Highly purified splenic nerve vesicles: Early post-mortem effects on ultrastructure

Summary

Electron microscopic analyses of whole pellet depths confirm the claim that large dense-core vesicles (750Å) can be isolated from bovine splenic nerve at a routine purity of 80–90%. After a minimal 10 min post-mortem delay at the slaughterhouse, essentially all vesicles in cold control preparations possess a homogeneous, finely granular, electron-dense matrix. This appearance is maintained after brief incubation with ATP at 30 °C, even though a newly discovered ATP-insensitive norepinephrine (NE) pool (20%) is rapidly and completely lost. Subsequent depletion of the remaining NE (80%) in the slower ATP-sensitive pool is paralleled by proportional decrements in vesicle matrix density.

In contrast, cold control vesicles in our earlier preparation with a 20–30 min post-mortem delay appear relatively depleted, but gain electron density after brief incubation with ATP at 30 °C. This effect can be duplicated in the present preparation by several procedures designed to mimic the additional post-mortem delay.

Ultrastructural events associated with NE depletion and vesicle degeneration begin with random dense granulation of the original finely granular matrix, followed by aggregation to form very electron-dense 200Å granules. The latter occur intravesicularly at first, but are released in increasing numbers as vesicles swell and rupture. Clusters of the 200Å granules give the appearance of dense cores both intra- and extravesicularly, particularly after certain fixation and staining procedures.