RESUMEN
Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [3H]-histamine uptake yielded a Km of 0.19 +/- 0.03 microM and a Vmax of 3.12 +/- 0.75 pmol X mg protein-1 X min-1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na+ with choline inhibited the initial rate of uptake showing a strong Na(+)-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K(+)-gradient (in > out) by high K+ or by K(+)-channel blockers, BaCl2, (100 microM), quinine (100 microM) or Sparteine (20 microM) produced also remarkable inhibitions in the uptake of [3H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 microM with a Ki value of 2.8 microM, indicating the specificity of the uptake. [3H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33 +/- 0.08 microM; and a Vmax of 2.65 +/- 0.35 pmoles x mg protein-1 x min-1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [3H]histamine efflux was sensitive to Na+ omission, ouabain treatment and high K+ or K+ channel blockers, resulting in marked elevations in the efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Astrocitos/metabolismo , Encéfalo/efectos de los fármacos , Histamina/farmacocinética , Bloqueadores de los Canales de Potasio , Potasio/farmacología , Sodio/deficiencia , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Gliotoxina/farmacología , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Técnicas In Vitro , Ratas , Ratas Wistar , TritioRESUMEN
The importance of cell-aggregation during retinoic acid-induced neural differentiation of embryonal carcinoma cells was studied on the PCC-7 cell line. These cells were chosen as they display low tendency for spontaneous aggregation, and they develop preferentially to neurons upon induced in vitro differentiation. Forced aggregation of these cells, in the absence of retinoic acid, did not result in development of neuron- or glial-like cells. Application of retinoic acid prior to or after the cell-aggregation did not result in neural tissue-like differentiation, either. Irreversible induction of neural development was achieved if cell-aggregation and retinonic acid acted simultaneously, and for a period longer than 48 h. Retinoic acid, on the other hand, was found to be toxic on non-aggregated PCC-7 cells. Our data suggest that cell to cell contacts alter the response of these cells to retinoic acid, and their close apposition is a prerequisite for the retinoic acid-induced neural differentiation.
Asunto(s)
Células Madre Neoplásicas/efectos de los fármacos , Neuronas/efectos de los fármacos , Tretinoina/farmacología , Animales , Anticuerpos Monoclonales , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Células Madre de Carcinoma Embrionario , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Ratones , Células Madre Neoplásicas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Células Tumorales Cultivadas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A multi-microelectrode culture chamber system was constructed for monitoring simultaneously morphological and electrophysiological development of neural cells in vitro. The setup consisted of a pattern of gold conductor lines evaporated onto a glass substrate and insulated with polyamide. The width of each electrode was 10 microns, and the distance between the electrodes was 60 microns. The electrode patterns were constructed and the uncovering of the electrode tips were carried out by photo-etching. This system allowed us to record spontaneous activities in both explant- and primary monolayer cultures of either rat or mouse spinal cords and forebrains, during neuronal regeneration and maturation.