Rumiko Hosoki

Depression of primary afferent‐evoked responses by GR71251 in the isolated spinal cord of the neonatal rat

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 μm to the spinal cord induced depolarization of the ventral root in normal artificial cerebrospinal fluid (CSF). The NK1 agonist, acetyl‐Arg6‐septide, and the NK3 agonist, senktide, at 0.01–3 μm, also had potent depolarizing actions whereas two NK2 agonists, β‐Ala8NKA4–10 and Nle10NKA4–10, showed little depolarizing effects at 1 μm. 3 GR71251 (0.3–3 μm) caused a rightward shift of the concentration‐response curves for SP, acetyl‐Arg6‐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 μm) had little effect on the depolarizing responses to NKB and senktide. 4 GR71251 (1–3 μm) 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 (3–10 μm). 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 μm) 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 μm). 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.

Pharmacological characterization of GR82334, a tachykinin NK1 receptor antagonist, in the isolated spinal cord of the neonatal rat.

Pharmacological characteristics of [D-Pro9,[spiro-gamma-lactam]Leu10,Trp11]physalaemin-(1-11) (GR82334), a tachykinin NK1 receptor antagonist, and its effects on slow depolarizing responses of lumbar ventral roots evoked by primary afferent stimulation were examined in isolated spinal cord preparations of neonatal rats. GR82334 (1-3 microM) caused dose-dependent rightward shifts of the concentration-response curves for substance P, substance P methyl ester, delta-aminovaleryl [Pro9,N-Me-Leu10]substance P-(7-11) (GR73632) and neurokinin A in normal artificial cerebrospinal fluid and those for substance P methyl ester, GR73632 and neurokinin A in the presence of tetrodotoxin. GR82334 (10 microM) did not evoke gamma-aminobutyric acid (GABA) release from spinal cords of neonatal rats, whereas [D-Pro9,[spiro-gamma-lactam] Leu10,Trp11]substance P (GR71251), another tachykinin NK1 receptor antagonist, induced a significant increase in GABA release. GR82334 (1-3 microM) markedly depressed the slow depolarizing response of ventral roots, referred to as slow ventral root potential, evoked by the stimulation of the contralateral dorsal root or the ipsilateral saphenous nerve. In contrast, cyclo[Gln,Trp,Phe,Gly,Leu,Met] (L-659,877, 1 microM), a selective tachykinin NK2 receptor antagonist, did not depress the saphenous nerve-evoked slow ventral root potential and did not antagonize the action of neurokinin A to induce ventral root depolarization. The present results provide further evidence for the involvement of substance P, neurokinin A and tachykinin NK1 receptors in the primary afferent-evoked slow ventral root potentials.

Effects of RP 67580, a tachykinin NK1 receptor antagonist, on a primary afferent-evoked response of ventral roots in the neonatal rat spinal cord.

1 The pharmacological characteristics of RP 67580, a non‐peptide tachykinin NK1 receptor antagonist, and its effects on a reflex response evoked by stimulation of primary afferent fibres, were examined in isolated neonatal spinal cord preparations of the rat. Potentials were recorded extracellularly from a lumbar ventral root and drugs were bath‐applied in normal artificial cerebrospinal fluid (CSF) or in the presence of tetrodotoxin (TTX). 2 In normal artificial CSF, RP 67580 (0.1‐0.3 μm) caused rightward shifts of the concentration‐response curves for substance P (SP), neurokinin A (NKA) and substance P methyl ester (SPOMe), an NK1selective agonist, with pA2 values of 7.25, 7.47 and 7.49, respectively. 3 In the presence of TTX (0.3 μm), RP 67580 also caused rightward shifts of the concentration‐response curves for SPOMe and NKA. The pA2 value of RP 67580 against SPOMe (6.75) was significantly lower than that against NKA (7.22). RP 67580 (0.3‐1 μm) did not cause a clear parallel shift of the concentration‐reponse curves for SP, and it depressed the depolarizations induced by low concentrations of SP, but slightly potentiated those induced by high concentrations of SP. 4 RP 67580 (1 μm) did not depress the depolarizing responses to bombesin, 1–glutamate, γ‐aminobutyric acid (GABA), thyrotropin‐releasing hormone and muscarine. RP 67580 (1 μm), however, depressed the response to acetylcholine in the presence of atropine and the response to nicotine. RP 68651 (1 μm), the enantiomer of RP 67580 devoid of activity at tachykinin NK1 receptors, also depressed the response to acetylcholine in the presence of atropine. 5 RP 67580 (1 μm) did not induce GABA release from the rat spinal cord. 6 In the neonatal gerbil spinal cord, the antagonist effects of RP 67580 (0.3‐1 μm) against SPOMe were much less potent than in the neonatal rat spinal cord. 7 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 μm) depressed the saphenous nerve‐evoked slow VRP. In contrast, RP 68651 (0.3 μm) had no effect on the slow VRP. 8 The results of the present study indicate that RP 67580 acts as a high affinity NK1 receptor antagonist in the neonatal rat spinal cord, although it also possesses an antinicotinic action. This study further suggests the existence of a subpopulation of tachykinin NK1 receptors that are activated by NKA and SPOMe, as well as by low concentrations of SP, and are sensitive to the antagonist action of RP 67580 in the neonatal rat spinal cord. This study also provides further evidence for the involvement of SP and NKA in the slow VRP evoked by C‐fibre stimulation in the neonatal rat spinal cord.

Pharmacological profiles of new orally active nonpeptide tachykinin NK1 receptor antagonists

Pharmacological profiles of new orally active amide-based tachykinin NK1 receptor antagonists, N-[3,5-bis(trifluoromethyl)benzyl]-5-(4-fluorophenyl)-7,8-dihydro-N,7-di methyl-8-oxo-1,7-naphthyridine-6-carboxamide (referred to as compound I) and two related compounds (compounds II and III), were compared with that of (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994), another nonpeptide tachykinin NK1 receptor antagonist. Compounds I, II, III and CP-99,994 caused parallel rightward shifts of the concentration-response curve of substance P in the guinea-pig ileum pretreated with atropine, mepyramine and indomethacin, with the pA2 values of 8.70, 7.56, 8.41 and 8.27, respectively. These antagonists did not alter the concentration-response curve of acetylcholine in the guinea-pig ileum nor that of neurokinin A in the rat vas deferens. Furthermore, contractile responses to senktide of the rat portal vein were not affected by these antagonists. In the isolated neonatal gerbil spinal cord pretreated with tetrodotoxin, substance P produced a dose-dependent depolarization of ventral roots. Compounds I, II, III and CP-99,994 caused parallel rightward shifts of the concentration-response curve of substance P in the spinal cord with the pA2 values of 7.07, 5.93, 6.40 and 7.26, respectively. In contrast, these antagonists did not affect the concentration-response curve of L-glutamate. These results suggest that compounds I, II and III are selective antagonists for tachykinin NK1 receptor both in peripheral tissues and the central nervous system.

Involvement of enzymatic degradation in the inactivation of tachykinin neurotransmitters in neonatal rat spinal cord

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 μm), arphamenine B (6 μm), bestatin (10 μm), captopril (10 μm) and thiorphan (0.3 μm). 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 μm) but not in the presence of both naloxone and a tachykinin receptor antagonist, GR71251 (5 μm). 4 Application of capsaicin (0.5 μm) 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.

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

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 microM), thiorphan (0.6 microM), bestatin (10 microM), arphamenine B (10 microM) and captopril (10 microM). Naloxone (0.5 microM) 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 [Met5]enkephalin in a dose dependent manner. This effect of [Met5]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 [Met5]enkephalin, whereas arphamenine B and captopril did not. Membrane fractions prepared from neonatal rat spinal cords showed degrading activities for [Met5]- and [Leu5]enkephalins and these activities were inhibited by the mixture of peptidase inhibitors. Among the five peptidase inhibitors, actinonin and thiorphan markedly inhibited the [Met5]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.

Enzymatic inactivation of tachykinin neurotransmitters in the isolated spinal cord of the newborn rat

A mixture of peptidase inhibitors increased the magnitude of the saphenous nerve-evoked slow depolarization of a lumbar ventral root and prolonged the similarly evoked inhibition of monosynaptic reflex (MSR) in the isolated spinal cord of the newborn rat in the presence of naloxone. The saphenous nerve-evoked MSR inhibition was curtailed by a tachykinin antagonist, GR71251, and after the treatment with GR71251, the peptidase inhibitor mixture no more prolonged the MSR inhibition. The present results suggest that enzymatic degradation plays a role in the termination of action of tachykinins released from primary afferents in the newborn rat spinal cord. The results provide a further support for the notion that tachykinins serve as neurotransmitters in the spinal cord of the newborn rat.

Tachykinin receptors on motoneurons in the spinal cords of neonatal rats, gerbils and hamsters

As a step to clarify the profiles of tachykinin receptors in the mammalian central nervous system, we examined the effects of various tachykinin receptor agonists and antagonists on motoneurons in isolated spinal cord preparations from rats, gerbils and hamsters. After treatment with tetrodotoxin, potential changes were recorded extracellularly from lumbar ventral roots at 27 degrees C. Bath-application of tachykinin NK1, NK3 receptor agonists produced depolarizing responses of ventral roots. In contrast, selective NK2 agonists exerted no or only marginal depolarizing action. Neurokinin A (NKA), however, exerted a distinct depolarizing action on motoneurons. Tachykinin NK1 receptor antagonists antagonized the actions of SPOMe and NKA in a competitive manner. The present results suggest that tachykinin NK1 and NK3 receptors are present on spinal motoneurons of newborn rats, gerbils and hamsters, and that NKA acts on the NK1 receptors.

Pharmacological Characterization of Receptors in the Spinal Cord of the Newborn Rat

The mammalian central nervous system (CNS) contains a considerable amount of neurokinin A (NKA) (Kanazawa et al., 1984; Arai and Emson, 1986; Takano et al., 1986; Tateishi et al., 1989). In contrast, blot-hybridization and RNase-protection analyses detected substantial amounts of mRNAs for NK1 and NK3 receptors, whereas mRNA for NK2 receptors was undetectable in the rat CNS (Tsuchida et al., 1990). This constitutes a remarkable transmitter-receptor mismatch, since NKA is generally assumed to be an endogenous ligand for the NK2 receptor. Does NKA act on NKl or NK3 receptors? In several places in the CNS, a definite excitatory action of NKA was observed (Matsuto et al., 1984; Innis et al., 1985; Kalivas et al., 1985; Xu et al., 1991). Furthermore, autoradiographic studies detected NKA binding sites in some regions in the CNS of the rat, e.g., cerebral cortex, solitary nucleus, and substantia gelatinosa of the spinal cord (Mantyh et al., 1984b, Mantyh et al., 1989; Quirion and Dam, 1985). These facts suggest the existence of tachykinin receptors responding to NKA.