Synaptic mechanism of methionine-enkephalin uptake

The actions of classical neurotransmitters at the synapse are rapidly terminated either by re-uptake or by enzymatic degradation. Enkephalins, like other neurotransmitters, have been demonstrated to be degraded by active proteinases present in the brain. However, it has been generally assumed that an uptake process is not operant for met-enkephalin. To confirm or disprove this assumption, we examined the problem of met-enkephalin uptake in rat brain synaptosomes. Male rat brains were collected in ice-cold sucrose (0.32M) and homogenized, and by differential centrifugation we prepared P2 pellet. The pellet was then incubated with 3H-met-enkephalin, the reaction terminated by dilution, and the reaction mixture filtered under vacuum. (1) Lysing the synaptosomes in ice cold water before or after incubation with met-enkephalin separates out the synaptic vesicles. The post-incubation lysis showed a market amount of radioactivity in the supernatant which contained the vesicles. The vesicles may thus be the sites of accumulation of met-enkephalin. Pre-incubation lysis produced markedly less accumulation of radioactivity. (2) Ca++ at various concentrations was found to be an activator of met-enkephalin uptake. (3) The rate of accumulation of met-enkephalin was found to be temperature sensitive; 37 degrees greater than 25 degrees greater than 0 degrees. (4) The degree of uptake varied with different concentrations of met-enkephalin and with time. That it is the met-enkephalin taken up and not the degraded fraction was confirmed by RIA of the material extracted from the synaptosomes. The evidence provided here suggests that there is an uptake system for met-enkephalin.