The presence of NK3 tachykinin receptors on rat uterus.

The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.     

Functional interaction between losartan and central tachykinin NK3 receptors in the conscious rat.

1. The cardiovascular and behavioural effects elicted by the intracerebroventricular (i.c.v.) injection of substance P (SP), neurokinin A (NKA), [MePhe7]neurokinin B ([MePhe7]NKB) or angiotensin II (AII) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for angiotensin AT1 (losartan and its active metabolite EXP 3174), angiotensin AT2 (PD 123,319) or tachykinin NK3 (R 486) receptors. 2. I.c.v. administration of 25 pmol AII evoked an increase in mean arterial blood pressure (MAP) and water intake behaviour, accompanied by a transient bradycardia, whereas 25 pmol [MePhe7]NKB caused a transient increase in MAP and heart rate (HR) concurrently with marked wet dog shake behaviour. At the same dose, SP and NKA were more potent than [MePhe7]NKB in increasing MAP and HR, but did not produce water intake or wet dog shake behaviours. 3. Losartan (650 pmol, i.c.v.) reduced significantly the cardiovascular and behavioural responses to AII or [MePhe7]NKB, but not to SP or NKA. While 65 pmol losartan was inactive, 260 pmol inhibited selectively the central effects of AII. Whereas EXP 3174 (6.5 nmol) blocked both AII and [MePhe7]NKB-mediated responses, the dose of 650 pmol blocked only the responses to AII. 4. The central responses to AII and [MePhe7]NKB were not affected by PD 123,319 (650 pmol). On the other hand, the [MePhe7]NKB-induced central effects were significnatly reduced by R 486 (650 pmol). The NK3-selective antagonist had no effect against AII. 5. This study provides functional evidence, to support earlier binding data, that losartan (and to some extent its active metabolite EXP 3174) interact with the tachykinin NK3 receptor in rat brain.(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.

     

Functional characterisation of hemokinin-1 in mouse uterus

The preprotachykinin gene Tac4 expressed in murine uterus and placenta is thought to encode a peptide RSRTRQFYGLM-NH(2), mouse hemokinin 1. We have examined the uterotonic effects of mouse hemokinin 1 and its N-terminally truncated analogue, mouse hemokinin 1(2-11) on mouse uterus. Mouse hemokinin 1(2-11) was equieffective with but slightly less potent than substance P in tissues from non-pregnant Swiss mice. On myometrium from Balb C mice primed with oestrogen the positions of concentration-response curves to substance P and the mouse hemokinins were similar to those of neurokinin A, but the maximum responses were lower. The tachykinin NK(1) receptor antagonist, 1-{2-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl}-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), reduced the effects of the agonists in tissues from both groups of mice. In myometria from late pregnant (Days 17-18) Balb C mice the responses to mouse hemokinin 1(2-11) were less potent than in those from oestrogen-primed mice. Human hemokinin 1, the human orthologue of mouse hemokinin 1, was more effective than mouse hemokinin 1(2-11), while endokinin D was inactive. Mouse hemokinin 1 effects were blocked by SR 140333 alone and in combination with ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968) but not by SR 48968 alone. Thus the mouse hemokinins are tachykinin NK(1) receptor-preferring uterotonic agonists in non-pregnant mice but lack action at the myometrial tachykinin NK(2) receptors present in late pregnant mice.     

Intrathecal histamine induces spinally mediated behavioral responses through tachykinin NK1 receptors

Intrathecal injection of histamine elicited a behavioral response consisting of scratching, biting and licking in conscious mice. Here, we have examined the involvement of substance P (SP) by using intrathecal injection of tachykinin neurokinin (NK)(1) receptor antagonists and SP antiserum. Histamine-induced behavioral response was evoked significantly 5-10 min after intrathecal injection and reached a maximum at 10-15 min. Dose-dependency of the induced response showed a bell-shaped pattern from 200 to 3200 pmol, and maximum effect was observed at 800-1000 pmol. The H(1) receptor antagonist, d-chlorpheniramine and pyrilamine but not the H(2) receptor antagonists, ranitidine and zolantidine, inhibited histamine-induced behavioral response. The NK(1) receptor antagonists, CP-99,994, RP-67580 and sendide, inhibited histamine-induced behavioral response in a dose-dependent manner. A significant antagonistic effect of [D-Phe(7), D-His(9)]SP (6-11), a selective antagonist for SP receptors, was observed against histamine-induced response. The NK(2) receptor antagonist, MEN-10376, had no effect on the response elicited by histamine. Pretreatment with SP antiserum resulted in a significant reduction of the response to histamine. No significant reduction of histamine-induced response was detected in mice pretreated with NK A antiserum. The present results suggest that elicitation of scratching, biting and licking behavior induced by intrathecal injection of histamine may be largely mediated by NK(1) receptors via H(1) receptors in the spinal cord.     

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)     

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

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

Differential role of tachykinin NK3 receptors on cholinergic excitatory neurotransmission in the mouse stomach and small intestine

Background and purpose:

Tachykinin NK₃ receptors are widely expressed in the mouse gastrointestinal tract but their functional role in enteric neuromuscular transmission remains unstudied in this species. We investigated the involvement of NK₃ receptors in cholinergic neurotransmission in the mouse stomach and small intestine.

Experimental approach:

Muscle strips of the mouse gastric fundus and ileum were mounted in organ baths for tension recordings. Effects of NK₃ agonists and antagonists were studied on contractions to EFS of enteric nerves and to carbachol.

Key results:

EFS induced frequency-dependent tetrodotoxin-sensitive contractions, which were abolished by atropine. The cholinergic contractions to EFS in the stomach were enhanced by the NK₃ antagonist SR142801, but not affected by the NK₃ agonist senktide or neurokinin B. The cholinergic contractions to EFS in the small intestine were not affected by SR142801, but dose-dependently inhibited by senktide and neurokinin B. This inhibitory effect was prevented by SR142801 but not by hexamethonium. SR142801, senktide or neurokinin B did not induce any response per se in the stomach and small intestine and did not affect contractions to carbachol.

Conclusions and implications:

NK₃ receptors modulate cholinergic neurotransmission differently in the mouse stomach and small intestine. Blockade of NK₃ receptors enhanced cholinergic transmission in the stomach but not in the intestine. Activation of NK₃ receptors inhibited cholinergic transmission in the small intestine but not in the stomach. This indicates a physiological role for NK₃ receptors in mouse stomach contractility and a pathophysiological role in mouse intestinal contractility.     

Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK₂R and NK₃R) and dog (NK₁R, NK₂R and NK₃R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs.The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca²⁺. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs.Data collected using the cloned dog NK₁R confirmed that the dog NK₁R displays similar pharmacology as the human and the gerbil NK₁R, but differs greatly from the mouse and the rat NK₁R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK₂R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK₃R likely influence the NK₃R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK₃R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK₁R and NK₃R.     

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

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

Functional characterization of kainate receptors in the mouse nucleus accumbens

Kainate receptors are abundantly expressed in the nucleus accumbens but their functional characterization and their role in synaptic transmission has not yet been investigated. Using patch-clamp recordings in mouse nucleus accumbens slices, we show the presence of functional kainate receptors activated by low concentrations of kainate (100-300 nM) in medium size neurons. These somatodendritic receptors are comprised of the GluR6 subunit, since they are absent in GluR6-deficient mice. Kainate receptors do not directly participate in glutamatergic synaptic transmission evoked by electrical stimulation of cortical afferent fibers in nucleus accumbens neurons. However, application of low concentrations of kainate inhibits cortico-accumbens synaptic transmission, by increasing synaptic failure rate and increasing variation coefficient, thus indicating a presynaptic site of action. Presynaptic kainate receptors are observed both in GluR6 and in GluR5-deficient mice, but are absent in mice devoid of both subunits. Hence, at variance with somatodendritic kainate receptors, presynaptic kainate receptors on cortical afferents are composed of both GluR5 and GluR6 kainate receptor subunits. These results indicate that different subtypes of kainate receptors, representing distinct pharmacological targets, should play important roles in the synaptic integration properties of nucleus accumbens neurons.     

Functional characterization of coronary vascular adenosine receptors in the mouse

Coronary responses to adenosine agonists were assessed in perfused mouse and rat hearts. The roles of nitric oxide (NO) and ATP-dependent K(+) channels (K(ATP)) were studied in the mouse. Resting coronary resistance was lower in mouse vs rat, as was minimal resistance (2.2+/-0.1 vs 3.8+/-0.2 mmHg ml(-1) min(-1) g(-1)). Peak hyperaemic flow after 20 - 60 s occlusion was greater in mouse. Adenosine agonists induced coronary dilation in mouse, with pEC(50)s of 9.4+/-0.1 for 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl carboxamidoadenosine (CGS21680, A(2A)-selective agonist), 9.3+/-0.1 for 5'-N-ethylcarboxamidoadenosine (NECA, A(1)/A(2) agonist), 8.4+/-0.1 for 2-chloroadenosine (A(1)/A(2) agonist), 7.7+/-0.1 for N(6)-(R)-(phenylisopropyl)adenosine (R-PIA, A(1)/A(2B) selective), and 6.8+/-0.2 for adenosine. The potency order (CGS21680=NECA>2-chloroadenosine>R-PIA>adenosine) supports A(2A) adenosine receptor-mediated dilation in mouse coronary vessels. 0.2 - 2 microM of the A(2B)-selective antagonist alloxazine failed to alter CGS21680 or 2-chloroadenosine responses. pEC(50)s in rat were 6.7+/-0.2 for CGS21680, 7.3+/-0.1 for NECA, 7.6+/-0.1 for 2-chloroadenosine, 7.2+/-0.1 for R-PIA, and 6.2+/-0.1 for adenosine (2-chloroadenosine>NECA=R-PIA>CGS21680> adenosine), supporting an A(2B) adenosine receptor response. NO-synthase antagonism with 50 microM N(G)-nitro L-arginine (L-NOARG) increased resistance by approximately 25%, and inhibited responses to CGS21680 (pEC(50)=9.0+/-0.1), 2-chloroadenosine (pEC(50)=7.3+/-0.2) and endothelial-dependent ADP, but not sodium nitroprusside (SNP). K(ATP) channel blockade with 5 microM glibenclamide increased resistance by approximately 80% and inhibited responses to CGS21680 in control (pEC(50)=8.3+/-0.1) and L-NOARG-treated hearts (pEC(50)=7.3+/-0.1), and to 2-chloroadenosine in control (pEC(50)=6.7+/-0.1) and L-NOARG-treated hearts (pEC(50)=5.9+/-0.2). In summary, mouse coronary vessels are more sensitive to adenosine than rat vessels. A(2A) adenosine receptors mediate dilation in mouse coronary vessels vs A(2B) receptors in rat. Responses in the mouse involve a sensitive NO-dependent response and K(ATP)-dependent dilation.     

Role of NK1 and NK2 receptors in mouse gastric mechanical activity

1. The aim of the present study was to examine the role of NK1 and NK2 receptors in the control of mechanical activity of mouse stomach. In this view, the motor effects induced by NK1 and NK2 receptor agonists and antagonists were analyzed, measuring motility as intraluminal pressure changes in mouse-isolated stomach preparations. In parallel, immunohistochemical studies were performed to identify the location of NK1 and NK2 receptors on myenteric neurons and smooth muscle cells. 2. Substance P (SP) induced biphasic effects: a contraction followed by relaxation; neurokinin A (NKA) and [beta-Ala8]-NKA(4-10), selective agonist of NK2 receptors, evoked concentration-dependent contractions, whereas [Sar9, Met(O2)11]-SP, selective agonist of NK1 receptors, induced concentration-dependent relaxation. 3. SR48968, NK2 receptor antagonist, did not modify the spontaneous activity and reduced the contractile effects induced by tachykinins without affecting the relaxation. SR140333, NK1 receptor antagonist, did not modify the spontaneous activity and antagonized the relaxant response to tachykinins, failing to affect the contractile effects. 4. The relaxation to SP or to [Sar9, Met(O2)11]-SP was abolished by tetrodotoxin (TTX) and significantly reduced by N(omega)-nitro-L-arginine methyl ester (L-NAME). 5. NK2-immunoreactivity (NK2-IR) was seen at the level of the smooth muscle cells of both circular and longitudinal muscle layers. NK1-immunoreactive (NK1-IR) neurons were seen in the myenteric ganglia and NK1/nNOS double labeling revealed that some neurons were both NK1-IR and nNOS-IR. 6. These results suggest that, in mouse stomach, NK1 receptors, causing relaxant responses, are present on nitrergic inhibitory myenteric neurons, whereas NK2 receptors, mediating contractile responses, are present at muscular level.     

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)     

Role of tachykinin NK(1) and NK(2) receptors in colonic sensitivity and stress-induced defecation in gerbils

The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK(1) and NK(2) receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK(1) and NK(2) receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2-3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19+/-6% inhibition, P<0.01) was slightly more effective than saredutant (10+/-9% inhibition, P<0.05). A combination of both compounds resulted in an additive effect (30+/-10% inhibition, P<0.01). The results demonstrate that NK(1) and NK(2) receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK(1)/NK(2) receptor antagonists may provide better clinical outcome than selective compounds.     

The human tachykinin NK1 (short form) and tachykinin NK4 receptor: a reappraisal

Excessive secretion of placental neurokinin B into the circulation during the third trimester of pregnancy is seen in women with preeclampsia. To determine a role for neurokinin B, we have used a number of different animal models to ascertain the expression of the three tachykinin receptors (NK1--both short and long forms, NK2 and NK3) and the putative human tachykinin NK4 receptor in the placenta. Human and rat placenta express all three classical tachykinin receptors. However, we failed to reveal the expression of the short tachykinin NK1 receptor or the tachykinin NK4 receptor in any of 24 human tissues examined including the placenta. We conclude that the proposed short form of the tachykinin NK1 receptor is a truncated genomic clone and that the human tachykinin NK4 receptor is in fact, the guinea pig tachykinin NK3 receptor.     

Possible involvement of tachykinin NK(1) and NMDA receptors in histamine-induced hyperalgesia in mice.

Intrathecal (i.t.) injection of histamine elicited a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of histamine (800 pmol) and returned to control level with 30 min. Hyperalgesia produced by histamine was inhibited dose-dependently by i.t. co-administration of the histamine H(1) receptor antagonist, d-chlorpheniramine, but not the histamine H(2) receptor antagonist, ranitidine. The tachykinin NK(1) receptor antagonists, (+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine] (CP-99,994), and [Tyr(6), D-Phe(7), D-His(9)]substance P-(6-11) (sendide), inhibited histamine-induced hyperalgesic response in a dose-dependent manner. A significant antagonistic effect of [D-Phe(7), D-His(9)]substance P-(6-11), a selective antagonist for substance P receptors, was observed against histamine-induced hyperalgesic response. The tachykinin NK(2) receptor antagonist, Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Lys-NH(2) (MEN-10,376), had no effect on hyperalgesia elicited by histamine. The competitive N-methyl-D-aspartate (NMDA) receptor antagonists, and D-(-)-2-amino-5-phosphonovaleric acid (D-APV), (+/-)-3-(2-carboxypiperazin-yl)propyl-1-phosphoric acid (CPP), the noncompetitive NMDA receptor antagonist dizocilpine (MK-801), and L-N(G)-nitro arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, markedly inhibited histamine-induced hyperalgesic response. The present results suggest that hyperalgesic response induced by i.t. injection of histamine may be mediated by tachykinin NK(1) receptors, but not NK(2) receptors in the spinal cord. In addition, spinal NMDA receptor-NO system may also contribute to elicitation of hyperalgesia following i.t. injection of histamine.     

Differential activation of the epithelial and smooth muscle NK1 receptors by synthetic tachykinin agonists in guinea-pig trachea

1. The presence of tachykinin NK₁ receptors have been shown in the epithelium and smooth muscle of guinea-pig airways. Previous data showed that substance P (SP), and the NK₁ receptor agonist, [Sar⁹, Met (O₂)¹¹]-SP, relax guinea-pig tracheal tube preparations by stimulation of epithelial NK₁ receptors and via nitric oxide (NO) release. However, the selective tachykinin NK₁ receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK₁ receptors of guinea-pig airway epithelium.
2. Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 μM) and the tachykinin NK₂ receptor antagonist, SR 48,968 ((S)-N-methyl N-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl)benzamide) (0.1 μM). Cumulative concentration-response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 μM).
3. Contractile responses to low concentrations (0.1–10 nM) of substance P (SP) and the selective agonist of NK₁ receptors, [Pro⁹]-SP, in non precontracted tracheae were higher in preparations pretreated with the NO-synthase inhibitor, N^G-monomethyl L-arginine (L-NMMA, 100 μM) than in preparations pretreated with its inactive enantiomer D-NMMA (100 μM). Tracheal tube preparations precontracted with ACh and pretreated with D-NMMA were relaxed by low concentrations of SP and [Pro⁹]-SP (0.1–10 nM). In contrast, after pretreatment with L-NMMA, SP and [Pro⁹]-SP contracted tracheae at all the concentrations tested.
4. Concentration-response curves to the NK₁ receptor agonists, SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) obtained in non-precontracted tracheae were similar in the presence of either D-NMMA or L-NMMA. SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D-NMMA. Concentration-response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L-NMMA or D-NMMA.
5. In guinea-pig tracheal tube preparations two groups of NK₁ receptor agonists can be distinguished: one group, including [Pro⁹]-SP, stimulator epithelial NK₁ receptors, the other group, including SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11), does not. One possible explanation for these findings and for the existence of compounds with a peculiar ‘septide-like'' pharmacological profile in the guinea-pig trachea could be the recently proposed phenomenon referred to as ‘agonist-directed receptor trafficking''.