RESUMEN
BACKGROUND: The alpha(2)-adrenoceptor agonist clonidine is used in combination with lidocaine for anaesthesia. Lidocaine inhibits axonal transport and neurite growth, whereas alpha(2)-adrenoceptor agonists have neurotrophic effects. Here we have investigated whether clonidine reduces lidocaine-induced inhibition of axonal transport in cultured mouse dorsal root ganglion neurones. METHODS: Axonal transport of organelles and neurite growth were assessed by video microscopy in cells treated with clonidine and lidocaine for 1 h. Stable responses were achieved within this period. RESULTS: Clonidine (10 and 100 microM) increased and lidocaine (10, 100 microM, and 1 mM) decreased axonal transport. The inhibitory effects of lidocaine were reduced by simultaneous treatment with clonidine. The actions of clonidine were antagonized by the alpha(2)-adrenoceptor antagonist yohimbine. Since clonidine was reported to block N-type channels, we further investigated the role of ion channels in the antagonistic action of clonidine on the lidocaine response. The action of lidocaine on axonal transport was not mimicked by the Na+ channel blocker tetrodotoxin and not blocked by the Na+ channel activator veratridine. The action of lidocaine was not blocked by the L-type Ca2+ channel blocker nifedipine, but was blocked by the N-type channel blocker omega-conotoxin MVIIA. These effects on axonal transport correlated with the effects on neurite growth. CONCLUSIONS: Inhibition of axonal transport induced by lidocaine, which may be mediated by N-type channel activation, can be blocked by clonidine. Clonidine may alleviate the effects of lidocaine on neuronal dysfunction.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Transporte Axonal/efectos de los fármacos , Clonidina/farmacología , Ganglios Espinales/efectos de los fármacos , Lidocaína/antagonistas & inhibidores , Anestésicos Locales/antagonistas & inhibidores , Anestésicos Locales/farmacología , Animales , Relación Dosis-Respuesta a Droga , Ganglios Espinales/fisiología , Lidocaína/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía por Video , Inhibición Neural/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Orgánulos/efectos de los fármacos , Orgánulos/fisiologíaRESUMEN
BACKGROUND/AIMS: Although chemical information on the dermis in vivo is highly important in skin research, an efficient method for gathering this information is yet to be developed. Here, we demonstrate that newly developed near-infrared (1064 nm) excited Raman spectroscopy is a powerful method for chemical analysis of human skin in vivo. METHODS: We used a laboratory-constructed Raman spectrometer equipped with a highly sensitive near-infrared detector (Hamamatsu Photonics), an optical fiber probe and a 1064 nm Nd:YAG laser. Raman spectra of porcine skin (in vitro) and human skin (in vivo) were measured with this spectrometer. RESULTS: The Raman spectrum of porcine skin measured from the outer side resembles that of the dermis more than that of the epidermis. The Raman spectra of human skin (cheek, forehead, inner forearm, outer forearm, palm) depend on the portion measured with the probe. The spectra of the forehead and inner forearm show larger lipid signals than that of the palm. CONCLUSIONS: The Raman spectrum of skin measured with the 1064 nm Raman system primarily reflects the chemical composition of the dermis. The 1064 nm excited Raman spectroscopy is useful for research of the dermis and skin appendages.
Asunto(s)
Dermis/química , Piel/química , Espectroscopía Infrarroja Corta/métodos , Espectrometría Raman/métodos , Adulto , Animales , Colágeno/química , Diseño de Equipo , Cara , Tecnología de Fibra Óptica , Humanos , Láseres de Estado Sólido , Masculino , Fibras Ópticas , Sebo/química , Espectroscopía Infrarroja Corta/instrumentación , Espectrometría Raman/instrumentación , Porcinos , Trioleína/química , Extremidad SuperiorRESUMEN
In primary sensory neurons, extracellular ATP plays important roles in nociception and afferent neurotransmission. Here we investigated the effects of ATP on axonal transport in cultured adult mouse dorsal root ganglion neurons using video-enhanced microscopy. Continuous application (26 min) of ATP (100 microM) significantly increased axonal transport of membrane-bound organelles in anterograde and retrograde directions. All neurons tested (n=5) responded to ATP. The number of transported organelles per min began to increase within 2 min and peaked at 11-14 min after the start of ATP application, and thereafter gradually declined. The peak values in both directions were approximately 140% of the initial values before application. The P2 receptor antagonist suramin (1 mM) completely blocked the effect of ATP. Uridine 5'-triphosphate (UTP; 100 microM) produced a similar effect to ATP, with peak values at 11 min reaching 140% in both directions (n=6). ADP (100 microM; n=5), alpha,beta-methylene ATP (100 microM; n=6), or 2-methylthio ATP (100 microM; n=5) had no effect on axonal transport. Our findings indicate that extracellular ATP is able to increase axonal transport in primary sensory neurons. The equal potency of ATP and UTP with no detectable response to ADP, alpha,beta-methylene ATP, or 2-methylthio ATP suggests the possible involvement of P2Y(2) receptors. Extracellular ATP may play an important role in the modulation of axonal transport in sensory neurons.
Asunto(s)
Adenosina Trifosfato/farmacología , Axones/efectos de los fármacos , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Orgánulos/efectos de los fármacos , Adenosina Difosfato/farmacología , Adenosina Trifosfato/agonistas , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Axones/fisiología , Células Cultivadas , Espacio Extracelular/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía por Video/métodos , Neuronas/fisiología , Orgánulos/fisiología , Factores de Tiempo , Uridina Trifosfato/farmacologíaRESUMEN
Histamine is important in mediating peripheral sensory information such as inflammation, allergic hypersensitivity, and itch. In the present study, using video-enhanced microscopy, we investigated the effect of histamine on axonal transport in cultured dorsal root ganglion (DRG) neurons of the mouse. Application of histamine (100 microM) reversibly reduced the number of particles transported within neurites in both anterograde and retrograde directions. The histamine H(1)-receptor agonist 2-thiazolylethylamine (100 microM) and the H(3)-receptor agonist R-alpha-methylhistamine (100 microM) also reduced anterograde and retrograde axonal transport, whereas the histamine H(2)-receptor agonist dimaprit (100-1000 microM) had no effect. The effect of histamine was partially blocked by pretreatment with H(1)-receptor antagonist pyrilamine (1 microM) or the H(3)-receptor antagonist thioperamide (1 microM). Pretreatment with a combination of pyrilamine (1 microM) and thioperamide (1 microM) completely blocked the response to histamine. The H(2)-receptor antagonist cimetidine (1 microM) was ineffective. These results suggest that histamine inhibits axonal transport of cultured mouse DRG neurons via the activation of H(1)- and H(3)-receptors.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Axones/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Histamina/farmacología , Neuronas Aferentes/efectos de los fármacos , Receptores Histamínicos/efectos de los fármacos , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Axones/ultraestructura , Células Cultivadas/citología , Células Cultivadas/metabolismo , Interacciones Farmacológicas/fisiología , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía por Video , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/efectos de los fármacos , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/efectos de los fármacos , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H3/efectos de los fármacos , Receptores Histamínicos H3/metabolismoRESUMEN
BACKGROUND: Axonal transport plays a critical role in supplying materials for a variety of neuronal functions such as morphogenetic plasticity, synaptic transmission, and cell survival. In the current study, the authors investigated the effects of the analgesic agent lidocaine on axonal transport in neurites of cultured mouse dorsal root ganglion neurons. In relation to their effects, the effects of lidocaine on the growth rate of the neurite were also examined. METHODS: Isolated mouse dorsal root ganglion cells were cultured for 48 h until full growth of neurites. Video-enhanced microscopy was used to observe particles transported within neurites and to measure the neurite growth during control conditions and in the presence of lidocaine. RESULTS: Application of 30 microM lidocaine immediately reduced the number of particles transported in anterograde and retrograde axonal directions. These effects were persistently observed during the application (26 min) and were reversed by lidocaine washout. The inhibitory effect was dose-dependent at concentrations from 0.1 to 1,000 microM (IC50 = 10 microM). In Ca2+-free extracellular medium, lidocaine failed to inhibit axonal transport. Calcium ionophore A23187 (0.1 microM) reduced axonal transport in both directions. The inhibitory effects of lidocaine and A23187 were abrogated by 10 microM KN-62, a Ca2+-calmodulin-dependent protein kinase II inhibitor. Application of such low-concentration lidocaine (30 microM) for 30 min reduced the growth rate of neurites, and this effect was also blocked by KN-62. CONCLUSIONS: Low-concentration lidocaine rapidly inhibits axonal transport and neurite growth via activation of calmodulin-dependent protein kinase II.
Asunto(s)
Anestésicos Locales/farmacología , Transporte Axonal/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Lidocaína/farmacología , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Ganglios Espinales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/efectos de los fármacos , Neuritas/fisiologíaRESUMEN
Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/farmacología , Ganglios Espinales/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Sustancia P/farmacología , Animales , Transporte Axonal/fisiología , Células Cultivadas , Ganglios Espinales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/fisiologíaRESUMEN
The effects of prostaglandin E(2) on axonal transport in cultured mouse dorsal root ganglion neurons were investigated by analysing the number of axonally transported particles under video-enhanced microscopy. Application of prostaglandin E(2) increased the number of particles transported in anterograde and retrograde directions. The EP(2) prostaglandin receptor agonist butaprost mimicked the effect of prostaglandin E(2), but the EP(1)/EP(3) prostaglandin receptor agonist 17-phenyl trinor prostaglandin E(2) and the EP(3) prostaglandin receptor agonist M&B 28767 had no effect. The membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP and the adenylate cyclase activator forskolin mimicked the effect of prostaglandin E(2). The protein kinase A inhibitor H-89 reversibly reduced the number of particles in both anterograde and retrograde directions. The effects of prostaglandin E(2) and dibutyryl cyclic AMP were blocked by H-89. Taken together with previous biochemical studies showing that prostaglandin E(2) increases cyclic AMP levels, the present results suggest that prostaglandin E(2) enhances axonal transport via the EP(2) receptor and cyclic AMP-dependent protein kinase A pathway. We further investigated the role of prostaglandin E(2) in neurite growth. Prostaglandin E(2) increased both the number of cells exhibiting neurites and the neurite growth rate, operating by a similar mechanism to stimulation of axonal transport. Prostaglandin E(2) may modulate axonal transport to supply materials for morphogenesis as well as other functions in sensory neurons.
Asunto(s)
Transporte Axonal , Dinoprostona/fisiología , Ganglios Espinales/ultraestructura , Neuritas/fisiología , Animales , Células Cultivadas , Dinoprostona/agonistas , Dinoprostona/farmacología , Ganglios Espinales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Transducción de Señal , Grabación en VideoRESUMEN
We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [(3)H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [(3)H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in (3)H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/farmacología , Mucinas/biosíntesis , Células Parietales Gástricas/efectos de los fármacos , Células Parietales Gástricas/enzimología , Fragmentos de Péptidos/farmacología , Animales , Anticuerpos , Benzoatos/farmacología , Péptido Relacionado con Gen de Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/inmunología , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Glucosamina/farmacocinética , Imidazoles/farmacología , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Células Parietales Gástricas/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Antro Pilórico/citología , Ratas , Ratas Wistar , TritioRESUMEN
The effects of ecabet sodium (ecabet), 12-sulfodehydroabietic acid monosodium salt, on gastric mucin biosynthesis in rat antrum were compared with those in the corpus. Intragastric administration of ecabet significantly increased [3H]glucosamine incorporation into antral mucin as well as into corpus mucin during five successive hours of organ culture. In contrast, mucin biosynthesis in either antrum or corpus was not susceptible to the addition of ecabet to the culture medium. Ecabet-induced stimulation of prostaglandin E2 production in the antrum was essentially the same as that seen in the corpus. In antrum treated with 100 mg/kg ecabet, immunoreactivity with three distinct anti-mucin monoclonal antibodies was found not only in the specific mucus-producing cells, but also in the secreted mucus present at the surface gel layer. These results suggest that ecabet enhances the mucin metabolism, and this stimulation occurs in both the corpus and antrum, suggesting that ecabet might be a useful tool for the further clarification of the regulatory mechanism of antral mucin synthesis.
Asunto(s)
Abietanos , Antiulcerosos/farmacología , Diterpenos/farmacología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Mucinas/biosíntesis , Animales , Dinoprostona/biosíntesis , Glucosamina/metabolismo , Inmunohistoquímica , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Estimulación QuímicaRESUMEN
We employed morphological and electrophysiological methods in order to elucidate mechanisms which are responsible for communication between cellular oscillators in the cultured rat suprachiasmatic nucleus, the site of the endogenous biological clock that regulates circadian rhythms in mammals. When a gap junction-permeable dye, Lucifer Yellow, was injected into single neurons in the suprachiasmatic nucleus culture, the dye was transferred to neighboring cells in a gap junction blocker-sensitive manner. Optical imaging of neural activity evoked by electrical stimulation in the culture revealed that the spread of depolarization was inhibited by gap junction blockers but not by a blocker of voltage-dependent Na(+) channels. Depolarization propagation was inhibited by muscimol, a GABA(A) receptor agonist, in a dose-dependent manner and the inhibition was reversed by bicuculline, a GABA(A) receptor antagonist. Furthermore, muscimol inhibited dye-transfer between neurons in the suprachiasmatic nucleus culture in a dose-dependent fashion.These independent lines of evidence suggest that the gap junction communication is involved in interneuronal communication in the suprachiasmatic nucleus slice culture and that the coupling state between neurons is not static but dynamically regulated via GABA(A) receptor systems.
Asunto(s)
Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Núcleo Supraquiasmático/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Bicuculina/farmacología , Técnicas de Cultivo , Electrofisiología , Colorantes Fluorescentes , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Halotano/farmacología , Isoquinolinas , Muscimol/farmacología , Octanoles/farmacología , Ratas , Ratas Wistar , Tetrodotoxina/farmacologíaRESUMEN
We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.
Asunto(s)
Transporte Axonal/fisiología , Ganglios Espinales/citología , Neuronas Aferentes/enzimología , Proteína Quinasa C/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Alcaloides , Animales , Transporte Axonal/efectos de los fármacos , Benzofenantridinas , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/ultraestructura , Fenantridinas/farmacología , Proteína Quinasa C/metabolismoRESUMEN
Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Ganglios Espinales/citología , Neuritas/efectos de los fármacos , Potasio/farmacología , Animales , Células Cultivadas , Electrofisiología , Ganglios Espinales/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Microscopía por Video , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/agonistas , Compuestos de Tetraetilamonio/farmacología , Veratridina/farmacologíaRESUMEN
Recent clinical and experimental studies suggest the effectiveness of lidocaine in blocking neuropathic pain. Because it has been demonstrated that the pathogenetic mechanisms of neuropathic pain involve morphological changes in afferent neuronal terminals onto spinal cord, we examined the effects of lidocaine on neurite growth in isolated mouse dorsal root ganglion cells in culture. Incubation for 2-42 h with various concentrations of lidocaine (0.006 mM, 0.6 mM, and 30 mM) reduced the number of cells exhibiting neurites. The effects were time- and dose-dependent. Lidocaine therefore may exert its pharmacological effect, at least in part, by changing neuronal structures derived from sensory neurons.
Asunto(s)
Ganglios Espinales/citología , Lidocaína/farmacología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Neuritas/fisiología , Neuritas/ultraestructura , Neuronas/citología , Neuronas/fisiologíaRESUMEN
The occurrence and distribution of neuropeptide-containing nerve fibres in the human circumvallate papillae were examined by the peroxidase-antiperoxidase immunolocalisation method using surgical specimens that had not been subjected to radiotherapy, and the abundance of neuropeptide-containing fibres was expressed as the percentage of total nerve fibres demonstrated by protein gene product (PGP) 9.5 immunoreactivity for a quantitative representation of these peptidergic fibres. Substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (IR) nerve fibres were densely distributed in the connective tissue core of the circumvallate papillae, and some SP and CGRP-IR fibres were associated with the taste buds. A moderate number of vasoactive intestinal polypeptide (VIP)-IR fibres and a few galanin (GAL)-IR fibres were also seen in the connective tissue core and subepithelial layer. There were, however, no VIP-IR or GAL-IR fibres associated with the taste buds. Neuropeptide Y (NPY)-IR fibres were few and were associated with the blood vessels. Within the epithelium of the circumvallate papillae, no peptidergic fibres were found, although a number of PGP 9.5-IR fibres were detected. The abundance of SP, CGRP, VIP, and GAL-IR fibres expressed as the percentage of total PGP 9.5 IR fibres was 25.35+/-3.45%, 22.18+/-3.26%, 10.23+/-1.18%, and 4.12+/-1.05%, respectively. The percentage of NPY-IR fibres was below 3%. In a deeper layer of the papillae, a few VIP, GAL, and NPY-IR ganglion cells were found, and VIP immunoreactivity was detected in a few cells of the taste buds. There was no somatostatin, leucine enkephalin, or methionine enkephalin immunoreactivity in the circumvallate papillae. These results suggest that the dense SP and CGRP-IR fibres within the connective tissue core of the human circumvallate papillae may be involved in the deep sensation of the tongue.
Asunto(s)
Fibras Nerviosas/química , Neuropéptidos/análisis , Lengua/química , Lengua/inervación , Adulto , Anciano , Biomarcadores/análisis , Péptido Relacionado con Gen de Calcitonina/análisis , Galanina/análisis , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Fibras Nerviosas/ultraestructura , Neuropéptido Y/análisis , Sustancia P/análisis , Papilas Gustativas/química , Tioléster Hidrolasas/análisis , Ubiquitina Tiolesterasa , Péptido Intestinal Vasoactivo/análisisRESUMEN
Salamander taste organs were recognized as oval cell clusters within the dorsal surface of the tongue. A moderate number of SP, CGRP, VIP, NPY, and GAL immunoreactive nerve fibers terminated in the cell clusters, and some of them penetrated into the basal half of the cell clusters. Around the glands, VIP, NPY, and GAL fibers were numerous, and SP and CGRP fibers were less numerous. Immunoreactivity of SOM and FMRF was not detected either in the nerve fibers associated with the cell clusters or those around the glands. These findings suggest that the chemosensory mechanisms of the salamander gustatory organs are under the control of peptidergic innervation. In addition, the present study indicates that the caudate taste organs are structurally primitive but functionally mature.
Asunto(s)
Fibras Nerviosas/química , Neuropéptidos/análisis , Papilas Gustativas/química , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , FMRFamida/análisis , Galanina/análisis , Neuropéptido Y/análisis , Somatostatina/análisis , Sustancia P/análisis , Urodelos , Péptido Intestinal Vasoactivo/análisisRESUMEN
Neuron requires a continual supply of materials synthesized in the cell body, for example a wide range of soluble proteins, membranous components, and various organelles. The transported materials are needed to replace constituents that turn over in the membrane and organelles of the fiber and also are needed to bring substances participating in energy metabolism. Other transported components are neurotransmitters or transmitter-related components supplied to the nerve terminals for the release and subsequent excitation of postsynaptic cells. Moreover, neurotropic substances and modulators are released from the nerve terminals to affect the functional state of the neuron. Conversely, some materials are conveyed back to the cell body. These include organelles, lysosomes, nerve growth factor, and selected small molecules such as adenosine, Ca2+, and some neurotransmitters. Axoplasmic transport is thought to be fundamental for a variety of neuronal cell functions. Thus it may be considered that axoplasmic transport relates to the dynamic physiological activity of neurons; in other words, axoplasmic transport is supposed to express the physiological activity of neurons. In turn, as in the case for many other physiological functions, axoplasmic transport is possibly controlled by neuronal, hormonal, and immunological systems. Since axoplasmic transport supplies neuron materials toward the synapses and back to the cell body, a feedback system of regulatory mechanisms of a variety of neuronal functions might be operated through axoplasmic transport pathways. Although axoplasmic transport is the important neuronal function, its regulation is poorly understood. In this review, we focus on the dynamics of organelle transport and its regulatory mechanisms mediated by neurotransmitters.
Asunto(s)
Transporte Axonal/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Microscopía por Video , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Modelos BiológicosRESUMEN
Noradrenaline (NA) is one of the most important neurotransmitters involved in the regulation of gonadotropin-releasing hormone (GnRH) secretion. In this study, the effects of NA on GnRH secretion, intracellular Ca2+ concentrations ([Ca2+]i), and membrane potentials were investigated in immortalized hypothalamic neurons (GT1-7) to determine the direct effects of NA on GnRH cells. Cells were perfused in a plastic minicolumn, and GnRH concentrations of the effluents were measured. NA increased the release of GnRH in a dose-dependent manner. Cells were loaded with a 4 microM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was calculated at 100-ms intervals. NA increased the [Ca2+]i responses of single GnRH cells dose-dependently. The NA-induced [Ca2+]i increase was attenuated in the absence of extracellular calcium and was blocked by the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. The cell membrane potential was recorded with a whole-cell patch clamp amplifier with glass-electrodes. NA induced membrane depolarization under current-clamp conditions. The depolarization was also inhibited by propranolol, but not by phentolamine. The results show that NA directly affects the membrane potential of GT1-7 cells via beta-adrenergic receptors and induces Ca2+ mobilization; these effects stimulate GnRH secretion.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/fisiología , Neuronas/metabolismo , Norepinefrina/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Antagonistas Adrenérgicos beta/farmacología , Calcio/metabolismo , Calcio/farmacología , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Electrofisiología , Hormona Liberadora de Gonadotropina/efectos de los fármacos , Hipotálamo/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Fentolamina/farmacología , Propranolol/farmacologíaRESUMEN
Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/fisiología , Animales , Bloqueadores de los Canales de Calcio , Canales de Calcio/fisiología , Comunicación Celular , Línea Celular Transformada , Estimulación Eléctrica , Electrofisiología , Colorantes Fluorescentes , Hipotálamo/fisiología , Ratones , Ratones Transgénicos , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Bloqueadores de los Canales de Sodio , Canales de Sodio/fisiología , Tetrodotoxina/farmacologíaRESUMEN
1. Intracellular recordings were obtained from supraoptic magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. Application of ATP and UTP, but not adenosine, produced TTX-insensitive depolarizations accompanied by increases of input conductance. 2. The P2X agonists alpha,beta-methylene ATP, beta,gamma-methylene ATP and 2-methylthio ATP mimicked the effects of ATP in > 77% of the cells tested. Depolarizing responses to ATP were reversibly inhibited by PPADS (pyridoxal-phosphate-6-azophenyl-2',4'- disulphonic acid; IC50 approximately 0.5 microM), a selective P2X antagonist. 3. The reversal potential of responses to ATP (-37 mV) was not strongly affected by intracellular Cl- injection or by removal of Cl- from the external solution. The reversal potential of responses to the most potent P2X agonist, alpha,beta-methylene ATP, was -29 mV. These values suggest the involvement of non-selective cationic channels, a finding which is consistent with the ionotropic cationic channel structure of cloned P2X purinoceptors. 4. The reversal potential of UTP-mediated responses (-33 mV) was also consistent with the involvement of non-selective cationic channels. Since cloned P2U receptors display homology with G-protein-coupled receptors, cationic channels modulated by UTP are probably different from those mediating P2X responses.