Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Development and differentiation of fetal rat sensory ganglia and spinal cord segments in vitro

Summary Fetal spinal ganglia and spinal cord segments with adhering spinal ganglia were explanted on collagen-coated coverslips. They were investigated with enzyme histochemical methods for the existence of hydrolases and dehydrogenases up to 54 days of cultivation.Alkaline phosphatase was located in arachnoid cells and in mantle cells (satellite cells). Butyrylcholinesterase and alpha-glycerophosphate-menadione-tetrazolium reductase were found in mantle cells. Acetylcholinesterase and indoxylesterase were active in the whole neuron; acid phosphatase and alpha-naphthylacetate esterase were restricted to the perikarya.During the process of cell differentiation in vitro alkaline phosphatase decreased in mantle cells. Acid phosphatase became diminished distinctly in spinal ganglion cells. The other neuronal enzymatic activities remained unaltered during the whole period of cultivation. Proliferated Schwann cells were conspicuous by their activity for butyrylcholinesterase. In newly formed myelin sheaths arylsulphatase was active. Lactate dehydrogenase was contained in the perineurium which had developed. Cultures of long duration could contain cytological formations which were reminiscent of sensory end-organs with respect to their enzyme patterns.The enzyme activities of nervous tissues in vitro in their approximation to the situation in situ are discussed.A preliminary report was presented at the 17th Meeting of the Association of German Neuropathologists and Neuroanatomists, in Freiburg, September 1972.The skilled technical assistance of Miss Johanna Sixel and Miss Charlotte Beyer is gratefully acknowledged.     

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.     

A study of the fluid uptake of rat kidney slices in vitro

Rat renal cortical and medullary tissues show a marked elevation of relative water content when immersed in "physiological solutions" containing sodium and chloride ions, or in equally concentrated solutions of monosaccharides. In contrast to this, no increase in relative water content occurs in isosmotic solutions of three disaccharides studied. It appears to be unlikely that the fluid uptake is a result of intracellular hypertonicity existing either physiologically or pathologically. A more satisfactory alternative hypothesis is that ingress of water accompanies entrance of solutes (ions, monosaccharide molecules) into the tissue cells.     

Kinetics of glial glutamine efflux and the mechanism of neuronal uptake studied in vivo in mildly hyperammonemic rat brain

Kinetics of glial glutamine (GLN) transport to the extracellular fluid (ECF) and the mechanism of GLN(ECF) transport into the neuron--crucial pathways in the glutamine-glutamate cycle--were studied in vivo in mildly hyperammonemic rat brain, by NMR and microdialysis to monitor intra- and extracellular GLN. The minimum rate of glial GLN efflux, determined from the rate of GLN(ECF) increase during perfusion of alpha-(methylamino)isobutyrate (MeAIB), which inhibits neuronal GLN(ECF) uptake by sodium-coupled amino-acid transporter (SAT), was 2.88 +/- 0.22 micromol/g/h at steady-state brain [GLN] of 8.5 +/- 0.8 micromol/g. Our previous study showed that the rate of glutamine synthesis under identical experimental conditions was 3.3 +/- 0.3 micromol/g/h. At steady-state glial [GLN], this is equal to its efflux rate to the ECF. Comparison of the two rates suggests that SAT mediates at least 87 +/- 8% (= 2.88/3.3 x 100%) of neuronal GLN(ECF) uptake. While MeAIB induced > 2-fold elevation of GLN(ECF), no sustained elevation was observed during perfusion of the selective inhibitor of LAT, 2-amino-bicyclo[1,1,2]heptane-2-carboxylic acid (BCH), or of d-threonine, a putative selective inhibitor of ASCT2-mediated GLN uptake. The results strongly suggest that SAT is the predominant mediator of neuronal GLN(ECF) uptake in adult rat brain in vivo.     

In vivo labeling of the central GABA uptake carrier with 3H-Tiagabine.

The in vivo binding of 3H-Tiagabine to the central GABA uptake carrier in mouse brain was characterized. 3H-Tiagabine in vivo bound to a single class of binding sites with a Kd = 72.5 nM and a Bmax = 640 pmol/g tissue. 3H-Tiagabine binding in vivo was regionally distributed within the CNS, and showed a good correlation with 3H-Tiagabine binding in vitro. Pharmacological characterization of 3H-Tiagabine binding in vivo revealed a binding site exhibiting specificity for GABA uptake inhibitors. Experiments examining the in vivo receptor occupancy of the GABA uptake carrier for a series of GABA uptake inhibitors revealed that 20-30% of the GABA uptake sites were occupied at the ED50 for inhibiting DMCM-induced clonic convulsions, while a 50-62% receptor occupancy in vivo was needed to inhibit rotarod performance. These data suggest that 3H-Tiagabine in vivo binding may be a useful method for assessing GABA uptake inhibitor penetration into the CNS, and may be a useful tool for studying the physiological regulation of the GABA uptake carrier.     

Pharmacological analysis of the ontogenetic traits of the functional receptor activity of glial and neuronal GABA uptake and GABA-T activity in the rat hippocampus

Studies have been made on the effect of GABA mimetics on epileptogenic focus in the hippocamp of growing rats. By the pattern of inhibition of this focus by these drugs, it is possible to evaluate the degree of functional maturation of the activity of GABA receptors, glial and neuronal uptake of GABA, and the activity of GABA-T. Higher sensitivity of GABA receptors in younger animals was found together with a higher functional role of glial uptake in GABA utilization. Functional activity of GABA-T and neuronal uptake of the mediator are of lower physiological significance in younger animals as compared to those in adults.     

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.     

Phagocytic cell responses to in vivo and in vitro exposure to the Lyme disease spirochete.

An experimental skin lesion induced in rabbits by the bite of infected adult Ixodes dammini showed dense dermal interstitial inflammatory cell infiltrates composed of mononuclear cells (histiocytes and lymphocytes) and granulocytes. The prevalence of phagocytic cells in this experimental lesion motivated a study on the interactions of macrophages and neutrophils with Lyme disease spirochetes. Interactions as measured by uptake of radiolabeled spirochetes and by indirect immunofluorescence were enhanced by opsonization of spirochetes with immune serum and not significantly decreased by heat inactivation of the same. Phagocytosis was inhibited by treatment of cells with Cytochalasin B. Adherence of opsonized spirochetes to neutrophils was decreased by blocking Fc receptors with heat-aggregated IgG, suggesting an important role for this receptor.     

Upregulation of cerebral cortical glutathione synthesis by ammonia in vivo and in cultured glial cells: the role of cystine uptake

Glutathione (GSH) is a major antioxidant in the brain and ammonia neurotoxicity is associated with oxidative stress. In this study, we show that intracerebral administration of ammonium chloride (“ammonia”, final concentration 5 mM) via a microdialysis probe, increases by 80% the glutathione content in cerebral cortical microdialysates, and tends to increase its content in striatal microdialysates. Treatment with ammonia in vitro dose-dependently increased the glutathione content in cultured cerebral cortical astrocytes and a C6 glioma cell line. Significant effects have been observed after 1 h (astrocytes) or 3 h (C6 cells) of exposure and were sustained up to 72 h of incubation. A gradual decrease of the GSH/GSSG ratio noted during 3 h (astrocytes) or 24 h (C6 cells) of exposure, was followed by an partial recovery after 24 h of incubation, the latter phase possibly reflecting increased availability of de novo synthesized glutathione. In our hands, cystine, the precursor for astrocytic glutathione synthesis, was transported to astrocytes almost exclusively by system X_AG⁻, while in C6 cells the transport engaged both system x_c⁻ (60% of uptake) and X_AG⁻ (40% of uptake). Ammonia in either cell type stimulated cystine uptake without changing the relative contribution of the uptake systems. The results are consistent with the concept of increased astrocytic glutathione synthesis as an adaptive response of the brain to ammonia challenge, and emphasize upregulation of cystine uptake as a factor contributing to this response.     

Azodicarbonamide inhibits T-cell responses in vitro and in vivo.

Azodicarbonamide was recently identified as a new anti-HIV agent that targets the zinc finger domains of the HIV-1 NCp7 nucleocapsid protein. Here, we demonstrate that azodicarbonamide inhibits in a dose-dependent manner the responses of purified human CD4+ T lymphocytes stimulated either by monoclonal antibodies against CD3 and CD28 or by allogeneic dendritic cells. These suppressive effects involve a direct action on the calcium mobilization machinery, as azodicarbonamide strongly inhibited the calcium influx induced either by antibodies against CD3 and CD28 or the chemokine RANTES, as well as by thapsigargin, an activator of depletion-activated calcium channels. In vivo, administration of azodicarbonamide into mice blunted their response to polyclonal T-cell activation induced by the injection of monoclonal antibody against CD3 and resulted in delayed rejection of skin allografts. In addition to its anti-HIV properties, azodicarbonamide is a new immunosuppressive agent that might have therapeutic applications in T cell-mediated inflammatory disorders.     

Induction of reactivation of herpes simplex virus in murine sensory ganglia in vivo by cadmium.

Herpes simplex viruses maintained in a latent state in sensory neurons in mice do not reactivate spontaneously, and therefore the factors or procedures which cause the virus to reactivate serve as a clue to the mechanisms by which the virus is maintained in a latent state. We report that cadmium sulfate induces latent virus to reactivate in 75 to 100% of mice tested. The following specific findings are reported. (i) The highest frequency of induction was observed after two to four daily administrations of 100 micrograms of cadmium sulfate. (ii) Zinc, copper, manganese, or nickel sulfate administered in equimolar amounts under the same regimen did not induce viral reactivation; however, zinc sulfate in molar ratios 25-fold greater than those of cadmium induced viral replication in 2 of 16 ganglia tested. (iii) Administration of zinc, nickel, or manganese prior to the cadmium sulfate reduced the incidence of ganglia containing infectious virus. (iv) Administration of cadmium daily during the first week after infection and at 2-day intervals to 13 days after infection resulted in the recovery from ganglia of infectious virus in titers 10- to 100-fold higher than those obtained from animals given saline. Moreover, infectious virus was recovered as late as 11 days after infection compared with 6 days in mice administered saline. (v) Administration of cadmium immediately after infection or repeatedly after establishment of latency did not exhaust the latent virus harbored by sensory neurons, inasmuch as the fraction of ganglia of mice administered cadmium and yielding infectious virus was similar to that observed in mice treated with saline. We conclude that induction of cadmium tolerance precludes reactivation of latent virus. If the induction of metallothionein genes was the sole factor required to cause reactivation of latent virus, it would have been expected that all metals which induce metallothioneins would also induce reactivation, which was not observed. The results therefore raise the possibility that in addition to inducing the metallothionein genes, cadmium inactivates the factors which maintain the virus in latent state.     

Acute and chronic leptin effect upon in vivo and in vitro rat thyroid iodide uptake

Leptin has stimulatory effects on the hypothalamic-pituitary-thyroid axis and on deiodinases activities. Here, we evaluated the effect of leptin injection upon in vivo and in vitro thyroid 125I uptake (RAIU). We designed two experiments: acute leptin (LepA) with a single dose of leptin (8 microg/100 g BW/sc), and chronic leptin (LepC), injected with the same dose of LepA, once a day, for 6 days. In parallel, control groups were saline-injected. For in vivo study, part of the animals were injected with 125I (3700 Bq) and killed after 15 or 120 min. In vivo thyroid RAIU was not changed in LepA animals. However, LepC animals showed higher in vivo thyroid RAIU (15 min:+130% and 120 min:+72%; p<0.05). For in vitro study, the other animals were killed and their thyroids were incubated with 125I. Thyroids explants from LepA and LepC groups presented lower thyroid 125I content (-32% and -29% p<0.05, respectively). The amount of our data suggest that, in vitro, leptin causes a direct inhibition of the rat thyroid RAIU, but in vivo, the effect of leptin was different according to the treatment period, which indicates that other indirect mechanisms are involved in the in vivo leptin chronic stimulation of the thyroid gland.