A kinetic study of stimulus-induced vesicle recycling in electromotor nerve terminals using labile and stable vesicle markers.

The kinetics of recovery, by recycling electromotor synaptic vesicles, of the biophysical parameters of the reserve population has been studied in perfused blocks of electric organ of Torpedo marmorata prestimulated in vivo, followed by density gradient separation of the extracted vesicles in a zonal rotor using labile (acetylcholine and ATP) and stable (proteoglycan) vesicle markers. Stimulation in vivo at 0.15 Hz for 3.3 h depleted tissue acetylcholine much less than stimulation at 1 Hz for 1 h but nevertheless generated a much larger pool of recycled vesicles that recovered more slowly. At the lower rate of stimulation, recovery of the biophysical characteristics of the reserve population by the recycled vesicles, identified by their content of newly synthesized transmitter, was essentially complete by 8 h. The stable proteoglycan marker was immunochemically assayed and was bimodally distributed in the vesicle-containing portion of the density gradient even in experiments with unstimulated or recovered tissue. The second peak corresponded with that of newly synthesized transmitter and was thus identified as containing the recycled vesicles. Its normalized acetylcholine/proteoglycan ratio was lower than that of the first peak, which is consistent with earlier findings that recycled vesicles, before recovery, are only partially loaded with transmitter. However, as expected, the proportion of total vesicular proteoglycan and acetylcholine associated with the recycled vesicle fraction was very much lower in preparations derived from unstimulated or recovered tissue than in those from recently stimulated tissue.

The density and free water of cholinergic synaptic vesicles as a function of osmotic pressure.

Synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo marmorata are among the most uniform subcellular organelles known and are osmotically sensitive. Changes in density accompanying osmotic perturbation have enabled changes in water content to be calculated; when referred to a standard state of known volume and water content, fractional and absolute water contents could be calculated for the perturbed states and compared with the fractional free water content as measured by the glycerol space. Under hyperosmotic conditions, discrepancies were found between these two estimates, the glycerol space falling more rapidly than the water space predicted from the density change. This is attributed to a failure of glycerol to displace water imbibed by the membrane as it collapses round an aqueous core of decreasing volume. 'Reserve' vesicles obeyed a relationship between density, osmotic load and osmolality derived for a perfect osmometer, and independent estimates of fractional free water content under standard conditions and osmotic load were made. The former of these agreed well with the glycerol space under standard conditions and the latter agreed with previous estimates of the osmotic load using morphological and analytical data and an assumed activity coefficient of 0.65. Finally, it was possible to model the interconversion of reserve and recycling vesicles more accurately than in previous work.

Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling.

In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP2-type vesicles lysed at (mean + S.E. (number of experiments] 332 +/- 14 (4) mosM and 50% of VP1-type vesicles lysed at 573 +/- 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.