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Levels of IMeurokinin A, Neurokinin B and Substance P in Rabbit Iris Sphincter Muscle†
Levels
of Neurokinin in Rabbit
A, Neurokinin
B and
Iris Sphincter
Muscles
Substance
P
Takashi TANIGUCHI, Motohatsu FUJIWARA, Yoshinori MASUO* and Ichiro KANAZAWA* Departmentof Pharmacology,Facultyof Medicine,KyotoUniversity,Kyoto606, Japan *Departmentof Neurology,Instituteof ClinicalMedicine,Universityof Tsukuba, Ibaraki305, Japan AcceptedOctober9, 1986 Abstract-We investigated the contents of neurokinin A, neurokinin B and substance P in the rabbit iris sphincter muscle, combining HPLC and radioimmunoassay, as our previous reports indicated that a slow component of neurogenic contractions of this muscle is most probably mediated by such tachykinins. The concentrations of these tachykinins were 44.3±8.7 fmol/mg protein, 35.3±10.2 fmol/mg protein and 186.8±29.8 fmol/mg protein (N=4), respectively. These results demonstrated that neurokinin A, neurokinin B and substance P are all present in the rabbit iris sphincter muscle. Substance P has been thought to be an excitatory neurotransmitter which functions in the spinal cord and in other areas of the nervous system (1). In addition to substance P, two novel tachykinins have recently been identified in mammals. Neurokinin A (also called substance K, neuromedin L) (2-4) and neurokinin B (also called neuromedin K) (2, 5) have been isolated from spinal cord. The rabbit iris sphincter muscle, which is highly sensitive to tachykinin, is innervated by the substance P-immunoreactive, trigeminal nerve (6), the stimulation of which causes a noncholinergic, nonadre nergic contraction (7, 8). Since this non cholinergic, nonadrenergic response was inhibited by a substance P antagonist, we considered its possible mediation by sub stance P or substance P-like peptide(s) released from the trigeminal nerve (9, 10). Recently, we have compared responses to substance P and to neurokinin A and at tempted to determine which of These tachykinins is most involved in the substance t This work was supported by a Grant-in-Aid for Special Project Research (No. 60126001, 61118001) from the Ministry of Education, Science and Culture, Japan.
P-immunoreactive nerve-mediated response in the rabbit iris sphincter muscle. These studies have suggested that noncholinergic, nonadrenergic contraction induced by electrical transmural stimulation is predomi nantly mediated by neurokinin A rather than by substance P, even though both these tachykinins are released concomitantly from the trigeminal nerve (11). The presence of substance P in rabbit iris-cilliary body rings has been reported (12), but there has been no report on the existence of neurokinin A in the iris sphincter muscle. In the present study, we examined the con tents of neurokinin A, substance P and related peptide, neurokinin B, in rabbit iris sphincter muscle, combining HPLC and radio immunoassay. Albino rabbits of either sex and weighing 2 to 3 kg were anesthetized with sodium pentobarbital (20 mg/kg, i.v.) and ex sanguinated. The eyes were removed within 1 5 min after death. One strip of iris sphincter muscle was cut from each eye. Tachykinins were measured according to the method of Kanazawa et al. (13). In brief, the two strips from each rabbit were pooled and homoge nized in a glass homogenizer in 3 ml of 1 N acetic acid containing 10 mM HCI, 0.1% Q
mercaptoethanol and 0.1% trifluoroacetic acid (TFA). After centrifugation, the super natant was injected into Sep-pak C18 and washed twice with 5 ml of 10% acetic acid. The peptide fraction was eluted with 0.1% TFA containing 50% acetonitrile. The separation of tachykinins prior to radio immunoassay was performed by HPLC. Eluates form Sep-pak were diluted with 0.1 % TFA and injected onto an Ultrasphere-ODS (4.6x250 mm) column pre-equilibrated with 0.1% TFA. Elution was initiated with a solution of 0.1% TFA+10% acetonitrile. The concentration of acetonitrile was linearly increased from 10% to 50% in 30 min and kept at 50% for another 10 min. The flow rate was 1 ml/min; each tube collected 0.5 ml of eluate. Neurokinin A was always eluted at fractions 43-45, substance P at 51-53, and neurokinin B at 55-57. Aliquots from each tube were evaporated to dryness in a freezing vacuum and processed for radioimmunoassay. 8Tyr-substance P and Bolton -Hunter neuro kinin A were radio-iodinated with 1251-Na by the chloramine-T method (14). The labelled compounds were applied to Sep pak C18 and eluted with a solution of 10% acetic acid containing acetonitrile. The radio immunoassay system for substance P was the same as described previously (14). The assay system for neurokinin A and B was essentially the same as that for substance P. Assay buffer was prepared as 50 mM barbitone buffer (pH 8.6) containing 5% human plasma and 0.1% 8-mercaptoethanol. Standard neurokinins or unknown sample (dissolved in 300 al of assay buffer) were incubated with anti-neurokinin A antiserum (final dilution of 1 : 150,000) at 4°C for 4 hr. Then, 1251-labelled Bolton-Hunter neuro kinin A (100 al in assay buffer) was added and further incubated at 4°C for 18-20 hr. One ml of Dextran T-70-coated charcoal
suspension in assay buffer without human plasma and 8-mercaptoethanol was added. After 30 min standing, the mixture was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant and precipitate were separated by decantation and counted. The same procedures were applied for measure ment of neurokinin B except using the standard curve for neurokinin B instead of that of neurokinin A. Since it is difficult to obtain highly specific antiserum against neurokinin A or neurokinin B, in the present study, the polyclonal antiserum against neurokinin A which can also cross-react 25% with neurokinin B was used in the radio immunoassay system following the separation of these tachykinins by HPLC. Protein content was determined by the method of Lowry et al. (15). The contents of neurokinin A, neurokinin B and substance P in the rabbit iris sphincter muscle are shown in Table 1. The content of substance P was higher than that of neurokinin A and neurokinin B. The contents of neurokinin A and B were almost the same. It has been reported that the substance P level of normal rabbit iris-cilliary body preparation was 414 pg/one preparation (12). According to the dimensional change, 186.8 fmol/mg protein would be equivalent to 534 pg/one iris sphincter muscle in this study. This value is close to the 414 pg/one iris cilliary body preparation. The contents of substance P and neurokinin A in the iris sphincter muscle and in rat cerebral cortex (13) were similar. It is also noted that the ratio of substance P to neurokinin A and the ratio of substance P to neurokinin B in the iris sphincter muscle were similar, although it has previously been reported that the ratio of substance P to neurokinin A was much higher than that of substance P to neurokinin B in rat brain and
spinal cord (13, 16) with the exception of the cerebellum (13) and the ventral part of the lumber cord (17). Therefore, it is interesting to note that the ratio of neurokinin A to neurokinin B was almost 1 :1 in the rabbit iris sphincter muscle. It is important to consider the results of Nawa et al. (18) which suggest the existence of two types of preprotachykinin mRNAs in bovine brain. They showed that 3-pre protachykinin mRNA contains not only the substance P sequence but also the neuro kinin A sequence, whereas a-preprotachy kinin mRNA lacks the latter sequence , containing only the one for substance P. The same group also reported that there are regional differences in the distribution of the relative levels of preprotachykinin mRNAs in the bovine brain and that there are a and 9 preprotachykinin mRNAs in the trigeminal ganglion (19). These reports suggests that substance P and neurokinin A are released from trigeminal nerve terminals since the rabbit iris sphincter muscle is considered to be innervated by the trigeminal nerve (6 , 9). Indeed, we found substance P and neurokinin A in the rabbit iris sphincter muscle in this study. As substance P and neurokinin A exhibit markedly different biological effects on the rabbit iris sphincter muscle (11) , such differences may be attributed to the existence of three types of tachykinin receptors, termed SP-P, SP-E and SP-K receptors (20). Acknowledgments: Thanks are due to Dr. S. Kimura of the Department of Biochemistry, Institute of Basic Medical Science, University of Tsukuba for pertinent advice. References 1 Otsuka, M. and Konishi, S.: Substance P and excitatory transmitter of primary sensory neurons. Cold Spring Harbor Symp. Quant. Biol. 40, 135-143 (1976) 2 Kimura, S., Okada, M., Sugita, Y., Kanazawa, I. and Munekata, E.: Novel neuropeptides, neurokinin a and (3, isolated form porcine spinal cord. Proc. Japan Acad. B 59, 101-104 (1983) 3 Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H.: Neuromedin L: a novel mammalian tachykinin identified in porcine spinal cord. Neuropeptide 4, 157-166 (1984) 4 Maggio, J.E., Sandberg, B.E.B., Bradley, C.V.,
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Iversen, L.L., Santikarn, S., Williams, B.H., Hunter, J.C. and Hanley, M.R.: Substance K: a novel tachykinin in mammalian spinal cord. In Substance P, Edited by Skrabanek, P. and Powell, D., p. 20, Bole Press, Dublin (1983) Kangawa, K., Minamino, N., Fukuda, A. and Matsuo, H.: Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 114, 533-540 (1983) Bill, A., Stjernshantz, J., Mandahl, A., Brodin, E. and Nillsson, G.: Substance P: Release of trigeminal nerve stimulation, effects in the eyes. Acta Physiol. Scand. 106, 371-373 (1979) Ueda, N., Muramatsu, I., Sakakibara, Y. and Fujiwara, M.: Noncholinergic, nonadrenergic contraction and substance P in rabbit iris sphincter muscle. Japan. J. Pharmacol. 31, 1071-1079 (1981 ) Leander, S., Hokanson, R., Rosell, S., Folkers, K., Sundler, F. and Tornqvist, K.: A specific sub stance P antagonist blocks smooth muscle contractions induced by non-cholinergic, non adrenergic stimulation. Nature 294, 467-469 (1981) Ueda, N., Muramatsu, I., Hayashi, H. and Fujiwara, M.: Trigeminal nerve: the possible origin of substance P-ergic response in isolated rabbit iris sphincter muscle. Life Sci. 31, 369 375 (1982) Fujiwara, M., Hayashi, H., Muramatsu, I. and Ueda, N.: Supersensitivity of the rabbit iris sphincter muscle induced by trigeminal dener vation: the role of substance P. J. Physiol. (Lond.) 350, 583-597 (1984) Ueda, N., Muramatsu, I., Taniguchi, T., Nakanishi, S. and Fujiwara, M.: Effects of neurokinin A, substance P and electrical stimulation on the rabbit iris sphincter muscle. J. Pharmacol. Exp. Ther. 239, (1986) (in press) Butler, J.M., Powell, D. and Unger, W.G.: Substance P levels in normal and sensorily denervated rabbit eyes. Exp. Eye Res. 30, 311 31 3 (1980) Kanazawa, I., Ogawa, T., Kimura, S. and Munekata, E.: Regional distribution of substance P, neurokinin a and neurokinin (3 in rat central nervous system. Neurosci. Res. 2, 111-120 (1984) Kanazawa, I. and Jesell, T.: Post mortem changes and regional distribution of substance P in the rat and mouse nervous system. Brain Res. 117, 362-367 (1976) Lowry, O.H., Rosebrough, J.N., Farr, A.L. and Randall, R.J.: Protein measurement with Folin
phenol reagent. J. Biol. Chem. 193, 265-275 (1951) 16 Minamino, N., Masuda, H., Kangawa, K. and Matsuo, H.: Regional distribution of neuromedin K and neuromedin L in rat brain and spinal cord. Biochem. Biophys. Res. Commun. 124, 731-738 (1984) 17 Ogawa, T., Kanazawa, I. and Kimura, S.: Regional distribution of substance P, neurokinin a and neurokinin 9 in rat spinal cord, nerve roots and dorsal root ganglia, and effects of dorsal root section or spinal transection. Brain Res. 359, 152-157 (1985) 18 Nawa, H., Hirose, T., Takashima, H., Kanayama,
S. and Nakanishi, S.: Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature 306, 32-36 (1983) 19 Nawa, H., Kotani, H. and Nakanishi, S.: Tissue specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 312, 729-734 (1984) 20 Buck, S.H., Burcher, E., Shults, C.W., Lovenberg, W. and O'Donohue, T.L.: Novel pharmacology of substance K-binding sites: A third type of tachykinin receptor. Science 226, 987-989 (1984)
of Neurokinin in Rabbit
A, Neurokinin
B and
Iris Sphincter
Muscles
Substance
P
Takashi TANIGUCHI, Motohatsu FUJIWARA, Yoshinori MASUO* and Ichiro KANAZAWA* Departmentof Pharmacology,Facultyof Medicine,KyotoUniversity,Kyoto606, Japan *Departmentof Neurology,Instituteof ClinicalMedicine,Universityof Tsukuba, Ibaraki305, Japan AcceptedOctober9, 1986 Abstract-We investigated the contents of neurokinin A, neurokinin B and substance P in the rabbit iris sphincter muscle, combining HPLC and radioimmunoassay, as our previous reports indicated that a slow component of neurogenic contractions of this muscle is most probably mediated by such tachykinins. The concentrations of these tachykinins were 44.3±8.7 fmol/mg protein, 35.3±10.2 fmol/mg protein and 186.8±29.8 fmol/mg protein (N=4), respectively. These results demonstrated that neurokinin A, neurokinin B and substance P are all present in the rabbit iris sphincter muscle. Substance P has been thought to be an excitatory neurotransmitter which functions in the spinal cord and in other areas of the nervous system (1). In addition to substance P, two novel tachykinins have recently been identified in mammals. Neurokinin A (also called substance K, neuromedin L) (2-4) and neurokinin B (also called neuromedin K) (2, 5) have been isolated from spinal cord. The rabbit iris sphincter muscle, which is highly sensitive to tachykinin, is innervated by the substance P-immunoreactive, trigeminal nerve (6), the stimulation of which causes a noncholinergic, nonadre nergic contraction (7, 8). Since this non cholinergic, nonadrenergic response was inhibited by a substance P antagonist, we considered its possible mediation by sub stance P or substance P-like peptide(s) released from the trigeminal nerve (9, 10). Recently, we have compared responses to substance P and to neurokinin A and at tempted to determine which of These tachykinins is most involved in the substance t This work was supported by a Grant-in-Aid for Special Project Research (No. 60126001, 61118001) from the Ministry of Education, Science and Culture, Japan.
P-immunoreactive nerve-mediated response in the rabbit iris sphincter muscle. These studies have suggested that noncholinergic, nonadrenergic contraction induced by electrical transmural stimulation is predomi nantly mediated by neurokinin A rather than by substance P, even though both these tachykinins are released concomitantly from the trigeminal nerve (11). The presence of substance P in rabbit iris-cilliary body rings has been reported (12), but there has been no report on the existence of neurokinin A in the iris sphincter muscle. In the present study, we examined the con tents of neurokinin A, substance P and related peptide, neurokinin B, in rabbit iris sphincter muscle, combining HPLC and radio immunoassay. Albino rabbits of either sex and weighing 2 to 3 kg were anesthetized with sodium pentobarbital (20 mg/kg, i.v.) and ex sanguinated. The eyes were removed within 1 5 min after death. One strip of iris sphincter muscle was cut from each eye. Tachykinins were measured according to the method of Kanazawa et al. (13). In brief, the two strips from each rabbit were pooled and homoge nized in a glass homogenizer in 3 ml of 1 N acetic acid containing 10 mM HCI, 0.1% Q
mercaptoethanol and 0.1% trifluoroacetic acid (TFA). After centrifugation, the super natant was injected into Sep-pak C18 and washed twice with 5 ml of 10% acetic acid. The peptide fraction was eluted with 0.1% TFA containing 50% acetonitrile. The separation of tachykinins prior to radio immunoassay was performed by HPLC. Eluates form Sep-pak were diluted with 0.1 % TFA and injected onto an Ultrasphere-ODS (4.6x250 mm) column pre-equilibrated with 0.1% TFA. Elution was initiated with a solution of 0.1% TFA+10% acetonitrile. The concentration of acetonitrile was linearly increased from 10% to 50% in 30 min and kept at 50% for another 10 min. The flow rate was 1 ml/min; each tube collected 0.5 ml of eluate. Neurokinin A was always eluted at fractions 43-45, substance P at 51-53, and neurokinin B at 55-57. Aliquots from each tube were evaporated to dryness in a freezing vacuum and processed for radioimmunoassay. 8Tyr-substance P and Bolton -Hunter neuro kinin A were radio-iodinated with 1251-Na by the chloramine-T method (14). The labelled compounds were applied to Sep pak C18 and eluted with a solution of 10% acetic acid containing acetonitrile. The radio immunoassay system for substance P was the same as described previously (14). The assay system for neurokinin A and B was essentially the same as that for substance P. Assay buffer was prepared as 50 mM barbitone buffer (pH 8.6) containing 5% human plasma and 0.1% 8-mercaptoethanol. Standard neurokinins or unknown sample (dissolved in 300 al of assay buffer) were incubated with anti-neurokinin A antiserum (final dilution of 1 : 150,000) at 4°C for 4 hr. Then, 1251-labelled Bolton-Hunter neuro kinin A (100 al in assay buffer) was added and further incubated at 4°C for 18-20 hr. One ml of Dextran T-70-coated charcoal
suspension in assay buffer without human plasma and 8-mercaptoethanol was added. After 30 min standing, the mixture was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant and precipitate were separated by decantation and counted. The same procedures were applied for measure ment of neurokinin B except using the standard curve for neurokinin B instead of that of neurokinin A. Since it is difficult to obtain highly specific antiserum against neurokinin A or neurokinin B, in the present study, the polyclonal antiserum against neurokinin A which can also cross-react 25% with neurokinin B was used in the radio immunoassay system following the separation of these tachykinins by HPLC. Protein content was determined by the method of Lowry et al. (15). The contents of neurokinin A, neurokinin B and substance P in the rabbit iris sphincter muscle are shown in Table 1. The content of substance P was higher than that of neurokinin A and neurokinin B. The contents of neurokinin A and B were almost the same. It has been reported that the substance P level of normal rabbit iris-cilliary body preparation was 414 pg/one preparation (12). According to the dimensional change, 186.8 fmol/mg protein would be equivalent to 534 pg/one iris sphincter muscle in this study. This value is close to the 414 pg/one iris cilliary body preparation. The contents of substance P and neurokinin A in the iris sphincter muscle and in rat cerebral cortex (13) were similar. It is also noted that the ratio of substance P to neurokinin A and the ratio of substance P to neurokinin B in the iris sphincter muscle were similar, although it has previously been reported that the ratio of substance P to neurokinin A was much higher than that of substance P to neurokinin B in rat brain and
spinal cord (13, 16) with the exception of the cerebellum (13) and the ventral part of the lumber cord (17). Therefore, it is interesting to note that the ratio of neurokinin A to neurokinin B was almost 1 :1 in the rabbit iris sphincter muscle. It is important to consider the results of Nawa et al. (18) which suggest the existence of two types of preprotachykinin mRNAs in bovine brain. They showed that 3-pre protachykinin mRNA contains not only the substance P sequence but also the neuro kinin A sequence, whereas a-preprotachy kinin mRNA lacks the latter sequence , containing only the one for substance P. The same group also reported that there are regional differences in the distribution of the relative levels of preprotachykinin mRNAs in the bovine brain and that there are a and 9 preprotachykinin mRNAs in the trigeminal ganglion (19). These reports suggests that substance P and neurokinin A are released from trigeminal nerve terminals since the rabbit iris sphincter muscle is considered to be innervated by the trigeminal nerve (6 , 9). Indeed, we found substance P and neurokinin A in the rabbit iris sphincter muscle in this study. As substance P and neurokinin A exhibit markedly different biological effects on the rabbit iris sphincter muscle (11) , such differences may be attributed to the existence of three types of tachykinin receptors, termed SP-P, SP-E and SP-K receptors (20). Acknowledgments: Thanks are due to Dr. S. Kimura of the Department of Biochemistry, Institute of Basic Medical Science, University of Tsukuba for pertinent advice. References 1 Otsuka, M. and Konishi, S.: Substance P and excitatory transmitter of primary sensory neurons. Cold Spring Harbor Symp. Quant. Biol. 40, 135-143 (1976) 2 Kimura, S., Okada, M., Sugita, Y., Kanazawa, I. and Munekata, E.: Novel neuropeptides, neurokinin a and (3, isolated form porcine spinal cord. Proc. Japan Acad. B 59, 101-104 (1983) 3 Minamino, N., Kangawa, K., Fukuda, A. and Matsuo, H.: Neuromedin L: a novel mammalian tachykinin identified in porcine spinal cord. Neuropeptide 4, 157-166 (1984) 4 Maggio, J.E., Sandberg, B.E.B., Bradley, C.V.,
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15
Iversen, L.L., Santikarn, S., Williams, B.H., Hunter, J.C. and Hanley, M.R.: Substance K: a novel tachykinin in mammalian spinal cord. In Substance P, Edited by Skrabanek, P. and Powell, D., p. 20, Bole Press, Dublin (1983) Kangawa, K., Minamino, N., Fukuda, A. and Matsuo, H.: Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 114, 533-540 (1983) Bill, A., Stjernshantz, J., Mandahl, A., Brodin, E. and Nillsson, G.: Substance P: Release of trigeminal nerve stimulation, effects in the eyes. Acta Physiol. Scand. 106, 371-373 (1979) Ueda, N., Muramatsu, I., Sakakibara, Y. and Fujiwara, M.: Noncholinergic, nonadrenergic contraction and substance P in rabbit iris sphincter muscle. Japan. J. Pharmacol. 31, 1071-1079 (1981 ) Leander, S., Hokanson, R., Rosell, S., Folkers, K., Sundler, F. and Tornqvist, K.: A specific sub stance P antagonist blocks smooth muscle contractions induced by non-cholinergic, non adrenergic stimulation. Nature 294, 467-469 (1981) Ueda, N., Muramatsu, I., Hayashi, H. and Fujiwara, M.: Trigeminal nerve: the possible origin of substance P-ergic response in isolated rabbit iris sphincter muscle. Life Sci. 31, 369 375 (1982) Fujiwara, M., Hayashi, H., Muramatsu, I. and Ueda, N.: Supersensitivity of the rabbit iris sphincter muscle induced by trigeminal dener vation: the role of substance P. J. Physiol. (Lond.) 350, 583-597 (1984) Ueda, N., Muramatsu, I., Taniguchi, T., Nakanishi, S. and Fujiwara, M.: Effects of neurokinin A, substance P and electrical stimulation on the rabbit iris sphincter muscle. J. Pharmacol. Exp. Ther. 239, (1986) (in press) Butler, J.M., Powell, D. and Unger, W.G.: Substance P levels in normal and sensorily denervated rabbit eyes. Exp. Eye Res. 30, 311 31 3 (1980) Kanazawa, I., Ogawa, T., Kimura, S. and Munekata, E.: Regional distribution of substance P, neurokinin a and neurokinin (3 in rat central nervous system. Neurosci. Res. 2, 111-120 (1984) Kanazawa, I. and Jesell, T.: Post mortem changes and regional distribution of substance P in the rat and mouse nervous system. Brain Res. 117, 362-367 (1976) Lowry, O.H., Rosebrough, J.N., Farr, A.L. and Randall, R.J.: Protein measurement with Folin
phenol reagent. J. Biol. Chem. 193, 265-275 (1951) 16 Minamino, N., Masuda, H., Kangawa, K. and Matsuo, H.: Regional distribution of neuromedin K and neuromedin L in rat brain and spinal cord. Biochem. Biophys. Res. Commun. 124, 731-738 (1984) 17 Ogawa, T., Kanazawa, I. and Kimura, S.: Regional distribution of substance P, neurokinin a and neurokinin 9 in rat spinal cord, nerve roots and dorsal root ganglia, and effects of dorsal root section or spinal transection. Brain Res. 359, 152-157 (1985) 18 Nawa, H., Hirose, T., Takashima, H., Kanayama,
S. and Nakanishi, S.: Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature 306, 32-36 (1983) 19 Nawa, H., Kotani, H. and Nakanishi, S.: Tissue specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 312, 729-734 (1984) 20 Buck, S.H., Burcher, E., Shults, C.W., Lovenberg, W. and O'Donohue, T.L.: Novel pharmacology of substance K-binding sites: A third type of tachykinin receptor. Science 226, 987-989 (1984)