Characterization of receptors for endothelins in the perfused arterial and venous mesenteric vasculatures of the rat.

1. Endothelin-1 and -3 induced marked arterial and venous constrictions in the perfused mesenteric vasculature of the rat with endothelin-3 being at least 20 times less active than endothelin-1, on both arterial and venous sides of the vasculature. 2. Two ETB selective agonists, BQ-3020 and IRL 1620 (500 pmol), induced weak constrictions of the venous mesenteric vasculature and were inactive in the arterial side at doses up to 1000 pmol. 3. In mesenteric vasculatures precontracted with either methoxamine (arterial side) or the thromboxane A2-mimetic, U46619 (venous side), acetylcholine or bradykinin produced vasodilations of both arterial and venous vessels, whereas endothelin-3 induced vasodilations only on the arterial side. 4. A selective ETA receptor antagonist, BQ-123, blocked, in a concentration-dependent and reversible fashion, the vasoconstrictions induced by endothelin-1 on both sides of the mesenteric circulation (IC50; arterial side: 0.013 microM; venous side: 0.032 microM). 5. In contrast, the vasodilator responses induced by endothelin-3 on the arterial side of the precontracted mesenteric vasculature were not affected by BQ-123. 6. The present study illustrates the presence of ETA receptors which are responsible for vasoconstriction by endothelins in the arterial and venous mesenteric vasculatures. Furthermore, we suggest that the vasodilations induced by endothelin-3 in the arterial vasculature uniquely, are ETB receptor-mediated.     

Neurokinins produce selective venoconstriction via NK-3 receptors in the rat mesenteric vascular bed.

The vasoactive properties of the neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and some selective analogues were assessed in the arterial and venous mesenteric beds of the rat. Although both sides of the mesenteric vasculature displayed endothelium-dependent relaxation in response to acetylcholine (ACh) or bradykinin (BK) (1 and 10 nmol), SP and the selective NK-1 analogue, [Sar9,Met(O2)11]SP were inactive. Of the three selective neurokinin agonists used, [Sar9,Met(O2)11]SP (NK-1), [beta-Ala8]NKA-(4-10) (NK-2) and [MePhe7]NKB (NK-3), only the latter induced a dose-dependent pressor effect in the venous mesenteric vasculature. Injections of SP and the selective NK-1 and NK-2 analogues at high doses (10 nmol), did not change the perfusion pressure in the mesenteric bed even when the mesenteric vasculature was treated with methylene blue (50 microM) to inhibit the effects of endothelium-derived relaxing factor (EDRF) or with NG-nitro-L-arginine (L-NNA) (20 microM) to inhibit the formation of EDRF or with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] (CHAPS 20 mM, 30 s) to remove the endothelial layer. In contrast, the vasoconstrictor effects of noradrenaline (NA), angiotensin II (ATII), NKB and [MePhe7]NKB on the venous side of the circulation were enhanced following treatment with L-NNA, methylene blue or CHAPS. The present results suggest that neurokinins act on the rat mesenteric bed by increasing the perfusion pressure of the venous vasculature via activation of NK-3 receptors. Neurokinins are inactive on the arterial mesenteric vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for kinins in the human isolated umbilical vein.

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) >> [Aib7]BK (pD2 6.38) >> desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys.     

Peptides and histamine release from rat peritoneal mast cells

Various vasoactive peptides were compared for their histamine releasing effects on rat mast cells. Neurotensin, substance P (SP), and kallidin were the most active natural peptides, followed by bradykinin; neurokinin A and B, bombesin, angiotensin and tuftsin were practically inactive. Several kinins and tachykinin-related peptides were tested in an attempt to characterize the receptors mediating histamine liberation. The order of potency of the kinins was the following: kallidin greater than [Tyr(Me)8]bradykinin = bradykinin greater than [desArg10]kallidin greater than desArg9-bradykinin, the same as that found in smooth muscle possessing receptors of the B2 type. Tachykinin-related peptides were potent stimulants and followed the order: [D-Tryp7,9,10]SP-(1-11) greater than [D-Pro2,D-Tryp7,9,10]SP-(1-11) greater than SP-(1-11) greater than SP-(1-9) greater than [D-Pro4,D-Tryp7,9,Leu11]SP-(4-11) greater than SP-(1-7) greater than SP-(4-11) greater than neurokinin A = neurokinin B, indicating that: (a) undecapeptide antagonists of SP behave as superagonists; (b) both N- and C-terminal portions of SP-(1-11) are essential for activity; and (c) receptors for the tachykinins mediating histamine release appear to be of the SP-P type.     

Substance P and capsaicin-induced mechanical hyperalgesia in the rat knee joint; the involvement of bradykinin B1 and B2 receptors.

1. Substance P (SP) and capsaicin induced a mechanical hyperalgesia when injected into rat knee joints. 2. The NK1 receptor antagonists CP 99994 (10-100 nmol) and RP 67580 (0.1-1 nmol) blocked the development of, and also reversed, SP-induced hyperalgesia. Capsaicin (10 nmol)-induced hyperalgesia was blocked by capsazepine (0.5-5 nmol). 3. Capsaicin-induced hyperalgesia was prevented and reversed by the NK1 receptor antagonists CP 99994 (100 nmol) and RP 67580 (1 nmol). 4. The bradykinin B2 receptor antagonist icatibant (5 pmol) blocked the development of both SP and capsaicin-induced hyperalgesia. Icatibant (100 pmol kg-1, i.v.) also reversed an established SP and capsaicin-induced hyperalgesia. 5. Both low dose SP (1 nmol) and capsaicin (1 nmol)-induced hyperalgesia were potentiated by the kininase II inhibitor captopril (100 micrograms). 6. The B1 receptor antagonists desArg9Leu8-bradykinin (BK) (0.5-5 nmol) and desArg10[Hoe 140] (5-50 pmol) only blocked the development of SP-induced hyperalgesia for 30 min after administration. desArg9Leu8-BK (10 nmol kg-1 i.v.) did not reverse an established SP-induced hyperalgesia. 7. Capsaicin-induced hyperalgesia was blocked by desArg9Leu8-BK (0.5 nmol) and this antagonist also reversed an established capsaicin-induced hyperalgesia. 8. Interleukin-1 receptor antagonist (IL-1ra 0.1 microgram) reduced the development of SP-induced hyperalgesia up to 4 h after administration, but did not reverse an established hyperalgesia. IL-1ra (0.1 microgram) also blocked the development of and reversed an established capsaicin-induced hyperalgesia. 9. Indomethacin pretreatment (1 mg kg-1, s.c.) did not reduce the development of either SP- or capsaicin-induced hyperalgesia but following indomethacin-pretreatment desArg9Leu8-BK (10 nmol kg-1, i.v.) failed to reverse a capsaicin-induced hyperalgesia. 10. In conclusion, both SP and capsaicin can induce behavioural hyperalgesia when injected into the knee joint of rats. In addition, blockade of NK1, bradykinin B1, B2 and IL-1 beta receptors can substantially modulate this hyperalgesia.     

BK2 but not BK1 receptors mediating contractile response in human umbilical arteries: role of thromboxane A2

Bradykinin receptors have been divided into B1 and B2 subtypes. The aim of this study on human umbilical arteries was: i) to compare the recognition properties of the mediating contractions of bradykinin receptors; and ii) to assess the possible role of thromboxane A2 in a bradykinin-induced contraction of smooth muscle. Umbilical arteries were dissected and mounted in organ baths for isometric measurement of force. Our results showed that the B1 agonist [Sar1dPhe8desArg9]-bradykinin had no effect on the concentration-response 10(-9)-3 x 10(-5) mM. Cumulative additions of bradykinin (10(-9)-3 x 10(-5) mM) and of the B2 agonist [Hyp3TyrMe8]-bradykinin (10(-9)-3 x 10(-5) mM) produced dose-dependent contractions. Dose response curves to bradykinin (10(-9)-3 x 10(-5) mM) were not significantly altered by the presence of B1 selective antagonist [des-Arg9, Leu8]-bradykinin (10(-5) mM), or by the selective B2 antagonist [Thi(5,8), D-Phe7]-bradykinin (10(-5) mM). However, Hoe 140 D-Arg-[Hyp3, Thi5,D-Tic7, Oic8]-bradykinin, an antagonist of B2 responses, significantly inhibited bradykinin-induced contraction. The responses to bradykinin were unaffected by indomethacin (10(-4) mM), dazoxiben (10(-5) mM) or even by nordihydroguaiaretic acid (10(-5) mM). However, bradykinin contractions were antagonized in a noncompetitive manner by quinacrine (10(-5) mM). These results showed that bradykinin contracts human umbilical arteries essentially through B2 receptors. Moreover, the responses to bradykinin are unlikely to be mediated by the cyclooxygenase/lipooxygenase pathway. The inhibitory effects of quinacrine may be due to a specific or nonspecific effect at a cellular level on smooth muscle contractility, or due to a direct action to block Ca2+ influx at membrane level.     

Evidence that tachykinins relax the guinea-pig trachea via nitric oxide release and by stimulation of a septide-insensitive NK1 receptor.

1. This study investigated the possibility that tachykinins relax the guinea-pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 microM) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl-N[4-(4-acetyl amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), 0.1 microM). 2. In the presence of the inactive enantiomer of an NO-synthase inhibitor, NG-monomethyl-D-arginine (D-NMMA, 100 microM), substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the selective NK1 receptor agonist, [Sar9, Met(O2)11]-SP, (0.1-10 nM) relaxed tracheal tube preparations. This relaxation was changed into a contraction by pretreatment with the NO-synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 microM). The effect of L-NMMA on SP- and [Sar9, Met(O2)11]-SP-induced responses was reversed by L-arginine (L-Arg, 1 mM), but not by D-Arg (1 mM). After removal of the epithelium SP, NKA and NKB and [Sar9, Met(O2)11]-SP (0.1-10 nM) evoked contractile responses in the presence of either L-NMMA (100 microM) or D-NMMA (100 microM). The effects of SP and [Sar9, Met(O2)11]-SP obtained in the presence of another NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) or its inactive enantiomer, NG-nitro-D-arginine methyl ester (D-NAME, 100 microM) were similar to those observed with L-NMMA or D-NMMA, respectively. 3. The selective NK1 receptor agonist, [pGlu6, Pro9]-SP(6-11) (septide, 0.1-10 nM) evoked contractile responses of tracheal tube preparations in the presence of either D-NMMA (100 microM) or L-NMMA (100 microM). The log concentration-response curve to septide obtained in the presence of L-NMMA was similar to that obtained in the presence of D-NMMA. [Sar9, Met(O2)11]-SP (0.1-10 nM) relaxed tracheal tube preparations precontracted with septide (1 microM), whereas septide (0.1 nM-1 microM) further contracted tracheal tube preparations precontracted with [Sar9, Met(O2)11]-SP (1 microM). 4. Relaxant and contractile responses evoked by SP, NKA, NKB and by [Sar9, Met(O2)11]-SP (0.1-10 nM) were not affected by a combination of the histamine H1 (pyrilamine, 1 microM) and H2 (cimetidine, 1 microM) receptor antagonists, but were abolished by the tachykinin NK1 receptor antagonist, CP-99,994 ((2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine, 1 microM), though not by its inactive enantiomer CP-100,263 (1 microM). Contractile responses evoked by septide (10 nM and 1 microM) were also abolished by CP-99,994 (1 microM) but not by CP-100,263 (1 microM). 5. These results demonstrate that tachykinins relax guinea-pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK1 receptors by a mechanism independent of histamine release. The NK1 receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)11]-SP but not to septide, and is pharmacologically distinct from the NK1 receptor that mediates contraction, which is stimulated by all the agonists, including septide.     

Study of SR 142801, a new potent non-peptide NK3 receptor antagonist on cardiovascular responses in conscious guinea-pig.

1. The cardiovascular responses to intravenous (i.v.) injection of natural tachykinins, substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective tachykinin (NK) receptor agonists, [Sar9, Met(O2)11]SP, [beta Ala8]NKA(4-10), [MePhe7]NKB and senktide were assessed in conscious, freely moving, guinea-pigs. 2. SP and [Sar9, Met(O2)11]SP (1-1000 pmol kg-1) induced dose-dependent decreases in mean arterial blood pressure (MAP) accompanied by increases in heart rate (HR). NKA evoked only weak hypotensive effects at high doses (3000 pmol kg-1) whereas [beta Ala8]NKA(4-10) (1-3000 pmol kg-1) had no effects. By contrast, NKB [MePhe7]NKB (1-10,000 pmol kg-1) and senktide (1-1000 pmol kg-1), produced dose-related hypertensive effects with the following rank order of potency: senktide > [MePhe7]NKB > NKB. Bradycardia occurred simultaneously with the increases in arterial pressure. 3. The pressor response to intravenous injection of senktide (300 pmol kg-1) was partially reduced by pretreatment with prazosin (0.71 mumol kg-1), or clonidine (0.38 mumol kg-1) and was completely inhibited by the combination of the two compounds. Atropine (1.5 mumol kg-1) suppressed the decrease in HR induced by senktide without altering the blood pressure response. These findings suggest that the blood pressure response to senktide is an indirect effect mediated by noradrenaline released from sympathetic nerve endings, whereas the bradycardia is of vagal reflex origin. 4. SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide), a potent and specific non-peptide NK3 receptor antagonist dose-dependently (0.46-4.6 mumol kg-1, i.v.; 4.6-46 mumol kg-1, p.o.) inhibited the cardiovascular effects of senktide and displayed a long-lasting inhibitory effect after oral administration. By contrast, SR 142806 (4.6 mumol kg-1, i.v.), the (R)-enantiomer of SR 142801 had no effect on the responses to senktide. SR 142801 at a high dose (15 mumol kg-1, i.v.) was inactive toward the [Sar9, Met(O2)11]SP-induced hypotension. 5. SR 142801 did not modify MAP in conscious guinea-pigs both after i.v. (4.6 and 15 mumol kg-1) and oral (46 and 150 mumol kg-1) administration, showing a lack of agonistic properties. However, a slight reduction in HR was observed only after i.v. injection. 6. In conclusion, these results show evident differences in the functional role of tachykinin receptors in the peripheral control of the cardiovascular system. Furthermore, a clear pressor effect of senktide, which was selectively blocked by SR 142801, was observed in conscious guinea-pigs. Hence, this antagonist appears suitable for investigating the functional role of NK3 receptors.     

Tachykinin receptors in the guinea-pig renal pelvis: activation by exogenous and endogenous tachykinins.

1. The contractile response to substance P, neurokinin A, selective agonists for the NK1, NK2 and NK3 tachykinin receptors and the activity of receptor-selective antagonists has been investigated in circular muscle strips of the guinea-pig isolated renal pelvis in the presence of indomethacin (3 microM). 2. Neurokinin A was the most potent agonist tested, being about 32 times more potent than substance P. The action of both substance P and neurokinin A was enhanced by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The selective NK2 receptor agonist [beta Ala8] neurokinin A (4-10), was slightly less potent and effective than neurokinin A itself. The selective NK1 receptor agonist [Sar9] substance P sulphone was effective at low (nM) concentrations but its maximal effect did not exceed 30% of maximal response to substance P or neurokinin A. The NK3-selective agonist [MePhe7] neurokinin B was effective only at high (microM) concentrations. 3. The pseudopeptide derivative of neurokinin A(4-10), MDL 28,564, displayed a clear-cut agonist character, although it was less potent than neurokinin A. 4. The responses to roughly equieffective (25-35% of maximal response) concentrations of [beta Ala8] neurokinin A (4-10), MDL 28,564 and [MePhe7] neurokinin B were antagonized to a similar extent by MEN 10,376 (3 microM), a selective NK2 tachykinin receptor antagonist, while the response to [Sar9] substance P sulphone was unchanged. 5. The response to [Sar9] substance P sulphone was inhibited by the NK1 receptor-selective antagonist, GR 82,334 (3 microM) while the response to [beta Ala8] neurokinin A (4-10) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Tachykinin NK1 and NK2 receptors mediate inhibitory vs excitatory motor responses in human isolated corpus cavernosum and spongiosum

Motor effects produced by tachykinins were studied in human isolated corpus spongiosum and cavernosum. In quiescent preparations neurokinin A caused potent contractions (pD(2)=8.3 - 7.9 respectively) prevented by the NK(2) receptor-selective antagonist nepadutant, whereas [Sar(9)]SP sulfone and senktide (NK(1) and NK(3) receptor-selective agonists) produced no effect or spare contractions. In KCl-precontracted corpus spongiosum septide (pD(2)=7.1) and [Sar(9)]SP sulfone (pD(2)=7.7) produced tetrodotoxin-resistant relaxations, abolished by the tachykinin NK(1) receptor-selective antagonist SR 140333. [Sar(9)]SP sulfone (1 microM) produced similar relaxations in precontracted corpus cavernosum. Electrical field stimulation (EFS) elicited tetrodotoxin-sensitive relaxations, which were additive to those produced by [Sar(9)]SP sulfone. N(omega)-nitro-L-arginine (L-NOARG) totally prevented both [Sar(9)]SP sulfone- and EFS-induced relaxations. These results show that tachykinin NK(1) and NK(2) receptors mediate opposite motor effects in human penile tissues, suggesting a possible modulatory role of tachykinins on smooth muscle tone in these organs.     

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.     

Characterization of the bradykinin receptor in the human nasal airway using the binding of [125I]-Hoe 140.

1. The aim of this study was to characterize the kinin receptor in the human nasal airway using [125I]-Hoe 140 binding to a membrane preparation from human nasal turbinates and to compare Ki values from binding displacement by antagonists with the functional effects of these drugs in vivo. We also investigated the effect of Hoe 140 ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), on bradykinin release into the nasal airway. 2. In a membrane preparation from human nasal turbinates removed during surgery, [125I]-Hoe 140 labelled a single, saturable binding site. The equilibrium dissociation constant (at 20 degrees C) for [125I]-Hoe 140 binding to the receptor was 0.46 +/- 0.08 nM. The Bmax was 0.136 +/- 0.003 pmol mg-1 protein and the Hill coefficient was 1.01 +/- 0.07. 3. The association rate constant for [125I]-Hoe 140 binding to the receptor was 0.20 +/- 0.06 nM-1 min-1 and the dissociation rate constant was 0.14 +/- 0.01 min-1. These values were determined at 4 degrees C. The equilibrium dissociation constant calculated from these rate constants was 0.70 nM. 4. Bradykinin and the B2 receptor antagonists, NPC 567, NPC 17731, NPC 17761, [1-adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, WIN 64338 and Hoe 140 displaced [125I]-Hoe 140 binding: the Ki values from binding displacement are consistent with values expected from a B2 receptor. The B1 agonist, [des-Arg9]-bradykinin and the B1 antagonist, [des-Arg9]-Hoe 140 failed to displace [125I]-Hoe 140 binding at concentrations up to 1 microM. 5. The bradykinin antagonist, Hoe 140, 10 to 200 micrograms, given by intranasal aerosol, produced a dose-related inhibition of the reduction in minimal nasal cross-sectional area (Amin) induced by bradykinin in normal subjects and by house dust mite antigen in subjects with allergic rhinitis to house dust mite. Hoe 140, 10 to 200 micrograms, also caused a dose-related inhibition of the release of albumin into the nasal cavity following challenge with bradykinin. 6. [1-Adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, 30 to 200 micrograms, caused a dose-related inhibition of the reduction in Amin and the release of albumin into the nasal cavity induced by bradykinin. NPC 567 ([D-Arg0, Hyp3, D-Phe7]-bradykinin) failed to inhibit the reduction in Amin or the release of albumin into the nasal cavity at a dose of 10 mg. 7. Challenge of allergic subjects with house dust mite antigen caused a significant elevation of the bradykinin concentration in nasal lavage fluid and a reduction in Amin. Hoe 140, 100 micrograms, prevented the antigen-induced reduction in Amin and also abolished the antigen-induced increase of bradykinin in nasal lavage fluid. 8. We conclude that there is a B2 bradykinin receptor in the human nasal airway which mediates nasal blockage and plasma extravasation induced by either bradykinin or antigen challenge. It is possible that Hoe 140 inhibits kallikrein in the human nasal airway as well as blocking the B2 receptor.     

Characterization of central and peripheral effects of septide with the use of five tachykinin NK1 receptor antagonists in the rat.

1. Effects of two tachykinin NK1 receptor selective agonists (septide and [Sar9, Met(O2)11]SP) were compared on the increases in mean arterial pressure (MAP), heart rate (HR) and motor behaviour following intracerebroventricular (i.c.v.) administration in unanaesthetized rat, and on the vascular permeability increases to intradermal (i.d.) injection in the anaesthetized rat. Moreover, five tachykinin NK1 receptor selective antagonists (LY303870, LY306740, LY303241, SR140333 and RP67580) were tested against the two agonists to compare their pharmacological profile. 2. [Sar9, Met(O2)11]SP and septide (10-100 pmol per rat, i.c.v.) were equipotent in increasing MAP and HR, yet they had dissimilar time-course. Both agonists increased dose-dependently face washing and sniffing while [Sar9, Met(O2)11]SP was the sole to produce grooming, septide was more potent than [Sar9, Met(O2)11]SP (6.5-650 pmol) in increasing vascular permeability. 3. For most centrally mediated responses, LY303870 and RP67580 were significantly more potent in inhibiting septide than [Sar9, Met(O2)11]SP. In some parameters, greater blockade was achieved when antagonists (particularly LY306740) were given 1 h instead of 10 min prior to i.c.v. septide. 4. All antagonists except LY303241 blocked dose-dependently the increases in vascular permeability to equipotent doses of [Sar9, Met(O2)11]SP and septide. LY303870 and LY306740 were more potent against septide. 5. The antagonism afforded by LY303870, LY306740 and LY303241 was stereoselective and only SR140333 was found to cause central and peripheral non specific effects. 6. The data confirm a distinct pharmacological profile for septide in vivo. RP67580 and LY306740 are currently the most valuable tachykinin NK1 receptor antagonists for in vivo studies in rat.     

Role of R-type calcium channels in the response of the perfused arterial and venous mesenteric vasculature of the rat to platelet-activating factor.

1. The vasoactive properties of platelet-activating factor (PAF) were studied in the arterial and venous vasculature of the rat double-perfused mesenteric bed. Although PAF (0.01-0.3 pmol) induced a dose-dependent vasodilatation of the arterial mesenteric vasculature, it triggered only vasoconstrictions on the venous side, with an intact endothelium as bradykinin induced a significant venodilatation. 2. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), a nitric oxide synthase inhibitor, markedly reduced the vasodilatation induced by PAF in the arterial mesenteric vasculature and potentiated the contractile responses of the venous side to the same agent. 3. The PAF antagonist, WEB-2170, markedly reduced the response to PAF on both sides of the mesenteric vasculature. However, the IC50 of WEB-2170 against PAF was reached at a much higher concentration (1 x 10(-8) M) on the arterial side than on the venous side (5.3 x 10(-11) M). Furthermore, a second antagonist of PAF receptors, SRI-63441, although being less potent on the venous vasculature than WEB-2170, was equipotent in antagonizing the venoconstriction and the arterial dilatation induced by PAF (IC50 of SRI-63441, arterial side: 2.9 x 10(-9) M; venous side: 3.1 x 10(-9) M). 4. The dual L- and R-calcium channel blocker, isradipine (PN 200-110), but not the L-type calcium channel blocker, nifedipine, markedly reduced the PAF-induced vasoactive properties on both sides of the mesenteric vasculature. 5. Our results illustrate the differential vasoactive properties of PAF in the mesenteric vasculature of the rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-dependent relaxant effect of neurokinins on rabbit aorta is mediated by the NK1 receptor.

Several neurokinins, namely substance P, neurokinin A, neurokinin B, [beta-Ala8]neurokinin A-(4-10) and senktide, were tested on noradrenaline-precontracted rabbit aortic rings to characterize the receptor mediating their endothelium-dependent relaxant effect in this preparation. CP-96,345, the new nonpeptide antagonist selective for the NK1 receptor, was also studied. Substance P, neurokinin A and neurokinin B, in that order of potency, were effective in relaxing precontracted rings, indicating the involvement of the NK1 receptor; [beta-Ala8]neurokinin A-(4-10) and senktide, which are selective agonists for NK2 and NK3 receptors, respectively, had no significant relaxant effect. The relaxant effects of substance P, neurokinin A and neurokinin B were competitively antagonized by nanomolar concentrations of CP-96,345. These findings support the view that the NK1 receptor mediates the endothelium-dependent relaxant effect of the neurokinins in rabbit aorta.     

Pharmacological characterization of a new highly potent B2 receptor antagonist (HOE 140: D-Arg-[Hyp3,Thi5,D-Tic7,Qic8]bradykinin).

HOE 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), a new B2 antagonist, was compared to R-493 (D-Arg[Hyp3-D-Phe7,Leu8]bradykinin) with respect to inhibition of the responses of seven isolated smooth muscle preparations to bradykinin. R-493 was found to exert: (a) high antagonistic activity on the rabbit jugular vein (pA2 of 8.86), (b) moderate activity on the rabbit aorta, guinea-pig ileum, hamster urinary bladder and human urinary bladder (pA2 of 5.76, 6.77, 7.16 and 7.15, respectively) and (c) a stimulatory effect on the guinea-pig trachea. On the other hand, HOE 140 showed identical apparent affinities (8.36-9.12) on all preparations except the rabbit aorta where it was inactive and the guinea-pig trachea where the compound was an antagonist (pA2: 7.42) without agonistic effect. HOE 140 is specific and selective for B2 receptors since it was inactive against angiotensin II, substance P, neurokinin A, desArg9-bradykinin, noradrenaline or acetylcholine in the various preparations. R-493 inhibited the contractile effects of bradykinin competitively, while HOE 140 was not competitive even at low concentrations (7.7 x 10(-9) M). These results demonstrate that HOE 140 is a potent B2 antagonist with high affinity, specific for kinin receptors and selective for the B2 receptor type, but is non-competitive. HOE 140 is the first bradykinin receptor antagonist that acts as such on the guinea-pig trachea without showing any agonistic activity.     

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.     

Structure-activity studies on bradykinin and related peptides: agonists.

1. Bradykinin, kallidin, T-kinin, [Hyp3]-bradykinin and several analogues were prepared by solid-phase synthesis and purified by high performance liquid chromatography. 2. The various peptides were tested for their abilities to relax the dog carotid and renal arteries, or to contract the rabbit jugular vein and aorta, in order to measure their activities on BK2 (the first three preparations) or BK1 (the rabbit aorta) receptors. The dog renal artery without endothelium was also used as a BK1 receptor system. 3. T-kinin was found to be less active than bradykinin, while the replacement of Pro3 with Hyp favoured BK2 receptor occupation. [Hyp3,Tyr(Me)8]-BK was found to be a selective BK2 receptor agonist. 4. Amidation or methylation of the C-terminal carboxyl decreased activity, while extension of the N-terminal with Sar or D-Arg increased affinity and selectivity for BK1 (Sar) and affinity for BK2 (D-Arg) receptors. Acetylation of N-terminal amide brought affinity down to 10% or less. 5. Replacement of the peptide bonds Phe8-Arg9 to protect from kininase I and II, decreased affinities slightly, but was incompatible with additional changes at the N-terminal or in the peptide bond Gly4-Phe5. 6. Substitution of C-terminal Phe in desArg9-BK (the BK1 receptor stimulant) with D-Phe increased potency and selectivity for BK1 receptors while protecting from carboxypeptidases. Sar[D-Phe8]desArg9-BK was found to be a potent and selective BK1 receptor agonist.