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1.
Neuroscience ; 310: 541-8, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26415767

RESUMO

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.


Assuntos
Gânglios Espinais/metabolismo , Peptídeos/metabolismo , Prurido/induzido quimicamente , Medula Espinal/metabolismo , Aminoácidos/química , Animais , Comportamento Animal/efeitos dos fármacos , Medula Cervical/metabolismo , Masculino , Camundongos , Pescoço/inervação , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/fisiologia , Pele/metabolismo
2.
Neuroscience ; 250: 622-31, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23912037

RESUMO

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.


Assuntos
Gânglios Sensitivos/fisiologia , Hormônios Hipotalâmicos/fisiologia , Hormônios Peptídicos/fisiologia , Ácido Acético , Animais , Interpretação Estatística de Dados , Gânglios Sensitivos/metabolismo , Hormônios Hipotalâmicos/metabolismo , Hormônios Hipotalâmicos/farmacologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Bulbo/efeitos dos fármacos , Bulbo/metabolismo , Medição da Dor/efeitos dos fármacos , Hormônios Peptídicos/metabolismo , Hormônios Peptídicos/farmacologia , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Medula Espinal/metabolismo , Medula Espinal/fisiologia
3.
Neuroscience ; 240: 155-62, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23470775

RESUMO

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.


Assuntos
Fibronectinas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/citologia , Contagem de Células , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Vias Neurais/fisiologia , Ratos , Ratos Sprague-Dawley , Estilbamidinas/metabolismo
4.
J Neuroendocrinol ; 25(2): 206-15, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22963497

RESUMO

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.


Assuntos
Hormônios Hipotalâmicos/metabolismo , Hormônios Peptídicos/metabolismo , Hormônios Hipofisários/isolamento & purificação , Reprodução/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Relação Dose-Resposta a Droga , Feminino , Fármacos para a Fertilidade/química , Fármacos para a Fertilidade/isolamento & purificação , Fármacos para a Fertilidade/metabolismo , Fármacos para a Fertilidade/farmacologia , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/isolamento & purificação , Hormônios Hipotalâmicos/farmacologia , Hormônio Luteinizante/sangue , Masculino , Dados de Sequência Molecular , Hormônios Peptídicos/genética , Hormônios Peptídicos/isolamento & purificação , Hormônios Peptídicos/farmacologia , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Hormônios Hipofisários/farmacologia , Ratos , Ratos Sprague-Dawley , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Homologia de Sequência de Aminoácidos
5.
Neuroscience ; 166(2): 455-63, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20056135

RESUMO

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.


Assuntos
Cálcio/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Hormônios Peptídicos/metabolismo , Somatostatina/metabolismo , Análise de Variância , Animais , Células Cultivadas , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Hormônios Peptídicos/farmacologia , Ratos , Ratos Sprague-Dawley , Somatostatina/farmacologia
6.
Neuroscience ; 159(2): 492-500, 2009 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-19373968

RESUMO

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.


Assuntos
Encéfalo/metabolismo , Peptídeo C/metabolismo , Animais , Encéfalo/anatomia & histologia , Peptídeo C/farmacologia , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Fura-2/metabolismo , Hipotálamo/citologia , Isótopos de Iodo/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Tiobarbitúricos/metabolismo
7.
J Neuroendocrinol ; 20(11): 1242-51, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18752653

RESUMO

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.


Assuntos
Cálcio/metabolismo , Glicopeptídeos/metabolismo , Hipotálamo , Neurônios/metabolismo , Animais , Pressão Sanguínea/fisiologia , Células Cultivadas , Frequência Cardíaca/fisiologia , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neurônios/citologia , Ocitocina/metabolismo , Precursores de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Vasopressinas/metabolismo
8.
Neuroscience ; 140(4): 1265-76, 2006 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-16650615

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Química Encefálica/fisiologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Animais , Sistema Nervoso Central/química , Sistema Nervoso Central/ultraestrutura , Masculino , Proteínas Associadas aos Microtúbulos/ultraestrutura , Ratos , Ratos Sprague-Dawley
9.
J Neurosci Res ; 83(6): 1106-17, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16511859

RESUMO

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.


Assuntos
Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Feminino , Hipotálamo/ultraestrutura , Imuno-Histoquímica/métodos , Masculino , Microscopia Imunoeletrônica/métodos , Ocitocina/metabolismo , Ratos , Ratos Sprague-Dawley , Ubiquitinas , Vasopressinas/metabolismo
10.
Brain Res Dev Brain Res ; 131(1-2): 93-102, 2001 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-11718840

RESUMO

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.


Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Neurônios Motores/química , Proteínas do Tecido Nervoso/análise , Estilbamidinas , Nervo Vago/citologia , Nervo Vago/crescimento & desenvolvimento , Fatores Etários , Animais , Especificidade de Anticorpos , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/imunologia , Comportamento Alimentar/fisiologia , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Masculino , Bulbo/citologia , Bulbo/crescimento & desenvolvimento , Neurônios Motores/enzimologia , Proteínas do Tecido Nervoso/imunologia , Ratos
11.
Neurosci Lett ; 305(1): 9-12, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11356295

RESUMO

The distribution of urotensin-II-immunoreactivity (irU-II) was studied in the rat brainstem and spinal cord with the use of an antiserum against the human urotensin II (U-II) peptide. A population of ventral horn neurons in the spinal cord, hypoglossal nucleus, dorsal motor nucleus of the vagus, facial motor nucleus, nucleus ambiguus, abducens nucleus and trigeminal motor nucleus exhibited irU-II of varying intensities. The number of irU-II motor neurons was higher in the lumbar segments as compared to that of cervical, thoracic and sacral segments. Double-labeling the sections with U-II- and choline acetyltransferase (ChAT)-antisera revealed that nearly all irU-II ventral horn and brainstem neurons were ChAT-positive. The result provides the first immunohistochemical evidence of the presence of irU-II in cholinergic motoneurons of the rat spinal cord and brainstem.


Assuntos
Tronco Encefálico/metabolismo , Medula Espinal/metabolismo , Urotensinas/metabolismo , Animais , Tronco Encefálico/citologia , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Região Lombossacral , Masculino , Neurônios Motores/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Distribuição Tecidual
12.
Neurosci Lett ; 294(3): 143-6, 2000 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-11072135

RESUMO

The distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was investigated in the rat spinal cords with the use of an antiserum against the CART peptide fragment 55-102. CART-LI fibers were concentrated in the superficial layers of the dorsal horn of all segments. In addition to CART-LI fibers, intensely labeled somata were detected in the intermediolateral cell column (IML) and other sympathetic preganglionic nuclei of the thoracolumbar segments. In the lumbosacral segments, CART-LI fibers but not somata were seen in the sacral parasympathetic nucleus. Double-labeling the spinal sections with choline acetyltransferase (ChAT)-antisera and CART-antisera revealed that the large majority of ChAT-positive somata in the sympathetic preganglionic nuclei were CART-positive, whereas ChAT-positive somata in the parasympathetic preganglionic nuclei were CART-negative. Our results show that CART-LI is selectively expressed in a population of sympathetic preganglionic neurons (SPNs), but not in parasympathetic preganglionic neurons (PPNs) of the rat.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Medula Espinal/metabolismo , Animais , Feminino , Gânglios Parassimpáticos/metabolismo , Gânglios Simpáticos/metabolismo , Região Lombossacral , Masculino , Ratos , Ratos Sprague-Dawley
13.
Biol Reprod ; 63(5): 1518-24, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11058560

RESUMO

Cocaine- and amphetamine-regulated transcript (CART) is a novel family of peptides, of which CART peptide fragments 55-102 and 62-102 are reported to be the endogenous, physiologically active peptides. Immunohistochemical studies with an antiserum directed against the CART peptide fragment 55-102 revealed CART-like immunoreactive (CART-LI) nerve fibers in the rat epididymis. The number was highest in the cauda epididymis and became progressively fewer toward the caput epididymis; the vas deferens exhibited an abundance of CART-LI fibers. Injection of the retrograde tracer Fluorogold (Fluorochrome, Inc., Englewood, CO) to the junction between the vas deferens and cauda epididymis labeled a large number of neurons in the major pelvic ganglion, some of which were CART-positive. Double-labeling the ganglion sections with tyrosine hydroxylase (TH) and CART antisera revealed that CART-LI and TH-LI were expressed in two distinct populations of ganglion cells. Some of the TH-LI cells in the ganglia, however, were covered with web-like CART-LI endings. The effects of CART peptide 55-102, referred to herein as CART, on anion secretion in the form of short circuit currents (Isc) were assessed in cultured epithelia. The CART (1 to 5 microM) applied to the basolateral or apical side of the cultured epithelia caused no significant responses on Isc, whereas lys-bradykinin (1 microM) produced a large Isc response in the same preparations. Our results show that CART-LI is present in a population of rat pelvic ganglion cells, which may give rise to CART-LI nerve fibers as observed in the vas deferens and the epididymis. The biological function of CART in the rat epididymis is not known, but it apparently is not involved in ion secretion across the epithelium.


Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Epididimo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Estilbamidinas , Animais , Células Cultivadas , Eletrofisiologia , Epididimo/efeitos dos fármacos , Epididimo/fisiologia , Corantes Fluorescentes , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Masculino , Proteínas do Tecido Nervoso/fisiologia , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo
14.
Eur J Pharmacol ; 399(2-3): 165-9, 2000 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-10884516

RESUMO

Immunohistochemical studies with the use of an antiserum against the cocaine- and amphetamine-regulated transcript (CART) peptide-(55-102) showed an abundance of CART-immunoreactive fibers in the mouse dorsal horn laminae I and II. A few CART-positive somata were scattered in the dorsal horn and around the central canal. Intrathecal injection of the CART peptide-(55-102) at doses 3, 10 and 100 ng caused a dose-dependent and significant decrease of paw withdrawal latency; whereas, saline injection was without significant effect. Our results provide the first evidence that CART-immunoreactive fibers are present in the dorsal horn and that the peptide administered intrathecally produces hyperalgesia, as assessed by paw withdrawal latency in mice.


Assuntos
Proteínas do Tecido Nervoso/farmacologia , Dor/prevenção & controle , Animais , Relação Dose-Resposta a Droga , Membro Posterior , Imuno-Histoquímica , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Dor/fisiopatologia , Medição da Dor , Fragmentos de Peptídeos/farmacologia , Tempo de Reação/efeitos dos fármacos , Medula Espinal/química
15.
J Biomed Sci ; 7(3): 213-20, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10810239

RESUMO

Endomorphin (Endo) 1 and 2, two tetrapeptides isolated from the bovine and human brain, have been proposed to be the endogenous ligand for the mu-opiate receptor. A multi-disciplinary study was undertaken to address the issues of localization, release and biological action of Endo with respect to the rat dorsal horn. First, immunohistochemical studies showed that Endo-1- or Endo-2-like immunoreactivity (Endo-1- or Endo-2-LI) is selectively expressed in fiber-like elements occupying the superficial layers of the rat dorsal horn, which also exhibit a high level of mu-opiate receptor immunoreactivity. Second, release of immunoreactive Endo-2-like substances (irEndo) from the in vitro rat spinal cords upon electrical stimulation of dorsal root afferent fibers was detected by the immobilized antibody microprobe technique. The site of release corresponded to laminae I and II where the highest density of Endo-2-LI fibers was localized. Lastly, whole-cell patch clamp recordings from substantia gelatinosa (SG) neurons of rat lumbar spinal cord slices revealed two distinct actions of exogenous Endo-1 and Endo-2: (1) depression of excitatory and/or inhibitory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (2) hyperpolarization of SG neurons. These two effects were prevented by the selective mu-opiate receptor antagonist beta-funaltrexamine. The localization of endomorphin-positive fibers in superficial layers of the dorsal horn and the release of irEndo upon stimulation of dorsal root afferents together with the observation that Endo inhibits the activity of SG neurons by interacting with mu-opiate receptors provide additional support of a role of Endo as the endogenous ligand for the mu-opiate receptor in the rat dorsal horn.


Assuntos
Oligopeptídeos/metabolismo , Vias Aferentes/fisiologia , Analgésicos Opioides/imunologia , Analgésicos Opioides/metabolismo , Animais , Anticorpos , Estimulação Elétrica , Feminino , Imuno-Histoquímica , Ligantes , Masculino , Potenciais da Membrana/efeitos dos fármacos , Oligopeptídeos/imunologia , Células do Corno Posterior/química , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/imunologia , Medula Espinal/química , Raízes Nervosas Espinhais/fisiologia , Substância Gelatinosa/efeitos dos fármacos , Distribuição Tecidual
16.
Neurosci Lett ; 283(2): 97-100, 2000 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-10739884

RESUMO

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was studied in the rat spinal cord, sympathetic ganglia and adrenal glands by immunohistochemical methods, utilizing a polyclonal antiserum raised against the CART peptide fragment 55-102. CART-LI was detected in nerve fibers and in basket-like terminals surrounding many postganglionic neurons of the superior cervical ganglion (SCG), stellate, paravertebral and prevertebral ganglia. Postganglionic neurons exhibited low or non-detectable levels of CART-LI. Surgical sectioning of the cervical sympathetic trunk for 6-7 days resulted in a nearly complete loss of CART-LI fibers and terminals in the SCG. In the adrenal gland, CART-LI nerve fibers formed a plexus underneath the capsule, some of which bifurcated and made a sharp turn toward the adrenal medulla, where clusters of chromaffin cells were intensely labeled. The detection of CART-LI in sympathetic ganglia and adrenal glands extends the previous observation of the presence of CART-LI in sympathetic preganglionic neurons and further supports the notion that CART peptide(s) may function as a signaling molecule in the sympatho-adrenal axis.


Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Gânglios Simpáticos/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Gânglios Simpáticos/citologia , Gânglios Simpáticos/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Fibras Nervosas/ultraestrutura , Proteínas do Tecido Nervoso/análise , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Gânglio Estrelado/citologia , Gânglio Estrelado/efeitos dos fármacos , Gânglio Estrelado/metabolismo , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/metabolismo
17.
Neurosci Lett ; 273(1): 25-8, 1999 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-10505643

RESUMO

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.


Assuntos
Oligopeptídeos/metabolismo , Receptores Opioides mu/metabolismo , Medula Espinal/metabolismo , Animais , Reações Cruzadas , Estimulação Elétrica , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Masculino , Fibras Nervosas/fisiologia , Células do Corno Posterior/metabolismo , Células do Corno Posterior/fisiologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia
18.
J Physiol ; 519 Pt 3: 657-67, 1999 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10457081

RESUMO

1. The short-circuit current (Isc) technique was used to study the role of 5-hydroxytryptamine (5-HT) in the regulation of anion secretion in cultured rat cauda epididymal epithelia. 2. 5-HT, the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) and the 5-HT2B-selective agonist alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) added basolaterally stimulated Isc in a dose-dependent manner with EC50 values of 0.4, 20 and 0.3 microM, respectively. No other agonists for 5-HT receptors had any effect. 3. The pattern of responses to 5-HT was biphasic. Pretreating the tissues with the 5-HT1B-selective antagonist isamoltane (200 microM) and the 5-HT2B-selective antagonist rauwolscine (200 microM) inhibited the rapid transient phase by 55 and 45 %, whereas the sustained phase could only be blocked by rauwolscine. 4. Removal of chloride or bicarbonate or both from the normal Krebs-Henseleit solution reduced the responses to 5-HT, 5-NOT and alpha-methyl-5-HT to varying degrees. The results suggest that 5-HT1B- and 5-HT2B-mediated responses were mainly due to chloride and bicarbonate secretion, respectively. 5. Manipulation of the cAMP and Ca2+ signal transduction pathways with chemical agents provided evidence that the responses to 5-HT were mediated through cAMP. 6. Piroxicam pretreatment abolished the Isc response to alpha-methyl-5-HT but not to 5-NOT, indicating that the 5-HT2B-mediated response, but not the 5-HT1B-mediated response, is dependent on prostaglandin synthesis. 7. Immunohistochemical studies showed that 5-HT-like immunoreactivity was detected in nerve fibres and in small granular cells surrounding the epididymal tubules. 8. It is suggested that the 5-HT released from serotonergic nerve endings and/or from mast cells regulates electrolyte and fluid secretion in the epididymis.


Assuntos
Ânions/metabolismo , Epididimo/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Sulfonamidas , Animais , Quelantes/farmacologia , Colforsina/farmacologia , Técnicas de Cultura , AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Epitélio/metabolismo , Iminas/farmacologia , Isoquinolinas/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1B de Serotonina , Receptor 5-HT2B de Serotonina , Receptores de Serotonina/efeitos dos fármacos , Tapsigargina/farmacologia
19.
Neurosci Lett ; 260(3): 161-4, 1999 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-10076892

RESUMO

Distribution of orexin-A-like immunoreactivity (ORX-LI) in rat brains was investigated with the use of a rabbit polyclonal antibody against the full length peptide orexin A. Virtually all the ORX-LI cell bodies were observed in the lateral hypothalamus at the level of median eminence. The large majority of ORX-LI neurons appeared spherical or fusiform, 20-30 microm in diameter and issued two to five cell processes with few secondary branchings. Numerous ORX-LI fibers were observed in subregions of the hypothalamus. ORX-LI cell processes were sparsely distributed in the cortex, hippocampus and thalamus. Many varicose ORX-LI cell processes were situated close to the 3rd and lateral ventricles, some of which appeared to be protruding into the lumen. As a corollary, orexin A may be released into the ventricles and interact with neurons in distant targets, in addition to influencing the activity of neurons with which ORX-LI axons make synaptic contacts.


Assuntos
Química Encefálica/fisiologia , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Neuropeptídeos/metabolismo , Animais , Axônios/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/citologia , Tamanho Celular , Feminino , Imuno-Histoquímica , Masculino , Fibras Nervosas/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Orexinas , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo
20.
Neuroscience ; 89(2): 317-21, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10077314

RESUMO

Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the mu-opiate receptor. Opioid compounds expressing mu-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunuoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective mu-opiate receptor antagonist beta-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.


Assuntos
Inibição Neural/fisiologia , Neurônios/fisiologia , Oligopeptídeos/metabolismo , Medula Espinal/metabolismo , Substância Gelatinosa/fisiologia , Animais , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Substância Gelatinosa/citologia , Substância Gelatinosa/efeitos dos fármacos
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