MDL 29913

1. In many species, both NK1 and NK2 tachykinin receptors appear to be important in mediating the contraction of airway smooth muscle. We have examined the distribution and characterization of receptors for tachykinins in rabbit airways using functional length tension studies, autoradiography and radioligand binding studies. 2. Contractile responses to tachykinins were elicited in four different areas of the respiratory tree--trachea, and three progressively more distal areas of the right bronchus. The NK2 receptor-preferring agonists, neurokinin A (NKA), neuropeptide gamma (NP gamma) and the NK2-selective [Lys5 MeLeu9, Nle10]-NKA(4-10) [NKA (4-10) analogue] produced similar contraction in all four areas. Substance P (SP) and the NK1-selective [Sar9,Met(O2)11]-SP (Sar-SP) exhibited a marked location-dependence in the magnitude of contraction, producing minimal contraction in the trachea and more proximal bronchi with contractions becoming progressively larger in the more distal airways. Senktide (which is selective for the NK3 receptor) produced negligible contraction in all areas. 3. The NK2-selective antagonist, MDL29,913, was a weak antagonist of NKA and NKA(4-10) analogue. At a concentration of 2 microM, it produced a small but significant shift in the response curve to NKA and a greater shift (8 fold) in the curve to NKA(4-10) analogue, but it had no effect on responses to Sar-SP. The non peptide NK1 receptor antagonist, CP-96,345, was also unexpectedly weak in this preparation. The pD2 value for Sar-SP was decreased 27 fold by CP-96,345 at a concentration of 1 microM, without alteration in the maximum response.4. Autoradiographic binding sites to ['251I]-NKA were sparse over smooth muscle in proximal airway preparations and markedly increased in density in the more distal airways. There was negligible binding over vascular smooth muscle and epithelium.5. Radioligand binding studies revealed binding to ['251I]-NKA which was 82% specific. The order of potency for inhibition of ['251I]-NKA binding was SP> = Sar-SP> NKA = NPy>CP-96,345> NKA(4-10) analogue >NKB>>>MEN 10207 (the NK2 subtype selective antagonist) >MDL 29,913> senktide. This profile indicates binding predominantly to NK, receptors.6. These results suggest that there are at least two types of tachykinin receptors in rabbit airways, a population of NK, receptors, the density of which is greatest in the periphery and, in addition, NK2 receptors which are uniformly distributed throughout the airways. These receptors have unusual characteristics in that the NK, antagonist, CP-96,345 and the NK2 antagonist, MDL 29,913 respectively exhibited only weak potency.

Functional and radioligand binding studies with selective agonists and antagonists were used to investigate tachykinin receptors in the human bladder. Strips of detrusor muscle were contracted by the tachykinins neurokinin A and neuropeptide gamma, and by the NK2 receptor selective agonists [Lys5,MeLeu9,Nle10]-NKA(4-10) and [Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4- 10), with pD2 values 8.2, 8.0, 8.1 and 7.1. [Sar9,Met(O2)11]-SP and senktide were ineffective agonists, indicating an absence of NK1 and NK3 receptors. The contractile responses to [Lys5,MeLeu9,Nle10]-NKA(4-10) were inhibited competitively by the NK2 receptor selective antagonists SR 48968, GR 94800 and MDL 29913, with pA2 values 9.1, 8.6 and 7.0. Specific binding of the new NK2 receptor selective radioligand [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) was saturable to a high affinity site (KD 2.3 nM.). Specific binding was inhibited by NK2 receptor agonists and antagonists, but not by NK1 and NK3 analogues, showing binding to NK2 receptors only. These data indicate that NK2 receptors may be involved in regulation of detrusor contractility in the human bladder.

Tachykinin receptors in guinea-pig airways were examined using radioligand binding techniques in lung homogenates, and using isolated bronchial segments. Binding of the NK1 selective radioligand 125I-labelled Bolton-Hunter [Sar9,Met(O2)11]substance P ([125I]BHSarSP) was saturable and of high affinity (KD, 0.26 nM). The rank potency order of competitors for [125I]BHSarSP binding was [Pro9]SP > CP 96345 >> septide > [pGlu6]SP(6-11) > RP 67580 > or = [DPro9,t beta Pro10(phi),Trp11]SP > [DPro9,t beta Pro10(CH2 phi),Trp11]physalaemin > or = GR82334 > or = 127I Bolton-Hunter neurokinin A (BHNKA). Septide had higher affinity than expected, and it was the only ligand to bind to two sites. Agonists interacting with NK2 receptors were more potent contractile agents than NK1 receptor agonists. Responses to BHNKA (pD2 8.4) were antagonized by MDL 29913 and MEN 10207, with pKB values 6.42 and 6.79, and also by SR 48968 and GR 94800, although this was not dose dependent. This agonist was also weakly inhibited by CP 96345 and RP 67580. These data demonstrate that BHNKA can interact with both NK1 and NK2 receptors. There was no relationship between the binding affinity of NK1 ligands in lung homogenates, with GR 82334 being notably weak, and their agonist or antagonist potency in bronchial smooth muscle.

NK-1 and NK-2 tachykinin receptors in guinea pig airways appear to have some unusual characteristics. The analog [pGlu6,Pro9] SP(6-11) (septide) may also act on atypical NK-1 receptors in guinea pig ileum. In this study, we used new tachykinin antagonists to investigate further the receptors in the guinea pig bronchus. In the presence of 1 microM indomethacin and phosphoramidon, the selective agonists [Sar9,Met(O2)11]-SP and [Pro9]-SP (both NK-1), [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK-2) and septide were full agonists, with pD2 values of 8.00, 7.78, 9.11 and 8.52, respectively on epithelium-intact preparations. Contractions to septide were unaffected by atropine (5 microM) and tetrodotoxin (1 microM). Denudation of epithelium significantly enhanced the potency of [Sar9,Met(O2)11]-SP and [Pro9]-SP but not of septide and [Lys5,MeLeu9,Nle10]-NKA(4-10). The potency order for NK-2-selective antagonists against [Lys5,MeLeu9,Nle10]-NKA(4-10) was GR 94800 > SR 48968 MDL 29913 > MEN 10207 (pA2 values 8.97, 8.73, 7.11 and 6.49, respectively). The NK-1 selective antagonists, OP 96345, GR 82334 and RP 67580 were weak or ineffective against [Sar9,Met(O2)11]-SP and [Pro9]-SP (pA2 6.69 or less), whereas they were more than one order of magnitude more potent against septide (pA2, 7.78, 7.48 and 6.58, respectively). In epithelium-denuded bronchi, the antagonist potency of GR 82334 was unchanged. These data indicate that septide interacts with tachykinin receptors in guinea pig bronchial smooth muscle in a manner different from that of [Sar9,Met(O2)11]-SP and [Pro9]-SP, and provide some evidence for heterogeneity of NK-1 receptors in the guinea pig airways.