Peptidases prevent mu-opioid receptor internalization in dorsal horn neurons by endogenously released opioids

To evaluate the effect of peptidases on mu-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and alpha-neoendorphin, but not endomorphins or beta-endorphin. The omission of any one inhibitor abolished Leu-enkephalin-induced internalization, indicating that all three peptidases degraded enkephalins. Amastatin preserved dynorphin A-induced internalization, and phosphoramidon, but not captopril, increased this effect, indicating that the effect of dynorphin A was prevented by aminopeptidases and neutral endopeptidase. Veratridine (30 microm) or 50 mm KCl produced MOR-1 internalization in the presence of peptidase inhibitors, but little or no internalization in their absence. These effects were attributed to opioid release, because they were abolished by the selective MOR antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) and were Ca(2+) dependent. The effect of veratridine was protected by phosphoramidon plus amastatin or captopril, but not by amastatin plus captopril or by phosphoramidon alone, indicating that released opioids are primarily cleaved by neutral endopeptidase, with a lesser involvement of aminopeptidases and dipeptidyl carboxypeptidase. Therefore, because the potencies of endomorphin-1 and endomorphin-2 to elicit internalization were unaffected by peptidase inhibitors, the opioids released by veratridine were not endomorphins. Confocal microscopy revealed that MOR-1-expressing neurons were in close proximity to terminals containing opioids with enkephalin-like sequences. These findings indicate that peptidases prevent the activation of extrasynaptic MOR-1 in dorsal horn neurons.