in a population of dorsal root ganglion (DRG) cells and in cell processes densely distributed to the superficial layers of the dorsal horn; irPNX cell processes were also detected in the skin. The retrograde tracer, Fluorogold, injected subcutaneously (s.c.) to the back of the cervical and thoracic spinal cord of mice, labeled a population of DRG, some of which were also irPNX. PNX-14 (2, 4 and 8 mg/kg) injected s.c.to the nape of the neck provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. The number of scratching bouts varied from 16 to 95 in 30 min, commencing within 5 min post-injection and lasted 10-15 min. Pretreatment of mice at -20 min with nalfurafine (20 µg/kg, s.c.), the kappa opioid receptor agonist, significantly reduced the number of bouts induced by PNX-14 (4 mg/kg) compared with that of saline-pretreated mice. Our results suggest that the peptide, PNX-14, serves as one of the endogenous signal molecules transducing itch sensation in the mouse.
Asunto(s)
Ganglios Espinales/metabolismo , Péptidos/metabolismo , Prurito/inducido químicamente , Médula Espinal/metabolismo , Aminoácidos/química , Animales , Conducta Animal/efectos de los fármacos , Médula Cervical/metabolismo , Masculino , Ratones , Cuello/inervación , Péptidos/química , Péptidos/farmacología , Péptidos/fisiología , Piel/metabolismoRESUMEN
Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 µg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 µg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 µg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.
Asunto(s)
Ganglios Sensoriales/fisiología , Hormonas Hipotalámicas/fisiología , Hormonas Peptídicas/fisiología , Ácido Acético , Animales , Interpretación Estadística de Datos , Ganglios Sensoriales/metabolismo , Hormonas Hipotalámicas/metabolismo , Hormonas Hipotalámicas/farmacología , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Bulbo Raquídeo/efectos de los fármacos , Bulbo Raquídeo/metabolismo , Dimensión del Dolor/efectos de los fármacos , Hormonas Peptídicas/metabolismo , Hormonas Peptídicas/farmacología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Médula Espinal/metabolismo , Médula Espinal/fisiologíaRESUMEN
Irisin is a recently identified myokine secreted from the muscle in response to exercise. In the rats and mice, immunohistochemical studies with an antiserum against irisin peptide fragment (42-112), revealed that irisin-immunoreactivity (irIRN) was detected in three types of cells; namely, skeletal muscle cells, cardiomyocytes, and Purkinje cells of the cerebellum. Tissue sections processed with irisin antiserum pre-absorbed with the irisin peptide (42-112) (1 µg/ml) showed no immunoreactivity. Cerebellar Purkinje cells were also immunolabeled with an antiserum against fibronectin type II domain containing 5 (FNDC5), the precursor protein of irisin. Double-labeling of cerebellar sections with irisin antiserum and glutamate decarboxylase (GAD) antibody showed that nearly all irIRN Purkinje cells were GAD-positive. Injection of the fluorescence tracer Fluorogold into the vestibular nucleus of the rat medulla retrogradely labeled a population of Purkinje cells, some of which were also irIRN. Our results provide the first evidence of expression of irIRN in the rodent skeletal and cardiac muscle, and in the brain where it is present in GAD-positive Purkinje cells of the cerebellum. Our findings together with reports by others led us to hypothesize a novel neural pathway, which originates from cerebellum Purkinje cells, via several intermediary synapses in the medulla and spinal cord, and regulates adipocyte metabolism.
Asunto(s)
Fibronectinas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/citología , Recuento de Células , Glutamato Descarboxilasa/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Miocardio/metabolismo , Miocardio/ultraestructura , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-Dawley , Estilbamidinas/metabolismoRESUMEN
Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin (SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatin-immunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive (irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the NST-antiserum with NST (1 microg/ml) but not with SST (1 microg/ml) abrogated irNST and vice versa. The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca(2+) imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in a population of hypothalamic neurons with two distinct profiles: (1) a fast and transitory increase in [Ca(2+)](i), and (2) an oscillatory response. Whereas, SST (100 nM) reduced the basal [Ca(2+)](i) in 21 of 61 hypothalamic neurons examined; an increase was not observed in any of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC(4)(3) showed that NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population of hypothalamic neurons. The result shows that NST and SST, though derived from the same precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons, resulting in diverse cellular activities.
Asunto(s)
Calcio/metabolismo , Hipotálamo/metabolismo , Neuronas/metabolismo , Hormonas Peptídicas/metabolismo , Somatostatina/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Inmunohistoquímica , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Hormonas Peptídicas/farmacología , Ratas , Ratas Sprague-Dawley , Somatostatina/farmacologíaRESUMEN
Amylin is a member of calcitonin or calcitonin gene-related peptide (CGRP) family. Immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion (DRG) and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the acetic acid-induced writhing test. Amylin (0.1, 0.5 and 1 mg/kg, intraperitoneally (i.p.) or 1-10 microg, intrathecally (i.t.)) reduced the number of writhes in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32), either by i.p. or i.t., antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 microg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were upregulated in the spinal cord of mice injected with acetic acid and the increase was attenuated by pretreatment with amylin (10 microg, i.t.). Collectively, our result demonstrates that irAMY is expressed in DRG neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive.
Asunto(s)
Amiloide/farmacología , Analgésicos/farmacología , Dolor/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-fos/metabolismo , Médula Espinal/efectos de los fármacos , Vísceras/efectos de los fármacos , Ácido Acético , Amiloide/administración & dosificación , Analgésicos/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Calcitonina/administración & dosificación , Calcitonina/farmacología , Relación Dosis-Respuesta a Droga , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos , Locomoción/efectos de los fármacos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Dolor/inducido químicamente , Dolor/metabolismo , ARN Mensajero/metabolismo , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Calcitonina/metabolismo , Receptores de Polipéptido Amiloide de Islotes Pancreáticos , Receptores de Péptidos/agonistas , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/metabolismo , Médula Espinal/metabolismoRESUMEN
The aims of the present study were to establish if nalfurafine, a kappa opioid agonist, inhibits compulsive scratching in mice elicited by the s.c. administration (behind the neck) of 5'-guanidinonaltrindole (GNTI), a kappa opioid antagonist; to assess if nalfurafine prevents c-fos expression provoked by GNTI or compound 48/80, two chemically diverse pruritogens; and to distinguish on the basis of neuroanatomy, those neurons in the brainstem activated by either GNTI-induced itch or formalin-induced pain (both compounds given s.c. to the right cheek). Pretreatment of mice with nalfurafine (0.001-0.03 mg/kg s.c.) attenuated GNTI (0.3 mg/kg)-evoked scratching dose-dependently. A standard antiscratch dose of nalfurafine (0.02 mg/kg) had no marked effect on the spontaneous locomotion of mice. Tolerance did not develop to the antiscratch activity of nalfurafine. Both GNTI and compound 48/80 provoked c-fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord and pretreating mice with nalfurafine inhibited c-fos expression induced by both pruritogens. In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. Our data indicate that the kappa opioid system is involved, at least in part, in the pathogenesis of itch; and that nalfurafine attenuates excessive scratching and prevents scratch-induced neuronal activity at the spinal level. On the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch.
Asunto(s)
Antipruriginosos/farmacología , Morfinanos/farmacología , Naltrexona/análogos & derivados , Prurito/tratamiento farmacológico , Receptores Opioides kappa/agonistas , Compuestos de Espiro/farmacología , p-Metoxi-N-metilfenetilamina/antagonistas & inhibidores , Vías Aferentes/efectos de los fármacos , Vías Aferentes/metabolismo , Vías Aferentes/fisiopatología , Analgésicos Opioides/farmacología , Analgésicos Opioides/uso terapéutico , Animales , Antipruriginosos/uso terapéutico , Biomarcadores/análisis , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Guanidinas , Masculino , Ratones , Morfinanos/uso terapéutico , Naltrexona/antagonistas & inhibidores , Antagonistas de Narcóticos/farmacología , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Dimensión del Dolor , Células del Asta Posterior/citología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Prurito/inducido químicamente , Prurito/fisiopatología , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Compuestos de Espiro/uso terapéutico , Resultado del TratamientoRESUMEN
With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.
Asunto(s)
Encéfalo/metabolismo , Péptido C/metabolismo , Animales , Encéfalo/anatomía & histología , Péptido C/farmacología , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electrofisiología/métodos , Fura-2/metabolismo , Hipotálamo/citología , Isótopos de Yodo/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Tiobarbitúricos/metabolismoRESUMEN
Copeptin is cleaved from the C-terminus of vasopressin (VP) prohormone. Immunohistochemical studies have revealed intense copeptin-immunoreactivity (irCOPT) in neurones of the rat hypothalamic nuclei, including paraventricular, supraoptic, suprachiasmatic, periventricular, and accessory secretory. Varicose cell processes emanated from irCOPT neurones, some of which projected caudally and traversed the internal layer of the median eminence, and terminated in the posterior pituitary. Double-labelling hypothalamic sections with copeptin antiserum and VP or oxytocin antiserum revealed an extensive overlapping of irCOPT and irVP neurones. The biological activity of human synthetic nonglycosylated copeptin or VP was evaluated in vivo and in vitro. Copeptin (1, 10, and 20 nmol/kg) injected i.v. caused no significant changes in the mean arterial pressure (MAP) and heart rate of urethane-anaesthetised rats. VP (0.1 nmol/kg) increased MAP, which was accompanied by a small decrease of the heart rate. The ratiometric fluorescence method was employed to assess changes in intracellular Ca2+ concentrations [Ca2+](i) which served as an index of the biological activity of peptides. VP (1 microM) markedly increased [Ca2+](i) of rat hypothalamic neurones or vascular smooth muscle cells, whereas copeptin (100 nm to 1 microM) caused a low amplitude, sustained increase of [Ca2+](i) in a population of hypothalamic neurones, but not in any of the vascular smooth muscle cells tested. The results obtained demonstrate that copeptin is expressed in VP neurones and that the peptide in the concentrations tested, although causing little or no detectable changes of blood pressure and heart rate in anaesthetised rats nor changes in [Ca2+](i) of cultured aortic smooth muscle cells, increases [Ca2+](i) in a small population (< 2%) of hypothalamic neurones tested, indicating that copeptin is biologically active in mammalian neurones.
Asunto(s)
Calcio/metabolismo , Glicopéptidos/metabolismo , Hipotálamo , Neuronas/metabolismo , Animales , Presión Sanguínea/fisiología , Células Cultivadas , Frecuencia Cardíaca/fisiología , Humanos , Hipotálamo/citología , Hipotálamo/metabolismo , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neuronas/citología , Oxitocina/metabolismo , Precursores de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Vasopresinas/metabolismoRESUMEN
Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.
Asunto(s)
Corteza Cerebral/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-Dawley , Tiobarbitúricos/metabolismoRESUMEN
Beacon-immunoreactive (B-ir) fibres and neurons in the hypothalamus of the domestic chick (Gallus domesticus) were studied using an immunohistochemical technique in order to verify the presence and elucidate the pattern of distribution of this novel peptide in an avian brain. B-ir neurons were seen in the n. supraopticus, pars ventralis and pars externus; n. magnocellularis preopticus, pars dorsalis, medialis and ventralis; n. preopticus periventricularis; n. suprachiasmaticus, pars medialis; n. ventrolateralis thalami. Only few B-ir cells were scattered in the most anterior part of the lateral hypothalamic area. B-ir fibres, appearing as thin punctuate structures, were seen mainly along the walls of the third ventricle and in the ventromedial hypothalamus. Labelled fibres and terminals were located in the external and internal zones of the anterior and posterior median eminence. In particular, fibre terminals were seen close to the capillary loops of the hypothalamo-hypophysial portal system. The anatomical data of the present study regarding the distribution of B-ir in the chick hypothalamus suggest that beacon may play a key role in the regulation of the neuroendocrine system by acting as a neuromodulator and/or neurotransmitter.
Asunto(s)
Pollos/anatomía & histología , Hipotálamo/anatomía & histología , Hipotálamo/química , Neuronas/inmunología , Animales , Química Encefálica , Femenino , Inmunohistoquímica/veterinaria , Masculino , Neuronas/químicaRESUMEN
We have previously demonstrated that GEC1 interacts with the kappa opioid receptor and GEC1 expression enhances cell surface expression of the receptor in Chinese hamster ovary cells. In this study, we generated an antiserum (PA629) directed against GEC1 in rabbits, characterized its specificity, and investigated distribution of GEC1 in tissues and in brain regions and spinal cord and its subcellular localization in hypothalamic neurons in the rat. Immunofluorescence staining demonstrated that PA629 recognized HA-GEC1 transfected into Chinese hamster ovary cells, but not HA-GABARAP or HA-GATE-16, although the three share high homology. Pre-incubation of PA629 with GST-GEC1, but not GST, abolished the staining. In immunoblotting, affinity-purified PA629 (PA629p) recognized GEC1, GABARAP and GATE-16. GEC1 migrated slower than GABARAP and GATE-16, with a M(r) of 16 kDa for GEC1 and M(r) of 14 kDa for GABARAP and GATE-16. Immunoblotting results showed that GEC1 level was higher in liver and brain than in lung and heart, and very low in kidney and skeletal muscle. GEC1 was present in all rat brain regions examined and spinal cord. Immunohistochemistry demonstrated that GEC1 immunoreactivity was distributed ubiquitously in the rat CNS with highly intense immunoreactivity in various brain nuclei and motor neurons of the spinal cord. Ultrastructural examination of neurons in the paraventricular nucleus of the hypothalamus showed that GEC1 was associated with endoplasmic reticulum and Golgi apparatus and distributed along plasma membranes and in cytosol. Coupled with our previous observation that GEC1 interacts with N-ethylmaleimide-sensitive factor, these findings strongly suggest that GEC1 functions in intracellular trafficking in the biosynthesis pathway and perhaps also the endocytic pathway. The widespread distribution of GEC1 suggests that GEC1 may be associated with many proteins, in addition to the kappa opioid receptor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Química Encefálica/fisiología , Encéfalo/metabolismo , Encéfalo/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/ultraestructura , Animales , Sistema Nervioso Central/química , Sistema Nervioso Central/ultraestructura , Masculino , Proteínas Asociadas a Microtúbulos/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.
Asunto(s)
Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Femenino , Hipotálamo/ultraestructura , Inmunohistoquímica/métodos , Masculino , Microscopía Inmunoelectrónica/métodos , Oxitocina/metabolismo , Ratas , Ratas Sprague-Dawley , Ubiquitinas , Vasopresinas/metabolismoRESUMEN
The correlation between nutrition and cardiovascular related disorders is a well-established fact. Previous work from our Laboratory has suggested a significant compromise of cardiovascular reflexes in conscious rats submitted to a low-protein (LP) diet. Our working hypothesis is that the basal level of mean arterial pressure (MAP), variability of the mean arterial pressure (VMAP), heart rate (HR) and variability of heart rate (VHR) are altered in rats submitted to a protein restricted diet. Two experimental groups were used: control group (normal protein 15%, NP) and malnourished group (low-protein 6%, LP). In order to verify the efficiency of the dietary restriction we measured body weight, total blood protein, plasma albumin, urea and glucose. Our experiments demonstrated that the malnourished rats presented augment levels of basal MAP (LP 122+/-2 mmHg vs. NP 113+/-1 mmHg) and of VMAP (LP 12.8+/-1.5mmHg vs. NP 9+/-1mmHg) when compared to the control group. We observed similar increased levels, in the malnourished group, for both HR (LP 429+/-8 bpm vs. NP 381+/-7bpm) and VHR (LP 67.6+/-8.3bpm vs. NP 44.4+/-4.9bpm). Our results suggest a correlation between the LP diet in Fisher rats and the increased basal levels of mean arterial pressure, HR and their respective variability.
Asunto(s)
Presión Sanguínea/fisiología , Dieta con Restricción de Proteínas/efectos adversos , Frecuencia Cardíaca/fisiología , Animales , Peso Corporal , Encéfalo/anatomía & histología , Masculino , Tamaño de los Órganos , Deficiencia de Proteína/fisiopatología , RatasRESUMEN
1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca(2+) three-fold had no effect on the MEPP frequency increase to 25 microM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca(2+) caused a 61% reduction in the MEPP frequency increase to 25 microM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 microM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP(3)) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 microM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP(3) and cyclic AMP/protein kinase A.
Asunto(s)
Neuronas Motoras/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Urotensinas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Rana pipiens , Urotensinas/fisiologíaRESUMEN
Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH(2), its C-terminal fragment (55-108)-NH(2), which is termed pCF-6; the rat CF-6, (33-108)-NH(2), its C-terminal fragment pCF-6, (55-108)-NH(2); and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2). Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2) caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.
Asunto(s)
ATPasas de Translocación de Protón Mitocondriales/síntesis química , Factores de Acoplamiento de la Fosforilación Oxidativa/síntesis química , Fragmentos de Péptidos/síntesis química , Presorreceptores/efectos de los fármacos , Secuencia de Aminoácidos , Anestesia , Anestésicos Intravenosos/administración & dosificación , Animales , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Proteínas Mitocondriales/síntesis química , Proteínas Mitocondriales/farmacología , ATPasas de Translocación de Protón Mitocondriales/farmacología , Datos de Secuencia Molecular , Peso Molecular , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Fragmentos de Péptidos/farmacología , Prazosina/farmacología , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Uretano/administración & dosificaciónRESUMEN
1. Whole-cell patch recordings were made from antidromically identified sympathetic preganglionic neurons (SPN) of immature rat spinal cord slices. Bath application of nociceptin (0.1-1 micromol/L) suppressed excitatory postsynaptic potentials (EPSP) and hyperpolarized a population of SPN; these effects were naloxone (1 micromol/L) insensitive. 2. Nociceptin suppressed the amplitude of EPSP without causing a concomitant change in glutamate-induced depolarizations, suggesting a presynaptic inhibitory action. 3. Analysis of current-voltage relationships showed that nociceptin hyperpolarized SPN by increasing an inwardly rectifying K+ current. 4. Intrathecal injection of nociceptin (3, 10 and 30 nmol) to urethane-anaesthetized rats dose-dependently reduced the mean arterial pressure and heart rate; these effects were not prevented by prior intravenous injection of naloxone (1 mg/kg). 5. Results from our in vitro and in vivo experiments suggest that nociceptin suppresses spinal sympathetic outflow either by attenuating excitatory synaptic responses or hyperpolarizing SPN.
Asunto(s)
Fibras Adrenérgicas/efectos de los fármacos , Fibras Adrenérgicas/fisiología , Inhibición Neural/fisiología , Péptidos Opioides/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiología , Animales , Fibras Autónomas Preganglionares/efectos de los fármacos , Fibras Autónomas Preganglionares/fisiología , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Espinales , Masculino , Inhibición Neural/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vasodilatadores/farmacología , NociceptinaRESUMEN
1. Whole-cell patch-clamp recordings were made from neurones of the dorsal motor nucleus of the vagus (DMNV), including Fluoro-gold-labelled parasympathetic preganglionic neurones (PPNs), in slices of the rat medulla. In the latter case, rats had received an I.P. injection of Fluoro-gold solution (10 microg) 2-3 days earlier. 2. Superfusion of orexin A or B (10-300 nM) caused a slow depolarization in approximately 30% of the DMNV neurones, including PPNs. Orexin-induced depolarizations, which persisted in TTX (0.5 microM)-containing Krebs solution, were reduced by 70% in a low-Na+ (26 mM) Krebs solution, indicating the involvement of Na+ ions. A significant change in orexin-induced depolarizations was not obtained in either a high-K+ (7 mM) or Cd2+ (100 microM) Krebs solution. 3. Inclusion of the hydrolysis-resistant guanine nucleotide GDP-beta-S in the patch solution significantly reduced the orexin A- or B-induced depolarizations. 4. Under whole-cell voltage-clamp conditions, the orexin-induced inward current declined with hyperpolarization, but did not reverse polarity in the potential range between -120 and 0 mV. In low-Na+ solution, the orexin-induced current was reduced, and the I-V curve reversed polarity at about -105 mV; the response was further reduced and the reversal potential shifted to -90 mV in a low-Na+, high-K+ Krebs solution. 5. It is concluded that the peptides orexin A and B, acting on orexin receptors, which are GTP-binding-protein coupled, are excitatory to DMNV neurones. In addition, more than one conductance, which may include a non-selective cation conductance and a K+ conductance, appears to be involved in the orexin-induced depolarization.
Asunto(s)
Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/fisiología , Proteínas Portadoras/farmacología , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/farmacología , Nervio Vago/fisiología , Animales , Tronco Encefálico/citología , Electrofisiología , Proteínas de Unión al GTP/fisiología , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/efectos de los fármacos , Neuronas/efectos de los fármacos , Orexinas , Ratas , Ratas Sprague-DawleyRESUMEN
The two recently isolated hypothalamic peptides orexin A and orexin B, also known as hypocretin 1 and 2, are reported to be important signaling molecules in feeding and sleep/wakefulness. Orexin-containing neurons in the lateral hypothalamus project to numerous areas of the rat brain and spinal cord including the intermediolateral cell column (IML) of the thoracolumbar spinal cord. An in vivo and in vitro study was undertaken to evaluate the hypothesis that orexins, acting on sympathetic preganglionic neurons (SPNs) in the rat spinal cord, increase sympathetic outflow. First, orexin A (0.3, 1, and 10 nmol) by intrathecal injection increased mean arterial pressure (MAP) and heart rate (HR) by an average of 5, 18, and 30 mmHg and 10, 42, and 85 beats/min in urethane-anesthetized rats. Intrathecal injection of saline had no significant effects. Orexin B (3 nmol) by intrathecal administration increased MAP and HR by an average of 11 mmHg and 40 beats/min. The pressor effects of orexin A were attenuated by prior intrathecal injection of orexin A antibodies (1:500 dilution) but not by normal serum albumin. Intravenous administration of the alpha(1)-adrenergic receptor antagonist prazosin (0.5 mg/kg) or the beta-adrenergic receptor antagonist propranolol (0.5 mg/kg) markedly diminished, respectively, the orexin A-induced increase of MAP and HR. Second, whole cell patch recordings were made from antidromically identified SPNs of spinal cord slices from 12- to 16-day-old rats. Superfusion of orexin A or orexin B (100 or 300 nM) excited 12 of 17 SPNs, as evidenced by a membrane depolarization and/or increase of neuronal discharges. Orexin A- or B-induced depolarizations persisted in TTX (0.5 microM)-containing Krebs solution, indicating that the peptide acted directly on SPNs. Results from our in vivo and in vitro studies together with the previous observation of the presence of orexin A-immunoreactive fibers in the IML suggest that orexins, when released within the IML, augment sympathetic outflow by acting directly on SPNs.
Asunto(s)
Proteínas Portadoras/farmacología , Ganglios Simpáticos/fisiología , Péptidos y Proteínas de Señalización Intracelular , Neuronas/fisiología , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Orexinas , Ratas , Ratas Sprague-Dawley , Tetrodotoxina/farmacologíaRESUMEN
Cocaine- and amphetamine-regulated transcript (CART) peptide, a family of neuropeptides, is shown to inhibit food intake upon intracerebroventricular injection to the rat. CART peptide-immunoreactivity (irCART) was detected in neurons of the dorsal motor nucleus of the vagus (DMNV) of postnatal day one (P1) rats, the earliest day examined. The number of labeled DMNV neurons reached the peak between P5 and P8 rats and gradually declined thereafter. Few irCART neurons were noted in the DMNV between P22 and P90 rats. Double-labeling the medullary sections from P5 and P8 rats with CART-antiserum and choline acetyltransferase (ChAT)-antiserum revealed that irCART neurons in the DMNV were ChAT-immunoreactive (irChAT), but not all irChAT neurons were irCART. Intraperitoneal injection of the retrograde tracer Fluorogold to P3 and P5 rats labeled DMNV neurons, the majority of which were also irCART. The number of irCART neurons in other regions of the brain and spinal cord generally showed an increase in adult rats as compared to that of the same regions in immature rats. Our result suggests that expression of irCART in DMNV neurons undergoes developmental changes such that few neurons appear to contain irCART in mature rats. As a corollary, CART may be a signaling molecule to the gastrointestinal tract during the critical period of early development.
Asunto(s)
Anfetamina/farmacología , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Neuronas Motoras/química , Proteínas del Tejido Nervioso/análisis , Estilbamidinas , Nervio Vago/citología , Nervio Vago/crecimiento & desarrollo , Factores de Edad , Animales , Especificidad de Anticuerpos , Colina O-Acetiltransferasa/análisis , Colina O-Acetiltransferasa/inmunología , Conducta Alimentaria/fisiología , Femenino , Colorantes Fluorescentes , Inmunohistoquímica , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/crecimiento & desarrollo , Neuronas Motoras/enzimología , Proteínas del Tejido Nervioso/inmunología , RatasRESUMEN
Inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine diphosphate-ribose (cADPR) are second messengers that enhance neurosecretion by inducing Ca(2+) release from smooth endoplasmic reticulum (SER). The putative intracellular messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), releases Ca(2+) from stores that are distinct from SER. Evidence is presented here that NAADP causes a concentration-dependent increase in quantal output that is associated with an increase in probability of transmitter release at the frog neuromuscular junction. This effect is mimicked by A23187, a Ca ionophore that promotes Ca(2+) entry at the plasmalemma. The response to NAADP is potentiated by IP(3) but antagonized by cADPR. Thapsigargin completely blocks IP(3) and cADPR responses and decreases but does not prevent the response to NAADP. We conclude that NAADP, whose receptors are widely distributed in the brain, enhances neurosecretion by releasing Ca(2+) from an internal store near the plasmalemma, possibly from synaptic vesicles in the releasable pool. These data also support the hypothesis of a two-pool model for Ca(2+) oscillations at the presynaptic site.