Inhibition of enkephalin degrading enzymes by metal chelating reagents

The N-phosphorylated dipeptide K2PO3-Leu-Phe (P-Leu-Phe) and the mercaptoacetylated dipeptides SHCH2CO-Leu-Phe and SHCH2CO-Phe-Leu are potent inhibitors of the enkephalin carboxydipeptidase (enkephalinase) activity from rat striatal synaptosomal membranes; IC50, 0.3 nM, 15 nM, and 70 nM, respectively. These metal chelating compounds also inhibit the enkephalin degrading aminopeptidase activity present in the soluble fraction of rat striatum, although higher concentrations of inhibitors are needed; IC50, 100 microM, 80 microM and 150 microM, respectively. The binding of Leu-enkephalin (Leu-Enk) to rat striatal synaptosomal membranes is enhanced when the enkephalinase activity or the aminopeptidase activity present in these membranes is inhibited. Higher binding is obtained when both enzymes are inhibited. Similarly, the inhibition of these activities in rat striatal slices, using either puromycin or secobarbital which inhibit the aminopeptidase and enkephalinase, respectively, increases the amount of enkephalin recovered following K+ depolarization by about 100 percent. P-Leu-Phe at 1 mM, a concentration which inhibits both enzymatic activities, increased the amount of enkephalin recovered by 10-fold. The intracerebroventricular administration of 60 micrograms P-Leu-Phe with 200 micrograms of Leu-Enk induced analgesia in 4 out of 5 rats while Leu-Enk alone was not effective.