Spinal action of neurokinins producing cardiovascular responses in the conscious freely moving rat: evidence for a NK-1 receptor mechanism

Summary This study was initiated to characterize the receptors which mediate the cardiovascular responses elicited by the intrathecal (i. th.) administration of neurokinins (NK) in the conscious freely moving rat. The dose response profile for substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) was determined over 0.065—65 nmol doses of the peptides. After i.th. administration at the T₈–T₁₀ thoracic level, only SP elicited a dose dependent pressor response. However, all NK elicited a dose dependent increase in heart rate (HR), and the following rank order of potency was observed: SP > NKA > NKB. SP (6.5 nmol) produced cardiovascular responses markedly greater than an equimolar dose of any of the seven SP fragments which were studied. The C-terminal sequences SP (4-11), [pGlu⁵]SP (5-11), [pGlu⁶]SP (6–11), and SP (7–11), as a group were slightly more potent than the N-terminal fragments, SP (1–4), SP (1–7) and SP (1–9) which were almost inactive. The NK-1 receptor selective agonists [Pro⁹, Met(O₂)¹¹]SP and [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP (4–11), produced pressor and positive chronotropic responses equal to or greater in intensity than SP. With up to 6.5 nmol of the NK-2 receptor selective agonist [Nle¹⁰]NKA (4–10), no dose dependent cardiovascular response was produced and the NK-3 receptor selective agonist senktide (succinyl-[Asp⁶, McPhe⁸]SP (6–11)), produced neither a cardiac nor pressor response when 6.5 nmol was administered. These results are consistent with the hypothesis that, receptors of the NK-1 subtype mediate the cardiovascular responses evoked by the spinal action of NK. Send offprint requests to R. Couture at the above address     

Demonstration of a neurokinin A receptor subtype in transfected fibroblasts

In competitive radioligand binding assays, the NK₂ receptor antagonists [Tyr⁵,D-Trp^6,8,9,Arg¹⁰]NKA(4–10) (MEN 10207) and [Tyr⁵,D-Trp^6,8,9,Arg¹⁰]NKA(3–10) (MEN 10208) had high and low affinity, respectively, in bovine stomach membranes and SKLKB82#3 cells, a murine fibroblast cell line transfected with a cDNA encoding for the bovine NK₂ receptor. These antagonists also had different affinities when inhibiting neurokinin A-induced polyphosphoinositide hydrolysis in SKLKB82#3 murine fibroblasts. Thus, the de novo protein expressed by the SKLKB82#3 murine fibroblasts may represent a distinct NK₂ receptor subtype.     

Structure-activity relationships of neurokinin A (4-10) at the human tachykinin NK(2) receptor: the role of natural residues and their chirality

A structure-activity study of neurokinin A (NKA) (4-10) was performed to investigate the importance of residue and chirality for affinity and efficacy at the NK(2) receptor in human colon circular muscle. Two series of NKA(4-10) analogues were produced with either L-alanine or the D-enantiomer substituted. Their activities were determined in vitro by means of radioligand binding and isolated smooth muscle pharmacology. NKA was more potent than NKA(4-10) at the human, unlike the rabbit, NK(2) receptor. The contractile response of NKA(4-10) was unaffected by N-terminal acetylation. L-Ala substitution of Asp(4), Val(7), Leu(9), and Met(10) caused an 8- to 80-fold decrease, and substitution of Phe(6) caused a 5000-fold decrease in binding affinity (P < 0.01). Positions Ser(5) and Gly(8) were not significantly affected. In functional studies, a similar pattern was observed. The replacement of residues with their respective D-enantiomer drastically reduced binding affinity and functional potency, particularly at positions 6 and 7 (P < 0.05). NKA(4-10) analogues L-Ala(6), L-Ala(8), D-Phe(6), D-Val(7), and D-Met(10) were partial agonists. An excellent correlation was observed between binding and functional data (r = 0.95). A retro-inverso analogue of NKA(4-10) was inactive. In conclusion, the side chains of Asp(4), Phe(6), Val(7), Leu(9), and Met(10) are structurally important features of NKA(4-10) for agonist activity, and changes in amino acid chirality are detrimental to binding affinity and functional activity. Overall, our data are broadly similar to those of previous studies in the rat. However, at the human NK(2) receptor, unlike the rat, [Ala(8)]NKA(4-10) was an antagonist.     

Effect of cyclopiazonic acid on contractions produced by tachykinin NK1 and NK2 receptor agonists in the circular muscle of guinea-pig colon

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK₁ and NK₂ receptor agonists, [Sar⁹]substance P (SP) sulfone and[βAla⁸]neurokinin A (NKA) (4–10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 μm ) and indomethacin (10 μm ). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nm ) was selected: [Sar⁹]SP sulfone (10 nm ) produced a biphasic contraction, the two amplitudes averaging 75 ± 2 and 43 ± 3% of the maximal response to KCl (80 mm ) at 1 and 15 min from application of the agonist, respectively. CPA (3 μm for 60 min) slightly reduced the phasic response to [Sar⁹]SP sulfone (16 ± 4% inhibition) and markedly suppressed the tonic component (89 ± 3% inhibition). 3 The contraction produced by [βAla⁸]NKA (4–10) (10 nm ) was more sustained than that induced by the NK₁ receptor agonist: it averaged 69 ± 5 and 73 ± 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [βAla⁸]NKA (4–10) (18 ± 7 and 21 ± 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK₁ and NK₂ receptor antagonists (SR 140333 and MEN 10627, respectively, 1 μm each) and of l -nitroarginine (100 μm ), KCl (40 mm ) produced a distinct phasic and tonic contraction which was suppressed by 1 mm nifedipine. CPA (3 μm ) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 μm nifedipine, the response to [βAla⁸]NKA (4–10) was slightly depressed (32 ± 6% inhibition) in its early component only, while the response to [Sar⁹]SP sulfone was abolished. CPA produced a slight inhibition (15 ± 9 and 33 ± 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [βAla⁸]NKA (4–10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [βAla⁸]NKA (4–10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar⁹]SP sulfone (0.1 μm for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 μm ) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar⁹]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK₁ receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK₂ receptor-mediated contractile responses.     

Inhibition of tachykinin-induced hypotension in dogs by CP-96,345, a selective blocker of NK-1 receptors

Summary The effects of substance P, neurokinin A, neurokinin B, [Sar⁹, Met(O₂)¹¹]-substance P, [Nle¹⁰]neurokinin A (4–10) and senktide {succinyl-[Asp⁶, McPhe⁸]-substance P (6–11)} on blood pressure and heart rate were studied in anesthetized dogs. Dose-dependent decreases in blood pressure and increases in heart rate were caused by each peptide except senktide. The latter elicited weak hypotensive or hypertensive responses at high doses. The order or potency was as follows: [Sar⁹, Met(O₂)¹¹]-substance P substance P > neurokinin ß₁ > neurokinin B > [Nle¹⁰]-neurokinin A (4–10) » senktide.CP-96,345, [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine] a selective NK-1 tachykinin receptor blocker, inhibited substance P-induced hypotension in a dose-related manner. R responses to each of the other peptides were inhibited by CP-96,345, 1.0 mg/kg (excluding senktide against which CP-96,345 was not tested). CP-96,344 (1.0 mg/kg i.v.) the 2R-3R enantiomer of CP-96,345 which does not block NK-1 receptors, had no effect on substance P-induced hypotension. We conclude that tachykinin-induced hypotension in dogs is mediated by NK-1 tachykinin receptors. Send offprint requests to J. W. Constantine at the above address     

Analogues of the C-terminal fragments of neurokinins with modifications at their C-terminal methionyl residue

Analogues of SP_4–11 have been synthesized in which the methionyl residue is replaced successively by the Glu(OCH₂CH₃), Glu(OBzl), Hse(CH₃) and Glu(CONHCH₃) residues, and analogues of NKA_4–10 and NKB_4–10 have been prepared in which the methionyl residue is replaced by the Hse(Bz1) and Hse(CH₃) residues, respectively. The SP_4–11 analogues were tested in three in vitro preparations representative of NK-1, NK–2 and NK-3 receptor types. Substitution of the SCH₃ group of the Met¹¹ side chain by the groups COOCH₂CH₃ and COOBzl has little affect on the agonist activity in NK-1 preparations, while in NK-2 the corresponding analogues are more potent than the parent octapeptide; that substituted with COOBzl being 8.2 times more potent than SP_4–11. In NK-3 preparations all analogues are weak agonists. The selectivity of all the analogues is reduced compared with the corresponding hexapeptide analogues. The SP_4–11 analogues, along with those of NKA_4–10, and NKB_4–11, were tested for their binding ability in the three receptor subtypes above. The SP_4–11 analogues show reduced affinity for NK-1 receptors, while the NKA_4–10 and NKB_4–10 analogues have almost the same affinities as NKA and NKB for NK-2 and NK-3 receptors, respectively. The effect of the lipophilicity of the Met¹¹ side chain, especially when a phenyl group is present in the side chain, at the NK-2 receptor is discussed.     

Structure-activity relationship of neurokinin A(4-10) at the human tachykinin NK(2) receptor: the effect of amino acid substitutions on receptor affinity and function

A structure-activity study of the neurokinin A (NKA) fragment NKA(4-10) was performed to investigate the importance of amino acid residues for receptor efficacy, potency and affinity at the NK(2) receptor in human colon circular muscle. Fourteen analogs of NKA(4-10) were produced with substitutions at positions 4, 5, 7, 9 and/or 10 of NKA. Their potencies were determined by in vitro contractile responses and affinities by radioligand binding using [125I]NKA. Functional potency was enhanced 8-fold by single amino acid substitutions with Lys(5) and MeLeu(9) but not significantly altered by substitutions Glu(4), Arg(5), His(5) and Nle(10). The multiply-substituted analogs [MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),(Tyr(7)),MeLeu(9),Nle(10)]NKA(4-10) displayed 6-9-fold increase in potency. Although [Arg(5),Nle(10)]NKA(4-10) was similar in potency to NKA(4-10), it was the only analog to show significantly reduced efficacy. All analogs were able to compete fully for [125I]NKA binding. [Lys(5),MeLeu(9)]NKA(4-10), [MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),Nle(10)]NKA(4-10) and analogs containing single substitutions with Glu(4), Arg(5), Lys(5) and MeLeu(9) displayed significantly higher affinity, whereas those with Nle(10) and [Glu(4),Nle(10)] substitutions showed significantly lower affinity than NKA(4-10). There was a positive correlation (r=0.63) between binding affinity and functional potency, which was markedly improved (r=0.95) by removal of three analogs: [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),Tyr(7),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),Tyr(I(2))(7),MeLeu(9),Nle(10)]NKA(4-10). These exhibited similar binding affinities to that of NKA(4-10) but were more potent in functional studies, possibly indicating a different mechanism of receptor interaction. In conclusion, substitution of Ser(5) with Lys, and/or N-methylation of Leu(9), were the most effective changes to increase functional and binding potency of NKA(4-10) at the human colon NK(2) receptor.     

Capillary permeability induced by intravenous neurokinins

Summary The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP > NKA > NKB) for neurokinins and (SP > [p-Glu⁶]SP(6–11) > SP(4–11) > [p-Glu⁵]SP(5–11) > SP(7–11)) for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu⁵, McPhe⁸, Sar⁹]SP(5–11) was slightly more potent than [p-Glu⁵]SP(5–11). The N-terminal fragments SP(1–4), SP(1–7) and SP(1–9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar⁹, Met(O₂)¹¹]SP and [-Ala⁴, Sar⁹, Met (O₂)¹¹]SP(4–11) were more potent than the NK-2 ([Nle¹⁰]NKA(4–10)) and NK-3 ([MePhe⁷]NKB and -Asp⁴, McPhe⁷NKB(4–10)) receptor selective agonists.Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of d-Arg⁰[Hyp³. d-Phe⁷]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar⁹, Met(O₂)¹¹]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP(4–11), SP(4–11) and [p-Glu⁶]SP(6–11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an incrase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP.These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT₁ and 5-HT₂ receptors), histamine (H1 receptors) and kinins (B₂ receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium. Send offprint requests to R. Couture at the above address     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15–200 μM) have been isolated and fully characterized. The cleavage product, GRF(4–44)-NH₂, and the isomer-ization product, [β-Asp³]GRF(1–44)-NH₂, from the degradation of GRF(1–44)-NH₂ in acidic solution and the corresponding products, GRF(4–29)-NH₂ and [β-Asp³]GRF(1–29)-NH₂, from the degradation of GRF(1–29)-NH₂ have been isolated and characterized. The products, [β-Asp⁸]GRF(1–44)-NH₂ and [Asp⁸]GRF(1–44)-NH₂, from the deamidation of GRF(1–44)-NH₂ at pH 8.0 and the corresponding products, [β-Asp⁸]GRF(1–29)-NH₂ and [Asp⁸]GRF(1–29)-NH₂, from the deamidation of GRF(1–29)-NH₂ have been isolated and characterized. AH the degradation products of GRF(1–44)-NH₂ and GRF(1–29)-NH₂ were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp³ and Asn⁸ and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4–5. At pH below the pK_a of the Asp³ side-chain (pH<4), cleavage at Asp³-Ala⁴ is the major route of degradation. For pH>4, isomerization of Asp³ to β-Asp³ (iso-Asp³) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp³ undergoes cleavage while the ionized (COO^-) form isomerizes. The more rapid deamidation of Asn⁸ to generate β-Asp⁸ and Asp⁸ in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH < 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn⁸ in GRF(1–29)-NH₂ at pH<2 to give exclusively [Asp⁸]GRF(1–29)-NH₂. The deamidation of Asn⁸ in GRF(1–29)-NH₂ at pH 8.0, 22–55°C, is relatively insensitive to temperature for T>37°C, possibly due to conformational constraints. Asp²⁵ and Asn³⁵ are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.     

Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinin-induced contractions of guinea-pig isolated trachea

1. The influence of epithelium removal and/or thiorphan on the effects of neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and related peptides on airway contractility was investigated on the guinea-pig isolated trachea. 2. Removing the tracheal epithelium significantly enhanced the sensitivity but not the maximum contractile responses to the peptides. 3. After removal of the epithelial layer, the shifts to the left of the log concentration response curves were greater for SP and SP-OMe (1.62 and 1.94 log units, respectively) than for two SP analogues substituted in position 9 namely [Pro9]SP sulfone and [beta-Ala4, Sar9]SP(4-11) sulfone (0.66 and 0.68 log units, respectively). The leftward shifts for compounds related to NKA or NKB lay between 0.58 and 0.73 log units. 4. The leftward shifts of the log concentration-response curves for SP, SP-OMe, [Pro9]SP sulfone, [beta-Ala4, Sar9]SP(4-11) sulfone and NKA were of similar magnitude after removal of the epithelium or after pretreatment with thiorphan (10(-5) M), an enkephalinase inhibitor, in the presence of epithelium. No significant additional shift of the curves to the left was observed with thiorphan plus epithelium removal. 5. The results obtained with the selective agonists for each of the three classes of neurokinin receptor (i.e NK1, NK2, NK3) suggest that the guinea-pig trachea contains receptors for SP and NKA but few if any for NKB. 6. It was concluded that neurokinins and related peptides (especially SP and analogues not substituted in position 9) are degraded by enkephalinase mainly located in the tracheal epithelium and that the addition of thiorphan or epithelium removal results in an inhibition or loss of enkephalinase activity, thereby increasing similarly the potencies of these peptides. It was, therefore, suggested that the supersensitivity to neurokinins produced by epithelium removal was due neither to the elimination of a permeability barrier nor to reduced production of a relaxant factor, but mainly to reduced peptide degradation.     

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [¹²⁵I]Bolton-Hunter [Sar⁹,Met(O₂)¹¹]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [¹²⁵I]Bolton-Hunter substance P ([¹²⁵I]BHSP) was saturable, of high affinity (K_d 0.3 nM) and was inhibited by SP (IC₅₀ 0.64 nM) > ranakinin < neurokinin A (NKA) $ SP(5–11) $ neuropeptide γ $ scyliorhinin II > scyliorhinin I $ [Sar⁹]-SP $ neurokinin B < physalaemin < carassin >> SP(7–11) < eledoisin $ SP(4–11) < SP(6–11). Binding was also inhibited by Gpp[NH]p $ GTPγS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC₅₀ 1.4 nM) > eledoisin < SP $ physalaemin $ ranakinin > SP(6–11) > scyliorhinin II $ neuropeptide γ > neurokinin B < NKA < scyliorhinin I $ SP(4–11) $ SP(5–11) > [Sar⁹]SP > SP(7–11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar⁹,Met(O₂)¹¹]SP, [Lys⁵,MeLeu⁹,Nle¹⁰]NKA(4–10) and senktide were weak or ineffective. There was a strong positive correlation between the pD₂ and pIC₅₀ values for mammalian tachykinins and analogues (r=0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar⁹,Met(O₂)¹¹]-SP(pD₂ 5.7) was approximately 25-fold less potent as an agonist than [Sar⁹]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg¹,D-Trp^7,9,Leu¹¹]-SP (spantide; 1 μM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 μM) were ineffective in both functional and binding studies. Tetrodotoxin (1 μM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [¹²⁵I]BHSP prefers SP and ranakinin. This toad “NK-1-like receptor” differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine. Received: 2 September 1997 / Accepted: 26 March 1998     

NMR and quenched molecular dynamics studies of superpotent linear and cyclic α-melanotropins

Conformational searching, computer simulations, synthesis and NMR are used on a variety of α melanocyte-stimulating hormone (α-MSH) analogues to understand the physical characteristics required for biological potency. Peptides I (AC-[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂), II (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂) and III (Ac-[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂ all show very similar conformational properties (backbone and side-chain torsional angles), and all display high biological potencies. The modeling results for these compounds are supported by the NMR data. Peptide IV (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂) appears to have a markedly different conformation and has decreased biological potency.     

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.     

Effects of tachykinins on uterine smooth muscle

1. Sensory nerves supplying the mammalian uterus have been shown to contain substance P (SP) and neurokinin (NK)A. This review presents some of the advances that have led to a greater understanding of the effects of tachykinins on uterine smooth muscle. 2. The cell-surface peptidase neprilysin (EC.3 24.11, endopeptidase 24.11, enkephalinase, CALLA, CD10) has been shown to play a major role in regulating the actions of tachykinins on both rat and human myometrium. Because this peptidase is known to be regulated by steroids and pregnancy, its effects may be of physiological relevance. 3. Tachykinins produce contractions of isolated myometrial preparations from non-pregnant rats and mice. The NK2 receptor mediates these effects in rat uterus, while the NK1 receptor may mediate these effects in the mouse uterus. 4. The effects of tachykinins have been examined on myometrial preparations obtained at Caesarean section from near-term pregnant women. In the presence of the peptidase inhibitors (thiorphan, captopril and bestatin), the mammalian tachykinins SP, NKA and NKB produced concentration-dependent uterine contractions. 5. The order of agonist potency NKA > SP = NKB suggested that NK2 receptors mediate uterine contractions in the human. This was confirmed using the stable analogues [Sar9,Met(O2)11]SP, [Lys5MeLeu9Nle10]NKA(4-10) and [N-MePhe7]NKB, which are NK1, NK2 and NK3 receptor selective, respectively. Only [Lys5MeLeu9Nle10]NKA(4-10) produced concentration-related contractions of human uterine smooth muscle. 6. The experimental findings described in the present review, taken together with results published previously in the literature, indicate that tachykinin peptides may play a physiological or pathophysiological role in regulating uterine smooth muscle activity. However, more extensive research will be required to confirm such a role for these peptides.     

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.     

Characterization of the peripheral action of neurokinins and neurokinin receptor selective agonists on the rat cardiovascular system

Summary The effects on mean arterial pressure (MAP) and heart rate (HR) of increasing doses (0.65–65 nmol/kg) of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were measured after intravenous (i. v.) injection in urethane anaesthetized rats. Neurokinins (NKs) elicited a vasodepressor effect with the following rank order of potency: SP (100%) > NKB (17.5%) > NKA (10%). The two undecapeptide NK-1 selective agonists, [Pro⁹, Met(O₂)¹¹]SP (787%) and [Sar⁹, Met(O₂)¹¹]SP (697%), evoked a significantly (P < 0.05) greater vasodepressor response than SP, while the potency of the octapeptide NK-1 selective agonist [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP (4–11) (316%) was not significantly different from SP. Conversely, the NK-2 selective agonist NKA (4–10) (<2%) caused only a small effect. The vasodepressor effect elicited by [MePhe⁷]NKB (112%) and [-Asp⁴, MePhe⁷]NKB (4–10) (92%), two NK-3 selective agonists, were not significantly different from that of SP. Senktide (1,095%) is the most potent NK-3 agonist, and is significantly (P < 0.01) more potent than SP. No cross-desensitization, of the vasodepressor response, was observed between NK-1 and NK-3 selective agonists. I. V. injection of 32.5 nmol/kg of NKA, NKA (4–10) and [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP (4–11) raised HR, while NKB and the NK-3 selective agonists produced a rapid and marked bradycardia. SP and the two undecapeptide, NK-1 selective agonists, produced an initial increase in HR and a latent long-lasting bradycardia. The bradycardia elicited by [Sar⁹, Met(O₂)¹¹]SP (32.5 nmol/kg) was blocked by methylatropine, hexamethonium, indomethacin and by treatment with capsaicin or compound 48/80. Although the bradycardia elicited by [-Asp⁴, MePhe⁷]NKB (4–10) (32.5 nmol/kg) was also blocked by hexamethonium, methylatropine, and by bilateral vagotomy, it remained unaffected after indomethacin, or in rats pretreated with either capsaicin or compound 48/80. The drop in MAP produced by the NK-1 and NK-3 agonists were reduced by hexamethonium, methylatropine and bilateral vagotomy (NK-3 agonist), but remained unaffected by indomethacin, capsaicin, and compound 48/80. The tachycardia to NKA (4–10) (65 nmol/kg) was blocked entirely by sotalol or metoprolol and potentiated by hexamethonium. Guanethidine and bilateral adrenalectomy (48 h) failed to affect the tachycardia induced by the agonist, whereas the combination of both treatments abolished the response. Rats sympathectomized with 6-hydroxydopamine (48 h) reduced the increase in HR to NKA (4–10) only at 1 min post-administration. These results support the hypothesis that the vasodepressor action of SP is mediated by the direct activation of NK-1 receptors on vascular endothelium. NKs can accelerate HR by releasing catecholamines through activation of NK-1 and/or NK-2 receptors, on noradrenergic fibers and adrenal chromaffn cells. Furthermore NKs can exert a depressor effect on the cardiovascular system by producing a vagal reflex (Bezold-Jarisch reflex), following either the activation of NK-3 receptors located on capsaicin-insensitive sensory fibers, or by promoting the release of prostaglandins from mast cells (via a non-receptor mechanism) which in turn will stimulate capsaicin-sensitive sensory fibers.     

Synthesis,activity on NK‐3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16

Abstract: Two analogs of a tachykinin family peptides – scyliorhinin II (ScyII): [Aib¹⁶]ScyII and [Sar¹⁶]ScyII were synthesized by the solid‐phase method using Fmoc chemistry. Conformational studies in water and DMSO‐d₆ on these peptides were performed using a combination of two‐dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar¹⁶]ScyII in water appeared to be more flexible, especially in the C‐terminal fragment. A better defined structure for this analog was obtained in DMSO‐d₆, in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3₁₀‐helix in the N‐terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib¹⁶]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3₁₀‐helix in the 13–18 fragment. A common structural motif was found in conformationally constrained Cys⁷?Cys¹³ fragment, which resembles the Greek letter ‘ω’. The differences in the solution structure of the C‐terminal fragment of the peptides studied are responsible for their specificity. [Aib¹⁶]ScyII showed 25% the agonistic activity of selective NK‐3 agonist – senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar¹⁶]ScyII appeared to be a full agonist of NK‐3 tachykinin receptor.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar¹,Tyr(Me)⁴]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with ?pA₂” values in the rat isolated uterus assay of ～ 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA₂ values of 6.7, 5.8, and < 5, while substitution of Phe for Tyr gave pA₂ values of 7.4, 6.7, and < 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar¹,Cha⁸]ANG II (7%), [Aib¹, Cha⁸]ANG II (12%) and [Des¹,Cha⁸]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

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.     

Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes

Background and purpose:

We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs).

Experimental approach:

The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E₂ (PGE₂) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP.

Key results:

Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol·L⁻¹-µmol·L⁻¹ concentration range, modulated COX-2 expression and PGE₂ release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar⁹, Met(O₂)¹¹]SP, [β-Ala⁸] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-κB concentration-dependently, with a maximum effect at 1 nmol·L⁻¹.

Conclusions and implications:

Human PMNs possess functional NK₁, NK₂ and NK₃ receptors, which mediate the induction of COX-2 expression and NF-κB activation by SP.