Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.

No abstract

The changes in quantal transmitter release induced by egg phosphatidylcholine liposomes with different internal ionic composition were examined at the frog neuromuscular junction by using conventional electrophysiological techniques. It was found that liposomes containing calcium or sodium ions increase both evoked and spontaneous transmitter release, while liposomes containing potassium do not. The results suggest that phosphatidylcholine liposomes are able to transfer their aqueous medium into the presynaptic nerve terminal. We report here the results of a study using ion-containing liposomes to modify quantal transmitter release from motor nerve terminals.A substantial amount of evidence exists suggesting that the liberation of packets of acetylcholine (1) (Amersham, Bucks, England) to the CaC12-containing media prior to ultrasonic irradiation. The irradiation was followed by a dialysis at 4VC against 50 ml of the low-calcium Ringer solution. The dialysis medium was changed 5 to 6 times during a total period of 16-24 hr. Under these conditions, it was found that the concentration of 45Ca in the dialysis bag reached a constant value. Because calcium ions do not bind to egg lecithin monolayers (12) or vesicles (13) in the concentration range used, it is possible to estimate the trapped volume.It was found to be 0.42 ,gl/,gmol of phosphatidylcholine. RESULTSEffect of ion containing liposomes on evoked transmitter release "Sodium/Calcium" Liposomes. The initial experiments were performed with liposomes containing standard Ringer solution whose calcium composition was increased to 25 mM. These liposomes were dialyzed overnight with Ringer solution containing 0.35 mM CaCl2. Addition of these liposomes to a neuromuscular preparation caused an increase in the EPP amplitude and the quantal content, which was easily detectable within 1-2 minutes. An example of such an experiment is shown in Fig. 1

SUMMARY1. Time series analysis of evoked transmitter release was performed at the frog neuromuscular synapse.2. Clumping of end-plate potentials with similar amplitude was found in the time domain. 3. At low quantal contents periodic oscillations were observed with a period of 14 sec.4. Clumping and oscillations are phenomena of presynaptic origin.5. The results are explained on the hypothesis that periodic fluctuations occur in Ca concentration inside the presynaptic nerve terminal.

The theme of this presentation has been to show that the control of transmitter release at the neuromuscular synapse is achieved by extracellular and intracellular calcium. For the fast information transfer represented by the end-plate potential, the electrochemical gradient for calcium across the presynaptic membrane and the associated calcium conductance seem to play the primary role. For slower processes such as tetanic and posttetanic potentiation, the combined effect of both sources for calcium determine the amount of transmitter liberated.