Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides

Abstract

The contents of [Met5]-enkephalin-Arg6-Phe7 (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37 degrees C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enk-hydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu5]-enkephalin (leu-enk), alpha- and beta-neoendorphins (alpha- and beta-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: alpha-neoend, beta-neoend, and dyn B.

Effect of peptidases on the ability of exogenous and endogenous neurokinins to produce neurokinin 1 receptor internalization in the rat spinal cord

“…NEN, DCP and aminopeptidases also efficiently degrade opioid peptides (Guyon et al, 1979;Chou et al, 1984;Yaksh & Chipkin, 1989;Hiranuma et al, 1998;Roques, 2000). Indeed, we found that inhibitors of NEN, DEC and aminopeptidase N increased 10 and 100 times, respectively, the potencies of dynorphin A and Leu-enkephalin to produce m-opioid receptor internalization (Song & Marvizon, 2003).…”

“…Inhibitors of NEN and DCP have been found to increase the release of SP (Duggan et al, 1992), and are routinely used to protect released SP against degradation (Malcangio & Bowery, 1993;Malcangio et al, 1997;Lever & Malcangio, 2002). Thiorphan and captopril were both used at 10 mM, a concentration at which they completely protect opioid peptides against degradation (Suzuki et al, 1997;Hiranuma et al, 1998;Song & Marvizon, 2003) and increase responses to SP and NKA in the spinal cord (Suzuki et al, 1994). Although some…”

Noxious mechanical stimulation evokes the segmental release of opioid peptides that induce μ-opioid receptor internalization in the presence of peptidase inhibitors

“…Another study (Kishioka et al, 1994) found that peptidase inhibitors substantially increased the analgesia produced by Metenkephalin, dynorphin or electroacupuncture. The high susceptibility of enkephalins (Guyon et al, 1979;Hiranuma et al, 1998b) and dynorphins (Hiranuma et al, 1998a;Numata et al, 1988) to peptidase degradation is well-known, and prompted the development of peptidase inhibitors as analgesics (Noble et al, 1992a;Noble et al, 1992b;Noble et al, 1997).…”

“…The intrathecal catheter was used to inject a mixture of peptidase inhibitors (at doses of 100 nmol each) onto the lumbar spinal cord. Peptidase inhibitors were amastatin, captopril and phosphoramidon, which inhibit, respectively, aminopeptidases, dipeptidyl carboxypeptidase and neutral endopeptidase, the three enzymes that cleave opioids in the spinal cord (Guyon et al, 1979;Hiranuma et al, 1998a;Hiranuma et al, 1998b;Numata et al, 1988;Song and Marvizon, 2003a). Noxious stimuli were applied 5 min after the intrathecal injection.…”

Great Increase in Antinociceptive Potency of [Leu5]Enkephalin After Peptidase Inhibition

“…It has been shown that when [Leu 5 ]enkephalin (LE) is incubated with an ileal or striatal membrane fraction for 60 min at 37°C in the presence of three peptidase inhibitors (PIs), amastatin (an aminopeptidase inhibitor), captopril (a dipeptidyl carboxypeptidase inhibitor), and phosphoramidon (an endopeptidase-24.11 inhibitor), approximately 98% of LE remains intact, while in the absence of the PI, the LE is completely hydrolyzed after incubation (1). This shows that LE is hydrolyzed, at least in the cerebral membrane preparation, by only three types of membrane-bound enzymes: amastatin-sensitive aminopeptidase(s) (AsA), captopril-sensitive dipeptidyl carboxypeptidase I (angiotensin I-converting enzyme, kininase II, EC 3.4.15.1) (CsD), and phosphoramidonsensitive endopeptidase-24.11 ("enkephalinase", EC 3.4.24.11) (PsE).…”