Jian-Zhong Guo

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.

Subtypes of tachykinin receptors on tonic and phasic neurones in coeliac ganglion of the guinea-pig

1 Intracellular recording techniques were used to investigate the characteristics of tachykinin receptors and their subtypes in tonic and phasic neurones, which constituted two major neuronal populations in the coeliac ganglion of the guinea‐pig. 2 In 95% of phasic neurones a long‐lasting after‐hyperpolarization (LAH), 5–8s in duration and 10–20 mV in amplitude, was observed following action potentials evoked by passing a train of depolarizing current pulses into the neurones. In contrast, LAH was observed in only 4% of tonic neurones. 3 In most tonic neurones, substance P (SP), neurokinin A (NKA) and senktide induced depolarizations, whereas in phasic neurones they usually inhibited LAH but rarely induced depolarization. 4 Tonic and phasic neurones were further classified into three groups based on their responses (depolarization for tonic neurones and LAH inhibition for phasic neurones) to these tachykinin receptor agonists: (1) neurones responsive to SP, NKA and senktide (71–78%); (2) those responsive to senktide but not to SP and NKA (12–23%) and (3) those not responsive to any of the three agonists (7–11%). 5 GR71251 (5 μM), an NK1‐selective tachykinin receptor antagonist, depressed the depolarization in tonic neurones and the LAH inhibition in phasic neurones induced by SP and NKA, but not those induced by senktide. 6 Selective NK2 receptor agonists, [Nle10]NKA4‐10, [β‐Ala8]NKA4‐10 and GR64349, were without effect in both tonic and phasic neurones. Furthermore, an NK2 receptor antagonist, L659,877, did not inhibit the depolarization induced by NKA, SP or senktide in tonic neurones. 7 It is suggested that NK1 and NK3 receptors are present on a large proportion of coeliac ganglion neurones. In tonic neurones both subtypes of tachykinin receptors are coupled to membrane depolarization, whereas in phasic neurones activation of these receptors leads to inhibition of LAH. The present study also suggests that NKA evokes the depolarization in tonic neurones and the LAH inhibition in phasic neurones via NK1, but not NK2 receptors.

Tachykininergic synaptic transmission in the coeliac ganglion of the guinea‐pig

1 The responses of coeliac ganglion neurones of the guinea‐pig to electrical stimulation of the mesenteric nerves and applications of tachykinin receptor agonists were investigated by use of intracellular recording techniques. 2 Ganglion neurones were classified into three groups based on firing patterns in response to a depolarizing current pulse: phasic (38% of the population), tonic (39%) and atypical (23%). In the majority of phasic neurones (91%) a long after‐hyperpolarization (LAH) lasting 5–8 s followed action potentials induced by a train of depolarizing current pulses. In contrast, LAH was rarely observed in tonic neurones (5%). 3 In most of tonic neurones (90%) slow excitatory post‐synaptic potentials (e.p.s.ps) lasting 3–10 min were evoked by repetitive electrical stimulation of the mesenteric nerves. Prolonged depolarizations were also evoked in most tonic neurones by applications of substance P (SP), neurokinin A (NKA) or senktide, a tachykinin NK3 receptor agonist. 4 In most of phasic neurones (73%), mesenteric nerve stimulation did not induce an obvious depolarization but induced a prolonged inhibition of LAH lasting 3–10 min. Bath‐applied tachykinin receptor agonists similarly induced an inhibition of LAH without causing depolarization in most of the phasic neurones. 5 GR71251 (5 μm), a tachykinin NK1 receptor antagonist, partially depressed the nerve‐evoked slow e.p.s.ps in tonic neurones and the nerve‐evoked LAH inhibition in phasic neurones. 6 Capsaicin (0.1–5 μm) induced a prolonged depolarization in tonic neurones and an inhibition of LAH in phasic neurones. 7 A mixture of peptidase inhibitors potentiated the depolarization and the LAH inhibition evoked by nerve stimulation, SP and NKA, but not those evoked by senktide. 8 It is concluded that tonic neurones respond to repetitive mesenteric nerve stimulation preferentially with slow e.p.s.ps and that phasic neurones respond preferentially with LAH inhibition. The present study further suggests that SP and NKA, released from axon collaterals of primary afferent neurones, produce slow e.p.s.ps in tonic neurones and the LAH inhibition in phasic neurones via NK1 receptors.

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.

Effects of a tachykinin NK3 receptor antagonist, SR 142801, studied in isolated neonatal rat spinal cord

Effects of a nonpeptide tachykinin NK3 receptor antagonist, SR 142801, were studied in the isolated spinal cord preparation of the neonatal rat. Potential changes were recorded extracellularly from a lumbar ventral root. Bath-application of neurokinin B induced a dose-dependent depolarization of the ventral root. SR 142801 caused rightward shifts of the concentration-response curve for neurokinin B with pA2 of 6.57, but did not affect the depolarizing responses to other agonists. Stimulation of a dorsal root evoked in the ipsilateral ventral root of the same segment monosynaptic and polysynaptic reflexes of fast time course which were followed by a slow depolarization (ipsilateral slow ventral root potential). SR 142801 depressed the ipsilateral slow ventral root potential. The present results indicate that SR 142801 is a specific antagonist for tachykinin NK3 receptors in the spinal cord and suggest that NK3 receptors are involved in primary afferent-evoked nociceptive responses of spinal neurones.

Involvement of NK1 receptors in synaptic transmission in the guinea pig coeliac ganglion

Using intracellular recording techniques, we examined the effects of tachykinin receptor agonists and antagonists on electrophysiologically identified tonic neurons of the isolated guinea pig coeliac ganglion. In most of the tonic neurons, substance P (SP), neurokinin A (NKA) and/or senktide induced a depolarization. The effects of SP and NKA were blocked by the NK1-selective antagonist, GR71251 (5 microM), but not by the NK2-selective antagonist, L659,877 (10 microM), whereas the effect of senktide was not affected by these antagonists. The NK1-selective agonists, [Sar9,Met(O)2(11)]SP and SP methyl ester, and the NK3-selective agonist, [MePhe7]neurokinin B, also evoked depolarizations in tonic neurons. By contrast, the NK2-selective agonists, [Nle10]NKA4-10, [beta-Ala8]NKA4-10 and GR64349, at 1 microM each, did not evoke any significant depolarizing response. Repetitive electrical stimulation of the mesenteric nerves induced slow excitatory postsynaptic potentials (EPSPs) in the majority of tonic neurons, which were depressed by GR71251 (5 microM). These results suggest that NK1 and NK3 receptors but not NK2 receptors are involved in the tachykinin-induced depolarization of tonic neurons, and that the NKA-induced response is due to the activation of NK1 receptors. This study also suggests the involvement of NK1 receptors in the slow EPSPs in tonic neurons.

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.

309 Involvement of NK1 receptors in tachykininergic synaptic transmission in the coeliac ganglion of the guinea pig

1. The responses of coeliac ganglion neurones of the guinea-pig to electrical stimulation of the mesenteric nerves and applications of tachykinin receptor agonists were investigated by use of intracellular recording techniques. 2. Ganglion neurones were classified into three groups based on firing patterns in response to a depolarizing current pulse: phasic (38% of the population), tonic (39%) and atypical (23%). In the majority of phasic neurones (91%) a long after-hyperpolarization (LAH) lasting 5-8 s followed action potentials induced by a train of depolarizing current pulses. In contrast, LAH was rarely observed in tonic neurones (5%). 3. In most of tonic neurones (90%) slow excitatory post-synaptic potentials (e.p.s.ps) lasting 3-10 min were evoked by repetitive electrical stimulation of the mesenteric nerves. Prolonged depolarizations were also evoked in most tonic neurones by applications of substance P (SP), neurokinin A (NKA) or senktide, a tachykinin NK3 receptor agonist. 4. In most of phasic neurones (73%), mesenteric nerve stimulation did not induce an obvious depolarization but induced a prolonged inhibition of LAH lasting 3-10 min. Bath-applied tachykinin receptor agonists similarly induced an inhibition of LAH without causing depolarization in most of the phasic neurones. 5. GR 71251 (5 microM), a tachykinin NK1 receptor antagonist, partially depressed the nerve-evoked slow e.p.s.ps in tonic neurones and the nerve-evoked LAH inhibition in phasic neurones. 6. Capsaicin (0.1-5 microM) induced a prolonged depolarization in tonic neurones and an inhibition of LAH in phasic neurones. 7. A mixture of peptidase inhibitors potentiated the depolarization and the LAH inhibition evoked by nerve stimulation, SP and NKA, but not those evoked by senktide. 8. It is concluded that tonic neurones respond to repetitive mesenteric nerve stimulation preferentially with slow e.p.s.ps and that phasic neurones respond preferentially with LAH inhibition. The present study further suggests that SP and NKA, released from axon collaterals of primary afferent neurones, produce slow e.p.s.ps in tonic neurones and the LAH inhibition in phasic neurones via NK1 receptors.