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1 The pharmacological profile of GR71251, a new tachykinin receptor antagonist, and its effect on the responses evoked by stimulation of primary afferent fibres were studied in isolated spinal cord preparations of neonatal rats. Potential changes were recorded extracellularly from a lumbar ventral root (L3-L5).2 Bath-application of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) at 0.01-3 AM to the spinal cord induced depolarization of the ventral root in normal artificial cerebrospinal fluid (CSF). The NK, agonist, acetyl-Arg6-septide, and the NK3 agonist, senktide, at 0.01-3JaM, also had potent depolarizing actions whereas two NK2 agonists, P-Ala8NKA4-10 and Nle'"NKA410, showed little depolarizing effects at 1 llM.3 GR71251 (0.3-3 lAM) caused a rightward shift of the concentration-response curves for SP, acetylArg6-septide and NKA with pA2 values of 6.14, 6.75 or 6.70, respectively. The effects of GR71251 were readily reversible within 15-30 min after its removal. By contrast, GR71251 (1-5 jAM) had little effect on the depolarizing responses to NKB and senktide.4 GR71251 (1-3 JAM) did not depress the depolarizing responses to bombesin, neuromedin B and gastrin-releasing peptide in normal artificial CSF. 5 Application of capsaicin to the spinal cord induced a depolarizing response, which was partially depressed by GR71251 (3-l10pM).6 In the isolated spinal cord preparation, intense electrical stimulation of a dorsal root evoked a slow depolarizing response of the contralateral ventral root of the same segment. A similar slow ventral root depolarization was evoked by electrical stimulation of the ipsilateral saphenous nerve in an isolated spinal cord-saphenous nerve preparation. GR71251 (0.3-10 IAM) dose-dependently depressed these slow ventral root potentials. 7 In the spinal cord-peripheral nerve preparation, conditioning stimulation of the saphenous nerve evoked an inhibition of the muscle nerve-evoked monosynaptic reflex lasting about 20 s. The late part of the inhibition was markedly depressed by GR71251 (1-3 JAM). 8 The present results indicate that GR71251 is a potent and specific antagonist for tachykinin receptors in the spinal cord. The present study further provides evidence for the involvement of SP and NKA in the slow ventral root depolarization and the prolonged inhibition of monosynaptic reflex that are evoked by primary afferent stimulation.

1 The possible involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters was examined in the spinal cord of the neonatal rat. 2 The magnitude of substance P (SP)-or neurokinin A (NKA)-evoked depolarization of a lumbar ventral root in the isolated spinal cord preparation was increased by a mixture of peptidase inhibitors, consisting of actinonin (6 ILM), arphamenine B (6 fLM), bestatin (10 giM), captopril (10 JM) and thiorphan (0.31M). The mixture augmented the response to NKA more markedly than that to SP. 3 In the isolated spinal cord-cutaneous nerve preparation, the saphenous nerve-evoked slow depolarization of the L3 ventral root was augmented by the mixture of peptidase inhibitors in the presence of naloxone (0.5 JM) but not in the presence of both naloxone and a tachykinin receptor antagonist, GR71251 (51M). 4 Application of capsaicin (0.5 JiM) for 6 min to the spinal cord evoked an increase in the release of SP from the spinal cord. The amount of SP released was significantly augmented by the mixture of peptidase inhibitors. 5 Synaptic membrane fractions were prepared from neonatal rat spinal cords. These fractions showed degrading activities for SP and NKA and the activities were inhibited by the mixture of peptidase inhibitors. The degrading activity for NKA was higher than that for SP and the inhibitory effect of the mixture for NKA was more marked than that for SP. Although some other fractions obtained from homogenates of spinal cords showed higher degrading activities for SP, these activities were insensitive to the mixture of peptidase inhibitors. 6 Effects of individual peptidase inhibitors on the enzymatic degradation of SP and NKA by synaptic membrane fractions were examined. Thiorphan, actinonin and captopril inhibited SP degradation, while thiorphan and actinonin, but not captopril, inhibited NKA degradation. The potency of the inhibition of each peptidase inhibitor was lower than that of the mixture. 7 The present results suggest that enzymatic degradation is involved in the inactivation of tachykinin neurotransmitters in the spinal cord of the neonatal rat.

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In the rat spinal cord-saphenous nerve preparation, electrical stimulation of the saphenous nerve at C-fibre strength evoked a prolonged depolarization of the ipsilateral L3 ventral root (slow VRP). RP 67580 (0.1-1 JM) depressed the saphenous nerve-evoked slow VRP. In contrast, RP 68651 (0.3 JAM) had no effect on the slow VRP.