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

Neuroscience Research, 2 (1984) 105-110

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Elsevier Scientific Publishers Ireland Ltd. NSR 00050

The excitatory action of the newly-discovered mammalian tachykinins, neurokinin g and neurokinin t, on neurons of the isolated spinal cord of the newborn rat Takayuki Matsuto ~, Mitsuhiko Yanagisawa ~, Masanori Otsuka ~, Ichiro K a n a z a w a 2 and Eisuke M u n e k a t a s IDepartment of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University,Bunkyo-ku, Tokyo 113; and 2Department of Neurology, Institute of ClinicalMedicine, and Slnstitute of Applied Biochemistry, University of Tsukuba, Ibaraki-ken 303 (Japan) (Received July 25th, 1984; Accepted August 20th, 1984)

Key words: neurokinin g - - neurokinin l / - - tachykinin - - substance P - - spinal cord SUMMARY The actions of two new mammalian tachykinins, neurokinin ,, and neurokinin ~, were examined using the isolated spinal cords of newborn i'ats. Depolarizing responses of spinal motoneurons were recorded extracellularly from the lumbar ventral root during application of neurokinin ~ or neurokinin fl at concentrations ranging from 3 x 10 - s M to 10 -~ M. The potencies of various tachykinins in depolarizing the motoneurons showed the following order: physalaemin > neurokinin ~ - k a s s i n i n - substance P > neurokinin ~. When the synaptic transmission in the spinal cord was blocked by tetrodotoxin, the depolarizing action of neurokinin ~ and neurokinin ~ was markedly reduced but not completely abolished. The depolarizing action of neurokinin ~ and neurokinin fl was depressed by a substance P antagonist, [D-Arg I, D-Pro 2, D-Trp 7"9, Leu I ~]SP. The possibility that neurokinin ~ and neurokinin fl act as neurotransmitters in the mammalian spinal cord is discussed.

Substance P (SP) exerts a potent excitatory action on certain spinal n e u r o n s 14'15. This and other lines of evidence strongly suggest that SP plays a role as neurotransmitter in certain primary afferent neurons (for review see Otsuka and Konishi~3). Until recently SP was the only known tachykinin occurring in the mammalian central nervous system. In 1983, however, two new tachykinins, i.e. neurokinin a (also known as substance K, or neuromedin L) and neurokinin/~ (also known as neuromedin K), were isolated from

Correspondence:Takayuki Matsuto, Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan. 0168-0102/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

106 mammalian spinal cords, and their structures were determined ~'6"8. A natural question which arises is whether or not these tachykinins act as neurotransmitters like SP. Like SP, neurokinin ~ and neurokinin fl exert a contracting activity on the guinea pig ileum2'5-7'~°'1~ and the rat vas deferens 2'7'~ and they are widely distributed in mammalian central nervous system4'9. In the present study, therefore, we examined their actions on spinal neurons of the newborn rat. The spinal cords of 0-3-day-old Wistar rats were used for the experiments. Under ether anesthesia, the spinal cord was isolated, hemisected sagittally and placed in a 0.2-ml bath perfused with artificial cerebrospinal fluid (CSF) which was oxygenated with a gas mixture of 95 ~o 02 and 5 ~o CO2. Perfusion rate was 5 ml/min, and the temperature was kept at 27 °C. The composition of the artificial CSF was given in a previous paper ~5, but in some experiments the concentrations of Mg 2÷ was increased up to 1.8mM. The potential changes generated in the motoneurons were recorded extracellularly from the L3-L5 ventral root with a suction electrode ~5. The tip of the suction electrode was a glass capillary, whose inner diameter fitted tightly about the recorded ventral roots (120-150 ~tm). Peptides were applied to the preparation either by perfusion or by brief-pulse injection into the perfusion system as described previously ~5. The SP antagonist, [D-Argl,D-Pro2,D-TrpV'9,Leu~]SP, was a gift from Prof. S. Rosell (Karolinska Institute) and from Dr. M, Fujino (Takeda Chemical Industries). Neurokinin 7 and neurokinin fl were synthesized as reported previously ~°. S P, kassinin and physalaemin were purchased from Protein Research Foundation (Osaka). When neurokinin ~ or neurokinin fl at concentrations ranging from 3.3 × 10 8 M to 10- 6 M was applied for 30 s by perfusion to the isolated spinal cord of the neonatal rat, depolarizing responses of spinal motoneurons were recorded extracellularly from the lumbar ventral root (Fig. 1A) ~°. The responses to neurokinin ~ and neurokinin were dependent on the concentrations, and the time courses of these responses were similar to those of the responses to SP except that the repolarization process of the responses to neurokinin fl was slower than that of the responses to SP or neurokinin (Fig. 1A¢). Fig. 1B shows the dose-response curves thus obtained for SP, neurokinin ~, and neurokinin fl as well as for other tachykinins such as physalaemin and kassinin. The relative potencies of these peptides are given in Table I. The order of potency of the peptides in depolarizing the motoneurons was: physalaemin > neurokinin fl - kassinin - SP > neurokinin ~. When the preparation was perfused with a medium containing tetrodotoxin (0.5 #M), the monosynaptic and polysynaptic reflexes induced by a single shock stimulation of the lumbar dorsal root were completely blocked. Under such conditions, neurokinin 7 and neurokinin fl at a concentration of 10 - 6 M still induced depolarizing responses of the ventral root, but the amplitude of the depolarization was much smaller than in normal medium. Several SP analogues have recently been reported to have antagonistic action toward SP, of which [D-Arg~,D-Pro2,D-Trp7,9,Leull]SP is one of the most potent TM. We examined the effect of this SP antagonist on the responses to SP, neurokinin 7 and

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Fig. 1. Responses to tachykinins recorded extracellularly from ventral roots of isolated spinal cords of newborn rats. A: responses of the ventral root L4 to SP (a), neurokinin ~t(b), and neurokinin fl (c). The peptides were applied for 30 s during the periods marked by horizontal bars under the records. Concentrations of the peptides were 10-7 M (left column) and 3.3 x 10-7 M (right column). B: dose-response

curves of tachykinins obtained in another preparation. Responses were recorded from ventral root L4 and each of the peptides was applied by perfusion for 30 s. O, SP; A, neurokinin ct; &, neurokinin fl; El, physalaemin; and III, kassinin. Ordinate: peak amplitude of the depolarization induced by the peptides. Abscissa: concentration of the peptides. TABLE I RELATIVE POTENCIES OF TACHYKININS Relative potencies were determined using 2 x 2 assay while the potency of SP in normal medium was taken as 1.00. Tyrode solution was used for guinea pig ileum. Peptides

Depolarization of lumbar ventral root

Contraction of guinea pig ileum After SP antagonist*

Substance P Neurokinin g Neurokinin fl Physalaemin Kassinin

1.00 0.22 3.52 9.49 3.16

1.00 0.13 0.50

0.207 0.027 0.034

* [D-Arg1,D-Pro2,D-TrpT'9,Leu]]]SPwas used at 5/~M. neurokinin ft. The peptides were a p p l i e d to the spinal c o r d in two different ways. First, when SP, neurokinin ~ or neurokinin fl was applied by brief pulse injection into the perfusion system, the depolarizing r e s p o n s e s to these p e p t i d e s were m a r k e d l y d e p r e s s e d by the S P antagonist (5 # M ) given b y perfusion (Fig. 2). After the r e m o v a l o f the antagonist, the r e s p o n s e s recovered within 2 0 - 4 0 min. O n the other hand, when SP,

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Fig. 2. Effects of [D-Argl,D-Pro2,D-TrpT.9,LeuH]SP on the depolarizing responses of ventral root L5 to tachykinins. A: responses to SP. B: responses to neurokinin ~ C: responses to neurokinin//. Each of the tachykinins was applied by brief-pulse injection into the perfusion system. The concentration of the solution of each of the tachykinins was 1/~M, and the durations of the pulses were 0.7 s for SP, &9 s for neurokinin g, and 0.6 s for neurokinin//. [D-Arg I,D-Pro2,D-TrpT'9,Leu 1i] SP was applied at 5 #m by perfusion during the 5-min period marked by a black horizontal bar. Ordinate: peak amplitude of the depolarization induced by the peptides. Abscissa: time.

neurokinin ~ or neurokinin ~ was applied to the preparation by perfusion for 30 s, and the SP antagonist (5/~M) was added to the perfusion medium during the 3-min period of pretreatment and the 30-s period of application of the agonist peptides, the responses to the peptides were either not affected or slightly potentiated by the SP antagonist. The contracting action of neurokinin ~ and neurokinin fl on guinea pig ileum was also markedly depressed by the SP antagonist at 5/~M (Table I)2. The present study showed that both neurokinin • and neurokinin fl have potent excitatory effect on neurons in the isolated spinal cord of the newborn rat. As is the case for SP t5, the depolarization of spinal motoneurons induced by neurokinin ~ and neurokinin fl is probably due to both the direct action on spinal motoneurons and the transsynaptic action through the spinal interneurons. In support of this, the present study showed that after the synaptic transmission was blocked by tetrodotoxin, the depolarization of spinal motoneurons induced by neurokinin 7 or neurokinin fl was depressed but not abolished. The SP antagonist, [D-Argl,D-Pro2,D-TrpT'9,Len I ~]SP, suppressed the response of the spinal motoneurons not only to SP but also to neurokinin ~ and neurokinin ft. Therefore the antagonist may be regarded as a tachykinin antagonist, and caution is needed when SP antagonists derived from SP analogues are used as experimental tools. In the present study, the responses to SP, neurokinin ~ and neurokinin/~ of the spinal motoneurons were depressed by the SP antagonist only when the tachykinins were applied by brief-pulse injection but not when the peptid¢s were applied by perfusion for 30 s. Therefore the main site of action of the taehykinins may be different, depending on the way of application.

109 Neurokinin ~ and neurokinin fl occur in considerable m o u n t s in the m a m m a l i a n spinal cord 4,9. Furthermore, the immunohistochemieal study of K a n a z a w a et al.4 suggested that these peptides m a y be concentrated in the nerve terminals in superficial layers of the spinal dorsal horn. Maggio and Hunter 9 reported the occurrence of kassinin-like immunoreactivity in rat dorsal root ganglia. These findings naturally raise the possibility that neurokinin ~ and neurokinin fl act as neurotransmitters in the spinal cord. Recently, N a w a et al. 12 reported that a m R N A of SP precursor contains a nucleotide sequence encoding neurokinin ~t. Therefore, neurokinin ~ m a y be released from certain primary afferent neurons as a neurotransmitter, alone or together with SP or some other co-transmitter. The present study also showed that SP, neurokinin ct and neurokinin fl are, though similar, slightly different in their pharmacological characters, such as potency and time course of the responses. Since these neurokinins and SP have a c o m m o n C-terminal sequence, these differences m a y be attributed to the sequences of the N-terminal side. If a neuron can produce more than one peptide that have similar but subtly different actions, and if the target cells have multiple types of receptors 3, the m o d e of peptidergic transmission could be extremely diverse depending on the chemistry of both pre- and postsynaptic elements.

ACKNOWLEDGEMENT Part of this work was supported by research grants from the J a p a n e s e Ministry of Education, Science and Culture (58480126, 59225003).

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110 8 Kimura, S., Okada, M., Sugita, Y., Kanazawa, I. and Munekata, E., Novel neuropeptides, neurokmin c~and/~, isolated from porcine spinal cord, Proc. Jap. Acad., 59(B) (1983) 101-104. 9 Maggio, J.E. and Hunter, J.C., Regional distribution of kassinin-like immunoreactivity in rat central and peripheral tissues and the effect of capsalcin, Brain Res., in press. 10 Munekata, E., Okada, M., Kimura, S., Sugita, Y., Kanazawa, I., Matsuto, T. and Otsuka, M., Neurokinin ~ and fl, synthesis and pharmacological properties, Chem. Lett., (1984) 1013-1016. 11 Nawa, H., Doteuchi, M., Igano, K., Inouye, K. and Nakanishi, S., Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile, Life Sei., 34 (1984) 1153-1160. 12 Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor, Nature (Lond), 306 (1983) 32-36. 13 Otsuka, M. and Konishi, S., Substance P - - the first peptide neurotransmitter? Trends Neurosci., 6 (1983) 317-320. 14 Otsuka, M., Konishi, S. and Takahashi, T., A further study of the motoneuron-depolarizing peptide extracted from dorsal roots of bovine spinal nerves, Proc. Jap. Acad., 48 (1972) 747-752. 15 Otsuka, M. and Yanagisawa, M., The effects of substance P and baclofen on motoneurones of isolated spinal cord of newborn rat, J. exp. Biol., 89 (1980) 201-214. 16 Rosell, S., Bj6rkroth, U., Xu, J.-C. and Folkers, K., The pharmacological profile of a substance P (SP) antagonist. Evidence for the existence of subpopulations of SP receptors, Actaphysiol. seand., 117 (1983) 445-449.