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1. A radiochemical plate assay is presented that allows a simultaneous evaluation of the capacity of cells in culture to degrade proteoglycan and collagen. Its principle consists of monitoring the release of soluble radioactive degradation products from Multiwell culture plates coated with dried reconstituted 3H-labelled-proteoglycan/14C-labelled-collagen mixed gels. The plates can also be used for the assay of proteolytic activities within enzyme solutions. 2. When cultured on the plates, rabbit synovial cells degrade collagen and proteoglycan almost simultaneously, owing to the secretion of collagenase and of a proteoglycan-degrading metal-dependent neutral proteinase.

Aerosolized substance P (0.1m, for 30 min) induced airway hyperresponsiveness in guinea-pigs 24 h after they were pre-treated with salbutamol (8.7 mmby aerosol for 10 min) and phosphoramidon (0.1 mmby aerosol for 10 min). This was displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine. A microvascular leakage hypersensitivity also occurred and was demonstrated by a potentiation of the plasma protein extravasation from bronchial vessels induced by histamine. The aim of this study was to investigate the effects of the non-peptide and potent tachykinin NK3receptor antagonist, SR 142801 (osanetant), in comparison with those of the tachykinin NK1and NK2receptor antagonists, SR 140333 (nolpitantium) and SR 48968 (saredutant) respectively, on substance P. When given once at 1 mg/kg ip 45 min before exposure to substance P, SR 142801 prevented both hyperresponsiveness to acetylcholine and the potentiation of histamine-induced increase in microvascular permeability. SR 142801 did not exhibit any tachykinin NK1or NK2antagonistic activity in experiments on guinea-pig isolated airways, in vitro or in vivo. The results suggest that tachykinin NK3receptors might be involved in these substance P-induced effects on airways.

Summary: The aim of this study was to demonstrate the involvement of neuropeptides in the increase in microvascular permeability induced by bradykinin in guinea-pig airways in vivo and to determine the type of receptor involved. Extravasation of intravenously injected Evans blue dye was used as an index of vascular permeability. Increase in plasma exudation induced by bradykinin (0.3 µg/kg, iv) was reduced or abolished by capsaicin (40 mg/kg, sc, 7 days before experiments), a drug which destroys neurokinins in the NANC nerve endings. (±)-CP-96,345 (3 mg/kg, iv), an antagonist of neurokinin NK₁-receptors, abolished the increase of vascular permeability induced by bradykinin and reduced or abolished the effects of substance P (0.3 µg/kg, iv). The higher dose of (±)-CP-96,345 (10 mg/kg, iv) completely blocked the effects of substance P, but it did not modify those of neurokinin A (100 µg/kg, iv). In contrast, SR 48968 (0.1 and 0.3 mg/kg, iv), an antagonist of neurokinin NK₂-receptors, reduced the increase of vascular permeability induced by neurokinin A without influencing the effects of bradykinin and substance P. These results demonstrate that a stimulation of the non-adrenergic non-cholinergic (NANC) nerves and a subsequent release of neuropeptides, especially of substance P, is involved in the effects of bradykinin. Copyright 1993, 1999 Academic Press

Several potent and selective antagonists for tachykinin receptors are now available and appear as powerful tools to investigate the physiological and pathological roles of tachykinins and to identify the type of receptor involved in their effect. Indeed, a lot of studies have shown that tachykinin NK2receptor antagonists (SR 48968, MEN 10627) are able to inhibit cough induced by citric acid, capsaicin or allergen challenge in the unanesthetized guinea-pig or mechanical stimulation of the trachea in the cat. The effects of tachykinin NK1receptor antagonists are still debated, whereas an inhibitory effect of SR 142801, a tachykinin NK3receptor antagonist, has been reported against citric acid-induced cough in the guinea-pig. Experiments with tachykinin receptor antagonists which do not cross the blood brain barrier suggest that the site of action of tachykinin receptor antagonists is most probably peripheral, but a central action, at least in an area not protected by the blood brain barrier, cannot be excluded. Finally, tachykinin NK2receptor stimulation seems to be involved in sensitisation of cough reflex.

The effects of SR142801, a nonpeptide tachykinin neurokinin (NK3) receptor antagonist, were investigated on the functional events linked to NK3 receptor activation by using Chinese hamster ovary (CHO) cells transfected with the human NK3 receptor. Radioligand binding conducted with [125I]iodohistidyl-[MePhe7]-NKB revealed a competitive inhibition by SR142801 and its (-)-antipode SR142806 with Ki values of 0.21 +/- 0.03 and 32.0 +/- 5.0 nM, respectively. NK3 agonists such as [MePhe7]-NKB and senktide stimulated inositol monophosphate formation with EC50 values of 2.0 +/- 1.4 and 2.1 +/- 0.7 nM, respectively. SR142801 antagonized the stimulatory effect of [MePhe7]-NKB (10(-8) M) with an IC50 of 14.3 +/- 2.6 nM and of senktide (10(-8) M) with an IC50 of 4.8 +/- 1.5 nM. The [3H]arachidonic acid release induced by either [MePhe7]-NKB (EC50 of 2.6 +/- 0.2 nM) or senktide (EC50 of 4.2 +/- 2.9 nM) also was inhibited by SR142801 with IC50 values of 16.1 +/- 0.5 and 8.0 +/- 1.7 nM, respectively. The cyclic AMP accumulation induced by 10(-7) M [MePhe7]-NKB (EC50 of 54 +/- 2 nM) also was antagonized by SR142801 with an IC50 value of 4.0 +/- 0.7 nM. These antagonistic effects were stereospecific and NK3 receptor specific because the (-)-antipode, SR142806, was much less effective than SR142801 in NK3 agonist-evoked responses, whereas the nonpeptide NK1 (SR140333) and NK2 (SR48968) receptor antagonists were almost inactive. The activity of SR142801 also was evaluated on the [Ca++]i increase induced by 10(-9) M [MePhe7]-NKB.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular mechanism of action of three chemically distinct nonpeptide antagonists, SR 140,333, FK 888, and RP 67,580, was compared with that of the previously characterized compound CP 96,345, using a series of chimeric constructs between their common target, the rat neurokinin (NK)-1 (substance P) receptor, and the homologous nonresponsive NK-3 (NKB) receptor. The ability of all four nonpeptide compounds to displace radiolabeled substance P from the NK-1 receptor and their ability to inhibit the peptide-induced increase in inositol phosphate turnover were critically dependent on structural elements located in an area from the middle of the second extracellular loop through transmembrane segments V and VI to the middle of the third extracellular loop of the NK-1 receptor. Dissection of the domain around the outer part of transmembrane segments V and VI into smaller segments demonstrated that the individual nonpeptide antagonists, in agreement with their distinct chemical structures, were dependent on different subepitopes within the common putative binding domain. Full NK-1-like susceptibility to SR 140,333, FK 888, and CP 96,345 could be transferred to the NK-3 receptor by exchange of transmembrane segments V and VI and adjacent parts with corresponding segments from the NK-1 receptor. For SR 140,333 and CP 96,345, almost the same effect could be achieved by transfer of two discontinuous segments around the top of transmembrane segments V and VI. RP 67,580 shared interaction sites with the other compounds around the top of transmembrane segment VI but appeared also to be dependent on transmembrane segment VII. It is concluded that four nonpeptide antagonists, despite overt chemical differences, appear to block NK-1 receptor function by interacting in distinct ways with a common site located spatially around the outer part of transmembrane segment VI.

The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK 1receptor agonist Ac [Arg 6,Sar 9,Met(O 2) 11]-substance P (6–11) induced shape change (EC 50= 3 and 6 nM, respectively), whereas the selective NK 2agonist [Nle 10]-Neurokinin A (4–10) and the selective NK 3agonist [MePhe 7]-Neurokinin B did not show any effect. Moreover, the specific NK 1receptor antagonist CP-96,345 selectively and dose-dependently counteracted the effect of substance P or of the NK 1receptor agonist (IC 50= 2 and 0.8 nM, respectively), whereas the selective NK 2receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK 1receptor agonist to become a proaggregating substance. These data therefore show that the NK 1receptor is solely involved in the neurokinin-induced shape change of rabbit platelets.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK1receptor agonist Ac [Arg6,Sar9,Met(O2)11]‐substance P (6–11) induced shape change (EC50= 3 and 6 nM, respectively), whereas the selective NK2agonist [Nle10]‐Neurokinin A (4–10) and the selective NK3agonist [MePhe7]‐Neurokinin B did not show any effect. Moreover, the specific NK1receptor antagonist CP‐96,345 selectively and dose‐dependently counteracted the effect of substance P or of the NK1receptor agonist (IC50= 2 and 0.8 nM, respectively), whereas the selective NK2receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK1receptor agonist to become a proaggregating substance. These data therefore show that the NK1receptor is solely involved in the neurokinin‐induced shape change of rabbit platelets.