SUMMARY1. The composition of the efflux from desheathed rabbit vagus nerve, loaded with radioactivity by incubation in [3H]adenosine, was studied at rest and during electrical activity and after application of inhibitors of ecto-enzymes and modifications of intermediary metabolism. In addition, the degradation ofexternally applied ATP and adenosine was examined.2.[3H]ATP applied to the incubation medium was degraded to ADP, AMP, adenosine and inosine. The hydrolysis to nucleosides was inhibited by a, ,8-methylene ADP; the appearance of AMP and nucleosides was slowed by fi, y-methylene ATP.Deamination of [3H]adenosine was blocked by 2-deoxycoformycin. 3. The effluent from resting and stimulated preparations showed the presence of large amounts of inosine and hypoxanthine, smaller amounts of adenosine and adenine and traces of nucleotides. The composition of the effluent was not significantly altered by addition of a, fl-methylene ADP; fi, y-methylene ATP or 2-deoxycoformycin.4. Application of glucose-free solutions caused a large release of adenosine instead of inosine and hypoxanthine and a small increase in resting and stimulated efflux of 3H. Addition of 2-deoxyglucose produced a large increase in resting efflux and increased liberation of adenosine. Cyanide, 2, 4-dinitrophenol, arsenate or salicylate increased the resting efflux of adenosine, inosine and hypoxanthine, and the effect of activity.5. It is concluded that electrical activity leads to release of adenosine, inosine and hypoxanthine, in various proportions depending on metabolic state, and that there is practically no liberation of nucleotides from nerve axons.

SUMMARY1. Influx of adenosine into rabbit non-myelinated nerve fibres was measured using [2-3H]adenosine. The uptake of radioactivity increased linearly with duration of incubation for up to 60 min and adenosine concentration up to 200 UM. The uptake at different adenosine concentrations showed a saturable component with a halfmaximal activation at 17-1 /M and a linear part.2. The radioactivity taken up was rapidly incorporated into AMP, ADP and ATP. Isotopic equilibrium between the nucleotides was achieved within 15 min.3. The uptake of 3H from 0-2 gM-adenosine was almost completely inhibited by addition of 200 gM-adenosine and to a similar extent by 200 /LM-tubercidin and AMP; a 70 % inhibition was found with ATP and ADP; a, , methylene-ADP had no effect.4. ATP, ADP and AMP added to the extracellular medium of a desheathed vagus were slowly hydrolysed.5. In preparations loaded with [2-3H]adenosine and then washed with adenosine and label-free solution there was a steady efflux of radioactivity amounting to 0 18 x 10-3/min. Addition of adenosine or tubercidin transiently increased the efflux. 6. Electrical stimulation caused an extra release of radioactivity. The extra fractional loss was 21-8 x 10-6/impulse in preparations that had rested for several hours; it decreased to 2-3 x 10-6/impulse when stimulation was applied after a 30 min rest.7. The radioactivity of the resting efflux and of the extra efflux after stimulation was found mostly in inosine and hypoxanthine; adenosine and adenine accounted for only 3 %, and the nucleotides for less than 1 % of the efflux.8. Adenosine added to the external medium of a desheathed nerve was slowly deaminated. 9. It is concluded that inosine and hypoxanthine found in the effluent from desheathed vagus nerve trunk result from release of these compounds from nerve fibres and not from extracellular breakdown of released ATP or adenosine.10. Electrical activity in non-myelinated nerve fibres of the nerve trunk thus causes the release of metabolites (inosine and hypoxanthine) together with small amounts of adenosine and adenine, while release of ATP and other nucleotides is almost completely absent.

We investigated apoptosis during early stages of in vitro differentiation of neuronal precursors generated by embryonic day 14 (E14) mouse striata stem cells. Differentiation was in conditions of suboptimal growth factor supply. Apoptosis reached 10-15% of cells and affected proliferating as well as postmitotic cells, including TUJ1-positive cells. Inhibition of apoptosis led to an increased proportion of TUJ1-positive cells generated by stem cells. K(+) current was reported to be related to apoptosis. Outward K(+) currents were present in differentiating neuronal precursors that were consistent with delayed rectifier and transient A-type currents. The amplitude of the delayed rectifier current varied during the first 4 days of stem cell differentiation. Current amplitude was greatly increased in the presence of staurosporine but reduced at elevated extracellular K(+) concentration. In addition, the amplitude of the current was significantly diminished by inhibiting several caspases, but not caspase 8. In Bax knock-out transgenic neuronal precursors, K(+) current was not decreased after the first day but at later stages of cell differentiation. At this early stage, apoptosis of proliferating cells and of TUJ1-positive cells was not reduced by the absence of Bax, but was by caspase 9 inhibition. Thus, activation of a delayed rectifier K(+) current in differentiating stem cells is related to apoptosis. Recordings of this current revealed that apoptosis at early stages of neuronal differentiation occurred in two phases that did not exhibit similar dependence on the proapoptotic protein Bax and that probably used different pathways.

SUMMARY1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes.2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs).3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2-6 + 0'4 ms and 6-0 + 1-1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0 7 + 0-1 pA for the mean amplitude of the elementary event, and 4'7 + 0-2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which MS 9242 R. GIROD AND OTHERS released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro.7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.8. The present results demonstrate (i) that synaptosomes in vitro are able to release ACh at a site that is very close to the myocyte receptor-rich membrane and (ii) that the molecular machinery ensuring pulsatile or quantal release of ACh at natural synapses remains functional after isolation of the nerve endings.

The structure of the rudimentary prostates of Ellobius lutescens is maintained intact after 6 days of organotypic culture in the absence of male hormones. Comparison with controls even shows a noticeable increase in the size of the epithelial cells. Adding male hormones to the culture medium does not modify the morphology of adult prostates, while it induces a sharp stimulation of immature prostates. In accordance with our previous results, these experiments show that the prostates of Ellobius lutescens lose their sensivity to androgens after puberty.