Mitsuhiko Yanagisawa

Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal cord‐tail preparation of the newborn rat.

1. The pharmacological profile of Spantide, [D‐Arg1, D‐Trp7,9, Leu11] substance P, as a substance P (SP) antagonist was examined in isolated spinal cords of newborn rats. Potential changes were recorded extracellularly from a lumbar ventral root (L1‐L5). Application of SP to the perfusion bath with a brief pressure pulse of 0.05‐0.8 s duration produced a dose‐dependent depolarization of the ventral root. Spantide in concentrations of 2‐16 microM depressed the depolarizing responses of the ventral root to SP in a concentration‐dependent manner. The log dose‐response curve of SP was shifted to the right in the presence of 16 microM‐Spantide by log 5. The responses to neurokinin A (NKA) and bombesin were similarly depressed by 16 microM‐Spantide whereas the responses to noradrenaline, gamma‐aminobutyric acid (GABA), neurotensin and thyrotrophin‐releasing hormone were not affected by 16 microM‐Spantide. 2. In an isolated spinal cord‐tail preparation of the newborn rat, brief pulse injection of capsaicin into the perfusion solution of the tail induced a depolarizing response in a lumbar ventral root (L3‐L5). This response probably represents a nociceptive C fibre reflex. 3. The capsaicin‐induced nociceptive reflex was markedly depressed by 16 microM‐Spantide and the reflex recovered its original amplitude and shape 30‐60 min after removal of Spantide. 4. The capsaicin‐induced nociceptive reflex was depressed by morphine (2 microM) and dynorphin (1‐13) (0.2 microM), and these effects were reversed by 1 microM‐naloxone. 5. In an isolated spinal cord preparation of the newborn rat, stimulation of a dorsal root with single or double shocks induced depolarizing responses of slow time course in both ipsilateral and contralateral ventral roots of the same segment. These slow depolarizing responses were also depressed by 16 microM‐Spantide. In contrast the monosynaptic reflex was not affected by 16 microM‐Spantide. 6. The present results suggest that SP and NKA are involved as neurotransmitters in the capsaicin‐induced nociceptive reflex in the isolated spinal cord‐tail preparation of the newborn rat.

The role of substance P as a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord.

1 In order to reveal the spinal reflexes involving the transmitter action of substance P (SP), the effects of capsaicin and an SP antagonist on the isolated spinal cord of the neonatal rat were studied. 2 When a single shock stimulus was given to a dorsal root (L3–L5) or a sciatic nerve, depolarizing responses of various time courses were recorded extracellularly from both ipsi‐ and contra‐lateral ventral roots of the corresponding segments. The reflex response recorded from the contralateral ventral root consisted of fast and slow components, which will be referred to as contralateral fast and slow ventral root potentials (v.r.ps). The latter contralateral slow v.r.p. had a time‐to‐peak of 2–5 s and lasted 10–30 s. 3 The threshold for the contralateral slow v.r.p. was about two times higher than that for the monosynaptic reflex, and it coincided with the threshold for activating the slow‐conducting afferent fibres. 4 The contralateral slow v.r.p. was abolished after the spinal cord was treated with capsaicin (1 μm for 30 min) in vitro. The contralateral slow v.r.p. was absent in the spinal cord derived from 4 day‐old rats that had received capsaicin (50 mg kg−1, s.c.) on the 2nd day of life. The contralateral fast v.r.p. and other reflexes of fast time courses remained unaltered after treatment with capsaicin in vitro or in vivo. 5 Administration of an SP antagonist, [d‐Arg1, d‐Pro2, d‐Trp7,9 Leu11]‐SP in concentrations of 5–16 μm depressed the contralateral slow v.r.p., but did not affect the monosynaptic reflex, the dorsal root potential and the contralateral fast v.r.p. [d‐Arg1, d‐Pro2, d‐Trp7,9, Leu11]‐SP (5 μm) markedly depressed the SP‐induced depolarizing response recorded from the ventral root whereas the responses to noradrenaline, 5‐hydroxytryptamine, neurotensin and thyrotrophin releasing hormone (TRH) were unaffected by the SP antagonist. The response of the ventral root to acetylcholine was slightly depressed by the antagonist. The SP antagonist at 5–10 μm did not exert any agonist action on the motoneurones. 6 The present results in conjunction with those of previous studies support the hypothesis that SP released from certain primary afferent fibres acts as a neurotransmitter, producing in dorsal horn neurones slow excitatory postsynaptic potentials which lead to the generation of the contralateral slow v.r.p.

Inhibitory effects of galanin on the isolated spinal cord of the newborn rat.

The effects of galanin, a 29-amino-acid peptide, on spinal reflexes were studied. In the isolated hemisected spinal cord of the newborn rat, galanin (0.1-5 microM) depressed the monosynaptic reflex that was induced by dorsal root stimulation and recorded from the corresponding ventral root. In the isolated spinal cord-tail preparation of the newborn rat, galanin (0.3-0.6 microM) depressed the nociceptive reflex that was induced by application of capsaicin to the tail and recorded from a lumbar ventral root. In both preparations the inhibitory effects of galanin were reversible and the full recovery of the reflexes was observed within 3-20 min after removal of the peptide. The mechanisms of action of galanin on the spinal reflexes and the physiological role of the peptide in the spinal cord are discussed.

Pharmacological properties of a C‐fibre response evoked by saphenous nerve stimulation in an isolated spinal cord‐nerve preparation of the newborn rat

1 An isolated spinal cord‐peripheral nerve preparation of the newborn rat was developed. In this preparation it is possible to record spinal reflexes from a lumbar ventral root in response to stimulation of the ipsilateral saphenous or obturator nerve. 2 Single shock, weak intensity stimulation of the saphenous nerve induced a fast conducted compound action potential in the L3 dorsal root and a fast depolarizing response in the ipsilateral L3 ventral root. As a stronger stimulus was applied to the saphenous nerve, a slowly conducted compound action potential appeared in the dorsal root and a slow depolarizing ventral root potential (v.r.p.) in the L3 ventral root. 3 Single shock stimulation of the obturator nerve induced a rapidly conducted compound action potential in the L3 dorsal root and monosynaptic and polysynaptic reflexes, with a fast time course, in the ipsilateral L3 ventral root. 4 The slow v.r.p. evoked by saphenous nerve stimulation was depressed by the tachykinin antagonist, [D‐Arg1, D‐Trp7,9, Leu11] substance P (spantide), 4–16 μm. The response recovered its original shape and size 30–60 min after the removal of this antagonist. 5 The saphenous nerve‐evoked slow v.r.p. was depressed by [Met5] enkephalin (0.1–1 μm), dynorphin (1–13)(0.2 μm) and morphine (1–2 μm), and these effects were reversed by naloxone (1 μm). 6 Two endogenous peptides, galanin (1–2 μm) and somatostatin (1–2.5 μm), inhibited the slow v.r.p. evoked by saphenous nerve stimulation, whereas another endogenous peptide, calcitonin gene‐related peptide (0.1‐0.5 μm), potentiated the slow v.r.p. The slow v.r.p. was also inhibited by γ‐aminobutyric acid (GABA, 20 μm) and muscimol (0.2 μm), and their effects were antagonized by bicuculline (1 μm). 7 The present results suggest that substance P and neurokinin A are involved in the saphenous nerve‐evoked C‐fibre response in the spinal cord of the newborn rat.

Tail-pinch method in vitro and the effects of some antinociceptive compounds.

Abstract An in vitro preparation for testing antinociceptive drugs is described. The preparation consists of isolated spinal cord, spinal nerve roots and functionally connected tail of the newborn rat. Noxious pressure stimulation given to the tail induced in a lumbar ventral root a depolarizing response of 1–2 mV lasting about 15–30 s, which is referred to as tail-pinch potential. Single shock stimulation of a lumbar dorsal root induced in the ipsilateral ventral root of the same segment monosynaptic and polysynaptic reflexes, which were followed by a depolarizing response lasting ca. 20 s. The latter slow response is reffered to as ipsilateral slow ventral root potential (VRP). Morphine, [Met 5 ]enkephalin [Leu 5 ]enkephalin and [D-Ala 2 ,Met 5 ]enkephalinamide depressed both the tail-pinch potential and the ipsilateral slow VRP, but did not exert any noticeable effect on the monosynaptic and polysynaptic reflexes. The effects of morphine and enkephalins were completely abolished by naloxone. Naloxone potentiated the tail-pinch potential and the ipsilateral slow VRP in the fresh preparations which had not been exposed to opiate compounds or enkephalins, suggesting that the endogenous enkephalinergic mechanism occurs in the spinal cord and modulates the incoming nociceptive signals. The present isolated spinal cord-tail preparation will be useful for studying actions of analgesic drugs and for screening compounds for analgesic effects.

Pain and neurotransmitters

Summary1.To study physiological roles of substance P (SP),γ-aminobutyric acid (GABA), enkephalins and other endogenous substances, we developed several kinds of isolated spinal cord preparations of newborn rats.2.In these preparations, various slow responses of spinal neurons evoked by stimulation of primary afferent C fibers were depressed by a tachykinin antagonist, spantide. These results together with many other lines of evidence suggest that SP and neurokinin A serve as pain transmitters in a subpopulation of primary afferent C fibers.3.Some C-fiber responses in various isolated spinal cord preparations were depressed by GABA, muscimol, and opioid peptides. In contrast, bicuculline (GABA antagonist) and naloxone (opioid antagonist) potentiated the “tail pinch potential,” i.e., a nociceptive response of the ventral root evoked by pinch stimulation of the tail in isolated spinal cord-tail preparation of the newborn rat. The latter results support the hypothesis that some primary afferents activate inhibitory spinal interneurons which release GABA and enkephalins as transmitters to modulate pain inputs.

Pharmacological profile of a tachykinin antagonist, spantide, as examined on rat spinal motoneurones

1 The pharmacological profile of a tachykinin antagonist, [d‐Arg1, d‐Trp7,9, Leu11] substance P (spantide), was studied on motoneurones of the isolated spinal cord of the newborn rat. For this purpose, potentials were recorded from a lumbar ventral root extracellularly and drugs were bath‐applied in the presence of tetrodotoxin (TTX). 2 Neurokinin A (NKA), a NK2‐receptor selective agonist, induced concentration‐dependent depolarizations, which were antagonized by spantide. Analyses of concentration‐response curves suggested a competitive type antagonism with a pA2 of 6.5. 3 Depolarizations induced by acetyl‐Arg6‐septide, a NK1‐receptor selective agonist, were also antagonized by spantide with a pA2 of 6.5. 4 Spantide (0.5–16 μm) had no depolarizing action on the ventral root in the presence of TTX. 5 Spantide antagonized the depolarizing action of substance P (SP) when SP was applied at low concentrations (0.1–0.3 μm) or by short duration pulses in artificial cerebrospinal fluid containing TTX, but much higher concentrations of spantide (4–10 μm) were needed to exert an antagonistic action against SP than against acetyl‐Arg6‐septide or NKA. 6 Thyrotrophin‐releasing hormone, l‐glutamate, GABA, and noradrenaline, also induced depolarizations of the ventral root in the presence of TTX but the responses to these agonists were not depressed by spantide (16 μm). 7 These results suggest that there is a subtype of tachykinin receptors on neonatal rat spinal motoneurones to which NKA, acetyl‐Arg6‐septide and spantide bind competitively with high affinity. The present results also suggest the existence on rat motoneurones of another class or other classes of tachykinin receptors that are less sensitive to the antagonistic action of spantide.

Identification by high-performance liquid chromatography of immunoreactive substance P released from isolated rat spinal cord ☆

An attempt was made to identify the immunoreactive substance P (SP) released from isolated rat spinal cords using reversed-phase high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA) for SP. Soaking the spinal cords of newborn rats in Krebs solution containing 90 mM K+ evoked a release of immunoreactive SP as well as of GABA and glycine in a calcium-dependent manner. Capsaicin also evoked a release of immunoreactive SP but not of GABA and glycine. The immunoreactive SP released from rat spinal cords by high K+ or capsaicin was analyzed by HPLC. A single peak was detected by RIA whose elution position coincided with that of synthetic SP.

The excitatory action of the newly-discovered mammalian tachykinins, neurokinin α and neurokinin β, on neurons of the isolated spinal cord of the newborn rat

The actions of two new mammalian tachykinins, neurokinin alpha and neurokinin beta, were examined using the isolated spinal cords of newborn rats. Depolarizing responses of spinal motoneurons were recorded extracellularly from the lumbar ventral root during application of neurokinin alpha or neurokinin beta at concentrations ranging from 3 X 10(-8) M to 10(-6) M. The potencies of various tachykinins in depolarizing the motoneurons showed the following order: physalaemin greater than neurokinin beta divided by kassinin divided by substance P greater than neurokinin alpha. When the synaptic transmission in the spinal cord was blocked by tetrodotoxin, the depolarizing action of neurokinin alpha and neurokinin beta was markedly reduced but not completely abolished. The depolarizing action of neurokinin alpha and neurokinin beta was depressed by a substance P antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP. The possibility that neurokinin alpha and neurokinin beta act as neurotransmitters in the mammalian spinal cord is discussed.

Substance P-Evoked release of GABA from isolated spinal cord of the newborn rat

Isolated spinal cords of newborn rats were perfused with artificial cerebrospinal fluid and the effects of substance P and its analogs on the release of endogenous GABA were examined. Application of substance P evoked a dose-dependent release of GABA from spinal cords. The threshold concentration of substance P for induction of a significant increase in the GABA release was 3 microM. The substance P-evoked GABA release was neither blocked by removal of Ca2+ from perfusion medium nor by tetrodotoxin. In contrast, the GABA release evoked by high K+ (90 mM) was abolished in Ca(2+)-free medium, and the GABA release evoked by veratridine (5 microM) was suppressed by tetrodotoxin (1 microM). A GABA uptake inhibitor, cis-4-hydroxynipecotic acid, markedly augmented the GABA release induced by high K+, but not that induced by substance P or veratridine. These results suggest the possibility that a carrier-mediated mechanism might be involved in the GABA release induced by substance P, as well as by veratridine, in the newborn rat spinal cord. Two N-terminal fragments of substance P, substance P free acid and substance P1-10 amide, as well as [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), evoked an increase in the GABA release, whereas substance P1-6, and a C-terminal fragment, substance P5-11 were inactive. Somatostatin and compound 48/80 also evoked a GABA release, which was independent of external Ca2+ and resistant to tetrodotoxin. [D-Pro4,D-Trp7,9,10]substance P4-11 (10-15 microM) inhibited the GABA release evoked by substance P, somatostatin and compound 48/80.(ABSTRACT TRUNCATED AT 250 WORDS)