Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats

1. We have developed and characterized a model of immediate hypersensitivity/inflammation of the urinary bladder in vivo induced by local application of ovalbumin (OA) in OA- sensitive female rats. Two parameters of the inflammatory response were assessed following OA challenge: plasma protein extravasation (PPE) and changes in smooth muscle reactivity. The former was estimated by measurement of Evans blue extravasation at 0.5, 2, 4, 8 and 24 h time point following in vivo challenge. Changes in reactivity were determined by measurement of isotonic tension responses of urinary bladder strips following OA challenge in vitro.
2. Acute in vivo intravesical OA challenge (10 mg in 0.3 ml saline) in actively sensitized female Wistar rats caused a time-dependent PPE in the urinary bladder which was biphasic with peak responses at 2–4 and 24 h.
3. The PPE response to acute OA challenge, above base-line, at 2 h was abolished by systemic capsaicin pretreatment (50 mg kg⁻¹, s.c., 4 days before use) (P<0.05) whilst the response at 24 h was unaffected. The 2 h time point was then used for further studies.
4. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg⁻¹, s.c. for 3 consecutive days), completely abolished the PPE response to OA challenge at the 2 h time point.
5. The tachykinin NK₁ receptor antagonist, SR 140333 (0.1 μmol kg⁻¹, i.v.), abolished the 2 h PPE response whilst the tachykinin NK₂ receptor antagonist MEN 11420 (0.1 μmol kg⁻¹, i.v.) appeared to reduce the response by approximately 50% but this did not reach significance. The bradykinin B₂ receptor antagonist, Hoe 140 (0.1 μmol kg⁻¹, i.v.), similarly to SR 140333, blocked the 2 h PPE response to OA, whereas the selective B₁ receptor antagonist B 9858 (0.1 μmol kg⁻¹, i.v.) had no significant effect. Inhibition of cyclo-oxygenase (COX) achieved by pretreatment with the COX inhibitor dexketoprofen (5.3 μmol kg⁻¹, i.v.) also blocked the PPE response, whilst the leukotriene receptor antagonist ONO 1078 (1 μmol kg⁻¹, i.v.) significantly reduced PPE by about 80%.
6. In the rat isolated urinary bladder OA (1 mg ml⁻¹) challenge produced a biphasic response with a rapidly achieved maximal contraction followed by a sustained contraction for approximately 25 min. In vitro capsaicin pretreatment (10 μM for 15 min) significantly attenuated the duration of the sustained contraction whilst having no effect on the maximum contractile response achieved. In vivo pretreatment of animals with compound 48/80 significantly attenuated (42%) the maximum contractile response. Combination of both treatments almost completely abolished the response. In vitro treatment with Hoe 140 (1 μM) had no significant effect on the response to OA and neither did ONO 1078 (1 μM).
7. These results show that both the early inflammatory response and alterations in smooth muscle reactivity to OA challenge in actively sensitized animals are dependent on mast cell degranulation and the activation of sensory C-fibres. Furthermore this model of allergic cystitis may be useful for investigating both the processes involved and potential novel therapies in the treatment of interstitial cystitis.

     

In vitro and in vivo characterization of NK3 receptors in the rabbit eye by use of selective non-peptide NK3 receptor antagonists

1. Inhibition of NK₃ receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK₃ receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK₃ receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists.
2. In vitro senktide (succinyl-[Asp⁹,MePhe⁸]-substance P (6-11) and [MePhe⁷]-neurokinin B ([MePhe⁷]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD₂=9.03±0.06 and mean n_H=1.2±0.02 (n=14). In contrast, [MePhe⁷]-NKB produced shallow log concentration-effect curves which often appeared biphasic (n_H=0.54±0.04, n=8), preventing the accurate determination of pD₂ values.
3. The contractile responses to the NK₃ receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((−)-(S)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide; 3–30 nM, pA₂=8.4, slope=1.8±0.3, n=4), SB 222200 ((−)-(S)-N-(α-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide; 30–300 nM, pA₂=7.9, slope=1.4±0.06, n=4) and SB 218795 ((−)-(R)-N-(α-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 μM apparent pK_B=7.4±0.06, n=6).
4. Contractile responses to the NK₃ receptor agonist [MePhe⁷]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 μM, n=8). In contrast, SB 223412 (30 and 300 μM, n=4) and SB 222200 (0.3 and 3 μM, n=4) inhibited responses to low concentrations (⩽1 nM), to a greater extent than higher concentrations (>1 nM) of [MePhe⁷]-NKB. Furthermore, log concentration-effect curves to [MePhe⁷]-NKB became steeper and monophasic in the presence of each antagonist.
5. SB 218795 (3 μM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK₃ receptors over NK₁ receptors in the rabbit.
6. In vivo, senktide (1, 10 and 25 μg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25±0.25 mm; basal pupillary diameter 7.75±0.48 mm; n=4). The onset of miosis was within 2–5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 μg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 μg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0±0.4 mm to 9.0±0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg⁻¹, i.v., i.e. 2.63 and 5.26 μmol kg⁻¹; maximum inhibition 100%; n=3–4), SB 223412 (0.5 and 1 mg kg⁻¹, i.v., i.e. 1.31 and 2.61 μmol kg⁻¹; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg−1, i.v., i.e. 1.26 and 2.52 μmol kg−1; maximum inhibition 78%; n=3), and the structurally distinct NK₃ receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5 mg kg⁻¹, i.v., i.e. 2.47 μmol kg⁻¹, maximum inhibition 92%; n=3).
7. Topical administration of senktide (25 μg; 29.7 nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P<0.05) between pupillary constriction obtained by topical or i.v. senktide, and topically administered atropine had no significant effect on responses to topical senktide (n=4).
8. [MePhe⁷]-NKB (125, 250 and 500 μg, i.v., i.e. 98.31, 196.62 and 393.24 nmol, respectively) also induced bilateral miosis in conscious rabbits (maximum pupillary constriction=4.13±0.30 mm; n=4), but in contrast to in vitro studies this agonist was approximately 100 fold less potent than senktide. [MePhe⁷]-NKB-induced miosis was inhibited by SB 222200 (5 mg kg⁻¹, i.v., i.e. 13.14 μmol kg⁻¹; maximum inhibition 69%; n=3).
9. In summary, SB 223412, SB 222200 and SB 218795 are potent and selective antagonists of NK₃ receptor-mediated contraction in the rabbit isolated iris sphincter muscle. In addition, NK₃ receptor agonist-induced miosis in conscious rabbits is a good in vivo correlate of the in vitro rabbit iris sphincter muscle preparation and appears to be a useful model for characterizing the pharmacodynamic profile and efficacy of structurally distinct NK₃ receptor antagonists, such as SB 222200, SB 223412, SB 218795 and SR 142801.

     

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips

1. Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC₅₀, 4.9 nM), the tachykinin NK₂-receptor selective agonist [β-Ala⁸]neurokinin A (4–10) ([β-Ala⁸]NKA (4–10)) (EC₅₀, 5.0 nM), neurokinin B (EC₅₀, 5.3 nM) and substance P (EC₅₀, 160 nM), but not by the tachykinin NK₁-receptor selective agonist [Sar⁹Met(O₂)¹¹] substance P, or the NK₃-receptor selective agonists, senktide and [MePhe⁷] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β-Ala⁸]NKA (4–10).
2. Atropine (1 μM) and tetrodotoxin (1 μM) did not significantly inhibit responses to [β-Ala⁸]NKA (4–10), neurokinin A, substance P or neurokinin B.
3. The newly developed non-peptide antagonists for tachykinin NK₂-receptors SR 48968, SR 144190 and its N-demethyl (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β-Ala⁸]NKA (4–10), with similar potency (Schild plot pA₂ values 9.4, 9.4 and 9.3, slope=1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pK_B) was 8.2. In addition, SR 144782 (0.1 μM) antagonized responses to neurokinin A, substance P and neurokinin B, with pK_B consistent with its affinity for tachykinin NK₂-receptors. The potent and selective NK₁ and NK₃-receptor antagonists, SR 140333 and SR 142801 (both 0.1 μM), failed to inhibit contractions induced by SP or NKB.
4. In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK₂-receptors, but not tachykinin NK₁- or NK₃-receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK₂-receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK₂-receptor might well account for the complexity of the observed interaction.

     

Excitatory motor and electrical effects produced by tachykinins in the human and guinea-pig isolated ureter and guinea-pig renal pelvis

1. In isolated tissue experiments, neurokinin A (NKA) produced concentration-dependent contraction of human and guinea-pig ureter (pD₂=6.7 and 7.2, respectively); an effect greatly reduced (>80% inhibition) by the tachykinin NK₂ receptor-selective antagonist MEN 11420 (0.1 μM). The tachykinin NK₁ and NK₃ receptor agonists septide and senktide, respectively, were ineffective.
2. Electrical field stimulation (EFS) of the guinea-pig isolated renal pelvis produced an inotropic response blocked by MEN 11420 (0.01–1 μM). In the same preparation MEN 11420 (0.1 μM) blocked (apparent pK_B=8.2) the potentiation of spontaneous motor activity produced by the NK₂ receptor-selective agonist [βAla⁸]NKA(4–10).
3. In sucrose-gap experiments, EFS evoked action potentials (APs) accompanied by phasic contractions of human and guinea-pig ureter, which were unaffected by tetrodotoxin or MEN 11420 (3 μM), but were blocked by nifedipine (1–10 μM). NKA (1–3 μM) produced a slow membrane depolarization with superimposed APs and a tonic contraction with superimposed phasic contractions. NKA prolonged the duration of EFS-evoked APs and potentiated the accompanying contractions. MEN 11420 completely prevented the responses to NKA in both the human and guinea-pig ureter.
4. Nifedipine (1–10 μM) suppressed the NKA-evoked APs and phasic contractions in both human and guinea-pig ureter, and slightly reduced the membrane depolarization induced by NKA. A tonic-type contraction of the human ureter in response to NKA persisted in the presence of nifedipine.
5. In conclusion, tachykinins produce smooth muscle excitation in both human and guinea-pig ureter by stimulating receptors of the NK₂ type only. NK₂ receptor activation depolarizes the membrane to trigger the firing of APs from latent pacemakers.

     

Effects of tachykinin NK1 receptor antagonists on vagal hyperreactivity and neuronal M2 muscarinic receptor function in antigen challenged guinea-pigs

1. The role of tachykinin NK₁ receptors in the recruitment of eosinophils to airway nerves, loss of inhibitory neuronal M₂ muscarinic receptor function and the development of vagal hyperreactivity was tested in antigen-challenged guinea-pigs.
2. In anaesthetized guinea-pigs, the muscarinic agonist, pilocarpine (1–100 μg kg⁻¹, i.v), inhibited vagally induced bronchoconstriction, in control, but not in antigen-challenged guinea-pigs 24 h after antigen challenge. This indicates normal function of neuronal M₂ muscarinic receptors in controls and loss of neuronal M₂ receptor function in challenged guinea-pigs. Pretreatment of sensitized guinea-pigs with the NK₁ receptor antagonists CP99994 (4 mg kg⁻¹, i.p.), SR140333 (1 mg kg⁻¹, s.c.) or CP96345 (15 mg kg⁻¹, i.p.) before antigen challenge, prevented M₂ receptor dysfunction.
3. Neither administration of the NK₁ antagonists after antigen challenge, nor pretreatment with an NK₂ receptor antagonist, MEN10376 (5 μmol kg⁻¹, i.p.), before antigen challenge, prevented M₂ receptor dysfunction.
4. Electrical stimulation of the vagus nerves caused a frequency-dependent (2–15 Hz, 10 V, 0.2 ms for 5 s) bronchoconstriction that was significantly increased following antigen challenge. Pretreatment with the NK₁ receptor antagonists CP99994 or SR140333 before challenge prevented this increase.
5. Histamine (1–20 nmol kg⁻¹, i.v.) caused a dose-dependent bronchoconstriction, which was vagally mediated, and was significantly increased in antigen challenged guinea-pigs compared to controls. Pretreatment of sensitized animals with CP99994 before challenge prevented the increase in histamine-induced reactivity.
6. Bronchoalveolar lavage and histological studies showed that after antigen challenge significant numbers of eosinophils accumulated in the airways and around airway nerves. This eosinophilia was not altered by pretreatment with the NK₁ receptor antagonist CP99994.
7. These data indicate that pretreatment of antigen-sensitized guinea-pigs with NK₁, but not with NK₂ receptor antagonists before antigen challenge prevented the development of hyperreactivity by protecting neuronal M₂ receptor function. NK₁ receptor antagonists do not inhibit eosinophil accumulation around airway nerves.

     

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 α2-adrenoceptors in the regulation of intestinal water transport

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP).
2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min⁻¹, i.a.) and i.v. injection of the selective α₂-adrenoceptor agonist UK 14,304 (1 μmol kg⁻¹, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg⁻¹, i.v). However, the selective α₁-adrenoceptor agonist phenylephrine (5 μmol kg⁻¹, i.v.) did not alter either the jejunal or ileal absorption rate.
3. The α₂-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg⁻¹, i.v.) and rauwolscine (10 μmol kg⁻¹, i.v.) decreased the basal absorption rate, while the α₁-adrenoceptor antagonist prazosin (3 μmol kg⁻¹, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg⁻¹) caused a significant antiabsorptive effect in the jejunum but not ileum.
4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg⁻¹, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.
5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α₂-adrenoceptors.
6. The subtype determination of α₂-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α₂-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min⁻¹).
7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC₅₀ values were 0.17 μmol kg⁻¹ in the jejunum and 0.22 μmol kg⁻¹ in the ileum.
8. The effect of UK 14,304 was blocked by the selective α_2A/D antagonist BRL 44408 and the non-selective α₂ antagonist yohimbine (each 10 μmol kg⁻¹). The selective α_2B/C antagonist ARC 239 (10 μmol kg⁻¹) did not affect the antisecretory action of UK 14,304. It is suggested that the α₂-adrenoceptors in the rat intestinal epithelium are the α_2D or α_2A-like subtype.

     

Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala⁸]NKA (4–10) and [MePhe⁷]neurokinin B (NKB) at T₁₃ thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK₁ (RP 67580), NK₂ (SR 48968) and NK₃ (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function.
2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min⁻¹, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min⁻¹, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min⁻¹, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.
3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min⁻¹, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min⁻¹, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min⁻¹, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK₂ ([β-Ala⁸]NKA(4-10)) and NK₃ ([MePhe⁷]NKB) receptors were devoid of renal effects.
4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK₁ receptors and the sympathetic renal nerve.

     

Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and anpirtoline,as evidenced by use of the corresponding new and selective receptor antagonists NAD-299 and NAS-181

1. Ejaculatory problems and anorgasmia are well-known side-effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5-HT_1A and 5-HT_1B receptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5-HT receptor subtypes.
2. The 5-HT_1A receptor agonist 8-OH-DPAT (0.25–4.00 μmol kg⁻¹ s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5-HT_1A receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD-299) (1.0 μmol kg⁻¹ s.c.). NAD-299 by itself (0.75–3.00 μmol kg⁻¹ s.c.) did not affect the male rat ejaculatory behaviour.
3. The 5-HT_1B receptor agonist anpirtoline (0.25–4.00 μmol kg⁻¹ s.c.) produced a dose-dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5-HT_1B receptor antagonist isamoltane (16 μmol kg⁻¹ s.c.) as well as by the new and selective antagonist (R)-(+)-2-(3-morpholinomethyl-2H-chromene-8-yl)oxymethylmorpholino methansulphonate (NAS-181) (16 μmol kg⁻¹ s.c.). Isamoltane (1.0–16.0 μmol kg⁻¹ s.c.) and NAD-181 (1.0–16.0 μmol kg⁻¹ s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non-selective 5-HT₁ receptor antagonist (−)-pindolol (8 μmol kg⁻¹ s.c.), did not antagonize the inhibition produced by anpirtoline.
4. The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5-HT_1A and 5-HT_1B receptors, respectively, suggesting that the SSRI-induced inhibition of male ejaculatory dysfunction is due to 5-HT_1B receptor stimulation.

     

Tachykinin NK2 receptors in the hamster urinary bladder: in vitro and in vivo characterization

We have characterized the contractile responses produced by stimulation of the tachykinin NK₂ receptor in the hamster urinary bladder in vitro and in vivo. In isolated bladder strips, neurokinin A (NKA, pD ₂ 7.40, E_max 71% of the response to 80 mM KCl) and the synthetic tachykinin NK₂ receptor selective agonist [βAla⁸]NKA(4–10) (pD ₂ 7.48, E_max 77% of the response to KCl) both induced a concentration-dependent contraction, whereas the tachykinin NK₁ and NK₃ receptor selective agonists, [Sar⁹]substance P sulfone and senktide, respectively, produced a negligible contractile effect. The bicyclic peptide antagonists MEN 11420 and MEN 10627 behaved as competitive antagonists of the response to [βAla⁸]NKA(4–10) with apparent pK _B values of 9.3 and 9.7, respectively. Comparable apparent pK _B values were estimated against NKA (pK _B 9.2 and 9.4 for MEN 11420 and MEN 10627, respectively). Under isovolumetric recording of the intravesical pressure, the nicotinic receptor agonist DMPP (0.6 μmol/kg i.v.) produced a phasic contraction of the hamster bladder in vivo that was abolished by hexamethonium (110 μmol/kg i.v.) or by surgical ablation of pelvic ganglia. In vivo [βAla⁸]NKA(4–10) (10 nmol/kg i.v.) induced a tonic-type sustained bladder contraction with superimposed high frequency and small amplitude (<12 mmHg) phasic contractions and, in about 70% of cases examined, a few high amplitude (>20 mmHg) phasic contractions. Hexamethonium abolished the high amplitude phasic contractions, indicating their reflex origin. In animals subjected to the ablation of pelvic ganglia, the urinary bladder response to [βAla⁸]NKA(4–10) was comparable to that observed after administration of hexamethonium. Moreover, hexamethonium did not affect the contractile responses to [βAla⁸]NKA(4–10) in ganglionectomized animals. MEN 10627 and MEN 11420 produced a dose-dependent and long-lasting inhibition of the contractile response to [βAla⁸]NKA(4–10): the least effective doses of the two antagonists were 30 and 3 nmol/kg i.v. for MEN 10627 and MEN 11420, respectively. An almost complete and long-lasting inhibition of the response to the agonist was produced at doses of 10 and 100 nmol/kg i.v. of MEN 11420 and MEN 10627. In urethane-anaesthetized hamsters the non-stop intravesical infusion of saline (50 μl/min) produced repetitive micturition cycles which were abolished by hexamethonium (110 μmol/kg i.v.) or by surgical removal of the pelvic ganglia. MEN 11420 (100 nmol/kg) had no significant effect on the volume-evoked micturition reflex in anaesthetized hamsters. In conclusion, the hamster urinary bladder is a suitable preparation for studying the action of tachykinin NK₂ receptor antagonists in vivo: in this species, the stimulation of tachykinin NK₂ receptors induces bladder contractions. Blockade of tachykinin NK₂ receptors does not appreciably modify the volume-evoked micturition reflex in this species. Received: 22 April 1998 / Accepted: 12 June 1998     

Signalling pathways in bradykinin- and nitric oxide-induced hypotension in the normotensive rat; role of K+-channels

1. Bradykinin and nitric oxide (NO) are potent hypotensive agents. In the present study, the role of K⁺-channels in the signalling pathways responsible for their hypotensive action was investigated in normotensive, anaesthetized rats. The rats were treated with ion-channel inhibitors before administration of bradykinin (2.8, 5.6, 28 and 56 nmol kg⁻¹, i.v.) followed in some of the protocols by nitroprusside (1.1, 3.5, 7, 14, and 28 nmol kg⁻¹, i.v.).
2. No attenuation of the hypotensive response to bradykinin was detected for inhibitors of the Na-K-Cl-cotransporter (30 μmol kg⁻¹ furosemide), the ATP-sensitive K⁺-channel (40 μmol kg⁻¹ glibenclamide), high conductance Ca²⁺-activated K⁺-channel (180 μmol kg⁻¹ tetraethylammonium, 54 μmol kg⁻¹ tetrabutylammonium, 35 nmol kg⁻¹ iberiotoxin, 35 nmol kg⁻¹ charybdotoxin) or the low conductance Ca²⁺-activated K⁺-channel (74 nmol kg⁻¹ apamin).
3. However, the voltage-sensitive K⁺-channel (I_A) inhibitor 4-aminopyridine (4.05–40.5 μmol kg⁻¹) induced a concentration-dependent (P<0.0001) attenuation of the hypotensive response (P<0.0001). Bradykinin had no effect on heart rate in anaesthetized rats and this observation was not altered by pretreatment with 4-aminopyridine.
4. 4-Aminopyridine (53 μmol kg⁻¹) also significantly attenuated the hypotensive response to nitroprusside (P<0.0003) without altering the heart rate concentration-response curve. Of the two Ca²⁺-activated K⁺-channel inhibitors tested on nitroprusside-induced hypotension, tetrabutylammonium induced a slight attenuation (P<0.0101), whereas iberiotoxin had no effect.
5. We therefore concluded that, although the acute hypotensive response to bradykinin in the normotensive rat is not mediated through nitric oxide synthesis, the hypotensive response to both agents was mediated through opening of voltage-sensitive K⁺-channels (I_A), resulting in a decrease in peripheral vascular resistance.

     

Effects of β2-agonist- and dexamethasone-treatment on relaxation and regulation of β-adrenoceptors in human bronchi and lung tissue

1. Long-term treatment with β₂-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to β-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with β₂-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied.
2. The effect of β₂-agonist incubation alone and after coincubation with dexamethasone on density and affinity of β-adrenoceptors was investigated by radioligand binding experiments.
3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent β-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73±4% in efficacy of isoprenaline to relax human bronchial smooth muscle.
4. After an incubation period of 60 min with 100 μmol l⁻¹ terbutaline, a significant decline in its relaxing efficacy (81±8%) and potency (by a factor 5.5) occurred.
5. Incubation with 30 μmol l⁻¹ isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4).
6. Coincubation of dexamethasone with isoprenaline (120 min; 30 μmol l⁻¹) preserved the effect of isoprenaline on relaxation (129±15%).
7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 μmol l⁻¹) resulted in a decrease in β-adrenoceptor binding sites (B_max) to 64±1.6% (P<0.05), while the antagonist affinity (K_D) for [³H]-CGP-12177 remained unchanged.
8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 μmol l⁻¹) or isoprenaline (30 μmol l⁻¹) plus dexamethasone (30 μmol l⁻¹) for 120 min did not lead to a significant change of B_max (160±22.1% vs 142.3±28.7%) or K_D (5.0 nmol l⁻¹ vs 3.5 nmol l⁻¹) compared to the controls.
9. In conclusion, pretreatment of human bronchi with β-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of β-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of β-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term β-adrenoceptor stimulation.

     

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound.
2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg⁻¹, p.o., 0.03 to 1 mg kg⁻¹, i.p. and 0.1 to 1 μg/rat, i.c.v.) increased urine output and urinary excretion of Na⁺, K⁺ and Cl⁻ and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats.
3. NT (0.1 μg/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 μg/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 μg/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis.
4. The NO synthesis inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME; 30 mg kg⁻¹, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg⁻¹, i.p.) but not D-arginine (1 g kg⁻¹, i.p.) restored the SR 48692-dependent increase in diuresis. L-NAME had no effect on furosemide-stimulated diuresis.
5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine.
6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692.
7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg⁻¹, i.p.) stimulated urine output.
8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na⁺, K⁺ and Cl⁻.

     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

1. Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 μM), produced a slowly-developing contraction (`on'' response) followed by a quick phasic `off '' contraction (`off peak'' response) and a tonic response (`off late'' response), averaging 16±2, 73±3 and 20±4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 μM; 15 min before) abolished the overall response to EFS (n=8).
2. Neither in vitro capsaicin pretreatment (10 μM for 15 min), nor guanethidine (3 μM, 60 min before) affected the excitatory response to EFS (n=5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. N^ω-nitro-L-arginine (L-NOARG, 100 μM, 60 min before) or naloxone (10 μM, 30 min before) significantly enhanced the `on'' response (294±56 and 205±25% increase, respectively; n=6–8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655±90% increase of the `on'' component, n=6, P<0.05).
3. The tachykinin NK₂ receptor-selective antagonist MEN 11420 (1 μM) almost abolished both the `on'' and `off late'' responses (P<0.01; n=5 each) to EFS, and reduced the `off-peak'' contraction by 55±8% (n=5, P<0.01). The subsequent administration of the tachykinin NK<sub>1</sub> receptor-selective antagonist GR 82334 (1 μM) and of the tachykinin NK<sub>3</sub> receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 μM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 μM, GR 82334 significantly reduced (by 68±9%, P<0.05, n=6) the `on'' response to EFS.
4. The contractile `off peak'' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 μM each) was abolished (P<0.01; n=6) by the administration of the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 30 μM). PPADS (30 μM) selectively blocked (75±9 and 50±7% inhibition, n=4 each) the contractile responses produced by 100 and 300 μM ATP.
5. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed.
6. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK₂ (and possibly NK₁) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

     

Vascular actions of octreotide in the portal hypertensive rat

1. We have investigated the actions of the somatostatin analogue octreotide in the portal hypertensive Wistar rat in vivo and in rat small mesenteric artery and aorta in vitro.
2. In small mesenteric artery, octreotide (0.1–0.3 μM) failed to produce any direct contraction, nor did it affect contractions to noradrenaline (NA, 10 μM) or endothelium-dependent relaxations to acetylcholine.
3. In rat aorta, octreotide (0.3 μM) and somatostatin (1 μM) failed to affect contractions to NA (1 μM), or concentration-contractile response curves to NA.
4. In rat vas deferens, octreotide and somatostatin significantly reduced contractile responses to electrical stimulation with pD₂ values (−log IC₅₀) of 8.19±0.10 (n=4) and 8.16±0.26 (n=4), respectively. Hence, the lack of effect of these agents in aorta or mesenteric artery was not due to lack of efficacy or inappropriate choice of concentration.
5. In the anaesthetized portal hypertensive rat, intravenous injection of octreotide (1–100 μg kg⁻¹) did not significantly affect systemic blood pressure, nor did it affect mesenteric vascular conductance as measured by laser doppler flow probes. However, octreotide (100 μg kg⁻¹) significantly reduced vascular conductance to 74.2±7.7% of control (n=6) in porto-systemic shunt vessels as measured by laser doppler flow probes.
6. Phenylephrine (1 μg kg⁻¹) significantly raised blood pressure and significantly decreased vascular conductance in both mesenteric (66.6±3.7% of control) and porto-systemic shunt vessels (58.7±10.0% of control).
7. It was concluded that octreotide has selective effects on porto-systemic shunt vessles in vivo in the portal hypertensive rat.

     

Opposite effects of CCKB agonists in grooming behaviour in rats: further evidence for two CCKB subsites

1. The hypothesis of the existence of two CCK_B receptor subsites, CCK_B1 and CCK_B2 corresponding probably to different coupling states of CCK_B receptors, was studied by measuring grooming behaviour in rats.
2. The B1 receptor agonist, BC197 (300 μg kg⁻¹, i.p.) produced a 45–50% decrease in grooming activity, which was prevented by both the CCK_B receptor antagonists CI-988 (20 μg kg⁻¹ i.p.) and L-365,260 (200 μg kg⁻¹, i.p.).
3. In contrast, 3, 10 and 30 μg kg⁻¹, i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150–190%). This effect was prevented by previous injection of 75 μg kg⁻¹ of L-365,260 while higher doses (200 μg kg⁻¹, i.p.) produced only a partial antagonism. Moreover, CI-988 (20 μg kg⁻¹, i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 μg kg⁻¹ of CI-988 tended to suppress the increase of grooming induced by BC264.
4. The effects of BC264 were prevented by the D₁ receptor (SCH 23390) and D₂ receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems.
5. This study brings additional evidence for the existence of the two CCK_B receptor subsites and suggests that particular attention should be focused on the selectivity of CCK_B receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.

     

RPR 106541, a novel,airways-selective glucocorticoid: effects against antigen-induced CD4+T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung

1. The effects of a novel 17-thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation.
2. In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4⁺ T lymphocytes and increased expression of mRNA for interleukin-4 (IL-4) and IL-5, but not interferon-γ (IFN-γ). These findings are consistent with an eosinophilia orchestrated by activated Th2-type cells.
3. RPR 106541 (10–300 μg kg⁻¹), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose-dependently inhibited cell influx into the airway lumen. RPR 106541 (100 μg kg⁻¹) caused a significant (P<0.01) (98%) inhibition of eosinophil influx and a significant (P<0.01) (100%) inhibition of neutrophil influx. RPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively.
4. When tested at a single dose (300 μg kg⁻¹), RPR 106541 and fluticasone each caused a significant (P<0.01) (100%) inhibition of CD4⁺ T cell accumulation in lung tissue. Budesonide (300 μg kg⁻¹) had no significant effect. RPR 106541 and fluticasone (300 μg kg⁻¹), but not budesonide (300 μg kg⁻¹), significantly (P<0.05) inhibited the expression within lung tissue of mRNA for IL-4. RPR 106541 (300 μg kg⁻¹) also significantly (P<0.05) inhibited expression of mRNA for IL-5.
5. The high topical potency of RPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

     

The protective effects of CP-060S on ischaemia- and reperfusion-induced arrhythmias in anaesthetized rats

1. CP-060S is a novel sodium and calcium overload inhibitor, and is also characterized as a calcium channel blocker. As these activities have each been shown independently to ameliorate ischaemia damage in the myocardium, the combination may synergistically exert cardioprotection. In this study, therefore, the protective effect of CP-060S against ischaemia- and reperfusion-induced arrhythmia was evaluated in anesthetized rats.
2. Rats were anaesthetized with pentobarbitone, and the left anterior descending coronary artery was occluded for either 5 min with subsequent reperfusion (a reperfusion-induced arrhythmia model) or 30 min without (an ischaemia-induced arrhythmia model). All drugs were intravenously administered 1 min before the onset of occlusion.
3. In the reperfusion-induced arrhythmia model, the animals in the vehicle-treated group exhibited ventricular tachycardia (VT) in 100%, ventricular fibrillation (VF) in 89%, and death caused by sustained VF in 56%. CP-060S (30–300 μg kg⁻¹) dose-dependently suppressed the incidences of arrhythmias. Significant decreases occurred at 100 μg kg⁻¹ in VF (incidence: 42%) and mortality (8%), and at 300 μg kg⁻¹ in VT (50%), VF (33%) and mortality (8%). This protective effect of CP-060S was 10 times more potent than that of a pure calcium channel blocker, diltiazem (30–1000 μg kg⁻¹) we tested, in terms of effective dose ranges. As both drugs decreased myocardial oxygen consumption estimated by rate-pressure product to a similar extent, the calcium channel blocking activity of CP-060S would not seem to be sufficient to explain its potency.
4. In the same model, co-administration of ineffective doses of diltiazem (300 μg kg⁻¹) and a sodium and calcium overload inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"R56865","term_id":"826971","term_text":"R56865"}}R56865 (100 μg kg⁻¹), produced significant suppression of VT (incidence: 62%), VF (46%) and mortality (8%). By contrast, co-administration of {"type":"entrez-nucleotide","attrs":{"text":"R56865","term_id":"826971","term_text":"R56865"}}R56865 at the same dose with CP-060S (300 μg kg⁻¹) did not add to the effect of a single treatment of CP-060S.
5. In the ischaemia-induced arrhythmia model, CP-060S (300 μg kg⁻¹) significantly decreased the incidence of VF from 75% to 29%, whereas diltiazem (1 mg kg⁻¹) was ineffective.
6. These results suggest that CP-060S inhibits both ischaemia- and reperfusion-induced arrhythmia. The combination of the calcium channel blocking effect and the calcium overload inhibition was hypothesized to contribute to these potently protective effects.

     

Localization of Fos-like immunoreactivity induced by the NK3 tachykinin receptor agonist,senktide, in the guinea-pig brain

1. The effects of intracerebroventricular (i.c.v.) administration of the NK₃ tachykinin receptor agonist, senktide (10 nmol each side), in guinea-pigs pretreated with the selective NK₃ tachykinin receptor antagonist, SR142801 (3 mg kg⁻¹ subcutaneous, s.c., 30 min before senktide), or its less active enantiomer, SR142806 (3 mg kg⁻¹ s.c. 30 min before senktide), on behaviour and on the distribution of Fos-like immunoreactivity (Fos-LI) in central neurones were investigated. Guinea-pigs were chosen for the study since they possess NK₃ tachykinin receptors with pharmacological characteristics similar to those in man.
2. Wet-dog shakes, but not locomotor activity, elicited by senktide i.c.v. were significantly reduced by SR142801 but not by SR142806, confirming the involvement of NK₃ tachykinin receptors in wet-dog shake behaviour.
3. Senktide induced increased numbers of Fos-LI neurones in the following brain areas: frontal, parietal and piriform cortex, the lateral septum, the CA1, CA2, subiculum and dentate gyrus of the hippocampus, most areas in the amygdala, thalamus and hypothalamus, medial geniculate nucleus and the ventral cochlear nucleus. Pretreatment with SR142801, but not with SR142806, before administration of senktide inhibited Fos-LI expression in the cingulate cortex, dentate gyrus of the hippocampus, some regions of the thalamus, hypothalamus and amygdala and the ventral cochlear nucleus.
4. The present results are the first demonstration that senktide induces Fos-LI in widespread areas of the guinea-pig brain. It is proposed that NK₃ tachykinin receptors may play a more extensive role in the control of diverse brain functions, including cortical processing, learning and memory, neuroendocrine and behavioural regulation, than is currently recognized.