Tachykinin NK1 receptor in the guinea-pig isolated proximal urethra: characterization by receptor selective agonists and antagonists.

1. The tachykinin receptor mediating contraction of the guinea-pig isolated proximal urethra has been characterized by use of receptor selective agonists and antagonists. All experiments were performed in the presence of peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each) in order to reduce peptide degradation. 2. The natural tachykinins, substance P and neurokinin A produced a concentration-dependent contraction of rings of the proximal urethra which approached the same maximum (about 50% of the response to 80 mM KCl). Substance P (EC50 155 nM) was slightly (3.6 times) more potent than neurokinin A (EC50 560 nM). 3. The tachykinin NK1 receptor selective agonist, [Sar9]substance P sulphone (EC50 62 nM), was slightly more potent than substance P and produced the same maximal response of natural tachykinins. The NK2 receptor selective agonist, [beta Ala8] neurokinin A(4-10), was active only at microM concentrations and its maximal effect did not exceed 20% of that to substance P or neurokinin A. The NK3 receptor selective agonist, senktide, was ineffective up to 30 microM. 4. The response to [Sar9]substance P sulphone was antagonized in a competitive manner by either (+/-)-CP 96,345 (pA2 7.75, slope - 1.10) or GR 82,334 (pA2 7.31, slope - 1.26), which are selective NK1 receptor antagonists, while it was unaffected (up to 10 microM) by MEN 10,376, a selective NK2 receptor antagonist. 5. The response to 10 microM [beta Ala8]neurokinin A (4-10) was abolished by either 0.2 microM (+/-)-CP 96,345 or 1 microM GR 82,334, suggesting the involvement of NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.     

Tachykinin receptors in the guinea-pig isolated bronchi.

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.     

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of a 'septide-sensitive' inflammatory response in rat skin.

1. Measurement of plasma protein extravasation induced by the natural tachykinins following intradermal administration in rat skin indicated equipotency between substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). The selective NK1 receptor agonist, [Sar9]SP sulphone was 10-100 times more potent than SP. The synthetic hexapeptide, septide, [pGlu6, Pro9]SP-(6-11), which has been proposed to act on a distinct NK1 receptor subtype/binding site was equipotent with [Sar9]SP sulphone. 2. The selective NK2 receptor agonist [beta Ala8]NKA(4-10) (0.1-1 nmol) and the selective NK3 receptor agonist, senktide (0.1-1 nmol) were both ineffective in producing oedema. The selective NK2 receptor antagonist, SR 48, 968 (0.3 mumol kg-1) had no significant inhibitory effects upon oedema induced by approximately equiactive doses of SP (0.2 nmol), septide (0.002 nmol), [Sar9]SP sulphone (0.002 nmol), or NKB (0.3 nmol). These results together suggest that neither NK2 nor NK3 receptors are involved in oedema formation in rat skin. 3. The non-peptide tachykinin NK1 receptor antagonist, RP 67,580 (1-3 mumol kg-1), inhibited plasma protein extravasation induced by septide (0.002 nmol) to a greater extent than that to SP (0.2 nmol). RP 67,580 (1 mumol kg-1) produced a significant inhibition of approximately 66% of the response to septide (0.002 nmol) only. Increasing the dose of RP 67,580 3 fold resulted in inhibition of the response to SP (0.2 nmol) and [Sar9]SP sulphone (0.002 nmol) by approximately 66% and 64% respectively with the response to septide being inhibited by approximately 70%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors.

1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin NK1 receptor subtypes in the rat urinary bladder.

1. Following the recent proposal that the selective agonist septide, ([pGlu6,Pro9]SP(6-11)), acts on a novel tachykinin receptor distinct from the ''classical'' NK1 receptor, the aim of the study was to investigate the possible heterogeneity of tachykinin NK1 receptors in the rat urinary bladder. 2. The synthetic tachykinin receptor agonists, septide (pD2 7.87) and [Sar9]substance P (SP) sulphone (pD2 7.64) produced concentration-dependent contractions of the rat isolated urinary bladder. 3. The NK1 receptor antagonists GR82,334, (+/-)-CP96,345, and RP67,580 competitively antagonized (slopes of Schild plot not significantly different from unity) the response to septide with the rank order of potency (pKB values in parentheses): RP 67,580 (7.57) > GR 82,334 (7.01) > (+/-)-CP 96,345 (6.80). The same antagonists were significantly less potent when tested against [Sar9]SP sulphone, while maintaining the same rank order of potency: RP 67,580 (7.00) > GR 82,334 (5.93) > (+/-)-CP 96,345 (< 6). The antagonists did not affect the concentration-response curve to bombesin. 4. To exclude the involvement of the NK2 receptor, a second series of experiments was performed in the presence of the potent nonpeptide NK2 receptor antagonist, SR 48,968. SR 48,968 (1 microM) produced a rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [beta Ala8]neurokinin A (NKA) (4-10). SR 48,968 did not significantly modify the response to SP, NKA, neurokinin B (NKB), neuropeptide K (NPK), neuropeptide gamma (NP gamma), SP(4-11), SP(6-11), septide or [Sar9]SP sulphone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of NK1 receptors and importance of the N-terminal sequence of substance P in the stimulation of protein secretion in rat parotid glands.

Recent in vitro studies have shown that the dose-response curve of substance P on [3H]protein secretion from rat parotid glands is biphasic. Such a response could result either from the activation of tachykinin receptors or from the amphiphilic character of substance P, since it has previously been shown that the N-terminal part of substance P may play an important role in the activation of phosphoinositides in rat parotid glands. To investigate these possibilities, we studied the effects of selective NK1, NK2, NK3 receptor agonists and C-terminal fragments of substance P and neurokinin A on protein secretion from rat parotid lobules. The poor activity of NK2 (neurokinin A-(4-10) and [beta-Ala8]neurokinin A-(4-10)) as well as of NK3 ([MePhe7]neurokinin B) selective agonists allowed us to rule out a possible involvement of NK2 and NK3 receptors in the parotid gland secretory process. Conversely, the selective NK1 receptor agonist, [Sar9,Met(O2)11]substance P, reproduced the biphasic dose-response curve for [3H]protein secretion typical of native substance P. However, a biphasic response was not observed with peptides deprived of the N-terminal moiety of substance P, such as substance P-(4-11) or [AcArg6,Sar9,Met(O2)11] substance P-(6-11). Our data therefore indicate that the [3H]protein secretion obtained with substance P results from the activation of NK1 receptors. Moreover, our data suggest that the N-terminal tripeptide of substance P is also active, and could stimulate different phospholipases either by acting through a second functional site on the NK1 receptor or by directly activating G-proteins.     

Tachykinin NK(3) receptor agonists induced microvascular leakage hypersensitivity in the guinea-pig airways.

Microvascular leakage hypersensitivity is a main component of neurogenic inflammation and of tachykinin effects.The aim of this study was to examine the ability of neurokinin B and of the tachykinin NK(3) receptor agonists, [MePhe(7)]neurokinin B or senktide, to potentiate when given by aerosol the microvascular leakage induced by histamine in guinea-pig airways and to compare their effects to those of tachykinin NK(1) (substance P, [Sar(9),Met(O(2))(11)]substance P) or tachykinin NK(2) (neurokinin A, [betaAla(8)]neurokinin A (4-10)) receptor agonists. Guinea-pigs were pretreated successively for 10 min with aerolized salbutamol and phosphoramidon; 15 min later, they were exposed for 30 min to an aerosolized solution of tachykinin receptor agonists; 24 h later, the animals were anaesthetized and vascular permeability was quantified by extravasation of Evans blue dye. Neurokinin B, [MePhe(7)]neurokinin B and senktide (3 x 10(-6)-3 x 10(-5)M) induced a potentiation of the effects of histamine on the vascular permeability in the trachea and main bronchi. Compared to other tachykinin NK(1) and NK(2) receptor agonists, the order of potency was: senktide>neurokinin B=[Sar(9),Met(O(2))(11)]substance P=[betaAla(8)]neurokinin A (4-10)=[MePhe(7)]neurokinin B>neurokinin A>substance P. The potentiation by [MePhe(7)]neurokinin B of histamine-induced microvascular leakage was abolished by the tachykinin NK(1) receptor antagonist SR140333 ([(S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)piperidin-3-yl]etyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane, chloride]) or the tachykinin NK(3) receptor antagonists SR 142801 ([(R)-(N)-(1-(3-(l-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide]) and SB 223412 ([(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide]). In conclusion, these results suggest that tachykinin NK(3) receptors might be involved in the potentiation of histamine-induced increase in microvascular permeability.     

Functional characterization of tachykinin NK1 receptors in the mouse uterus

1. Contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of oestrogen-treated mice. 2. In the presence of thiorphan (3 microM), captopril (10 microM), and bestatin (10 microM), substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-related contractions of uterine preparations. The order of potency was SP > or =NKA>NKB. 3. Neither atropine (0.1 microM) nor l-NOLA (100 microM), nor indomethacin (10 microM) alone or in combination with either ranitidine (10 microM) or mepyramine (10 microM), affected responses to SP. These findings indicate that SP actions are not mediated or modulated through the release of acetylcholine, nitric oxide, prostanoids or histamine. 4. In the presence of peptidase inhibitors, the tachykinin NK(1) receptor-selective agonist [Sar(9)Met(O(2))(11)]SP, produced a concentration-dependent contractile effect. The tachykinin NK(2) and NK(3) receptor-selective agonists [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) and [MePhe(7)]NKB were relatively inactive. The potencies of SP analogues in which Glu replaced Gln(5) and/or Gln(6) were similar to that of SP. 5. The tachykinin NK(1) receptor-selective antagonist, SR140333 (10 nM), alone or combined with the tachykinin NK(2) receptor-selective antagonist, SR48968 (10 nM), shifted log concentration curves to SP, NKA and NKB to the right. SR140333 (10 nM) reduced the effect of [Sar(9)Met(O(2))(11)]SP. SR48968 did not affect responses to SP or [Sar(9)Met(O(2))(11)]SP, but reduced the effect of higher concentrations of NKA and shifted the log concentration-response curve to NKB to the right. The tachykinin NK(3) receptor-selective antagonist, SR 142801 (0.3 microM), had little effect on responses to SP and NKB. 6. We conclude that the tachykinin NK(1) receptor mediates contractile effects of SP, NKA and NKB and [Sar(9)Met(O(2))(11)]SP in myometrium from the oestrogen-primed mouse. The tachykinin NK(2) receptor may also participate in the responses to NKA and NKB.     

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a ''NK2A'' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.     

The influence of the initial stretch and the agonist-induced tone on the effect of basal and stimulated release of EDRF.

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific neurokinin receptors mediate plasma extravasation in the rat knee joint.

1 Plasma extravasation in the rat knee joint was induced by intra-articular injection of neurokinins and specific neurokinin receptor agonists. 2 Pronounced plasma extravasation was produced by substance P (SP, 4-185 microM) and to a lesser extent by neurokinin-B (NKB, 83-413 microM), whereas neurokinin-A (NKA, 88-440 microM) and calcitonin gene-related peptide (CGRP, 26-130 microM) had no significant effect. 3 The specific neurokinin1 receptor agonist [Sar9, Met(O2)11]-substance P (NK1 agonist) in doses of 0.4-70 microM appeared to be more potent than SP in eliciting plasma extravasation. The neurokinin2 receptor agonist [Nle10]-neurokinin A4-10 (NK2 agonist) was not effective at 70 microM but produced a small and significant effect at 330 microM, whereas the neurokinin3 receptor agonist [MePhe7]-neurokinin B (NK3 agonist) was without effect at 40 microM or 400 microM. 4 Injections of SP or NKA into the synovial cavity of the rat knee were equally effective in producing marked plasma extravasation in remote sites such as the forelimb and hindlimb paws. 5 Co-administration experiments showed that the effects of SP were synergistic with NKA or the NK1 receptor agonist, but not with CGRP or the NK2 receptor agonist. 6 The rank order of potency was NK1 agonist greater than or equal to SP greater than NKB greater than NK2 agonist suggesting that NK1 receptors mediate plasma extravasation in the rat knee joint.     

Neurokinin3-receptors are linked to inositol phospholipid hydrolysis in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation.

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of longitudinal muscle from guinea-pig ileum. 2. Substance P, neurokinin A and neurokinin B induced a concentration-dependent accumulation of total [3H]-inositol phosphates in the presence of 12 mM lithium with similar maximal responses and EC50 values. 3. The selective NK1-receptor agonist, substance P methyl ester, and the selective NK3-receptor agonist succ-[Asp6, MePhe8]-SP(6-11) (senktide) also stimulated [3H]-inositol phosphate formation with maximum responses of 50.69 +/- 0.96 and 45.64 +/- 1.17% relative to 10 microM substance P, respectively. Substance P methyl ester was approximately equipotent with substance P, whereas senktide was approximately 100 times more potent. 4. When added together, maximally effective concentrations of substance P methyl ester and senktide gave responses that were fully additive. In contrast, responses to substance P and neurokinin B were not additive. 5. The stimulation of [3H]-inositol phosphate formation by substance P, neurokinin B and senktide was not affected by atropine (2 microM) or tetrodotoxin (TTX, 0.3 microM). 6. The contractile effect of senktide was inhibited completely by TTX and partially blocked by atropine. Contractions induced by substance P methyl ester were not changed in the presence of TTX or atropine. 7. [D-Pro4, D-Trp7,9,10]-SP(4-11) competitively antagonized the action of substance P methyl ester on inositol phospholipid hydrolysis and contraction, but had no significant effect on senktide-induced inositol phospholipid breakdown or contraction. 8. These results suggest that NK3-receptors in the guinea-pig ileum are coupled to inositol phospholipid hydrolysis.     

Identification of both NK1 and NK2 receptors in guinea-pig airways.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurokinin B- and specific tachykinin NK(3) receptor agonists-induced airway hyperresponsiveness in the guinea-pig

1. The aim of this study was to determine whether neurokinin B (NKB) or specific agonists of tachykinin NK(3) receptors, [MePhe(7)]NKB and senktide, were able to induce airway hyperresponsiveness in guinea-pigs. The effects of these compounds were compared to those of substance P (SP), neurokinin A (NKA) and the preferential tachykinin NK(1) ([Sar(9), Met(0(2))(11)]SP) or NK(2) ([betaAla(8)]NKA (4-10)) receptor agonists. 2. In guinea-pigs pretreated with phosphoramidon (10(-4) M aerosol for 10 min) and salbutamol (8.7x10(-3) M for 10 min), all tachykinins administrated by aerosol (3x10(-7) to 10(-4) M) induced airway hyperresponsiveness 24 h later, displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine (i.v.). The rank order of potency was: [betaAla(8)]NKA (4-10)>NKA=NKB=senktide=[MePhe(7)]NKB=[Sar(9),Met(0(2))(11)]SP>SP. 3. Airway hyperresponsiveness induced by [MePhe(7)]NKB was prevented by the tachykinin NK(3) (SR 142801) and NK(2) (SR 48968) receptor antagonists. 4. Bronchoconstriction induced by tachykinins administered by aerosol was also determined. SP, NKA, NKB and the tachykinin NK(1) and NK(2) receptor agonist induced bronchoconstriction. The rank order of potency was: NKA=[betaAla(8)]NKA (4-10)>NKB=SP=[Sar(9), Met(0(2))(11)]SP. Under similar conditions, and for concentrations which induce airway hyperresponsiveness, senktide and [MePhe(7)]NKB failed to induce bronchoconstriction. 5. It is concluded that tachykinin NK(3)-receptor stimulation can induce airway hyperresponsiveness and that this effect is not related to the ability of tachykinins to induce bronchoconstriction.     

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist

The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2相似文献