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     

Nociceptin protects capsaicin-sensitive afferent fibers in the rat urinary bladder from desensitization

Chemical stimulation of primary afferent nerves in the rat urinary bladder in vivo with topical capsaicin (1 microg in 50 microl saline) determines a dual motor response, consisting of a contractile effect mediated by tachykinins released from sensory nerves in the bladder wall and a transient activation of a bladder-to-bladder micturition reflex organized at the supraspinal level (chemoceptive micturition reflex). Both responses undergo complete desensitization upon repeated applications of capsaicin. The i.v. administration of the novel neuropeptide nociceptin (100 nmol/kg) produced a long-lasting protection from capsaicin desensitization of afferent nerves which mediate the chemoceptive micturition reflex. In fact a chemoceptive micturition reflex could be repeatedly evoked by topical capsaicin in nociceptin-pretreated rats. In sharp contrast, nociceptin did not influence the development of desensitization of the local response to capsaicin, corresponding to the 'efferent' function of capsaicin-sensitive afferent neurons. These results suggest that the afferent and 'efferent' function of capsaicin-sensitive primary afferent neurons (CSPANs) in the rat bladder are differentiated by nociceptin. Alternative mechanisms underlying this phenomenon are discussed.     

The role of the capsaicin-sensitive innervation of the rat urinary bladder in the activation of micturition reflex

Capsaicin applied on the serosal surface of the urinary bladder in urethane-anaesthetized rats produces two distinct types of motor effects: a tetrodotoxin-, hexamethonium- and lidocaine-insensitive 'tonic' contraction and a series of tetrodotoxin-, hexamethonium- and lidocaine-sensitive rhythmic contractions. Both 'tonic' and rhythmic contractions are abolished by bladder denervation indicating their neurogenic origin. The rhythmic but not the 'tonic' component of the contractile effect of capsaicin is abolished by spinal cord transection indicating activation of a supraspinal micturition reflex. The motor effects of topical capsaicin are unaffected by pretreatment with indomethacin or diphenhydramine plus cimetidine. Pretreatment with a large dose of subcutaneous (SC) capsaicin increases both volume and pressure threshold for micturition while amplitude of micturition contraction is unaffected. Moreover the spinal somatovesical reflex elicited by pinching of the perineal skin is unaffected by capsaicin-desensitization. The intracerebroventricular (ICV) administration of capsaicin reproduces the effects of SC capsaicin on the bladder response to saline filling. Rats pretreated with ICV capsaicin are as sensitive as controls in reacting to noxious heat (hot plate test) while the wiping response to instillation of capsaicin into one eye was abolished. These findings provide functional evidence for the presence in the rat urinary bladder of a capsaicin-sensitive innervation which subserves a sensory function in relaying volume/pressure information from detrusor muscle to central nervous system. Information carried through these capsaicin-sensitive fibers appears to be relevant for initiation of a supraspinal vesico-vesical micturition reflex. Functional evidence indicates that these fibers may terminate at supraspinal level.     

Effects of nonsteroidal antiestrogens in the in vitro rat uterus.

We have studied the effect of nonsteroidal antiestrogens on rat uterine contractions induced by oxytocin (8 nmol/l), methacholine (10 mumol/l), prostaglandin F2 alpha (1 mumol/l), KCl (60 mmol/l) and CaCl2 (6 mmol/l). In a concentration-dependent way, the antiestrogens tamoxifen, clomiphene, nafoxidine and ethamoxytriphetol inhibited the amplitude and frequency of the oxytocin-induced contractions and the contraction produced by CaCl2. At a concentration of 30 mumol/l the four drugs inhibited the contractions induced by methacholine and prostaglandin F2 alpha. They also relaxed the tonic contraction to KCl in a concentration-dependent way. This action was partially counteracted by CaCl2 (0.1-10 mmol/l). Bay k 8644 (0.3 nmol/l to 3 mumol/l) only partially reversed the inhibition by ethamoxytriphetol (0.1 mmol/l) of CaCl2 (6 mmol/l)-induced contractions. The steroidal antiestrogen, ICI 164,384, which lacks agonist activity, had an inhibitory effect (44 +/- 4%, n = 7) on KCl-induced contractions only at a concentration of 0.1 mmol/l. However, the quaternary analogue of tamoxifen (tamoxifen ethyl bromide) produced 86 +/- 3% relaxation of the KCl-induced contracture (IC50 1.52 +/- 0.1 mumol/l, n = 10) and this effect was counteracted by addition of CaCl2. Taken together the results indicate that the inhibitory effects of nonsteroidal antiestrogens on rat uterine contractions could be mediated by an action to block Ca2+ entry through an agonist action on extracellular estrogen receptors.     

Effect of a calcitonin gene-related peptide antagonist (CGRP8-37) on skin vasodilatation and oedema induced by stimulation of the rat saphenous nerve.

1. The effect of the calcitonin gene-related peptide antagonist (CGRP8-37, 400 nmol kg-1, i.v.) on the increased blood flow induced by calcitonin gene related peptide (CGRP), vasodilator prostaglandins, and topical capsaicin was measured with a laser Doppler blood flow meter in rat abdominal skin. 2. The saphenous nerve was electrically stimulated and the effect of CGRP8-37 (400 nmol kg-1, i.v.) on the increased blood flow (measured by laser Doppler flowmetry) and oedema formation (measured by the extravascular accumulation of [125I]-albumin) was investigated in the rat hind paw. 3. CGRP8-37 (400 nmol kg-1, i.v.) had no effect on basal cutaneous blood flow at uninjected sites and sites injected with Tyrode buffer, but acted selectively to inhibit the increased blood flow induced by intradermal CGRP (10 pmol/site, P < 0.05), but not that induced by prostaglandin E2 (PGE2, 300 pmol/site) or carba-prostacyclin (cPGI2, 100 pmol/site). 4. Capsaicin (0.1-33 mM), applied topically, acted in a dose-related manner to increase blood flow. CGRP8-37 (400 nmol kg-1, i.v.) almost totally inhibited blood flow induced by capsaicin (10 mM; P < 0.05) but did not significantly inhibit blood flow induced by a higher dose of capsaicin (33 mM). 5. The increased blood flow induced by short stimulation of the saphenous nerve (10 V, 1 ms, 2 Hz for 30 s) was inhibited by 76%, 5 min after i.v. CGRP8-37 (400 nmol kg-1, i.v., P < 0.05). 6. A longer (5 min) electrical stimulation of the saphenous nerve caused oedema formation, in addition to increased blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of topical capsaicin on rat urinary bladder motility in vivo

The effects of topical capsaicin on urinary bladder motility were investigated following saline-induced distension of the bladder wall in urethane-anaesthetized rats and compared to the effects of topical substance P and acetylcholine. Capsaicin and substance P produced similar excitatory effects in both quiescent and rhythmically contracting bladders, i.e., a TTX resistant tonic contraction followed by a series of rhythmic, TTX sensitive, phasic contractions. Acetylcholine, in doses equieffective in producing TTX resistant contractions was less effective than capsaicin or substance P in triggering neurogenic rhythmic contractions of bladder muscle. Atropine pretreatment prevented the neurogenic component of the excitatory effect of both capsaicin and substance P. Repeated applications of capsaicin but not of substance P led to desensitization. Bladders of animals pretreated (4 days before) with a large dose of s.c. capsaicin developed insensitivity to topical capsaicin and a larger volume of saline was required to trigger neurogenic rhythmic contractions of the detrusor muscle. These results suggest that capsaicin acts by interfering with the mechanism(s) regulating the threshold for the micturition reflex to occur.     

The effect of nifedipine on spontaneous, drug-induced and reflexly-activated contractions of the rat urinary bladder: evidence for the participation of an intracellular calcium store to micturition contraction

1. The effect of nifedipine on spontaneous and stimulated motility of the rat urinary bladder has been investigated in vitro (isolated detrusor strips) and in vivo (micturition reflex). 2. Nifedipine inhibited tone and spontaneous activity of the isolated rat bladder, its effect being greater in indomethacin-treated preparations. Nifedipine suppressed the KCl induced phasic and tonic contraction and inhibited by 60-80% the carbachol- or ATP- induced contractions. Nifedipine reduced by about 70% amplitude of the nerve-mediated bladder contractions. 3. Exposure to Ca free medium containing EDTA suppressed tone and spontaneous activity of the rat bladder. In these conditions the response to KCl or ATP was rapidly abolished while a response to carbachol was still evident even after a long exposure to the Ca free medium. 4. In vivo, nifedipine affected reflex micturition e.g. increased volume threshold and slightly reduced amplitude of micturition contraction. In addition, nifedipine reduced voiding efficiency e.g. increased residual volume after micturition. These effects were evident following ligation of the ureters because in normal conditions nifedipine induced a marked diuresis which masked its effect on volume threshold. 5. These findings indicate that in the rat urinary bladder Ca from both intra- and extracellular pools is mobilized during spontaneous or stimulated contractions. Mobilization of an intracellular Ca pool by cholinomimetics or other neurotransmitter(s) may be responsible for the nifedipine-resistant component of the voiding contraction in vivo.     

The response of non-pregnant rat myometrium to oxytocin in Ca-free solution.

The contractile response of the longitudinal muscle of non-pregnant rat myometrium to oxytocin (0.2-20 nM) consisted of a phasic and a tonic component. Ca-removal abolished the phasic component but a tonic contraction could be evoked without reduction of amplitude for 50 h. Exceptionally, the tonic contraction also disappeared gradually in Ca-free medium containing 2 mM EGTA. When oxytocin was repeatedly applied in the absence of Ca, the response became at first progressively larger before reaching a steady state. Transient addition of Ca to the medium reduced the size of the subsequent oxytocin contraction. In Ca-free medium, the tissue lost Ca slowly, but it still contained 40 mumol kg-1 after 6 h and roughly 1 mumol kg-1 wet weight after 24 h exposure. 45Ca efflux was marginally increased by oxytocin (20 nM). Caffeine (5-30 mM) produced no contraction, but slightly reduced the resting tension and strongly inhibited the oxytocin response both in the presence and in the absence of Ca. Caffeine also blocked the contraction induced by Ca added to Ca-free 40 mM K solution. However, pretreatment with caffeine (30 mM) had no effect on the following oxytocin response. A calmodulin antagonist, trifluoperazine (1-10 microM) suppressed strongly the Ca-induced contraction, but had only a weak effect on the oxytocin response in Ca-free medium. Chlorpromazine (10-100 microM) and fluphenazine (10-30 microM) had similar effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

An investigation into the mechanism of capsaicin-induced oedema in rabbit skin.

1. Oedema formation induced by intradermal capsaicin has been studied in rabbit skin. The effect of the anti-inflammatory steroid dexamethasone and also of a range of known inhibitors of oedema formation have been investigated in order to elucidate mechanisms involved in capsaicin-induced oedema formation. 2. Oedema formation, in response to intradermally-injected test agents, was measured by the local extravascular accumulation of intravenously injected 125I-labelled albumin. In separate experiments skin blood flow was assessed by the clearance of intradermally-injected 133xenon. 3. Oedema formation induced by intradermal histamine (3 nmol) and bradykinin (1 nmol), when in the presence of vasodilator doses of calcitonin gene-related peptide (CGRP) (3 pmol) or prostaglandin E1, (PGE1) (10 pmol), was significantly inhibited (P < 0.01) in rabbits pretreated with intravenous dexamethasone (3 mg kg-1, -4 h). In contrast dexamethasone had no effect on capsaicin (3 mumol)-induced oedema formation or, on capsaicin (30-100 nmol)-induced blood flow. 4. Oedema formation observed in response to intradermal capsaicin (3 mumol) was significantly inhibited (P < 0.01) when the selective capsaicin antagonist, ruthenium red (3 nmol) was co-injected. This suggests that the mechanism of capsaicin-induced oedema involves activation of sensory nerves. However, oedema was not inhibited when capsaicin was co-injected with the neurokinin NK1 receptor antagonist, RP67580 (10 nmol), the NK2 antagonist SR48960 (10 nmol) or the CGRP antagonist CGRP8-37 (300 pmol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antinociceptive profile of the pseudopeptide B2 bradykinin receptor antagonist NPC 18688 in mice.

1. The purpose of this study was to investigate the topical and systemic anti-hyperalgesic effect of the newly-developed pseudopeptide B2 receptor antagonist, NPC 18688, in different models of nociception in mice. 2. Given systemically 30 min beforehand, NPC 18688 (10-300 nmol kg-1, i.p.) caused no agonist effect, but produced a dose-related and significant inhibition of abdominal constrictions caused by intraperitoneal injection of acetic acid (0.6%), acetylcholine (ACh, 4.5 mg kg-1) or kaolin (50 mg kg-1). The calculated mean ID50s and the percentages of maximal inhibitions (MI) for these effects were: 77, 34 and > 300 nmol kg-1 and 65 +/- 6, 70 +/- 5 and 40 +/- 3%, respectively. The anti-hyperalgesic effect of NPC 18688 (100 nmol kg-1, i.p.) occurred rapidly (30 min) and lasted for at least 150 min. Hoe 140 (3-30 nmol kg-1, i.p.) given 30 min beforehand also inhibited, in a graded manner, acetic acid and ACh-induced writhing, with mean ID50s and MI of 6 and 9 nmol kg-1 and 56 +/- 7 and 62 +/- 6%, respectively. 3. NPC 18688 (10-300 nmol kg-1, i.p.) caused a graded inhibition of both phases of formalin (2.5%)-induced pain, its effects being more potent in relation to the second phase of the formalin test. The calculated mean ID50s and the MI were > 300 and 60 nmol kg-1 and 20 +/- 3 and 60 +/- 5% against the first and second phases of formalin-induced nociception, respectively. NPC 18688 at the same doses also inhibited, in a dose-related manner, formalin-induced paw oedema (MI of 35 +/- 3%). 4. When injected locally in the mouse paw, NPC 18688 (2, 10 and 20 nmol/paw) had no agonist activity. However, when co-injected with formalin NPC 18688 (2-20 nmol/paw), it produced significant inhibition of both phases of formalin response, with MI of 40 +/- 3 and 33 +/- 2%, respectively. NPC 18688 at 10 nmol/paw also significantly inhibited formalin-induced paw oedema (25 +/- 2%). 5. Given intraperitoneally, NPC 18688 (30-300 nmol kg-1) determined a graded inhibition of the nociceptive response caused by intraplantar injection of capsaicin (1.6 micrograms/paw) (40 +/- 2%). However, NPC 18688 (up to 300 nmol kg-1, i.p.), given 30 min beforehand, had no significant analgesic effect when analyzed in the tail flick and in the hot plate pain models, nor did it change the performance of animals in the rota rod test. 6. The action of NPC 18688 was quite selective for the B2 receptor, and like Hoe 140, (1 to 100 nmol kg-1, i.p.) it caused graded inhibition of bradykinin (BK, 3 mol/paw)-induced increase in mouse paw volume, with mean ID50s of 61 and 6 nmol kg-1, respectively. In addition, at 100 nmol kg-1, the dose at which NPC 18688 significantly antagonized BK (3 nmol)-mediated rat paw oedema in naive animals, it had no significant effect on des-Arg9-BK (100 nmol/paw)-induced oedema in paws that had been desensitized to BK. NPC 18688 (210 nmol kg-1), like Hoe 140 (230 nmol kg-1) given s.c. 30 min beforehand, completely abolished BK (28 nmol)-induced hypotension, without affecting the fall of mean arterial blood pressure induced by i.v. injection of ACh (2 nmol kg-1). Finally, NPC 18688 (1 microM) did not affect ACh-mediated contraction in the guinea-pig ileum or toad rectus abdominii in vitro. 7. These results demonstrate that the newly-developed and selective pseudopeptide B2 receptor antagonist, NPC 18688, although less potent than the available second generation of B2 peptide BK receptor antagonists, exhibits topical and long-lasting systemic anti-hyperalgesic properties when analysed in several models of nociception in mice, making it a useful tool for investigating the participation of BK and related kinins in physiological and pathological processes. Finally, this new class of selective pseudopeptide B2 receptor antagonist may constitute a new strategy for developing the third generation of potent and long-lasting orally-active non-peptide BK antagonists, which may be useful for the management of clinical disorders involving BK and relate     

Involvement of tachykinins in plasma extravasation induced by bradykinin and low pH medium in the guinea-pig conjunctiva.

1. The effect of bradykinin, capsaicin, substance P and low pH medium on plasma extravasation in the guinea-pig conjunctiva has been studied. Evans blue dye was measured in the conjunctiva after local instillation of the agents into the conjunctival sac. 2. Bradykinin (2-50 nmol), capsaicin (20-50 nmol) and substance P (0.5-5 nmol) caused a dose-dependent increase in plasma extravasation with the following order of potency: substance P > bradykinin = capsaicin. The effect of capsaicin (50 nmol) and substance P (5 nmol) was abolished by the tachykinin NK1 receptor antagonist, CP-99,994 (8 mumol kg-1, i.v.) (P < 0.01), whereas CP-100,263 (8 mumol kg-1, i.v.) the inactive enantiomer of CP-99,994 was without effect. CP-99,994 inhibited by 70% (P < 0.01) the effect of bradykinin. 3. The kinin B2 receptor antagonist, Hoe 140 (icatibant, 10 nmol kg-1, i.v.) abolished the response to bradykinin (50 nmol) (P < 0.01), but did not affect the responses to capsaicin (50 nmol) or substance P (5 nmol). Plasma extravasation induced by low pH medium (pH 1) was abolished by CP-99,994 (P < 0.01) and by Hoe 140 (P < 0.01). 4. The present findings suggest that: endogenous or exogenous tachykinins increase plasma extravasation in the guinea-pig conjunctiva by activation of NK1 receptors; bradykinin-induced plasma extravasation is mediated by tachykinin release from sensory nerve endings; low pH media cause plasma extravasation via release of kinins that by activation of B2 receptors release tachykinins from sensory nerve endings.     

BaCl2- and 4-aminopyridine-evoked phasic contractions in the rat vas deferens.

1. The actions of BaCl2 and 4-aminopyridine, blockers of K+ channels, on the mechanical activity of the epididymal half of the rat vas deferens were investigated. 2. Both BaCl2 and 4-aminopyridine dose-dependently evoked phasic contractions. High extracellular potassium (35-40 mM) caused a tonic contraction but abolished the BaCl2- and 4-aminopyridine-induced phasic activity and reduced the BaCl2-induced sustained component of contraction, but increased the 4-aminopyridine-induced tonic contraction. 3. Omission of calcium from the extracellular medium totally abolished the 4-aminopyridine-induced response but only reduced the mean amplitude of phasic contractions induced by BaCl. 4. Procaine (10 mM), an inhibitor of internal calcium release, completely abolished the phasic activity and reduced the sustained contraction induced by BaCl2. The remaining tone was abolished by nifedipine (1 microM). 5. Tetraethylammonium (1 mM) suppressed the amplitude of the BaCl2-induced phasic contractions, and induced a biphasic increase in tonic tension. 6. The BaCl2-induced responses were resistant to prazosin (1 microM), yohimbine (3 microM), propranolol (3 microM) or atropine (3 microM); in contrast, the 4-aminopyridine-induced activity was effectively inhibited by prazosin (1 microM) attenuated by yohimbine (1 microM) and atropine (1 microM) but not by propranolol (3 microM). The 4-aminopyridine-induced response was abolished by pretreatment of the vas deferens with 6-hydroxydopamine (0.5 mM). 7. The results indicate that the BaCl2-evoked activity in the vas deferens was mainly due to blockade of Ba(2+)-sensitive K+ channels on the smooth muscle plasma membrane. Subsequent calcium entry through the depolarized plasma membrane was needed to trigger generation of phasic contractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

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

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

Tachykinin inhibition of acid-induced gastric hyperaemia in the rat.

1. Primary afferent neurones releasing the vasodilator, calcitonin gene-related peptide, mediate the gastric hyperaemic response to acid back-diffusion. The tachykinins neurokinin A (NKA) and substance P (SP) are located in the same neurones and are co-released with calcitonin gene-related peptide. In this study we investigated the effect and possible role of tachykinins in the acid-evoked gastric vasodilatation in urethane-anaesthetized rats. 2. Gastric acid back-diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 M HCl, increased gastric mucosal blood flow by 60-90%, as determined by the hydrogen clearance technique. NKA and SP (0.14-3.78 nmol min-1 kg-1, infused intra-aortically) inhibited the gastric mucosal hyperaemic response to acid back-diffusion in a dose-dependent manner, an effect that was accompanied by aggravation of ethanol/acid-induced macroscopic haemorrhagic lesions. 3. The inhibitory effect of NKA (1.26 nmol min-1 kg-1) on the acid-induced gastric mucosal vasodilatation was prevented by the tachykinin NK2 receptor antagonists, MEN 10,627 (200 nmol kg-1) but left unaltered by the NK1 receptor antagonist, SR 140,333 (300 nmol kg-1) and the mast-cell stabilizer, ketotifen (4.6 mumol kg-1). 4. Under basal conditions, with 0.05 M HCl being perfused through the stomach, NKA (1.26 nmol min-1 kg-1) reduced gastric mucosal blood flow by about 25%, an effect that was abolished by SR 140,333 but not MEN 10,627 or ketotifen. 5. SR 140,333, MEN 10,627 or ketotifen had no significant effect on basal gastric mucosal blood flow nor did they modify the gastric mucosal hyperaemic reaction to acid back-diffusion. 6. The effect of NKA (1.26 nmol min-1 kg-1) in causing vasoconstriction and inhibiting the vasodilator response to acid back-diffusion was also seen when blood flow in the left gastric artery was measured with the ultrasonic transit time shift technique. 7. Arginine vasopressin (AVP, 0.1 nmol min-1 kg-1) induced gastric mucosal vasoconstriction under basal conditions but was unable to inhibit the dilator response to acid back-diffusion. 8. These data show that NKA has two fundamentally different effects on the gastric circulation. Firstly, NKA reduces gastric blood flow by activation of NK1 receptors. Secondly, NKA inhibits the gastric hyperaemic response to acid back-diffusion through an NK2 receptor-mediated mechanism. These two tachykinin effects appear to take place independently of each other since they are mediated by different receptors. This concept is further supported by the inability of AVP to mimic tachykinin inhibition of the gastric vasodilator response to acid back-diffusion.     

Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the rat lower urinary tract

Summary 1. The effects of capsaicin, substance P (SP) and neurokinin A (NKA) on motor activity and vascular permeability was investigated in the rat lower urinary tract (bladder dome and neck, proximal urethra and ureters). 2. Capsaicin produced contractions of the rat bladder dome and neck and of the proximal urethra in vitro, which were unaffected by tetrodotoxin and abolished by ganglionectomy. SP and NKA were almost equipotent in producing a contraction of the rat isolated bladder dome or neck and urethra. However, the maximal response to NKA was about twice that of SP on the urethra and bladder neck. 3. Capsaicin did not affect motility of the unstimulated rat isolated ureter, while NKA or SP activated rhythmic contractions, NKA being about 850 times more potent than SP. Either capsaicin or field stimulation produced a transient inhibition of the NKA-activated rhythmic contractions of the rat isolated ureter which was prevented by capsaicin-desensitization. 4. The capsaicin-(1 M) or field stimulation-induced inhibition of NKA-activated rhythmic contractions of the rat isolated ureter were unaffected by removal of pelvic ganglia but abolished by cold storage (72 h at 4°C). 5. Intravenous capsaicin induced an inflammatory response (Evans blue leakage) in the bladder, proximal urethra and ureters in vivo. Plasma extravasation was greater in the ureters, urethra and bladder neck than in the dome. SP, NKA and histamine produced a dose-dependent dye leakage in all segments of the rat urinary tract, the response being slightly greater in the bladder neck than in the dome. 6. The capsaicin-induced inflammatory response was abolished by systemic capsaicin-desensitization and reduced, to a variable extent, by pelvic ganglionectomy, in the various tissues examined. Topical application of tetrodotoxin on the bladder dome failed to affect the capsaicin-induced plasma extravasation in the urinary bladder. 7. These findings indicate that chemoceptive, capsaicin-sensitive nerves are present throughout the whole rat lower urinary tract and their activation determines a variety of visceromotor responses and an increase of vascular permeability. In various instances the response to capsaicin may be explained by the action of tachykinins but some effects may involve other sensory neuropeptides. Send offprint requests to C. A. Maggi at the above address     

Four motor effects of capsaicin on guinea-pig distal colon.

The motor effects of capsaicin on the guinea-pig distal colon have been investigated in vivo and in vitro. Capsaicin (0.1-10 micrograms kg-1 i.v.) produced a transient relaxation which was reduced by pretreatment with capsaicin itself, atropine, hexamethonium, phentolamine or guanethidine and almost abolished by tetrodotoxin (TTX). Topically applied capsaicin produced a transient inhibition of tone and spontaneous activity prevented by topically applied TTX. In isolated preparations of distal colon, capsaicin produced a transient, TTX- and atropine-sensitive contraction which was followed by a depression of the contractile activity. The depressant effect was unaffected by atropine plus guanethidine but was greatly reduced by TTX, indicating activation of intramural non-adrenergic, non-cholinergic (NANC) mechanisms. The depressant effect on the first exposure to capsaicin (1 microM) was greater than that produced by a second, third or fourth exposure. In preparations excised from capsaicin-pretreated animals, capsaicin (1 microM) only produced an inhibitory effect on spontaneous contractions. Desensitization did not occur to this inhibitory effect. In preparations pre-exposed to capsaicin (1 microM, 1 h before), capsaicin (1-30 microM) produced a concentration-related inhibition of spontaneous contractions (IC50 = 19 microM) and of the high K+-induced tonic contraction (IC50 = 23 microM). A similar effect on spontaneous motility was produced by capsaicin in colonic segments excised from capsaicin-pretreated guinea-pigs (IC50 = 16 microM) or guinea-pigs treated with TTX (IC50 = 20 microM). It is concluded that, in vivo, capsaicin activates inhibitory reflexes, presumably due to stimulation of primary afferent fibres. This effect involves, at least in part, activation of sympathetic nerves to this organ. The contractile effect of capsaicin on the isolated colon involves activation of intramural cholinergic neurones, whereas the TTX-sensitive component of the inhibitory effect involves either release of an inhibitory transmitter through an axon reflex arrangement or activation of NANC neurones. In addition, at high concentrations capsaicin produces a direct depression of smooth muscle contraction.     

Haemodynamic and abdominal motor reflexes elicited by neurotensin in anaesthetized guinea-pigs.

1. Single intraperitoneal (i.p.) injections of neurotensin (NT) (0.14- 140 nmol kg-1) in anaesthetized guinea-pigs were found to trigger transient abdominal wall contractions (TAWC) accompanied by relatively sustained increases of systemic blood pressure (BP) and heart rate (HR). The modification of the latter NT effects by various drugs and surgical manipulations was examined to obtain some insight into the nature of, and possible relationship between, these responses. 2. The abdominal motor response (i.e. TAWC) to i.p. NT (14 nmol kg-1) was inhibited by prior i.v. injection of the guinea-pigs with pancuronium (0.27 mumol kg-1), morphine (1.5 and 15 mumol kg-1), clonidine (0.34 mumol kg-1), by concomitant i.p. injection of procaine 2% w/v, or by acute spinalization. It was potentiated by naloxone (2.8 and 28 mumol kg-1), but not affected by i.v. injection of autonomic drugs (i.e. pentolinium, prazosin, yohimbine and atropine), by capsaicin desensitization, or by acute bilateral cervical vagotomy. In spinalized animals a sustained abdominal wall contraction (SAWC) was unmasked, which was resistant to i.v. morphine, clonidine or baclofen but suppressed by i.v. pancuronium or i.p. lignocaine 2% w/v. 3. Haemodynamic responses to i.p. NT were not affected by i.v. pancuronium, morphine, naloxone, atropine, or by vagotomy. They were inhibited by i.v. pentolinium or clonidine (BP, HR), i.v. prazosin (BP), i.p. procaine 2% w/v (BP, HR), capsaicin desensitization or acute spinalization (BP, HR). Yohimbine (i.v.) potentiated BP and HR increases caused by i.p. NT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Large conductance Ca2+ -activated K+ channel activation with NS1619 decreases myogenic and neurogenic contractions of rat detrusor smooth muscle

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips pre-contracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20mM). In the presence of elevated KCl (60 mM KCl), the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5-50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.     

Specific receptors for bradykinin-induced cardiac sympathetic chemoreflex in the dog

Bradykinin (BK, 0.03-1 microgram), capsaicin (1 microgram) or potassium chloride (KCl, 13 mumol) applied to the epicardium of the left ventricle of anaesthetized, open-chest dogs, caused reflex tachycardia and pressor effects, whereas des-Arg9-BK (1-100 micrograms), a selective bradykinin B1-receptor agonist, failed to produce any cardiovascular response. Superfusion of the epicardium with a selective B1-receptor antagonist, des-Arg9-[Leu8]BK (50-100 micrograms/min) had no effect on reflex responses to epicardial BK (0.03-0.1 microgram). However, the selective B2-receptor antagonist, D-Arg-[Hyp3,Thi5,8,D-Phe7]BK (10-25 micrograms/min) abolished the reflex effects of 0.03 and 0.1 microgram BK and reduced by 50 to 70% the responses to 1 microgram BK. Another B2-receptor antagonist [Thi6,9,D-Phe8]kallidin (10-50 micrograms/min) also reduced (30-70%) responses to 1 microgram BK. The antagonism was reversible and specific for BK since reflex responses to epicardial application of either capsaicin or KCl were not affected. The results indicate that BK interacts with B2-receptors, probably located on terminals and/or axons of sympathetic afferents supplying the dog heart, to activate a cardiac sympathetic chemoreflex.