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 microM 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 microM, also had potent depolarizing actions whereas two NK2 agonists, beta-Ala8NKA4-10 and Nle10NKA4-10, showed little depolarizing effects at 1 microM. 3. GR71251 (0.3-3 microM) 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 microM) had little effect on the depolarizing responses to NKB and senktide. 4. GR71251 (1-3 microM) 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 microM). 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 microM) 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 microM) or by short duration pulses in artificial cerebrospinal fluid containing TTX, but much higher concentrations of spantide (4-10 microM) 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 microM). 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 microM) caused rightward shifts of the concentration-response curves for substance P (SP), neurokinin A (NKA) and substance P methyl ester (SPOMe), an NK1-selective agonist, with pA2 values of 7.25, 7.47 and 7.49, respectively. 3. In the presence of TTX (0.3 microM), 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 microM) did not cause a clear parallel shift of the concentration-response 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 microM) did not depress the depolarizing responses to bombesin, L--glutamate, gamma-aminobutyric acid (GABA), thyrotropin-releasing hormone and muscarine. RP 67580 (1 microM), however, depressed the response to acetylcholine in the presence of atropine and the response to nicotine. RP 68651 (1 microM), the enantiomer of RP 67580 devoid of activity at tachykinin NK1 receptors, also depressed the response to acetylcholine in the presence of atropine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Functional characterization of tachykinin NK1 receptors in the mouse uterus

1. Contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of oestrogen-treated mice. 2. In the presence of thiorphan (3 microM), captopril (10 microM), and bestatin (10 microM), substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-related contractions of uterine preparations. The order of potency was SP > or =NKA>NKB. 3. Neither atropine (0.1 microM) nor l-NOLA (100 microM), nor indomethacin (10 microM) alone or in combination with either ranitidine (10 microM) or mepyramine (10 microM), affected responses to SP. These findings indicate that SP actions are not mediated or modulated through the release of acetylcholine, nitric oxide, prostanoids or histamine. 4. In the presence of peptidase inhibitors, the tachykinin NK(1) receptor-selective agonist [Sar(9)Met(O(2))(11)]SP, produced a concentration-dependent contractile effect. The tachykinin NK(2) and NK(3) receptor-selective agonists [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) and [MePhe(7)]NKB were relatively inactive. The potencies of SP analogues in which Glu replaced Gln(5) and/or Gln(6) were similar to that of SP. 5. The tachykinin NK(1) receptor-selective antagonist, SR140333 (10 nM), alone or combined with the tachykinin NK(2) receptor-selective antagonist, SR48968 (10 nM), shifted log concentration curves to SP, NKA and NKB to the right. SR140333 (10 nM) reduced the effect of [Sar(9)Met(O(2))(11)]SP. SR48968 did not affect responses to SP or [Sar(9)Met(O(2))(11)]SP, but reduced the effect of higher concentrations of NKA and shifted the log concentration-response curve to NKB to the right. The tachykinin NK(3) receptor-selective antagonist, SR 142801 (0.3 microM), had little effect on responses to SP and NKB. 6. We conclude that the tachykinin NK(1) receptor mediates contractile effects of SP, NKA and NKB and [Sar(9)Met(O(2))(11)]SP in myometrium from the oestrogen-primed mouse. The tachykinin NK(2) receptor may also participate in the responses to NKA and NKB.     

Substance P enhances the release of endogenous serotonin from rat ventral spinal cord

The effect of the tachykinin neuropeptides, substance P (SP), neurokinin A (NKA) and the neurokinin B (NKB) receptor agonist, senktide, on the potassium-evoked release of endogenous serotonin (5-hydroxytryptamine, 5-HT) was investigated in superfused tissue slices of rat ventral spinal cord, where 5-HT is known to coexist with SP. Endogenous 5-HT was assayed by HPLC with electrochemical detection. The evoked release of 5-HT was significantly enhanced by 10(-4) M SP (190% increase) and 10(-5) M SP (74% increase) but not by 10(-6) M SP, NKA (10(-5) and 10(-4) M) and senktide (10(-5) and 10(-4) M) had no significant effect on the 5-HT release. The results suggest that, in the rat ventral spinal cord, where most of the 5-HT and SP is stored in the same nerve endings. SP but not NKA nor NKB potentiates the evoked release of 5-HT in a dose-dependent manner.     

Effects of subchronic treatment with imipramine, zimelidine and alaproclate on regional tissue levels of substance P- and neurokinin A/neurokinin B-like immunoreactivity in the brain and spinal cord of the rat

The effects of subchronic (14 day) treatment with the inhibitors at the uptake of monoamines, zimelidine, alaproclate and imipramine, on regional levels of substance P (SP) and other tachykinins in tissue in the central nervous system of the rat were studied by radioimmunoassay. In the ventral spinal cord, in which substance P is known to exist together with 5-hydroxytryptamine (5-HT), in the terminals of descending neurones, treatment with the selective inhibitors of the uptake of 5-HT zimelidine (2 X 10 mumol/kg p.o.) or alaproclate (2 X 10 mumol/kg or 2 X 20 mumol/kg p.o.), increased the level of substance P-like immunoreactivity (SP-LI). The effect of alaproclate appeared to be dose-dependent. After treatment with imipramine (2 X 10 mumol/kg p.o.) only a tendency to increased levels of substance P-like immunoreactivity spinal cord was seen. Treatment with alaproclate, at the highest dose level, also elevated the concentration of neurokinin A/neurokinin B-like immunoreactivity (NKA/NKB-LI) in the ventral spinal cord. In the frontal cortex, in which separate monoaminergic and tachykinin-containing neurones interact, treatment with imipramine reduced the levels of SP-LI and NKA/NKB-LI, while treatment with alaproclate had the opposite effect. In the periaqueductal grey matter, treatment with zimelidine and alaproclate increased the levels of SP-LI and NKA/NKB-LI, while treatment with imipramine increased only the level of NKA/NKB-LI. In conclusion, subchronic treatment of rats with inhibitors of the uptake of monoamines induced changes in levels of tachykinin in frontal cortex, periaqueductal grey and spinal cord. The selective inhibitors of the uptake zimelidine and alaproclate, had similar effects on levels of tachykinin, while the inhibitor of the uptake of 5-HT and noradrenaline, imipramine induced changes in the frontal cortex, which were qualitatively different from the effects of zimelidine and alaproclate. Furthermore, the levels of different tachykinins were not always changed in parallel by the same treatment.     

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), 5-8 s 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 microM), 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, [beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)     

The excitatory and inhibitory modulation of primary afferent fibre-evoked responses of ventral roots in the neonatal rat spinal cord exerted by nitric oxide.

1. We investigated the role of nitric oxide (NO) in modulating spinal synaptic responses evoked by electrical and noxious sensory stimuli in the neonatal rat spinal cord in vitro. 2. Potentials were recorded extracellularly from a ventral root (L3-L5) of the isolated spinal cord preparation or spinal cord-saphenous nerve-skin preparation of 0- to 2-day-old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or by noxious skin stimulation. 3. In the spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced mono-synaptic reflex followed by a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of NO gas-containing medium (10(-4)- 10(-2) dilution of saturated medium) and NO donors, 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC12, 3-300 microM), S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 3-300 microM) and S-nitroso-L-glutathione (GSNO, 3-300 microM), produced an inhibition of the slow VRP and a depolarization of ventral roots. Another NO donor, 3-morpholinosydononimine (SIN-1, 30-300 microM), also depressed the slow VRP but did not depolarize ventral roots. These agents did not affect the mono-synaptic reflex. 4. In the spinal cord-saphenous nerve-skin preparation, application of capsaicin (0.1-0.2 microM) to skin evoked a slow depolarizing response of the L3 ventral root. This slow VRP was depressed by NOC12 (10-300 microM) and SIN-1 (100-300 microM). When the concentration of NOC12 was increased to 1 mM, spontaneous synaptic activities were augmented and the depressant effect of NOC12 on the slow VRP became less pronounced. 5. A NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide( carboxy- PTIO, 100-300 microM) prevented the depressant effect on the dorsal root-evoked slow VRP and ventral root depolarizing effects of NO donors. Carboxy-PTIO increased spontaneous synaptic activities and markedly potentiated the slow VRP. A NO synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 0.03-1 microM), but not D-NAME (0.03-1 microM), also markedly potentiated the slow VRP and this effect was reversed by L-arginine (300 microM). 6. 8-Bromo-cyclic guanosine 3': 5'-monophosphate (8-Br-cyclic GMP, 100-300 microM) produced both an inhibition of the slow VRP and a depolarization of ventral roots. A cyclic GMP-dependent protein kinase inhibitor, KT5823 (0.3 microM), partly inhibited the depressant effects of NO donors and 8-Br-cyclic GMP on the dorsal root-evoked slow VRP. In contrast, KT5823 did not inhibit the depolarizing effects of NO donors. 7. Perfusion of the spinal cord with medium containing tetrodotoxin (0.3 microM) and/or low Ca2+ (0.1 mM)-high Mg2+ (10 mM) markedly potentiated the depolarizing effect of NO donors. The SNAP-evoked depolarization in the tetrodotoxin-containing low Ca(2+)-high Mg2+ medium was significantly inhibited by excitatory amino acid receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM). 8. The present study suggests that inhibitory and excitatory mechanisms meditated by the NO-cyclic GMP cascade are involved in the primary afferent fibre-evoked nociceptive transmission in the neonatal rat spinal cord. The inhibitory mechanism, but not the excitatory mechanism, appears to be partly mediated by cyclic GMP-dependent protein kinase. It is also suggested that Ca(2+)-independent release of excitatory amino acid neurotransmitters contributes to the depolarizing response to NO of ventral roots.     

Muscarinic excitatory and inhibitory mechanisms involved in afferent fibre-evoked depolarization of motoneurones in the neonatal rat spinal cord.

1. The involvement of acetylcholine and muscarinic receptors in spinal synaptic responses evoked by electrical and noxious sensory stimuli was investigated in the neonatal rat spinal cord in vitro. 2. Potentials were recorded extracellularly from a ventral root (L3-L5) of the isolated spinal cord, spinal cord-cutaneous nerve, and spinal cord-skin preparations of 1- to 4-day-old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or the cutaneous saphenous nerve, or by noxious skin stimulation. 3. Single shock stimulation of supramaximum intensity of a dorsal root induced a mono-synaptic reflex in the corresponding ventral root. Bath-application of the muscarinic agonists, muscarine (0.3-30 microM) and (+)-cis-dioxolane (0.1-100 microM), produced an inhibition of the mono-synaptic reflex and a depolarization of motoneurones. Other muscarinic agonists, arecoline (10 nM-10 microM) and oxotremorine (10 nM-1 microM), inhibited the mono-synaptic reflex with little or no depolarization of motoneurones. Repetitive stimulation of the saphenous nerve at C-fibre strength induced a slow depolarizing response lasting about 30 s of the L3 ventral root. This slow ventral root potential (VRP) was also inhibited by arecoline (10 nM-10 microM) and oxotremorine (10 nM-1 microM). 4. In the spinal cord-saphenous nerve-skin preparation, a slow VRP was evoked by application of capsaicin (0.5 microM), bradykinin (3 microM), or noxious heat (47 degrees C) to skin. This slow VRP was depressed by the muscarinic agonists, arecoline (3 microM) and oxotremorine (1 microM). 5. Of the (+)-cis-dioxolane-induced inhibition of mono-synaptic reflex and motoneurone depolarization, the M2 antagonists, AF-DX 116 (0.1-1 microM) and methoctramine (100-300 nM), preferentially blocked the former response, whereas the M3 antagonists, 4-DAMP (3-10 nM) and p-F-HHSiD (0.3-3 microM), preferentially blocked the latter response. AF-DX 116 (0.1-1 microM) and methoctramine (100-300 nM) also effectively antagonized the arecoline- and oxotremorine-induced inhibition of the slow VRP. The pA2 values of AF-DX 116 and methoctramine against the arecoline-induced inhibition of the mono-synaptic reflex were both 6.79, and that of 4-DAMP against the (+)-cis-dioxolane-induced motoneurone depolarization was 8.16. 6. In the spinal cord-cutaneous nerve preparation, the saphenous nerve-evoked slow VRP was augmented by the anticholinesterase, edrophonium (5 microM). AF-DX 116 (1 microM) and methoctramine (100 nM) also potentiated the slow VRP, whereas 4-DAMP (10 nM) depressed the response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurokinin-alpha injected into the ventral tegmental area elicits a dopamine-dependent behavioral activation in the rat

Neurokinin-alpha (NKA) and substance P (SP), neuropeptides of the tachykinin family, have been identified in dopaminergic areas of rat brain. It has previously been shown that SP microinjected into the ventral tegmental area (VTA), site of the dopaminergic A10 (DA-A10) cell bodies, causes a behavioral activation characteristic of dopamine agonists. The present experiment measured open field behavior following bilateral VTA injections of NKA (0.02, 0.2, 2.0 micrograms/0.5 microliters). NKA induced a dose-dependent behavioral activation at lower concentrations of NKA than previously reported with SP. Medium and high doses of NKA produced significant increases in locomotion and rearing in both the center and periphery of the open field. Grooming decreased with dose, although this effect was not significant. In a second experiment, the behavioral activation by NKA (2.0 micrograms) was blocked by pretreatment with haloperidol (0.2 mg/kg), confirming that the NKA-induced effect is mediated by dopamine. Although the VTA contains both SP and NKA, receptors binding NKA exist here in greater density than those binding SP. Thus NKA may be the tachykinin in this region that preferentially interacts with DA-A10 neurons mediating behavioral arousal.     

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of excitatory amino acid responses by tachykinins and selective tachykinin receptor agonists in the rat spinal cord.

1. The effects of tachykinins and agonists selective for the three subtypes of neurokinin (NK) receptor have been tested on spinal neuronal responses both to the excitatory amino acids (EAAs) NMDA, AMPA and kainate, and to noxious heat stimuli. The agonists were applied by microiontophoresis in in vivo experiments in alpha-chloralose-anaesthetized, spinalized rats. 2. The NK1-selective agonist, GR 73632, enhanced responses to all three EAAs similarly, whilst the NK2-selective agonist, GR64349, reduced responses to AMPA and kainate without affecting those to NMDA, and the NK3 selective agonist, senktide, enhanced responses to AMPA and kainate. 3. The endogenous ligands substance P (SP) and neurokinin A (NKA) both enhanced responses to NMDA with little effect on responses to kainate, whereas neurokinin B (NKB) selectively enhanced responses to kainate without affecting those to NMDA. 4. The effects of GR73632 on EAA responses showed some differences between the dorsal and ventral horn, with more selectivity towards enhancement of NMDA responses in the ventral horn, but a smaller maximum effect. 5. Background activity was significantly enhanced by GR73632, GR64349, SP and NKA but not by senktide or NKB. GR73632 had the greatest effect on background firing, but this action was variable between cells and was related both to the location within the spinal cord and to the degree of spontaneous activity prior to GR73632 administration. 6. Responses to noxious heat were enhanced consistently only by NKA. 7. These data show that selective agonists for the tachykinin receptors are capable of modulating EAA responses differentially.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differences of the contractile responses to acetylcholine and neurokinin A in rabbit airway: heterogeneous distribution of the metabolic enzymes.

1. Experiments were designed to determine whether differences exist in the sensitivity to muscarinic and tachykinin agonists in rabbit airways. 2. The rank order of sensitivity (pD2 value) to acetylcholine was: trachea > proximal bronchus > distal bronchus, whereas no regional difference was observed in the sensitivity to carbamylcholine which is resistant to acetylcholinesterase. 3. Acetylcholinesterase activity was greater in the distal than in the proximal airway. 4. In the absence of the peptidase inhibitor, phosphoramidon, the pD2 values of neurokinin A (NKA) and substance P (SP) in trachea were significantly greater than that in bronchus, whereas no regional difference was observed in the NK1 selective agonist, substance P methyl ester (SPOMe). 5. Application of phosphoramidon (10 microM) to avoid peptide degradation abolished the regional difference of the pD2 values of SP. 6. In conclusion, regional differences in sensitivities to acetylcholine and NKA in the rabbit airway were suggested to be due to distribution to the metabolic enzymes of these drugs.     

Bradykinin-induced activation of nociceptors: receptor and mechanistic studies on the neonatal rat spinal cord-tail preparation in vitro.

1. The effects of bradykinin on nociceptors have been characterized on a preparation of the neonatal rat spinal cord with functionally connected tail maintained in vitro. Administration of bradykinin to the tail activated capsaicin-sensitive peripheral fibres and evoked a concentration-dependent (EC50 = 130 nM) depolarization recorded from a spinal ventral root (L3-L5). 2. The response to bradykinin was unaffected by the peptidase inhibitors, bestatin (0.4 mM), thiorphan (1 microM), phosphoramidon (1 microM) and MERGETPA (10 microM) or by the presence of calcium blocking agents, cadmium (200 microM) and nifedipine (10 microM). 3. Inhibition of cyclo-oxygenase with indomethacin (1-5 microM), aspirin (1-10 microM) and paracetamol (10-50 microM) consistently attenuated responses to bradykinin. 4. The effect of bradykinin was mimicked by the phorbol ester PDBu, an activator of protein kinase C. The response to bradykinin was attenuated following desensitization to PDBu but desensitization to bradykinin did not induce a cross-desensitization to PDBu. The protein kinase C inhibitor staurosporine (10-500 nM) consistently attenuated the effects of PDBu and bradykinin. 5. Bradykinin responses were reversibly enhanced by dibutyryl cyclic AMP (100 microM). However dibutyryl cyclic GMP (0.5 mM) and nitroprusside (10 microM) produced prolonged block of responsiveness to bradykinin. Prolonged superfusion with pertussis toxin did not affect responses to bradykinin. 6. The B1-receptor agonist des Arg9-bradykinin (10-100 microM) was ineffective alone or after prolonged exposure of the tail to lipopolysaccharide (100 ng ml-1) or epidermal growth factor (100 ng ml-1) to induce B1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

'Sensory-efferent' neural control of mucus secretion: characterization using tachykinin receptor antagonists in ferret trachea in vitro.

1. We characterized the tachykinin receptor(s) mediating 'sensory-efferent' neural control of release of 35SO4-labelled macromolecules (mucus) from ferret trachea in vitro in Ussing chambers using selective tachykinin antagonists. Secretion was induced by substance P (SP), neurokinin A (NKA), capsaicin, the NK1 tachykinin receptor agonist [Sar9, Met(O2)11]substance P ([Sar9]SP), or acetylcholine (ACh), or by electrical stimulation of nerves. Antagonists used were FK888 and L-668,169, selective for the NK1 receptor, SR 48968, selective for the NK2 receptor, and FK224, a dual antagonist at NK1 and NK2 receptors. The selectivity of FK888 and SR 48968 was examined on NKA-induced contraction of ferret tracheal smooth muscle in vitro. 2. SP (1 microM) increased mucus secretion by 695% above vehicle controls. FK888 (0.1 microM-30 microM) inhibited SP-induced secretion in a dose-dependent manner, with complete inhibition at 10 microM and an IC50 of 1 microM. L-668,169 (1 microM) also completely inhibited SP-induced secretion. 3. NKA (1 microM) significantly increased mucus secretion by 271% above baseline, a response which was completely inhibited by FK888 (10 microM) or L-668,169 (microM). Secretion induced by ACh (10 microM: 317% above baseline) was not inhibited by FK888 but was inhibited by atropine. Capsaicin (10 microM)-induced secretion (456% above vehicle controls) was significantly inhibited by FK888 and by L-668,169 (111% and 103% inhibition respectively). 4. Electrical stimulation (50 V, 10 Hz, 0.5 ms, 5 min) increased mucus output above baseline (increased by 12 to 26 fold), a response blocked by tetrodotoxin (0.1 microM). FK888 (10 microM) inhibited the increase in secretion due to electrical stimulation by 47%.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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 studied. 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. 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. The contralateral slow v.r.p. was abolished after the spinal cord was treated with capsaicin (1 microM 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. Administration of an SP antagonist, [D-Arg1, D-Pro2, D-Trp7,9 Leu11]-SP in concentrations of 5-16 microM 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 microM) 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 microM did not exert any agonist action on the motoneurones. 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.