Effects of TAK-637, a tachykinin receptor antagonist, on the micturition reflex in guinea pigs

The effects of a new tachykinin NK(1) receptor antagonist, (aR, 9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10, 11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g] [1, 7]naphthyridine-6,13-dione (TAK-637), on the micturition reflex were compared with those of drugs used for abnormally frequent micturition or incontinence. TAK-637 showed a characteristic effect on the distension-induced rhythmic bladder contractions in guinea pigs. The systemic administration of TAK-637 decreased the number but not the amplitude of the distension-induced rhythmic bladder contractions. A similar effect was observed in animals in which the spinal cord had been severed. TAK-637 also inhibited the micturition reflex induced by topical application of capsaicin onto the surface of bladder dome. From these results, it is concluded that TAK-637 inhibits sensory transmissions from the bladder evoked by both physiological and nociceptive stimuli by blocking tachykinin NK(1) receptors, possibly at the level of the spinal cord. On the other hand, the other drugs such as oxybutynin, tolterodine, propiverine, and inaperisone showed no effects on the frequency of the distension-induced rhythmic bladder contractions but decreased the contraction amplitude. Therefore, TAK-637 may represent a new class of drugs, which would be effective for abnormally frequent micturition without causing voiding difficulties due to decreased voiding pressure.     

MEN 11,420, a peptide tachykinin NK2 receptor antagonist, reduces motor responses induced by the intravesical administration of capsaicin in vivo

This study investigates the role of tachykinin NK₁ and NK₂ receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 μmol/kg, i.v.), a non-peptide NK₁ receptor antagonist, abolished urinary bladder contractions induced by the selective NK₁ receptor agonist [Sar⁹]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK₂ receptor agonist [?Ala⁸]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK₂ receptor antagonist, abolished bladder contractions induced by [?Ala⁸]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar⁹]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 μmol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 μmol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 μmol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK₂ receptors. Received: 11 February 1997 / Accepted: 17 April 1997     

Effect of colchicine on micturition reflex in rats

Intracerebral or intraspinal cord, but not intraperitoneal, injection of low doses of colchicine in rats induces specific toxic symptoms. This paper deals in particular with the effect of colchicine on micturition. After the injection of 5–25 μg/rat in cerebral ventricle or 2.5–20 μg/rat intraspinal cord, bladder content was markedly increased, due to a dramatic urine retention. Time of latency of vesical retention was related to the dose and to the route of colchicine administration. Cystometrographic analyses were performed in control and treated rats at various intervals of time after the injection: bladder tone, as expressed by the ΔP/ΔV ratio, monitored from 12 to 120 hr after colchicine injection, decreased more and more during time, suggesting that the observed vesical hypotonicity is an irreversible phenomenon.     

Effects of specific tachykinin receptor antagonists on citric acid-induced cough and bronchoconstriction in unanesthetized guinea pigs

We compared the effects of a tachykinin NK₁ receptor antagonist, FK888 (N²-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl- -prolyl]-N-methyl-N-phenylmethyl-3-(2-naphtyl)- -alaninamide), and a tachykinin NK₂ receptor antagonist, SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenyl-piperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]), on citric acid-induced cough and bronchoconstriction in conscious guinea pigs. FK888 and SR48968 inhibited the cough dose dependently. Combination of FK888 and SR48968 showed a small additive effect compared with that of FK888 or SR48968 alone. SR48968 but not FK888 inhibited the bronchoconstriction dose dependently. These results indicate that tachykinin NK₁ receptors as well as tachykinin NK₂ receptors are involved in the citric acid-induced cough response. The antitussive activity of the tachykinin NK₁ receptor antagonist appeared not to depend on the anti-bronchoconstrictor effects.     

Possible site of action of TAK-637, a tachykinin NK(1) receptor antagonist, on the micturition reflex in guinea pigs

TAK-637((aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10, 11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g] [1,7]naphthyridine-6,13-dione) is a novel tachykinin NK(1) receptor antagonist that has been shown to inhibit the micturition reflex in guinea pigs. The aim of this study was to clarify its mechanism of action in guinea pigs. TAK-637 inhibited the spinal vesico-vesical reflex induced by electrical stimulation of the proximal cut end of the pelvic nerve in spinal animals, but not bladder contractions induced by electrical stimulation of the distal cut end of the nerve. Furthermore, TAK-637 had no effect on carbachol- or electrical field stimulation-induced contractions of isolated bladder muscle strips in an organ bath, whereas drugs used for abnormally frequent micturition inhibited both contractions. These results suggest that TAK-637 inhibits the micturition reflex by acting, at least in part, on the spinal cord, and its mechanism of action clearly differs from those of antimuscarinics or spasmolytics.     

The subtypes of muscarinic receptors for neurogenic bladder contraction in rats

We evaluated in vivo functional selectivity profiles for muscarinic M(2) and M(3) subtypes of four muscarinic antagonists: Compound A (a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity), darifenacin, (a muscarinic M(3) receptor antagonist); methoctramine (a muscarinic M(2) receptor antagonist) and tolterodine (a nonselective muscarinic receptor antagonist), and compared the inhibition potency on distention-induced bladder contraction in rats. In an in vivo functional study, Compound A (0.03-10 mg/kg, i.v.) showed antimuscarinic activity with high selectivity for M(3) (salivation) over M(2) (bradycardia) (>100-fold). Darifenacin (0.01-0.3 mg/kg, i.v.) showed only slight selectivity for M(3) over M(2) (3.7-fold). Methoctramine (0.003-1 mg/kg, i.v.) showed the reverse selectivity profile (0.077-fold). Tolterodine (0.003-0.3 mg/kg, i.v.) showed less selectivity (1.2-fold). Compound A at M(3) inhibitory doses (0.1 and 0.3 mg/kg, i.v.) showed inhibition in a distention-induced neurogenic bladder contraction model, and its maximal inhibitory effects were about 60% at an even higher dose (3 mg/kg). Methoctramine at M(2) inhibitory doses (0.03 and 0.1 mg/kg, i.v.) did not significantly affect distention-induced bladder contraction. When tolterodine and darifenacin caused inhibition of distention-induced bladder contraction, its maximal inhibitory effects were similar to that of Compound A. Therefore, these findings suggest that Compound A would be an excellent pharmacological tool to give a better understanding of which subtypes of muscarinic receptors act in bladder function so far, and muscarinic M(3), but not M(2), receptors mainly mediate the cholinergic component of distention-induced bladder contraction.     

Multiple tachykinin binding sites in peripheral tissues and in brain

Tachykinin binding sites in guinea pig urinary bladder (GPUB), rat salivary gland (RSG), hamster urinary bladder (HUB), rat vas deferens (RVD) and rat cerebral cortex (RCC) were compared using ¹²⁵I-Bolton Hunter conjugates of substance P (¹²⁵I-BHSP), eledoisin (¹²⁵I-BHE) and neurokinin A (¹²⁵I-BHNKA). In typical SP-P tissues (GPUB, RSG) and in RCC, SP was the most potent displacer of ¹²⁵I-BHSP and [Glp⁶, D-Pro⁹]-SP(6–11) was 90 times less active than [Glp⁶, L-Pro⁹]-SP(6–11) while SP methyl ester (SPOMe) was 5–10 times more active than the Bolton Hunter conjugate of SPOMe (I-BHSPOMe). On the other hand, in typical SP-E tissues (HUB, RVD), neurokinin A was most potent in displacing ¹²⁵I-BHE and [Glp⁶,D-Pro⁹]-SP(6–11) was over 300 times more active than [Glp⁶,L-Pro⁹]-SP(6–11) while SPOMe was 160 times less active than I-BHSPOMe. In rat cerebral cortex, the rank order of potency of tachykinins and related analogues in displacing ¹²⁵I-BHE was distinct from that of peripheral SP-E sites, with neurokinin B being the most potent displacer, and SPOMe was over 1 000 times more active than I-BHSPOMe; ¹²⁵I-BHE binding sites in CNS may represent a third category of tachykinin receptor, designated SP-N.     

速激肽受体拮抗剂对豚鼠组胺反应的影响

目的：探讨速激肽与组胺（Ｈｉｓ）反应的关系。方法：观察速激肽受体拮抗剂对豚鼠Ｈｉｓ的整体和离体的呼吸道和心血管效应。结果：单用或合用速激肽ＮＫ－１受体拮抗剂ＣＰ－９６３４５（１ｍｇ·ｋｇ－１，ｉｐ）及ＮＫ－２受体拮抗剂ＳＲ－４８９６８（１ｍｇ·ｋｇ－１，ｉｐ）均可显著降低清醒豚鼠吸入Ｈｉｓ气雾的气道反应性。ＣＰ－９６３４５（１ｍｇ·ｋｇ－１，ｉｖ）可显著降低静脉注射Ｈｉｓ引起麻醉豚鼠支气管和心房的伊文思蓝渗出，ＳＲ－４８９６８（１ｍｇ·ｋｇ－１，ｉｖ）则对肺内压升高有较弱的抑制作用，两药对平均动脉压降低无明显作用。在豚鼠的离体气管和支气管平滑肌标本，ＣＰ－９６３４５（１μｍｏｌ·Ｌ－１）及ＳＲ－４８９６８（１μｍｏｌ·Ｌ－１）对Ｈｉｓ的Ｅｍａｘ及ｐＤ２无明显作用。结论：速激肽部分参与了豚鼠的Ｈｉｓ炎症反应，速激肽受体拮抗剂有抗炎作用。     

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.     

Acetylcholine and tachykinin receptor antagonists attenuate wood smoke-induced bronchoconstriction in guinea pigs

To study the mechanisms of wood smoke-induced bronchoconstriction, we measured total lung resistance (R_L) and dynamic lung compliance (C_dyn) in anesthetized and mechanically ventilated guinea pigs. Airway exposure to various doses of wood smoke (lauan wood; 5, 10, and 15 breaths) resulted in a dose-dependent increase in R_L and decrease in C_dyn. The smoke-induced changes in R_L and C_dyn were significantly attenuated by pretreatment with atropine, CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine; a tachykinin NK₁ receptor antagonist], and SR-48,968 [(S)-N-methyl-N(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)-butyl)benzamide; a tachykinin NK₂ receptor antagonist] in combination, atropine alone, and SR-48,968 alone, but were not significantly affected by pretreatment with the inactive enantiomers of CP-96,345 and SR-48,968, CP-96,345 alone, indomethacin (a cyclooxygenase inhibitor), and MK-571 [((3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl((3-dimethyl amino-3-oxo-propyl)thio)methyl)propanoic acid; a leukotriene D₄ receptor antagonist]. The activity of airway neutral endopeptidase, a major enzyme for tachykinin degradation, was not significantly influenced by wood smoke during the development of bronchoconstriction. We conclude that: (1) both cholinergic mechanisms and endogenous tachykinins, but not cyclooxygenase products or leukotriene D₄, play an important role in the acute bronchoconstriction induced by wood smoke, and (2) the contribution of tachykinins to this airway response is primarily mediated via the activation of tachykinin NK₂ receptors, but is not associated with inactivation of the airway neutral endopeptidase.     

Synergistic role of muscarinic acetylcholine and tachykinin NK-2 receptors in intestinal peristalsis

It is known that tachykinins (substance P, neurokinin A) participate in the excitatory neural pathways subserving peristaltic motor activity in the intestine. The aim of the present study was to elucidate the types of tachykinin receptor (NK-1 or NK-2) involved in peristalsis by the use of receptor subtype-selective antagonists. Peristaltic motility in isolated segments of the guinea-pig ileum was induced by pumping fluid into the oral end of the intestinal segment. By way of the intraluminal pressure the compliance of the intestinal wall during the preparatory phase and the pressure threshold to trigger the emptying phase of peristalsis were recorded. The tachykinin antagonists were used at concentrations that were at least 30 times in excess of the equilibrium dissociation constants which had previously been evaluated with receptor subtype-selective agonists on the guineapig ileum circular muscle. The NK-1 selective antagonist CP-96,345 (0.3 M) had a slight stimulant influence on peristalsis, whereas the NK-2 selective antagonists MEN-10,376 (10 M), GR-94,800 (0.3 M) and SR-48,968 (0.1 M) led to a small inhibition of motor activity. However, when given after exposure of the ileum to a threshold concentration of atropine (5–20 nM) causing little depression of peristalsis, the tachykinin NK-2 receptor antagonists invariably abolished peristalsis. This synergistic interaction was not seen when SR-48,968 was administered after the ileal segments had been exposed to concentrations of hexamethonium, isoproterenol or calcitonin gene-related peptide that by themselves caused a slight inhibition of peristalsis only. CP-96,345 was without effect on peristalsis when it was applied in the presence of a threshold concentration of atropine. These findings indicate that transmission via tachykinin NK-2, but not NK-1, receptors synergizes with cholinergic transmission via muscarinic receptors in the relay of excitatory enteric pathways subserving intestinal peristalsis. Correspondence to: P. Holzer at the above address     

Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder

We compared the density and function of M₂ and M₃ muscarinic acetylcholine receptor subtypes in the urinary bladder of young adult (3 months) and old (23 months) male Wistar rats. Old rats had a reduced density of muscarinic receptors (96±10 vs. 156±21 fmol/mg protein), but competition experiments with the M₃-selective darifenacin did not indicate alterations in the relative roles of M₂ and M₃ receptors, with the former being more abundant. The amount of immunodetectable -subunits of various G-proteins potentially linked to muscarinic receptor function was unchanged. The potency of carbachol to contract bladder strips was also unaltered; its maximum effects as well as those of a single KCl concentration were unchanged if raw data or those corrected for strip length were analysed, but somewhat reduced when those corrected for strip weight were analysed. Antagonistic effects of atropine, the M₂-selective Ro 320–6206 and the M₃-selective darifenacin were unchanged. Agonistic effects of the M₃-sparing agonist 4-(2-oxo-2,3-dihydro-benzoimidazol-1-yl)-[1,4]bipiperidinyl-1-carboxylic acid ethyl ester were similarly poor in young and old rats. Additional experiments were concomitantly performed in submandibular glands from the same animals. While total muscarinic receptor density in submandibular glands was not significantly affected by age (56±5 vs. 61±4 fmol/mg protein), the relative contribution of M₃ receptors significantly declined from 68±3% to 57±2% based upon darifenacin competition curves. We conclude that aged Wistar rats express fewer muscarinic receptors in their urinary bladder, but there is no change in the relative abundance of M₂ and M₃ receptors; this is accompanied by only minor if any alterations in receptor responsiveness. In contrast, submandibular gland expresses similar receptor numbers in young and old rats, but slightly fewer M₃ receptors in old animals.     

Effect of SR 142801 on nitric oxide-dependent and independent responses to tachykinin NK3 receptor agonists in isolated guinea-pig colon

We have determined the ability of the novel nonpeptide tachykinin (TK) NK₃ receptor antagonist, SR 142801, [(S) -(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide] in inhibiting the nitric oxide (NO)-independent prejunctional inhibition of cholinergic twitches and the NO-dependent relaxation produced by the NK₃ receptor selective agonist, senktide, in the circular muscle of the guinea-pig proximal colon. Under moderate load (10 mN) and isometric recording of mechanical activity, single pulse electrical field stimulation (EFS) produced atropine- and tetrodotoxin-sensitive twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of NK₁ and NK₂ receptor antagonists (SR 140333 0.01 M and GR 94800 0.1 M, respectively) the NK₃ receptor selective agonist, senktide (EC₅₀ 33 pM) and the NK₃ receptor preferring natural TK, neurokinin B (NKB, EC₅₀ 13 pM) produced a concentration-dependent slowly developing inhibition of cholinergic twitches. Senktide (1 nM) did not affect the contractile response to acetylcholine (1 gM) indicating that depression of evoked twitches occurs prejunctionally. The inhibitory effect of senktide was 'unaffected when evoked in the presence of the cyclooxygenase inhibitor (S)-ketoprofen (10 M), guanethidine (10 M), naloxone (0.3 M), the GABA_B receptor antagonist 2-hydroxysaclofen (10 M) or the combined application of the adenosine A₁ and A₂ receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (10 M) and 3,7-dimethyl-l-propargylxanthine (30 M) respectively.In the presence of NK₁ and NK₂ receptor antagonists, the NO-synthase inhibitor l-nitroarginine (L-NOARG 30-100 M) did not affect twitch inhibition induced by senktide (EC₅₀ 33 pM). The response to NKB (EC₅₀ 95 pM) was slightly reduced by L-NOARG, yet the bulk of the inhibitory effect of both agonists on cholinergic twitches was substantially independent of NO generation. SR 142801 (0.1–0.3 M) produced a moderate rightward shift of the concentration-response curve to senktide without depression of the Emax to the agonist, yielding an apparent pK_B value of 7.65.Under low resting tone (3 mN) and isotonic recording of mechanical activity, mucosa-free circular muscle strips from the guinea-pig proximal colon gained a high intrinsic tone suitable for testing the response to relaxant agents. In the presence of atropine (1 M), guanethidine (3 M), SR 140333 (0.01 [M) and GR 94800 (0.1 LM), senktide (EC₅₀ 50 pM) produced a concentration-dependent relaxation of the strips, which was blocked by L-NOARG. SR 142801 (0.01–0.1 M) produced a large rightward shift of the L-NOARG-sensitive concentration-response curve to senktide yielding an apparent pKB value of 8.62. Under isometric recording condition, SR 142801 (0.1 M) did not affect twitch inhibition produced by 3 nM clonidine. Under isotonic recording condition, SR 142801 did not affect the L-NOARG-sensitive relaxation produced by EFS.The present results indicate that NK₃ receptor stimulation produces a NO-dependent relaxation of the guinea-pig colon and a substantially NO-independent prejunctional inhibition of cholinergic twitches. The variable affinities of SR 142801 in antagonizing various senktide-induced neuromodulatory effects in the guinea-pig intestine suggest a possible intraspecies heterogeneity of NK₃ receptors in the enteric nervous system.     

Anxiolytic activity of tachykinin NK2 receptor antagonists in the mouse light-dark box

The tachykinin NK₂ receptor antagonists, GR100679 (0.02–200 μg/kg s.c.) and (±)-SR48968 (0.05–5.0 μg/kg s.c.), dose-dependently increased the time which mice spent in the light side of the light-dark box. There was no evidence of sedation or other overt behaviours. The amplitudes of these effects were similar to that evoked by diazepam (1.75 mg/kg s.c.). These results indicate a disinhibitory action of NK₂ antagonists on suppressed behaviours in a novel aversive environment. This suggests an involvement of NK₂ receptors in anxiety-related behaviours.     

Enhancement of neurokinin A-induced smooth muscle contraction in human urinary bladder by mucosal removal and phosphoramidon: relationship to peptidase inhibition

Neurokinin A (NKA) is potent in contracting the human detrusor muscle. Here, we have investigated whether these contractile responses are influenced by the presence of the mucosa, by the peptidase inhibitor phosphoramidon or by possible modulators, prostaglandins and nitric oxide. Contractile responses to neurokinin A were unaffected by indomethacin or N-omega-nitro-L-arginine, but were significantly reduced in strips containing mucosa. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11 (neprilysin, CD10), was ineffective at 10 microM, but at 100 microM, significant increase in the maximum response was achieved by neurokinin A in detrusor strips with and without mucosa. In immunohistochemical studies, neutral endopeptidase immunoreactivity occurred in peripheral nerve trunks in the detrusor and in a fibrous meshwork in the subepithelial lamina propria. Our data indicate that neutral endopeptidase is present in bladder mucosa and detrusor, and support the concept that this metalloprotease and/or related enzymes are important in regulating the actions of tachykinins.     

The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats

1. We have investigated the effect of nociceptin on the micturition reflex evoked by distension or topical application of capsaicin on the urinary bladder of urethane-anaesthetized rats.
2. Nociceptin produced a dose-dependent (3–100 nmol kg⁻¹ i.v.) transient suppression of the distension-evoked micturition reflex: its effect was not modified by guanethidine (68 μmol kg⁻¹ s.c.) nor by bilateral cervical vagotomy, alone or in combination, and by naloxone (1.2 μmol kg⁻¹ i.v.).
3. Nociceptin (100 nmol/kg i.v.) slightly (about 30%) inhibited the contractions of the rat bladder produced by pre- or postganglionic electrical stimulation of the pelvic nerve.
4. Nociceptin almost totally abolished the reflex component of the response to topical capsaicin (1 μg in 50 μl).
5. In the rat isolated bladder, submaximal contractions produced by electrical field stimulation were slightly reduced (25±4% inhibition) by 1 μM nociceptin. Nociceptin did not affect the contraction of the rat bladder induced by acetylcholine (10 μM) or ATP (1 mM).
6. These findings indicate that nociceptin exerts a naloxone-resistant suppression of the volume-evoked micturition reflex which involves inhibition of transmitter release from postganglionic bladder nerves. An inhibitory effect on bladder afferent nerves is also suggested.

     

Role of adenosine A2A receptor signaling in the nicotine-evoked attenuation of reflex cardiac sympathetic control

Baroreflex dysfunction contributes to increased cardiovascular risk in cigarette smokers. Given the importance of adenosinergic pathways in baroreflex control, the hypothesis was tested that defective central adenosinergic modulation of cardiac autonomic activity mediates the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate (HR) to increases or decreases in blood pressure (BP) evoked by i.v. doses (1-16 μg/kg) of phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious rats; slopes of the curves were taken as measures of baroreflex sensitivity (BRS). Nicotine (25 and 100 μg/kg i.v.) dose-dependently reduced BRS_SNP in contrast to no effect on BRS_PE. BRS_SNP was also attenuated after intracisternal (i.c.) administration of nicotine. Similar reductions in BRS_SNP were observed in rats pretreated with atropine or propranolol. The combined treatment with nicotine and atropine produced additive inhibitory effects on BRS, an effect that was not demonstrated upon concurrent exposure to nicotine and propranolol. BRS_SNP was reduced in preparations treated with i.c. 8-phenyltheophylline (8-PT, nonselective adenosine receptor antagonist), 8-(3-Chlorostyryl) caffeine (CSC, A_2A antagonist), or VUF5574 (A₃ antagonist). In contrast, BRS_SNP was preserved after blockade of A₁ (DPCPX) or A_2B (alloxazine) receptors or inhibition of adenosine uptake by dipyridamole. CSC or 8-PT abrogated the BRS_SNP depressant effect of nicotine whereas other adenosinergic antagonists were without effect. Together, nicotine preferentially impairs reflex tachycardia via disruption of adenosine A_2A receptor-mediated facilitation of reflex cardiac sympathoexcitation. Clinically, the attenuation by nicotine of compensatory sympathoexcitation may be detrimental in conditions such as hypothalamic defense response, posture changes, and ventricular rhythms.     

Occurrence and pharmacological characterization of four human tachykinin NK2 receptor variants

Tachykinin NK(2) receptor antagonists are potentially beneficial in treating various disorders including irritable bowel syndrome, urinary incontinence, depression and anxiety. The current study evaluates the frequency of single nucleotide polymorphisms (SNPs) in the human NK(2) receptor gene (TACR2). In addition, the potency of the endogenous peptide agonist neurokinin A (NKA), and the small molecule antagonists saredutant (NK(2)-selective) and ZD6021 (pan-NK antagonist) at the various NK(2) receptor protein variants were determined. The TACR2 gene was sequenced from 37 individuals. Two amino acid changing SNPs encoding the NK(2) receptor variants Ile23Thr and Arg375His were found. The frequency of the four possible protein variants differed between populations. Site-directed mutagenesis was performed introducing either SNP or both SNPs into the TACR2 gene and the constructs were transfected into CHO cells. NKA-evoked increases in intracellular Ca(2+) were monitored by FLIPR. The potency of saredutant and ZD6021 was evaluated by their ability to inhibit NKA-induced increases in intracellular Ca(2+). NKA evoked increases in intracellular Ca(2+) with a potency ranging between 1 and 5nM in CHO cells expressing the different constructs. Saredutant and ZD6021 blocked NKA-evoked increases in intracellular Ca(2+) with pK(b) values ranging between 8.8-9.3 and 7.9-8.7, respectively. The current study demonstrates that polymorphisms leading to the Ile23Thr and Arg375His amino acid exchanges are highly prevalent in the human TACR2 gene. These polymorphisms however do not appear to affect the potency of the endogenous agonist NKA or the small molecule antagonists saredutant and ZD6021 with respect to intracellular Ca(2+) signalling.     

Alleviation of wood smoke-induced lung injury by tachykinin receptor antagonist and hydroxyl radical scavenger in guinea pigs

We recently reported that wood smoke inhalation initially (within 5 min) causes airway injury and subsequently produces both airway and parenchymal injury after a delay (within 2 h). In this study, we investigated the mediator mechanisms of this delayed smoke-induced lung injury in 126 anesthetized and artificially ventilated guinea pigs who received challenges of either air or 40 tidal breaths of wood smoke. Two hours after inhalation, wood smoke produced various injurious responses, including increases in alveolar-capillary permeability, microvascular permeabilities, and histological injury scores, in airway and parenchymal tissues. Pre-treatment given before smoke challenge with CP-96,345 [a tachykinin NK1 receptor antagonist; (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-aza bicyclo(2.2.2.)-octan-3-amine], dimethylthiourea (a hydroxyl radical scavenger), or a combination of these two drugs largely alleviated both the airway and parenchymal responses, whereas pre-treatment with SR-48,968 [a tachykinin NK2 receptor antagonist; (S)-N-methyl-N(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)-butyl)benzamide] or a combination of CP-96,344 and SR-48,965 (inactive enantiomers) failed to do so. Post-treatment given at 5 min after smoke challenge with CP-96,345 or dimethylthiourea significantly alleviated the parenchymal responses, while having no effect on the airway responses. Pre-treatment with dimethylthiourea prevented the smoke-induced reduction in airway neutral endopeptidase activity (an enzyme for tachykinin degradation). We concluded that (1) tachykinins and hydroxyl radical play important roles in producing smoke-induced delayed lung injury in guinea pigs, and both may be involved in the spread of injury from the airways to the pulmonary parenchyma, and (2) the contribution of tachykinins is mediated via the activation of tachykinin NK1 receptors, and is associated with the hydroxyl radical-induced inactivation of airway neutral endopeptidase.     

Pharmacological evaluation of alpha and beta human tachykinin NK(2) receptor splice variants expressed in CHO cells

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK(2) receptor splice variant named beta isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the alpha isoform. Moreover, neurokinin A was unable to inhibit the [(3)H]SR48968 binding to the beta isoform up to microM concentrations. In cells expressing the human tachykinin NK(2) receptor beta isoform, contrary to those expressing the alpha isoform, natural or selective tachykinin receptor agonists (1 microM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca(2+)](i). The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca(2+)](i) increase in cells expressing the alpha or beta isoform of the human tachykinin NK(2) receptor. The present data indicate that the human tachykinin NK(2) receptor beta isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK(2) receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands.