Impaired IL-1β-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice

Tachykinin NK₁ receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air-pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin-1β (IL-1β, 10 ng into the air-pouch) is altered in NK₁ receptor knockout mice compared to wild type controls. Air-pouches were washed following a 4 h IL-1β treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL-1β was significantly attenuated in NK₁ receptor +/− (40% reduction) and −/− mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine-induced neutrophil chemoattractant (CINC, 0.3 μg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK₁ receptor +/− and −/− mice compared to wild type controls. In conclusion NK₁ receptors play a significant role in the cellular response to IL-1β in a model of inflammation.     

Inhibition by SR 140333 of NK1 tachykinin receptor-evoked, nitric oxide-dependent vasodilatation in the hamster cheek pouch microvasculature in vivo.

1. This study investigated tachykinin-evoked vasodilatation in the microvasculature of the hamster cheek pouch in vivo. Arterioles and venules were observed by intravital microscopy with video recording, and vasodilatation and constriction, defined as changes in blood vessel diameter, measured by image analysis. All agents were applied topically by superfusion. None of the agents tested had a significant effect on venule diameter. 2. When arterioles were preconstricted (by ca. 50%) with endothelin-1 present in the superfusing medium, substance P (0.3-30 nM) was a potent vasodilator, being 10 fold more active than both neurokinin A and the NK1 receptor-selective agonist, substance P methyl ester. The NK2 receptor-selective agonist, [beta-Ala8]-NKA(4-10)(0.1-10 microM) was active only at high concentrations, and the NK3 receptor-selective agonist senktide (0.1-10 microM) was virtually inactive (n = 8 hamsters). Dilatation evoked by tachykinins and analogues was rapid in onset (< 0.5 min) and readily reversible. 3. At low concentrations (1-10 nM), the non-peptide tachykinin NK1 receptor antagonist SR140333 ((S)1-(2-[3(3,4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)pi peridin-3- yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octone, chloride) had no effect on the diameter of preconstricted arterioles per se, but potently inhibited dilator responses to substance P methyl ester (apparent pKB 9.9 +/- 0.2; n = 5 hamsters, n = 10 estimates). SR140333 (10 nM) did not inhibit submaximal dilator responses evoked by human alpha calcitonin gene-related peptide (alpha CGRPh; 1.0 nM; P > 0.05; n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Further evidence for the existence of NK2 tachykinin receptor subtypes.

1. We have evaluated the biological activity of a number of neurokinin A (4-10), (NKA (4-10)) analogues in the endothelium-deprived rabbit isolated pulmonary artery (RPA) and hamster isolated trachea (HT), two tissues rich in different NK2 receptor subtypes. 2. MDL 28,564, a pseudopeptide selective for NK2 receptor sites, behaved as a full agonist in the RPA, while in the HT it competitively antagonized NKA or [beta Ala8]-NKA (4-10) contractile effects. 3. The peculiar behaviour of MDL 28,564 in the RPA and HT may be explained neither by a difference in receptor reserve between the two organs (the reserve being three times greater in RPA than in the HT) nor by a different affinity for the two receptor subtypes (identical dissociation constants, pKA or pKB, calculated in the RPA and in the HT). On the other hand, MDL 28,564 displayed a very different intrinsic efficacy for the two receptor subtypes. 4. The novel peptides MEN 10,295 ([Trp7, beta Ala8]-NKA-(4-10)) and MEN 10,296 ([Tyr5, Trp7, beta Ala8]-NKA-(4-10] behaved as weaker agonists than MDL 28,564 in the RPA, but retained appreciable agonist activity also in the HT. 5. The novel peptides: MEN 10,282 ([Tyr5, D-Trp6,8, Trp9, Arg10]-NKA-(4-10], MEN 10,449 ([diI-Try5, D-Trp6,8,9, Arg10]-NKA-(4-10] and the cyclic hexapeptide L 659,877 (cyclo [Leu-Met-Gln-Trp-Phe-Gly]) behaved as competitive antagonists against NKA contractile effects both in the RPA and HT. MEN 10,282 and MEN 10,449 were unable to distinguish between the NK2 receptor subtypes, having almost the same affinity in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo evidence for the involvement of tachykinin NK3 receptors in the hexamethonium-resistant inhibitory transmission in the rat colon

In urethane-anaesthetized rats, moderate colonic distension (0.5 ml) induced reflex rhythmic contractions (5 mm Hg amplitude and 1.1 cycles/min frequency). Senktide (1–10 nmol/kg, i.v.), a tachykinin NK₃ receptor selective agonist, transiently suppressed distension-induced contractions. SR 142,801 (1–10 gmol/kg, i.v.), a non-peptide tachykinin NK₃ receptor antagonist, had no effect on distension-induced contractions but prevented the inhibitory effect of senktide.Infusion of N--nitro-1-arginine methyl esther hydrochloride (L-NAME, 20 mol/ml/h, i.v.) increased the amplitude of colonic contractions and decreased the inhibitory effect of senktide.Hexamethonium (15 mol/ml/h, i.v.) or atropine (1 mol/ml/h, i.v.) inhibited the distension-induced contractions. In hexamethonium- or atropine-treated rats, senktide (10 nmol/kg) transiently and selectively enhanced the amplitude of contractions. Also SR 142,801 (10 mol/kg), but not its inactive enantiomer SR 142,806, increased both amplitude and frequency of contractions.During continuous infusion of L-NAME and hexamethonium or atropine both frequency and amplitude of distension-induced colonic contractions were higher than when in hexamethonium or atropine only. Senktide (10 nmol/kg) had no effect and SR 142,801 (10 pmol/kg) produced a slight enhancement of colonic contractions.Infusion of sodium nitroprusside (3 mol/ml/h, i.v.) decreased amplitude and frequency of distension-induced contractions. SR 142,801 had no effect in the presence of the nitric oxide (NO) donor.We conclude that tachykinins acting through NK₃ receptors exert at least four different actions on colonic motility activated by distension: (1) a hexamethonium resistant, NO-dependent, suppressant effect on contractions; (2) a hexamethonium-sensitive, NO-independent inhibitory effect on the amplitude of contractions; (3) a hexamethonium-resistant, NO-independent inhibitory effect on the amplitude of contractions and (4) a hexamethonium resistant and L-NAME-sensitive excitatory effect on amplitude of contractions. The prevalent inhibitory effect evoked in normal conditions along with the excitatory activity induced by SR 142,801 on hexamethonium-resistant colonic motility indicates that tachykinins, acting through neuronal NK₃ receptors, activate NO-dependent and NO-independent inhibitory neurotransmission in the rat colon.     

Endogenous modulation of excitatory amino acid responsiveness by tachykinin NK1 and NK2 receptors in the rat spinal cord.

1. The effects of selective tachykinin (neurokinin, NK) NK1 and NK2 receptor antagonists have been examined on spinal neurones in alpha-chloralose anaesthetized, spinalized rats. They were tested for effects on responses both to excitatory amino acids (EAA) and to noxious heat stimuli. They were also tested for their ability to reverse the actions of selective NK agonists. 2. The NK1-selective antagonists GR82334 (peptide) and CP-99,994 (non-peptide), when applied by microiontophoresis, both reduced responses to kainate > AMPA > NMDA. Intravenous CP-99,994 (3 mg kg-1) also reduced responses to kainate but had inconsistent effects on nociceptive responses. 3. GR82334, applied microiontophoretically, reduced the enhancement by the selective NK1 agonist, GR73632 of both responses to EAAs and background activity. Systemic CP-99,994 (< or = 10 mg kg-1) failed to reverse the effects of GR73632. 4. The selective peptide NK2 antagonist, GR103537, had no consistent effects on responses to EAAs when applied by iontophoresis. In contrast, the non-peptide NK2 antagonist, GR159897, administered systemically (0.5-2 mg kg-1, i.v.) enhanced responses to kainate (but not NMDA); responses to noxious heat were enhanced only weakly. 5. Iontophoretically-administered GR103537 attenuated the effects of the NK2 agonist GR64349, which selectively reduced responses to kainate compared to those to NMDA. Systemically administered GR159897 (< or = 2 mg kg-1, i.v.) caused little antagonism of the effects of GR64349. 6. The data indicate that under these conditions the non-peptide antagonists are not reliable at reversing the actions of selective NK agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

A pharmacological study of NK1 and NK2 tachykinin receptor characteristics in the rat isolated urinary bladder.

1. We have estimated potencies of tachykinin receptor agonist and antagonist analogues in order to determine the recognition characteristics of tachykinin receptors mediating phasic contractile responses of the rat isolated urinary bladder in vitro. 2. The NK1-selective synthetic agonists, substance P methyl ester and GR73632, the synthetic NK2-selective agonists [beta-Ala8]-NKA(4-10) and GR64349, and the mammalian tachykinins, neurokinin A and neurokinin B, were assayed relative to substance P and were found to be approximately equipotent. The NK3-selective agonist, senktide, was inactive (10 microM). 3. Potencies of all these agonists were not significantly different (P > 0.05) when experiments were carried out in the presence of the neutral endopeptidase inhibitor, phosphoramidon, and the kininase II inhibitor, enalaprilat (both 1 microM). 4. The NK1-selective antagonist, GR82334, inhibited responses to substance P methyl ester in a competitive manner in the rat urinary bladder and the rat ileum, and also in the guinea-pig ileum. Markedly different pKB estimates were obtained in the rat bladder (6.38) and rat ileum (6.56) compared to the guinea-pig ileum (7.42). GR82334 (3 microM) was inactive against responses of the rat bladder to [beta-Ala8]-NKA(4-10). 5. The NK1-selective antagonist (+/-)-CP-96,345 also inhibited responses of the rat bladder and guinea-pig ileum to substance P methyl ester; however, in the rat bladder at 1 microM, this antagonist reversibly inhibited responses both to the NK2-selective agonist [beta-Ala8]-NKA(4-10) and to the muscarinic agonist carbachol (P < or = 0.01), thus showing evidence of some non-selective depressant actions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct evidence for the interaction of neurokinin A with the tachykinin NK(1) receptor in tissue.

Neurokinin A (NKA) is a tachykinin peptide that binds with high affinity to the tachykinin NK(2) receptor. Recent homologous binding studies, however, have shown that neurokinin A is also a high-affinity ligand for the tachykinin NK(1) receptor. In this report, we demonstrate that a photoreactive neurokinin A analogue specifically labels the NK(1) receptor in rat submandibular gland membranes and show via bioassay that neurokinin A is a potent stimulator of salivary secretion. Through the use of specific non-peptide antagonists in both photolabeling and functional assays, we unequivocally demonstrate that neurokinin A can specifically interact with the NK(1) receptor in vivo and elicit NK(1) receptor-mediated physiological responses.     

Characterization of NK1 and NK2 tachykinin receptors in guinea-pig and rat bronchopulmonary and vascular systems.

1. NK1 and NK2 tachykinin receptors were characterized in guinea-pig and rat bronchopulmonary systems and in the vasculature of the rat by use of radioligand binding and/or functional studies. 2. The radioligands for NK1 and NK2 receptors ([3H]-SP and [3H]-pNKA, respectively) did not label tachykinin receptors in homogenates of rat lungs or bronchi. In contrast, in the guinea-pig, [3H]-SP bound with high affinity to these tissues (KD = 0.23 +/- 0.08 nM and 0.34 +/- 0.05 nM, for lungs and bronchi, respectively). The total number of binding sites was 4.6 fold greater in bronchus (Bmax = 135 +/- 27 fmol mg-1 protein) than in lung homogenates (Bmax = 29.3 +/- 0.1 fmol mg-1 protein). Furthermore, this binding was markedly displaced by CP-96,345 (pKi = 9.5 +/- 0.1) and RP 67580 (pKi = 7.6 +/- 0.1), antagonists of NK1 receptors, slightly displaced by SR 48968 (pKi = 6.6 +/- 0.1), but not affected by actinomycin D or L-659,877, antagonists of NK2 receptors. Specific binding of [3H]-pNKA, detected in guinea-pig bronchi (KD = 5.2 +/- 0.1 nM, and Bmax = 203 +/- 19 fmol mg-1 protein) but not in lungs, was similarly (40 to 53%) displaced by RP 67580 (1 microM), CP-96,345 (10 and 100 nM) or SR 48968 (10 and 100 nM). The displacement approximately doubled (87 to 91%) when SR 48968 (10 nM) was combined with either RP 67580 (1 microM) or CP-96,345 (10 nM), but not when RP 67580 was combined with CP-96,345. 3. In urethane-anaesthetized guinea-pigs, i.v. injections of the NK1 receptor agonists SP, [Pro9]-SP, [Sar9,Met(O2)11]-SP and septide, as well as the NK2 receptor agonists NKA and [Lys5,MeLeu9,NLeu10]-NKA(4-10) (0.1-10 micrograms kg-1, i.v.), dose-dependently increased lung inflation pressure. The most potent of these peptides were septide and [Lys5, MeLeu9,NLeu10]-NKA(4-10) (EC50 = 0.38 +/- 0.07 and 0.07 +/- 0.02 microgram kg-1, respectively). Interestingly, septide was 130 fold less potent than SP in displacing [3H]-SP from its binding sites in the guinea-pig lung, whereas it was 14 fold more potent than SP as a bronchoconstrictor. RP 67580 (0.3-5 mg kg-1, i.v.) and CP-96,345 (0.01-3 mg kg-1, i.v.) dose-dependently reduced the bronchoconstriction produced by the NK1 receptor agonists.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of tachykinin NK1 or PAF receptor blockade on the lung injury induced by scorpion venom in rats.

In cases of severe human scorpion envenoming, lung injury is a common finding and frequently the cause of death. In the rat, two distinct mechanisms account for oedema following the intravenous injection of the venom -- acute left ventricular failure resulting from a massive release of catecholamines and an increase in pulmonary vascular permeability. In the present work, we investigated the effects of a tachykinin NK1 receptor antagonist (CP96,345, the dihydrochloride salt of (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)methyl)-1-az abicycol[2.2.2]octan-3-amine) and its 2 R-3 R inactive enantiomer (CP96,344) on the acute lung injury induced by the i.v. injection of Tityus serrulatus venom in rats. Lung injury was assessed by evaluating the extravasation of Evans blue dye in the bronchoalveolar lavage fluid and in the lung of venom-treated and control animals. The effects of the platelet-activating factor (PAF) receptor antagonist WEB2170 (2-methyl-1-phenylimidazol[4,5c]pyridine) were evaluated for comparison. The i.v. injection of the venom induced the extravasation of Evans blue in the bronchoalveolar lavage fluid and into the left lung. Pretreament with the tachykinin NK1 receptor antagonist CP96,345, but not CP96,344, inhibited Evans blue dye extravasation in the bronchoalveolar lavage fluid and in the lung by 96% and 86%, respectively. The PAF receptor antagonist WEB2170 inhibited the increase in vascular permeability in the bronchoalveolar lavage fluid by 60% and had no effect on the extravasation to the lung parenchyma of venom-injected animals. In addition to abrogating lung injury, pretreatment of rats with CP96,345, but not CP96,344 or WEB2170, decreased by 70% the mortality induced by the venom. This is the first study to show the relevance of the tachykinin NK1 receptor in mediating lung injury and mortality in animals injected with the neurotoxic T. serrulatus venom. Blockade of the tachykinin NK1 receptor may represent an important strategy in the treatment of patients with signs of severe envenoming and clearly deserves further studies.     

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.     

Central anti-hypertensive effect of tachykinin NK3 receptor antagonists in rat

Tachykinins are involved in the central autonomic control of blood pressure. In the present study, we examined the i.c.v. cardiovascular effects of several tachykinin receptor antagonists in awake spontaneously hypertensive rats (SHR, 15 weeks old). Results showed that two tachykinin NK(3) receptor antagonists (R-820: 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl and SB 222200: (S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide) caused a sustained and dose-dependent reduction of blood pressure when injected i.c.v. but not i.v. The stereoselective anti-hypertensive effect of SB 222200 peaked at 3 h and faded at 6 h post-injection (if injected at 07:00 h) or had a slower onset and peaked at 8 h post-injection (if injected at 13:00 h). The effect of R-820 was maximal at 24 h and lasted up to 48 h post-injection. Both antagonists failed to alter blood pressure in normotensive Wistar-Kyoto rats (WKY) and heart rate was not affected in both strains. The anti-hypertensive effect of SB 222200 was not associated with changes in plasma levels of catecholamines and vasopressin and it remained unchanged in SHR subjected to acute bilateral nephrectomy. In contrast, blood pressure was not affected by tachykinin NK(1) (RP 67580: (+/-) 7,7-diphenyl-2[1-imino-2(2-methoxy-phenyl)-ethyl]perhydroisoindol-4-one(3aR,7aR)) and NK(2) (SR 48968: (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide) receptor antagonists. Data suggest that brain tachykinin NK(3) receptors are implicated in the maintenance of hypertension in SHR. Hence, these receptors may represent promising therapeutic target in the treatment of arterial hypertension.     

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis

1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor.     

Evidence for neurokinin-3 receptor-mediated tachykinin release in the guinea-pig ileum

Contraction of longitudinal muscle strips of the guinea-pig ileum induced by the selective NK3 receptor agonist, succ-[Asp6,MePhe8]SP-(6-11) (senktide), were completely inhibited by tetrodotoxin and partially blocked by atropine. The atropine-resistant contraction was markedly reduced if the smooth muscle tachykinin receptors were either desensitized with the selective NK1 agonist substance P methyl ester, or blocked with the tachykinin receptor antagonist [D-Pro4,D-Trp7,9,10]SP-(4-11). These results suggest that activation of NK3 receptors on enteric neurones results in acetylcholine and tachykinin release.     

Central tachykinin NK3 receptors in the inhibitory action on the rat colonic propulsion of a new tachykinin, PG-KII.

The inhibitory action of the natural selective tachykinin NK3 receptor agonist, PG-KII, (pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2), on colonic propulsion was studied in rats after central administration. Intracerebroventricular injection of PG-KII (0.1, 1, 10 and 100 ng/rat) produced a dose-related inhibition of colonic propulsion, measured as the increase in the mean expulsion time of a 5-mm glass bead placed in the distal colon. At the same doses as PG-KII, the selective tachykinin NK3 receptor agonist, senktide, (succ-[Asp6-MePhe8] substance P-(6-11)), induced a similar dose-related inhibition. Conversely, substance P (0.1, 1 and 10 microg/rat), a tachykinin NK1-preferring receptor agonist, had weaker antipropulsive effects, neurokinin A (0.1, 1 and 10 microg/rat), a tachykinin NK2-preferring receptor agonist, at the highest dose used only slightly inhibited colonic propulsion and neurokinin B (0.1, 1 and 10 microg/rat), a tachykinin NK3-preferring receptor agonist, left propulsion unchanged. Pretreatment with the selective tachykinin NK3 receptor antagonist, 3-indolycarbonyl-Hyp-Phg-N(me)-Bzl, referred as to R820 (6.2 microg/rat), prevented PG-KII-induced colonic antipropulsion, whereas the tachykinin NK1 receptor antagonist, (S)-1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pi peridin-3-yl] ethyl)-4-phenyl-1-azoniabicyclo[2.2.2] octane chloride, referred to as SR 140,333 (1 microg/rat), and the tachykinin NK2 receptor antagonist, ([Tyr5,D-Trp6,8,9, Arg10] neurokinin A-(4-10)), referred to as Men 10,376 (5 microg/rat), left it unchanged. These findings show that of the tachykinins tested, PG-KII and senktide are the most potent central inhibitors of colonic propulsion in the rat, suggesting that the central tachykinin NK3 receptor system plays an inhibitory role in modulating colonic transit. As well as confirming the selectivity of PG-KII for tachykinin NK3 receptors, we show that PG-KII provides useful information about the physiological role of central tachykinin NK3 receptors and that glass bead expulsion test is a reliable non-invasive in vivo method for evaluating the tachykinin NK3 receptor selectivity of new synthetic or natural tachykinins.     

Inhibition of shock-induced foot tapping behaviour in the gerbil by a tachykinin NK1 receptor antagonist

The selective tachykinin NK1 receptor antagonist, 2-(R)-(1-(R)-3,5-Bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine (MK-869), has been recently described as a novel therapeutic approach for anxiety/depression. A frequently used model to establish the central nervous system (CNS) activity of tachykinin NK1 receptor antagonists is the inhibition of NK1 agonist-induced foot tapping in gerbils. In the present study, we demonstrate that foot tapping can also be induced in most, but not all, gerbils by footshock and associated cues. MK-869 (0.3-3 mg/kg, i.p.) dose-dependently blocked this foot tapping response. This effect was further shown to be due to selective NK1 receptor blockade, since (2S,3S)-cis-3(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994; 3 mg/kg, i.p.) inhibited foot tapping, whereas its less active enantiomer (2R,3R)-cis-3(2-methoxybenzylamino)-2-phenylpiperidine (CP-100,263; 3 mg/kg, i.p.) had no effect. Diazepam (1-10 mg/kg, i.p.) also inhibited foot tapping, whereas fluoxetine (10-30 mg/kg, i.p.) markedly increased this behaviour. The present data support the view that foot tapping in the gerbil is a behavioural response to an aversive stimulus, and is robustly inhibited by two NK1 receptor antagonists. The data support a role for tachykinin NK1 receptor antagonists as novel anxiolytic/antidepressants.     

A tachykinin NK1 receptor antagonist attenuates the 4 beta-phorbol-12-myristate-13-acetate-induced nociceptive behaviour in the rat

Antinociceptive effect of a tachykinin NK1 receptor antagonist ezlopitant [(2S,3S-cis)-2-(diphenylmethyl)-N-{(2-methoxy, 5-isopropylphenyl)methyl}-1-azabicyclo[2.2.2]octan-3-amine] was investigated in the 4beta-phorbol-12-myristate-13-acetate (PMA)-induced nociceptive test in the rat. Intraplantar injection of PMA-induced paw-licking and flinching behaviour lasted up to 120 min and was accompanied by inflammatory reactions, such as swelling and invasion of granulocytes. Pretreatment with resiniferatoxin [200 microg/kg, subcutaneous (s.c.)] blocked the PMA-induced nociceptive behaviour, suggesting that vanilloid VR1 receptor-expressing primary sensory neurons play a major role in this response. Subcutaneous pretreatment with ezlopitant (0.3-30 mg/kg) and morphine (0.3-6 mg/kg) caused a dose-dependent inhibition of the behaviour. Ezlopitant (3-30 mg/kg) given subcutaneously after PMA injection also significantly attenuated the behavioural response. When administered intrathecally, ezlopitant and a nonselective glutamate receptor antagonist MK-801 had an inhibitory effect, whereas CJ-12,191, an inactive isomer of ezlopitant, was unaffected. These results suggest that spinal tachykinin NK1 receptors contribute to processing of ongoing pain associated with peripheral inflammation.     

Comparative behavioural profile of centrally administered tachykinin NK1, NK2 and NK3 receptor agonists in the guinea-pig.

1. The NK1 tachykinin receptor agonists, septide, [Sar9,Met(O2)11]SP and [Pro9]SP produced locomotor hyperactivity (10-20 min) when injected intracerebroventricularly (i.c.v.) in the guinea-pig. The most potent in eliciting this hyperactivity was septide (from 0.63 to 5 micrograms), compared to [Sar9,Met(O2)11]SP, which was active at 2.5 and 5 micrograms and [Pro9]SP which induced a non-significant increase even at 10 micrograms. 2. Wet-dog shakes were elicited by septide, [Sar9,Met(O2)11]SP and [Pro9]SP injected by the i.c.v. route in the guinea-pig. [Sar9,Met(O2)11]SP, active from 0.16 to 2.5 micrograms was more potent than septide (active at 1.25 micrograms) and [Pro9]SP (active at 0.63 micrograms) in eliciting such behaviour. To a lesser extent, grooming was also observed after injection of these agonists. 3. The NK2 tachykinin receptor agonist, [Lys5,MeLeu9,Nle10]NKA(4-10), up to the dose of 10 micrograms i.c.v. had no effect in the guinea-pig. It neither modified locomotor activity nor induced a characteristic behavioural response. At higher doses (20 micrograms), some toxic effects were noted. 4. The NK3 tachykinin receptor agonist, senktide, contrasts with the NK1 receptor agonists in that it elicited only wet-dog shakes, at doses ranging from 0.32 to 1.25 micrograms. It neither modified locomotor activity (1 microgram) nor induced grooming (up to 5 micrograms) in the guinea-pig. 5. To our knowledge, these results are the first demonstration that the guinea-pig could be useful to differentiate tachykinin agonists on the basis of their behavioural profile, distinct from those obtained in mice and rats.     

Electroconvulsive shock increases tachykinin NK(1) receptors, but not the encoding mRNA, in rat cortex

Recent studies have suggested that the substance P (tachykinin NK(1)) receptor may be a pharmacological target for the treatment of mood disorders. Here, the effects of electroconvulsive shock on tachykinin NK(1) receptor gene expression in the rat brain was investigated. Rats received either a single electroconvulsive shock or five shocks on alternate days. Quantitative autoradiography with [(125)I]Bolton Hunter-substance P, and in situ hybridisation histochemistry, were used to measure tachykinin NK(1) receptor-binding site densities and mRNA abundance, respectively. Densities of tachykinin NK(1) receptor-binding sites were significantly increased in the cerebral cortex following repeated electroconvulsive shock compared to sham treated animals. Densities remained unchanged in the hippocampus, striatum and amygdala. Neither single nor repeated electroconvulsive shock altered tachykinin NK(1) receptor mRNA in the brain regions examined. Hence, repeated electroconvulsive shock increases tachykinin NK(1) receptors in the rat brain in a regionally specific way. Upregulation of receptor-binding sites without a change in mRNA indicates that translational or post-translational mechanisms underlie this process.     

Lack of evidence for epsilon-opioid receptors in the rat vas deferens

Experiments were performed to test the hypothesis that the field-stimulated rat vas deferens preparation contains opioid receptors, other than of mu-type, which mediate part of the inhibitory effect of beta-endorphin. The Piebald Viral Glaxo strain of rats was used. The reported finding that delta-opioid receptors are present in Sprague-Dawley rat vas deferens, the effects of which are greatly enhanced in reduced calcium concentrations, could not be replicated in the rat strain used. Reducing the calcium concentration from 2.5 to 1.25 mM improved the response to opioid drugs: all full agonists were about 10 times more potent, the partial agonist normorphine became able to inhibit the twitch completely, and morphine (which behaves as a competitive antagonist in 2.5 mM Ca2+) appeared to behave as a partial agonist. The pA2 values for antagonism by naloxone in low calcium of the mu-selective peptide [D-Ala2,MePhe4,Gly(ol)5]enkephalin and other mu- or delta-selective agonists were consistent with an action at mu-receptors only. The value for beta-endorphin was slightly but significantly lower. A similar small discrepancy was found with two other competitive antagonists. The discrepancy remained in the presence of the peptidase inhibitors thiorphan, bestatin and bacitracin. Responses to both [D-Ala2,MePhe4,Gly(ol)5]enkephalin and beta-endorphin were attenuated by the irreversible antagonists beta-funaltrexamine and beta-chlornaltrexamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs

1. The role of endogenous tachykinins on guinea-pig colonic propulsion was investigated by using potent and selective tachykinin NK₁ and NK₂ receptor antagonists. Colonic propulsion and contractions were determined by means of a balloon-catheter device, inserted into the rectum of guanethidine (68 μmol kg⁻¹, s.c., 18 and 2 h before)-pretreated, urethane-anaesthetized guinea-pigs. Propulsion of the device (dynamic model) was determined by measuring the length of the catheter expelled during 60 min filling of the balloon (flow rate 5 μl  min⁻¹).
2. In control conditions the tachykinin NK₁ receptor antagonist SR 140333 (1 μmol kg⁻¹, i.v.) did not affect either colonic propulsion or the amplitude of contractions. The tachykinin NK₂ receptor antagonists MEN 10627 and MEN 11420 (1 μmol kg⁻¹, i.v.) increased colonic propulsion at 10 min (+120% and 150%, respectively) but at 60 min the effect was significant only for MEN 10627 (+84%). SR 48968 (1 μmol kg⁻¹, i.v.) did not significantly enhance the colonic propulsion. None of these tachykinin NK₂ receptor antagonists modified the amplitude of colonic contractions. In contrast, both atropine (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹) and hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished propulsion (81% and 87% inhibition, respectively) and decreased the amplitude of contractions (68% inhibition for either treatment).
3. In atropine-treated animals (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹), apamin (30 nmol kg⁻¹, i.v.) restored colonic propulsion (+416%) and increased the amplitude of contractions (+367% as compared to atropine alone). Hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished the apamin-induced, atropine-resistant colonic propulsion (97% inhibition) and reduced the amplitude of the atropine-resistant contractions (52% inhibition).
4. The apamin-induced, atropine-resistant colonic propulsion was inhibited by SR 140333 (−69% at 1 μmol kg⁻¹), SR 48968 (−78% at 1 μmol kg⁻¹), MEN 11420 (−59% at 1 μmol kg⁻¹) and MEN 10627 (−50% at 1 μmol kg⁻¹), although the latter effect was not statistically significant. The combined administration of SR 140,333 and MEN 10,627 (1 μmol kg⁻¹ for each antagonist) almost completely abolished colonic propulsion (90% inhibition). The amplitude of colonic contractions was also reduced by SR 140333 (−42%), SR 48968 (−29%), MEN 11420 (−45%) but not by MEN 10627 (−16%). The combined administration of SR 140333 and MEN 10,627 reduced the amplitude of contractions by 47%. SR 140603 (1 μmol kg⁻¹, i.v.), the less potent enantiomer of SR 140333, was inactive.
5. In control animals, apamin (30 nmol kg⁻¹, i.v.) enhanced colonic propulsion (+84%) and increased the amplitude of contractions (+68%), as compared to the vehicle. Hexamethonium (55 μmol kg⁻¹, i.v. plus infusion of 17 μmol h⁻¹) inhibited propulsion (86% inhibition) and decreased the amplitude of contractions (49% inhibition). SR 140333, SR 48968, MEN 11420, MEN 10627, or the coadministration of SR 140333 and MEN 10627 had no effect.
6. In a separate series of experiments, the mean amplitude of colonic contractions was also recorded under isovolumetric conditions through the balloon-catheter device kept in place at 75 mm from the anal sphincter (static model). In control conditions, neither SR 140333 nor MEN 11420 modified the amplitude of contractions. In atropine-pretreated guinea-pigs, SR 140333 and MEN 11420 (0.1–1 μmol kg⁻¹) dose-dependently decreased the amplitude of contractions. In apamin- and atropine-pretreated animals, only the highest (1 μmol kg⁻¹) dose of SR 140333 or MEN 11420 significantly decreased the amplitude of contractions. The inhibitory potency of atropine (0.3–1 μmol kg⁻¹) was similar in apamin-pretreated animals and in controls.
7. It was concluded that, in anaesthetized guinea-pigs, endogenous tachykinins, acting through both NK₁ and NK₂ receptors, act as non-cholinergic excitatory neurotransmitters in promoting an apamin-evoked reflex propulsive activity of the distal colon.