Enzymatic inactivation of enkephalin neurotransmitters in the spinal cord of the neonatal rat

Abstract

The possible involvement of enzymatic degradation in the inactivation of enkephalins in the spinal cord of neonatal rats was investigated electrophysiologically and biochemically. In an isolated spinal cord–saphenous nerve preparation, electrical stimulation of the saphenous nerve evoked a slow depolarization lasting 20–30 s of the ipsilateral L3 ventral root. This slow depolarization was depressed by a mixture of peptidase inhibitors, consisting of actinonin (10 μM), thiorphan (0.6 μM), bestatin (10 μM), arphamenine B (10 μM) and captopril (10 μM). Naloxone (0.5 μM) not only reversed this effect of the mixture of peptidase inhibitors but also potentiated the slow depolarization beyond the pre-control level. In an isolated spinal cord preparation, electrical stimulation of a lumbar dorsal root evoked a slow depolarization of the contralateral ventral root of the same segment. This slow depolarization was depressed by application of [Met 5]enkephalin in a dose dependent manner. This effect of [Met 5]enkephalin was markedly potentiated by addition of the mixture of peptidase inhibitors. Among the five peptidase inhibitors, actinonin, thiorphan or bestatin alone potentiated the depressant effect of [Met 5]enkephalin, whereas arphamenine B and captopril did not. Membrane fractions prepared from neonatal rat spinal cords showed degrading activities for [Met 5]- and [Leu 5]enkephalins and these activities were inhibited by the mixture of peptidase inhibitors. Among the five peptidase inhibitors, actinonin and thiorphan markedly inhibited the [Met 5]enkephalin-degrading activity while bestatin was less effective. Arphamenine B and captopril were ineffective. The present results suggest that enzymatic degradation by peptidases plays a role in the termination of the transmitter action of enkephalins in the neonatal rat spinal cord. The present results, together with our previous results on the enzymatic degradation of tachykinins in a study in which we used the same preparations, suggest that similar but distinct combinations of peptidases are involved in the inactivation of enkephalin and tachykinin neurotransmitters.