RESUMEN
The phenotypic differentiation between oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus is relevant to understanding how several physiological and pharmacological challenges affect their electrical activity. Although the firing patterns of OT and VP neurons, both in vivo and in vitro, may appear different from each other, much is assumed about their characteristics. These assumptions make it practically impossible to obtain a confident phenotypic differentiation based exclusively on the firing patterns. The presence of a sustained outward rectifying potassium current (SOR) and/or an inward rectifying hyperpolarization-activated current (IR), which are presumably present in OT neurons and absent in VP neurons, has been used to distinguish between the two types of MNCs in the past. In this study, we aimed to analyze the accuracy of the phenotypic discrimination of MNCs based on the presence of rectifying currents using comparisons with the molecular phenotype of the cells, as determined by single-cell RT-qPCR and immunohistochemistry. Our results demonstrated that the phenotypes classified according to the electrophysiological protocol in brain slices do not match their molecular counterparts because vasopressinergic and intermediate neurons also exhibit both outward and inward rectifying currents. In addition, we also show that MNCs can change the relative proportion of each cell phenotype when the system is challenged by chronic hypertonicity (70% water restriction for 7 days). We conclude that for in vitro preparations, the combination of mRNA detection and immunohistochemistry seems to be preferable when trying to characterize a single MNC phenotype.
Asunto(s)
Potenciales de Acción/fisiología , Neuronas/metabolismo , Oxitocina/metabolismo , ARN Mensajero/metabolismo , Núcleo Supraóptico/metabolismo , Vasopresinas/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Dieta , Expresión Génica , Masculino , Microtomía , Neuronas/clasificación , Neuronas/citología , Neuronas/efectos de los fármacos , Oxitocina/genética , Técnicas de Placa-Clamp , Fenotipo , ARN Mensajero/genética , Ratas , Ratas Wistar , Análisis de la Célula Individual , Sodio en la Dieta/farmacología , Núcleo Supraóptico/citología , Núcleo Supraóptico/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Vasopresinas/genética , Privación de AguaRESUMEN
Increases in plasma osmolality enhance nitric oxide (NO) levels in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and modulate the secretion of both vasopressin (VP) and oxytocin (OT). In this paper, we describe the effects of hypertonicity on the electrical properties of MNCs by focusing on the nitrergic modulation of their activity in this condition. Membrane potentials were measured using the patch clamp technique, in the presence of both glutamatergic and GABAergic neurotransmission blockers, in coronal brain slices of male Wistar rats. The recordings were first made under a control condition (295 mosm/kg H2O), then in the presence of a hypertonic stimulus (330 mosm/kg H2O) and, finally, with a hypertonic stimulus plus 500 µM L-Arginine or 100 µM N-nitro-L-Arginine methyl ester hydrochloride (L-NAME). Hypertonicity per se increased the firing frequency of the neurons. L-Arginine prevented the increase in fire frequency induced by hypertonic stimulus, and L-NAME (inhibitor of nitric oxide synthase) induced an additional increase in frequency when applied together with the hypertonic solution. Moreover, L-Arginine hyperpolarizes the resting potential and decreases the peak value of the after-hyperpolarization; both effects were blocked by L-NAME and hypertonicity and/or L-NAME reduced the time constant of the rising phase of the after-depolarization. These results demonstrate that an intrinsic nitrergic system is part of the mechanisms controlling the excitability of MNCs of the SON when the internal fluid homeostasis is disturbed.
Asunto(s)
Núcleo Basal de Meynert/metabolismo , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Concentración Osmolar , Núcleo Supraóptico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Arginina/farmacología , Núcleo Basal de Meynert/citología , Fenómenos Electrofisiológicos/fisiología , Inhibidores Enzimáticos/farmacología , Soluciones Hipertónicas/farmacología , Soluciones Hipotónicas/farmacología , Técnicas In Vitro , Cinética , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Núcleo Supraóptico/citologíaAsunto(s)
Hiperesplenismo/sangre , Derivación Portosistémica Quirúrgica , Derivación Esplenorrenal Quirúrgica , Adulto , Anciano , Femenino , Hemorragia Gastrointestinal/etiología , Pruebas Hematológicas , Humanos , Hipertensión Portal/etiología , Hipertensión Portal/cirugía , Parasitosis Hepáticas/complicaciones , Masculino , Persona de Mediana Edad , Pancitopenia/etiología , Esquistosomiasis mansoni/complicaciones , Enfermedades del Bazo/complicacionesRESUMEN
Twelve women with iron-deficiency anemia due to chronic loss of blood, but free from any other pathology that might alter the immune response, were studied. The patients were tested for cell immunity both in vitro, by B and T lymphocyte quantitation and by blastic transformation of the lymphocytes with phytohemagglutinin (PHA), and in vivo, by dinitrochlorobenzene (DNCB), tuberculin, trychophytine and varidase skin tests. The same tests were repeated after iron therapy and also applied to a group of 12 normal control subjects. The percent of T lymphocytes increased significantly from 55.1 to 66.0% after treatment, while the absolute values did not change. There was a significant decrease in both the number and percent of "null" lymphocytes after treatment. The percent and absolute number of B lymphocytes did not change after treatment. Blastic transformation indices were within the normal range both before and after treatment. Seven women who were DNBC-negative before treatment became DNBC-positive after iron therapy. Of the 5 patients who were tuberculin-negative before treatment, 2 became positive after iron therapy. Reactivity to trychophytine was observed in 3 patients before treatment as compared to 5 afterwards. Reactivity to varidase increased from 4 to 6 patients upon iron therapy. On the basis of changes in immunological reactivity observed after iron replenishment, we conclude that iron deficiency is an important factor in the genesis of the immunological alterations occurring in iron-deficiency anemia.