Potentiation of enkephalin action by peptidase inhibitors in rat locus ceruleus in vitro

Intracellular recordings were made from locus ceruleus (LC) neurons in slices of rat pons. It has been shown previously that opioids inhibit firing and hyperpolarize LC neurons by activating mu-opioid receptors. Concentration-response curves were constructed by measuring the hyperpolarization, or outward current at -60 mV, caused by various opioid agonists added to the superfusing solution. Peptidase inhibitors (kelatorphan, bestatin and thiorphan) each increased the potency of Met-enkephalin, Leu-enkephalin and metorphamide but had no effect of their own on the LC neurons. Kelatorphan (20 μM) produced a 7-fold shift to the left of the Met-enkephalin dose-response curve. Higher concentrations produced no further shift. Thiorphan (3 μM) and bestatin (20 μM) each had a smaller potentiating effect on the Met-enkephalin hyperpolarization. The effects of Tyr-D-Ala-Gly-MePhe-Gly-ol and noradrenaline were not potentiated by these peptidase inhibitors. Electrical stimulation produced an inhibitory synaptic potential that was prolonged by cocaine (10 μM) and blocked by the alpha-2 adrenoceptor antagonist idazoxan (1 EmM). After addition of idazoxan, hyperpolarizing synaptic potentials could be evoked even in the presence of kelatorphan (20 μM). These results indicate that peptide metabolism in the intact brain slice can account for a substantial decrease in the sensitivity of LC neurons to exogenously applied opioid peptides; even after peptidase inhibition, no evidence could be obtained for hyperpolarizing synaptic potentials due to the release of endogenous opioids.