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A novel, selective radioligand, [125I]-[Lys5, Tyr(I2)7, Meleu9, Nle10]-NKA(4–10), for the tachykinin NK-2 receptor
46 (1993) 455-457 © 1993 Elsevier SciencePubhshers B.V All nghts reserved0167-0115/93/$06 00
455
Regulatory Peptutes,
REGPEP 01493
A novel, selective radioligand, [125I]-[LysS,Tyr(I2)7,MeLeu 9, Nlel°]-NKA(4-10), for the tachykinin NK-2 receptor Tim B adgery-Parker a, Xiang-Ping Zeng a, Solange Lavielle b and Elizabeth Burcher
a
a School of Physwlogy and Pharmacology, Umverslty of New South Wales, Sydney (Austraha) and b CNRS URA 493, Pans (France) K e y words:
Tachykinin; NK-2 receptor; Neuropeptide ~,; Selective radiohgand, Rat gastric fundus
Summary A new radlohgand, [125I]-[LysS,Tyr(I2)7,MeLeu9,Nlel°]-NKA(4-10), based on the selective agonist [LysS,MeLeu9,Nlel°]-NKA(4-10) has been developed. Binding in rat fundus membranes was displaced by NPT>NKA>-[LysS,MeLeu9,Nlel°]-NKA(4-10)>neuropeptlde K>[LysS,Tyr(I2) 7,MeLeu9,Nlel°]-NKA(410) > SP > [ Sar9,Met(O2) 11]-SP ~ senktide, indicating binding to NK-2 receptors. Prehmmary studies demonstrated high specific binding in membranes from rat urinary bladder, duodenum and colon Specific binding In rat brain and lung was negligible, and binding m a range of guinea-pig tissues was no more than 35 ~o spectfic. These data may indicate species differences in NK-2 receptors.
Tachykinins are a group of structurally related peptides with many similar potent actions, Including contraction of smooth muscle. In addition to the endogenous tachykinins substance P (SP), neurokinln A (NKA) and neuroklnin B (NKB), two N-terminally extended forms of NKA, neuropeptide K and neuropeptlde ), (NPv), also exist. All naturally occurring tachykinins are somewhat unselective at thetr receptors' the SP-preferring NK-1 receptor, the NKA and NPT-preferring NK-2 receptor and the
Correspondence to" E. Burcher, School of Physiologyand Pharmacology, Umversltyof New South Wales, Sydney2033, Australia.
NKB-preferrlng NK-3 receptor. Recently, selective radioligands for the NK-1 and NK-3 receptors based on selective agonists [1-3] have been developed, to replace older radlohgands based on endogenous, unselective tachyklnins. No selective radioligand has been avadable for the NK-2 receptor The only commercially avmlable NK-2 receptor preferring radioligand, [125I]-NKA, is somewhat unselective and brads to NK-1 sites in guinea-pig lung [4]. Here, we report the synthesis and binding characteristics of the radloligand [ 125I]-[LysS,Tyr(I2)7,MeLeu9,Nlel°]NKA(4-10), based on the selective NK-2 receptor agonist [ LysS,MeLeu9,Nlel°]-NKA(4-10). The tyrosyl derivative of the tachykinin NK-2 selectwe agonist [LysS,MeLeu9,Nlel°]-NKA(4-10)
456
was lodlnated and the product purified usmg reverse phase HPLC. The radiohgand, identified as []25I][ LysS,Tyr(i2)7, MeLeug,Nle m]_NKA(4_ 10), eluted at 29% acetonitrile. Homogenates from fresh rat gastrtc fundus were prepared as prewously described [1]. The homogenates were ultimately resuspended (final concentration 2%) in Tns HC1 (pH 7.4, 25°C), MnC12 (3 raM), BSA (0.02%), and chymostatm (4 #g/ml), and incubated for 60 min at 25°C with radmllgand. Non-specific binding was defined using 1 #M [LysS,Tyr (I2)7,MeLeug,Nlel°]-NKA(4-10). Binding data were processed using computer programs EBDA and LIGAND. In fundus, binding of [ 125I]-[ LysS,Tyr(I2)7,MeLeu9,Nlel° ]-NKA(4-10) was saturable, reversible and to a single population of high affimty sites of K D 1 3 nM, with Bm,~,, 4 2 fmol/mg wet weight of tissue. Spectfic binding (8404) of [ ]251]-[LysS,Tyr(I2)7,MeLeug,Nlem]-NKA(4 - 10) was mhlbited [ 13 ] by NP ? > NKA > [ Lys 5,MeLeu 9, Nle m]-NKA(4-10) > neuropept]de K > [ Lys 5, Tyr(I2) 7, MeLeu9,Nlem]-NKA(4 - 10) > SP > [ Sar9, Met(O2)n]-SP,>senktlde, indicating binding to NK-2 receptors. The ineffectiveness of NK-1 and NK-3 selective peptldes demonstrates the NK-2 selectwlty of the radmligand The chemmal equxvalent of the radmhgand, [127I]-[LysS,Tyr(I2)7,MeLeu 9, Nlem]-NKA(4 - 10), was a potent, full agonlst m contractmg the isolated fundus strip and gulnea-ptg bronchus [ 13,14] In some prehminary studies, specific binding of 50 pM [ a25I]-[ LysS,Tyr(I2)V,MeLeug,Nlem]-NKA(4 10) was lnvesUgated m homogenates (2.5-3%) of several rat and guinea-pig tissues. Membranes were prepared as above, in mcubatton buffer containmg chymostatln (4 #g/ml), leupeptln (4 #g/ml), phosphoramidon (10 #M), bestatm (10/aM), captoprtl (1 /~M) and diprotmm A (10 #M). Nonspecific binding was defined by 10 #M [LysS,Tyr(I2)7,MeLeu9,Nlem] NKA(4-10). This prehmmary screen demonstrated vartable capacity for binding m different organs Spemfic binding in rat Ussues was' bladder (82%), duodenum (73 %), colon (68 %), vas deferens (66 %), kidney (47 %), submandibular gland (46 %), with lung
(22 % ) and brain (14 ~o) showing only weak specific binding (Fig 1A) Specific binding m guinea-pig hssues was lower than m most rat tissues bladder (39 % ), ileum (38 %), gall bladder (35 ° o), lung (32 ° o), kidney (31%), duodenum (165o) and brmn (19°o) (Fig. 1B). Since specific binding of [125I]-[LysS,Tyr(I2) 7, MeLeu9,Nle]°]-NKA(4-10) an these tissues has not been charactensed, we cannot conclude that this is defining NK-2 receptors excluswely. However, zero specific binding of this selective radioligand would be expected if no NK-2 receptors were present. Differences between gmnea-p~g and rat may reflect spemes differences m NK-2 receptors. In gumea-pig lung,
2400[
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m 400
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Fig 1 Binding of [12sI]-[LysS,Tyr(I2)7, MeLeug,NIe]°]-NKA(410) to homogenates of rat (A) and guinea-pig (B) Ussues Open bars, total binding, hatched bars, non-specific binding defined by 10 #M [LysS,Tyr(I2)7,MeLeug,Nle I°]-NKA(4-10) D, duodenum, C, colon. UB, unnary bladder, VD, vas deferens, K, kidney, SG, submandlbular gland, B, brmn, L, lung, GB, gall bladder, I, deum
457
specific binding of high affinity (K D 1.3 nM) but low capacity, has been characterized to NK-2 receptors [ 14]. In addition to fundus, several rat tissues (bladder, vas deferens, duodenum and colon), showed good binding capacity for [125I]-[LysS,Tyr(I2) 7, MeLeu9,-Nlel°]-NKA(4-10), and the presence of NK-2 receptors m these ttssues has been well described previously [5-7] Notably, specific binding (dpm/mg tissue) was approx. 60-100~/o greater in rat unnary bladder than in duodenum or vas deferens, consistent with this organ showing the highest level of NK-2 receptor mRNA expression in the rat [8]. In rat brain, the negligible specific binding of [125I][LysS,Tyr(I2)7,MeLeu9,Nlel°]-NKA(4-10) is in accordance wtth the absence of NK-2 receptors from the adult of this species [9] and with the very low levels of NK-2 mRNA expression [8]. Binding of NKA-recognising radioligands in submandibular gland has earlier been charactensed to NK-1 sites [ 10], and no expression of NK-2 receptor mRNA is found here [8], in spite of the high saalogoglc potency of N P K and NPy [11,12]. In conclusion, we have developed a novel and highly selective radioligand for the NK-2 receptor This radtollgand shows high aJ~nlty binding in some peripheral tissues from the rat and should prove to be a selective and inexpenswe radioligand for the NK-2 receptor. Further work is reqmred to characterise its binding sttes in peripheral tissues.
Acknowledgement This study was supported by the National Health and Medical Research Council of Australia.
References 1 Lew, R., Geraghty, D P , Drapeau, G , Regoh, D and Burcher, E, Bmdmg characteristics of [ ~25I]-Bolton-Hunter [ S ar9,Met(O2) t i].substance P, a new selective radiohgand for the NK1 receptor, Eur J Pharmacol, 184 (1990) 97-108
2 Mussap, C_J_ and Burcher, E , [125I]-Bolton-Hunter scyhorhlnm II a novel, selective radiohgand for the tachykmm NK3 receptor m rat brim, Peptides, 11 (1990) 827-836 3 Guard, S, Watson, S P , Maggao, J E , Too, H - P and Wathng, K J , Pharmacological analysis of [3H]-senktlde binding to NK 3 tachykmln receptors in guinea-pig deum longitudinal muscle-myentenc plexus and cerebral cortex membranes, Br J Pharmacol, 99 (1990) 767-773 4 Geraghty, D P , Mussap, C J and Burcher, E., Radtolodlnated substance P, neuroklnm A, and eledolsm brad predominantly to NK1 receptors in gumea-plg lung, Mol Pharmacol, 41 (1992) 147-153 5 Burcher, E , Buck, S H., Lovenberg, W and O'Donohue, T.L, Charactenzatlon and autora&ographlc localization of multiple tachyklmn binding sites m gastrointestinal tract and bladder, J Pharmacol Exp Ther, 236 (1986) 819-831 6 Lee, C -M, Campbell, N J , Wllha.ms, B.J and Iversen, L L , Muluple tachykinln binding sites m peripheral tissues and in brain, Eur J Pharmacol, 130 (1986) 209-217 7 Bergstrom, L , Beaujouan, J - C , Torrens, Y, Saffroy, M , Glowmskl, J , Lavlelle, S_, Chassaing, G , Marquet, A_, D'Orleans-Juste, P , Dlon, S and Regoll, D , 3H-Neurokmm A labels a speexfic tachyklnln-bmdmg site in the rat duodenal smooth muscle, Mol Pharmacol., 32 (1987) 764-771 8 Takeda, Y and Krause, J E., Pharmacological and molecular biological studies on the &verslty of rat tachyklnm NK-2 receptor subtypes in rat CNS, duodenum, vas deferens, and unnary bladder, Ann N_Y Acad Scl, 632 (1991) 479-482 9 Saffroy, M , Beaujouan, J -C, Torrens, Y., Besseyre, J , Bergstrom, L_ and J Glowmskh J , Locahzataon of tachykmm binding sites (NKL, NK2, NK 3 llgands) in the rat brain, Peptides, 9 (1988) 227-241 10 Buck, S_H and Butcher, E , The rat submaxillary gland contams predominantly P-type tachyklnln binding sites, Peptxdes, 6 (1985) 1079-1084_ 11 Takeda, Y and Krause, J E , Neuropeptlde K potently stimulates salivary gland secretion and potentiates substance P-induced sahvatxon, Proc Natl Acid Scl USA, 86 (1989a) 392-396 12 Takeda, Y and Krause, J E , ~,-Preprotachykmm-(72-92)peptade amlde potentiates substance P-reduced salivation, Eur J Pharmacol, 161 (1989)267-271. 13 Burcher, E , Badgery-Parker, T_, Zeng, X.-P and Lawelle, S, CharactensaUon of a novel, selectwe radlollgand, [~25I][LysS,Tyr(I2)7,MeLeu 9, NIe~°]-NKA(4-10), for the tachyklnm NK-2 receptor m rat fundus, Eur J Pharmacol, (in press) 14 Zeng, X -P, Lawelle, S and Burcher, E , Evidence for tachykinln NK-2 receptors m guinea-pig airways from binding and functional studies, using [x25I]-[LysS,Tyr(I2)7,MeLeu9,NleI°]NKA(4-10), NeuropepUdes, (m press).
455
Regulatory Peptutes,
REGPEP 01493
A novel, selective radioligand, [125I]-[LysS,Tyr(I2)7,MeLeu 9, Nlel°]-NKA(4-10), for the tachykinin NK-2 receptor Tim B adgery-Parker a, Xiang-Ping Zeng a, Solange Lavielle b and Elizabeth Burcher
a
a School of Physwlogy and Pharmacology, Umverslty of New South Wales, Sydney (Austraha) and b CNRS URA 493, Pans (France) K e y words:
Tachykinin; NK-2 receptor; Neuropeptide ~,; Selective radiohgand, Rat gastric fundus
Summary A new radlohgand, [125I]-[LysS,Tyr(I2)7,MeLeu9,Nlel°]-NKA(4-10), based on the selective agonist [LysS,MeLeu9,Nlel°]-NKA(4-10) has been developed. Binding in rat fundus membranes was displaced by NPT>NKA>-[LysS,MeLeu9,Nlel°]-NKA(4-10)>neuropeptlde K>[LysS,Tyr(I2) 7,MeLeu9,Nlel°]-NKA(410) > SP > [ Sar9,Met(O2) 11]-SP ~ senktide, indicating binding to NK-2 receptors. Prehmmary studies demonstrated high specific binding in membranes from rat urinary bladder, duodenum and colon Specific binding In rat brain and lung was negligible, and binding m a range of guinea-pig tissues was no more than 35 ~o spectfic. These data may indicate species differences in NK-2 receptors.
Tachykinins are a group of structurally related peptides with many similar potent actions, Including contraction of smooth muscle. In addition to the endogenous tachykinins substance P (SP), neurokinln A (NKA) and neuroklnin B (NKB), two N-terminally extended forms of NKA, neuropeptide K and neuropeptlde ), (NPv), also exist. All naturally occurring tachykinins are somewhat unselective at thetr receptors' the SP-preferring NK-1 receptor, the NKA and NPT-preferring NK-2 receptor and the
Correspondence to" E. Burcher, School of Physiologyand Pharmacology, Umversltyof New South Wales, Sydney2033, Australia.
NKB-preferrlng NK-3 receptor. Recently, selective radioligands for the NK-1 and NK-3 receptors based on selective agonists [1-3] have been developed, to replace older radlohgands based on endogenous, unselective tachyklnins. No selective radioligand has been avadable for the NK-2 receptor The only commercially avmlable NK-2 receptor preferring radioligand, [125I]-NKA, is somewhat unselective and brads to NK-1 sites in guinea-pig lung [4]. Here, we report the synthesis and binding characteristics of the radloligand [ 125I]-[LysS,Tyr(I2)7,MeLeu9,Nlel°]NKA(4-10), based on the selective NK-2 receptor agonist [ LysS,MeLeu9,Nlel°]-NKA(4-10). The tyrosyl derivative of the tachykinin NK-2 selectwe agonist [LysS,MeLeu9,Nlel°]-NKA(4-10)
456
was lodlnated and the product purified usmg reverse phase HPLC. The radiohgand, identified as []25I][ LysS,Tyr(i2)7, MeLeug,Nle m]_NKA(4_ 10), eluted at 29% acetonitrile. Homogenates from fresh rat gastrtc fundus were prepared as prewously described [1]. The homogenates were ultimately resuspended (final concentration 2%) in Tns HC1 (pH 7.4, 25°C), MnC12 (3 raM), BSA (0.02%), and chymostatm (4 #g/ml), and incubated for 60 min at 25°C with radmllgand. Non-specific binding was defined using 1 #M [LysS,Tyr (I2)7,MeLeug,Nlel°]-NKA(4-10). Binding data were processed using computer programs EBDA and LIGAND. In fundus, binding of [ 125I]-[ LysS,Tyr(I2)7,MeLeu9,Nlel° ]-NKA(4-10) was saturable, reversible and to a single population of high affimty sites of K D 1 3 nM, with Bm,~,, 4 2 fmol/mg wet weight of tissue. Spectfic binding (8404) of [ ]251]-[LysS,Tyr(I2)7,MeLeug,Nlem]-NKA(4 - 10) was mhlbited [ 13 ] by NP ? > NKA > [ Lys 5,MeLeu 9, Nle m]-NKA(4-10) > neuropept]de K > [ Lys 5, Tyr(I2) 7, MeLeu9,Nlem]-NKA(4 - 10) > SP > [ Sar9, Met(O2)n]-SP,>senktlde, indicating binding to NK-2 receptors. The ineffectiveness of NK-1 and NK-3 selective peptldes demonstrates the NK-2 selectwlty of the radmligand The chemmal equxvalent of the radmhgand, [127I]-[LysS,Tyr(I2)7,MeLeu 9, Nlem]-NKA(4 - 10), was a potent, full agonlst m contractmg the isolated fundus strip and gulnea-ptg bronchus [ 13,14] In some prehminary studies, specific binding of 50 pM [ a25I]-[ LysS,Tyr(I2)V,MeLeug,Nlem]-NKA(4 10) was lnvesUgated m homogenates (2.5-3%) of several rat and guinea-pig tissues. Membranes were prepared as above, in mcubatton buffer containmg chymostatln (4 #g/ml), leupeptln (4 #g/ml), phosphoramidon (10 #M), bestatm (10/aM), captoprtl (1 /~M) and diprotmm A (10 #M). Nonspecific binding was defined by 10 #M [LysS,Tyr(I2)7,MeLeu9,Nlem] NKA(4-10). This prehmmary screen demonstrated vartable capacity for binding m different organs Spemfic binding in rat Ussues was' bladder (82%), duodenum (73 %), colon (68 %), vas deferens (66 %), kidney (47 %), submandibular gland (46 %), with lung
(22 % ) and brain (14 ~o) showing only weak specific binding (Fig 1A) Specific binding m guinea-pig hssues was lower than m most rat tissues bladder (39 % ), ileum (38 %), gall bladder (35 ° o), lung (32 ° o), kidney (31%), duodenum (165o) and brmn (19°o) (Fig. 1B). Since specific binding of [125I]-[LysS,Tyr(I2) 7, MeLeu9,Nle]°]-NKA(4-10) an these tissues has not been charactensed, we cannot conclude that this is defining NK-2 receptors excluswely. However, zero specific binding of this selective radioligand would be expected if no NK-2 receptors were present. Differences between gmnea-p~g and rat may reflect spemes differences m NK-2 receptors. In gumea-pig lung,
2400[
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UB
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SG
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m 400
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UB
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Fig 1 Binding of [12sI]-[LysS,Tyr(I2)7, MeLeug,NIe]°]-NKA(410) to homogenates of rat (A) and guinea-pig (B) Ussues Open bars, total binding, hatched bars, non-specific binding defined by 10 #M [LysS,Tyr(I2)7,MeLeug,Nle I°]-NKA(4-10) D, duodenum, C, colon. UB, unnary bladder, VD, vas deferens, K, kidney, SG, submandlbular gland, B, brmn, L, lung, GB, gall bladder, I, deum
457
specific binding of high affinity (K D 1.3 nM) but low capacity, has been characterized to NK-2 receptors [ 14]. In addition to fundus, several rat tissues (bladder, vas deferens, duodenum and colon), showed good binding capacity for [125I]-[LysS,Tyr(I2) 7, MeLeu9,-Nlel°]-NKA(4-10), and the presence of NK-2 receptors m these ttssues has been well described previously [5-7] Notably, specific binding (dpm/mg tissue) was approx. 60-100~/o greater in rat unnary bladder than in duodenum or vas deferens, consistent with this organ showing the highest level of NK-2 receptor mRNA expression in the rat [8]. In rat brain, the negligible specific binding of [125I][LysS,Tyr(I2)7,MeLeu9,Nlel°]-NKA(4-10) is in accordance wtth the absence of NK-2 receptors from the adult of this species [9] and with the very low levels of NK-2 mRNA expression [8]. Binding of NKA-recognising radioligands in submandibular gland has earlier been charactensed to NK-1 sites [ 10], and no expression of NK-2 receptor mRNA is found here [8], in spite of the high saalogoglc potency of N P K and NPy [11,12]. In conclusion, we have developed a novel and highly selective radioligand for the NK-2 receptor This radtollgand shows high aJ~nlty binding in some peripheral tissues from the rat and should prove to be a selective and inexpenswe radioligand for the NK-2 receptor. Further work is reqmred to characterise its binding sttes in peripheral tissues.
Acknowledgement This study was supported by the National Health and Medical Research Council of Australia.
References 1 Lew, R., Geraghty, D P , Drapeau, G , Regoh, D and Burcher, E, Bmdmg characteristics of [ ~25I]-Bolton-Hunter [ S ar9,Met(O2) t i].substance P, a new selective radiohgand for the NK1 receptor, Eur J Pharmacol, 184 (1990) 97-108
2 Mussap, C_J_ and Burcher, E , [125I]-Bolton-Hunter scyhorhlnm II a novel, selective radiohgand for the tachykmm NK3 receptor m rat brim, Peptides, 11 (1990) 827-836 3 Guard, S, Watson, S P , Maggao, J E , Too, H - P and Wathng, K J , Pharmacological analysis of [3H]-senktlde binding to NK 3 tachykmln receptors in guinea-pig deum longitudinal muscle-myentenc plexus and cerebral cortex membranes, Br J Pharmacol, 99 (1990) 767-773 4 Geraghty, D P , Mussap, C J and Burcher, E., Radtolodlnated substance P, neuroklnm A, and eledolsm brad predominantly to NK1 receptors in gumea-plg lung, Mol Pharmacol, 41 (1992) 147-153 5 Burcher, E , Buck, S H., Lovenberg, W and O'Donohue, T.L, Charactenzatlon and autora&ographlc localization of multiple tachyklmn binding sites m gastrointestinal tract and bladder, J Pharmacol Exp Ther, 236 (1986) 819-831 6 Lee, C -M, Campbell, N J , Wllha.ms, B.J and Iversen, L L , Muluple tachykinln binding sites m peripheral tissues and in brain, Eur J Pharmacol, 130 (1986) 209-217 7 Bergstrom, L , Beaujouan, J - C , Torrens, Y, Saffroy, M , Glowmskl, J , Lavlelle, S_, Chassaing, G , Marquet, A_, D'Orleans-Juste, P , Dlon, S and Regoll, D , 3H-Neurokmm A labels a speexfic tachyklnln-bmdmg site in the rat duodenal smooth muscle, Mol Pharmacol., 32 (1987) 764-771 8 Takeda, Y and Krause, J E., Pharmacological and molecular biological studies on the &verslty of rat tachyklnm NK-2 receptor subtypes in rat CNS, duodenum, vas deferens, and unnary bladder, Ann N_Y Acad Scl, 632 (1991) 479-482 9 Saffroy, M , Beaujouan, J -C, Torrens, Y., Besseyre, J , Bergstrom, L_ and J Glowmskh J , Locahzataon of tachykmm binding sites (NKL, NK2, NK 3 llgands) in the rat brain, Peptides, 9 (1988) 227-241 10 Buck, S_H and Butcher, E , The rat submaxillary gland contams predominantly P-type tachyklnln binding sites, Peptxdes, 6 (1985) 1079-1084_ 11 Takeda, Y and Krause, J E , Neuropeptlde K potently stimulates salivary gland secretion and potentiates substance P-induced sahvatxon, Proc Natl Acid Scl USA, 86 (1989a) 392-396 12 Takeda, Y and Krause, J E , ~,-Preprotachykmm-(72-92)peptade amlde potentiates substance P-reduced salivation, Eur J Pharmacol, 161 (1989)267-271. 13 Burcher, E , Badgery-Parker, T_, Zeng, X.-P and Lawelle, S, CharactensaUon of a novel, selectwe radlollgand, [~25I][LysS,Tyr(I2)7,MeLeu 9, NIe~°]-NKA(4-10), for the tachyklnm NK-2 receptor m rat fundus, Eur J Pharmacol, (in press) 14 Zeng, X -P, Lawelle, S and Burcher, E , Evidence for tachykinln NK-2 receptors m guinea-pig airways from binding and functional studies, using [x25I]-[LysS,Tyr(I2)7,MeLeu9,NleI°]NKA(4-10), NeuropepUdes, (m press).