The rat brain P
2 fraction was suspended in Krebs Ringer bicarbonate buffer containing 20 μM bacitracin and incubated at 37°C for 10 min under an atmosphere of 95% O
2-5% CO
2. Incubation was terminated by centrifugation at 4°C and 10, 000×g for 10 min. The supernatant was designated as the S
1 fraction, and from the pellet, the S
2 to S
4 fractions were collected by repeated suspension, incubation, and centrifugation. The radioimmunoassays of each S fraction revealed the spontaneous release of [
5Met] and [
5Leu]-enkephalins at the ratio of 2 to 1. The peptide contents gradually decreased from S
1 to S
4, but the release tended to become constant in S
3 and S
4. Thus, the effects of some ions and drugs on the release were compared at the stage of obtaining the S
3 fraction. The release of both peptides were significantly stimulated in 50 mM KCl buffer, and the stimulatory effect appears to be dependent on Ca
2+ concentration. Veratrine and A23187 were also effective stimulants of the release. On the other hand, neither spontaneous nor K
+-stimulated release of enkephalins was affected by morphine (1 μM), naloxone (1 μM), kyotorphin (1 or 10 μM), and Li
+ (50 mM). Similar results were obtained with the release of
3H-noradrenaline taken up in vitro by the P
2 fraction. The usability of the P
2 fraction as an in vitro model for the study of stimulus-coupled release of enkephalins was discussed with some limitations found herein.
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