Highly purified splenic nerve vesicles: Early post-mortem effects on norepinephrine content and pools

Summary

Isolated large dense-core vesicles from bovine splenic nerve can be prepared at a purity comparable to that of adrenomedullary vesicles. Improved methods coupled with a short post-mortem delay of ∼10 min, from the moment of sacrifice at the slaughterhouse to chilling the nerves, yield vesicles containing 11–12 μg norepinephrine (NE)/mg protein (range 5–18) at 80–90% purity. In the upper range, therefore, the vesicles contain 100 nmol NE/mg protein uncorrected for purity, which is more than an order of magnitude higher than published values for splenic nerves from other laboratories. The extrapolated estimate for contentin vivo is 200 nmol/mg protein.

The shorter post-mortem delay has revealed a second more labile pool of NE accounting for 20% of the vesicle NE content, and 50% if extrapolated to zero post-mortem delay. It has a half-life of 4–5 min for net NE loss at 30 °C and is relatively insensitive to the presence of ATP in a medium containing 0.5–1.0 μg 1-NE/ml. This rapid single exponential component is in addition to the well-known slower ATP-sensitive component, which was the only one found in earlier preparations with a 20–30 min post-mortem delay. Characteristics of the latter can be reproduced in the present preparation after an unavoidable slow slaughter, and by several other procedures designed to mimic the additional post-mortem delay.