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.     

Inhibitory action of capsaicin on cholinergic responses induced by GABAA agonists in the guinea-pig ileum

Pretreatment of the guinea-pig ileum with capsaicin resulted consistently in depression of the neurogenic cholinergic contractions induced by the GABAA receptor agonists 3-aminopropane sulphonic acid (3-APS) and muscimol. Since capsaicin acts mainly by releasing and depleting substance P from its stores in intestinal nerves, it is likely that substance P plays a role in the response caused by GABAA-mimetic compounds, On the whole, our results suggest that excitatory responses to 3-APS and muscimol result from both direct and indirect activation of intrinsic intestinal cholinergic neurons innervating smooth muscle cells.     

Intracisternal glycine activates the micturition reflex in urethane-anaesthetized rats

Intracisternal glycine, but not GABA, activates a series of neurogenic rhythmic contractions of the urinary bladder in urethane-anaesthetized rats. This effect of glycine was prevented by strychnine but not by bicuculline, indicating the involvement of specific glycinergic receptors. The effects of glycine were also prevented by either atropine or haloperidol suggesting an involvement of cholinergic and monoaminergic excitatory neurotransmission to the bladder.     

Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder

Capsaicin (0.03-3 microM) induces contractions of the rat isolated bladder which are unaffected by either atropine (3 microM) or tetrodotoxin (0.5 microM). In the presence of capsaicin (0.1 microM) an enhancement of field stimulation-induced contractions was observed. Capsaicin-desensitization did not modify the height of these. The neurogenic nature of the capsaicin-induced contractions was proved by the observation that 'chronic' (48 h) denervation prevented, while 'acute' (2 h) denervation did not modify the effect of capsaicin. Denervated bladders maintained their responsiveness to acetylcholine but not to field stimulation. Isolated bladders from rat pups (1-2 days old) did not respond to capsaicin while strong contractile responses to acetylcholine or field stimulation were obtained in these preparations. In bladders from two week old animals, capsaicin produced similar contractions to those observed in preparations from adult animals. The bladders from rats receiving a high dose of capsaicin (50 mg kg-1 s.c.) at birth were heavier than those of their age-matched, vehicle-treated controls. Isolated bladders from 2 month old animals pretreated with capsaicin at birth were unresponsive to capsaicin while responsiveness to acetylcholine, substance P or field stimulation was unaffected compared with that of vehicle-treated controls. These experiments provide evidence that a capsaicin-sensitive innervation exists in the rat urinary bladder which undergoes a postnatal development at end organ level.     

Further studies on the mechanisms of the tachykinin-induced activation of micturition reflex in rats: evidence for the involvement of the capsaicin-sensitive bladder mechanoreceptors

The relative ability of substance P, neurokinin A, neurokinin B and kassinin to activate the micturition reflex was investigated in urethane-anaesthetized rats. When administered topically neurokinin A, neurokinin B and kassinin were 14, 36 and 280 times, respectively, more potent than substance P to activate micturition. On the other hand substance P, neurokinin A and kassinin were practically equipotent (and neurokinin B was about 3-4 times less potent than substance P) to stimulate the contraction of the rat isolated bladder and to potentiate the contractions induced by electrical field stimulation. This indicates that neither a direct action on muscle cells nor a potentiating effect on efferent neurotransmission can account for the rank order of potency of tachykinins for activation of the micturition reflex. The ability of topical tachykinins to activate the micturition reflex was largely impaired in 2 months old rats pretreated with capsaicin (50 mg/kg s.c.) on their second day of life, indicating that integrity of the capsaicin-sensitive bladder mechanoreceptors is essential for the production of this effect. These findings indicate that an NK-B receptor, possibly located on sensory nerves in the bladder wall, participates in the tachykinin-induced activation of reflex micturition.     

Pupillary constriction by bradykinin and capsaicin: mode of action

The application of bradykinin or capsaicin to the rabbit eye evoked strong miosis. The effect could be prevented by pretreatment of the eye with tetrodotoxin (TTX) or a substance P (SP) antagonist. However, the miotic response could be elicited despite TTX or the SP antagonist if the dose of capsaicin or bradykinin was increased. Bradykinin and capsaicin contracted the isolated rabbit sphincter pupillae muscle. The contraction produced by bradykinin and capsaicin was unaffected by TTX but reduced by specific SP antagonists. This indicates that bradykinin and capsaicin exert their effects on the isolated sphincter muscle through the release of SP but independent of neuronal conduction. In vivo, the situation seems to be different. The finding that TTX is capable of blocking the miotic response to moderate doses of bradykinin and capsaicin suggests that the effect on the eye under these circumstances is dependent upon a normal impulse traffic.     

Effects of capsaicin on vascular smooth muscle

Acute administration of capsaicin in vitro produced either vascular smooth muscle contraction (cat middle cerebral artery) or smooth muscle relaxation (guinea pig carotid artery and thoracic aorta). Prior in vivo treatment with capsaicin abolished the relaxation response of guinea pig vessels to acute capsaicin. Instead a contractile response was seen after chronic capsaicin treatment, suggesting that the relaxation response produced by capsaicin is due to release of a vasodilator substance. Substance P caused relaxation in both cat cerebral arteries and the guinea pig thoracic aorta, an effect which was abolished or reduced by endothelial damage. However, responses to acute capsaicin were not altered by endothelial damage, suggesting that substance P does not mediate the relaxation response to acute capsaicin administration. Exposure to capsaicin in vitro did not affect the neurogenic vasodilator response of cat cerebral arteries and did not alter substance P levels. Therefore, it was concluded that the acute effect of capsaicin is composed of two components. A contractile response is most likely due to direct effects on vascular smooth muscle, while a relaxation response is attributed to release of an as yet unidentified bioactive substance distinct from substance P.     

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     

Motor and inflammatory effect of hyperosmolar solutions on the rat urinary bladder in relation to capsaicin-sensitive sensory nerves

1. Intravesical administration of hyperosmolar NaCl or urea solutions produced a concentration-dependent stimulatory action on the micturition reflex in urethane-anesthetized rats. This effect was not modified in rats pretreated with capsaicin as adults (50 mg/kg s.c. 4 days before). 2. Hyperosmolar NaCl also produced Evans blue leakage (plasma extravasation) in the rat bladder. This effect was greatly reduced by extrinsic bladder denervation and in rats desensitized to capsaicin as newborns but not as adults. 3. Cumulative addition of NaCl produced a concentration-dependent increase in tone and biphasic effects on neurogenic contractions of the rat isolated bladder. These effects were not modified by in vitro capsaicin desensitization. 4. These findings do not support the idea that true osmoreceptors are present in the rat urinary bladder. The neurogenic component of the inflammatory response to hyperosmolar NaCl could involve activation of a subpopulation of bladder sensory fibers susceptible to the neurotoxic action of capsaicin in the early postnatal period only.     

Tachykinin-independent activity of capsaicin on in-vitro lamb detrusor

The capsicum alkaloid capsaicin is an afferent fibre exciter. In the vesical bladder, capsaicin acts by releasing peptides stored in afferent fibres. The aim of this work was to verify the activity of capsaicin on in-vitro lamb urinary bladder and to ascertain whether this alkaloid evokes peptide release. Capsaicin relaxed about 80% of the lamb detrusor muscle preparations tested and contracted about 20%. Whereas neurokinin A and substance P antagonists, administered alone or together, left the contractile responses to capsaicin unchanged, atropine and tetrodotoxin totally inhibited contraction. Ruthenium red and indometacin abolished contractions and relaxation. The substance P and neurokinin A antagonists and the NO-synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) left relaxation unchanged; conversely, the calcitonin gene-related peptide antagonist alpha h-CGRP (8-37) abolished this response. These results suggest that capsaicin relaxes lamb detrusor muscle not through tachykinins but by releasing CGRP from afferent fibres. Our observation that indometacin blocks the capsaicin response in in-vitro lamb urinary bladder also suggests a role of prostanoids.     

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.     

Sensitivity to indomethacin of tetrodotoxin-resistant contractions of smooth muscle from the base of rabbit bladder.

Tetrodotoxin (TTX) reduced the contractions to field stimulation of strips of rabbit bladder base by 58% of control (at 40 Hz), and increased the spontaneous activity occurring between the evoked responses. The TTX-resistant contractions resembled the spontaneous activity in that they were of comparable size and poorly sustained; in the presence of indomethacin, TTX produced a significantly greater reduction (to 13% of control at 40 Hz), of the evoked contractions. Indomethacin abolished spontaneous activity in the presence and absence of TTX, but did not affect evoked responses in strips that were not exposed to TTX. The results imply that a prostaglandin-like substance may potentiate residual evoked responses in TTX-treated strips, but does not contribute to field stimulation-induced contractions in untreated bladder base smooth muscle.     

Neuropeptide action on the guinea-pig bladder; a comparison with the effects of field stimulation and ATP

A comparison was made between the effects of several neuropeptides and ATP as possible mediators of the non-adrenergic, non-cholinergic excitatory response in detrusor strips from the guinea-pig urinary bladder. Both substance P and vasoactive intestinal polypeptide produced contractions of the guinea-pig bladder, but the form of the atropine-resistant neurogenic excitation was mimicked more precisely by ATP. Neither methionine enkephalin nor leucine enkephalin had a prominent direct action on the smooth muscle (up to 100 microM) and did not significantly modify the cholinergic or non-cholinergic components of the response elicited by field stimulation. A proteolytic enzyme, chymotrypsin (10 U/ml), antagonised the excitatory effect of substance P, but not that of the non-adrenergic, non-cholinergic excitatory response or ATP. The slow excitation elicited by a high concentration of vasoactive intestinal polypeptide (10 microM), in contrast to responses elicited by ATP or field stimulation, was attenuated by preincubation with the structurally related polypeptide PHI, which was itself inactive (up to 10 microM). The present observations argue against a role for the peptides studied as neuromuscular transmitters in the detrusor but do not preclude such a role for ATP.     

Enhancement by nitric oxide of neurogenic contraction in the mouse urinary bladder

In isolated detrusor strips from the mouse urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine and ATP. The aim of this study is to investigate the possible role of nitric oxide (NO) involved in the neurogenic detrusor contraction. Repetitive electrical field stimulation evoked muscle contractions of the isolated mouse detrusor strips, which could be abolished by tetrodotoxin (TTX). NO donors including sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) as well as exogenous NO increased, while hemoglobin and NO synthase inhibitor N^G-nitro-L-arginine decreased the neurogenic detrusor contractions. The addition of L-arginine reversed the inhibitory effect of N^G-nitro-L-arginine. SNP failed to affect the contractions induced by carbachol and α,β-methylene ATP. These findings suggest that NO potentiated the excitatory neuromuscular transmission in electrically stimulated detrusor strips from the mouse. Received: 15 July 1997 / Accepted: 4 September 1997     

Evidence for the involvement of endogenous substance P in the motor effects of capsaicin on the rat urinary bladder

[pro4,trp7,9,Leu11]SP-(4-11), a substance P (SP) antagonist, selectively antagonized contractions produced by either capsaicin or SP on the rat isolated urinary bladder. These experiments provide direct evidence indicating that the motor effects of capsaicin on rat urinary bladder are attributable, at least in part, to the release of endogenous SP.     

Excitatory and inhibitory urinary bladder reflexes induced by stimulation of cervicovaginal capsaicin-sensitive sensory fibers in rats

We have investigated the effect of intravaginal application of capsaicin on micturition reflex in female rats. Urinary bladder contractility was measured by transurethral pressure recording at isovolumetric and subthreshold conditions in anaesthetized rats. The intravaginal application of capsaicin (15 mug/50 mul rat) induced reproducible bladder phasic contractions, without desensitization upon repeated applications, that were blocked by intravenous atropine (1 mg/kg) or hexamethonium (5 mg/kg) and prevented by removal of paracervical ganglia or systemic capsaicin pretreatment (125 mg/kg, s.c.). The inhibition of sympathetic transmission by guanethidine (30 mg/kg, s.c.) produced significant increase of the bladder reflex contractions activated by intravaginal capsaicin. Intravenous administration of the TRPV1 antagonist, capsazepine (3 mg/kg), significantly reduced the excitatory reflex response to capsaicin. Intravaginal administration of capsaicin (15 mug/50 mul), during distension-induced reflex bladder contractions, produced a transient block of reflexes, unaffected by guanethidine pretreatment. In conclusion, the stimulation of capsaicin-sensitive sensory nerve endings in the rat cervix-vagina induced a dual excitatory or inhibitory bladder response in anaesthetized female rats depending on the degree of bladder distension.     

Pharmacological modulation of the contractile response to toluene diisocyanate in the rat isolated urinary bladder.

1. Toluene diisocyanate produced concentration-dependent contractions of the rat isolated urinary bladder. 2. The contractions were tetrodotoxin-resistant and were abolished by previous exposure of the strips to capsaicin. 3. Indomethacin (5 microM) and ruthenium red (30 microM) inhibited toluene diisocyanate-induced contractions. Responses expressed as a percentage of the response obtained with substance P, 30 nM, were respectively 141.6 +/- 24.8% and 20.1 +/- 5.1% in control and indomethacin-treated strips (P less than 0.005); 123.0 +/- 30.2% and 14.0 +/- 6.5% in control and ruthenium red-treated strips (0.01 less than P less than 0.05). 4. These results suggest that toluene diisocyanate-induced contractions of the rat isolated bladder are the result of the release of cyclo-oxygenase products which may act by activating the capsaicin receptor.     

Effects of topically applied capsaicin cream on neurogenic inflammation and thermal sensitivity in rats

The effects of capsaicin cream on neurogenic inflammation and thermal nociceptive threshold were investigated in rats. Firstly, for topical application of capsaicin cream to hind paw, we shaped boots from dental cement to prevent the animals from licking off the drug. Capsaicin cream (1%) led to significant increases in the amounts of Evans blue and substance P (SP) released into the perfusate, and the former response was significantly suppressed by pretreatment with RP67580, an NK1-receptor antagonist, but not by treatment with an NK2-receptor antagonist. Subsequent electrical stimulation of the sciatic nerve resulted in a significant reduction in Evans blue and SP extravasation 24 h after topical application of capsaicin cream. On the other hand, when capsaicin cream was repeatedly applied to both hind paws once a day, withdrawal latency for noxious heat stimulation decreased after 24 h, and this thermal hyperalgesia was reversed 3 days later. These results suggest that capsaicin cream initially affects neurogenic inflammation mechanisms and then blocks the pain transmission mechanism.     

Pharmacological characterization of the excitatory innervation to the guinea-pig urinary bladder in vitro: evidence for both cholinergic and non-adrenergic-non-cholinergic neurotransmission.

1 Field stimulation of strips of guinea-pig isolated urinary bladder with 5 s trains at 0.1 to 15 Hz resulted in frequency-dependent, reproducible contractions. 2 At concentrations of 1 and 4 x 10(-7) M and 1 x 10(-6) M, atropine produced a variable, partial inhibition of contractions at all frequencies but was most effective at frequencies of 3 Hz or more. 3 Tetrodotoxin (TTX), 5 x 10(-7) M, inhibited contractions at all frequencies by 80 to 90%. 4 Physostigmine, 2 x 10(-6) M, significantly enhanced the contractile response to frequencies of less than 10 Hz but did not enhance responses resistant to inhibition by atropine. Hexamethonium, 1 x 10(-4) M, slightly enhanced the contractile response to frequencies of 4 Hz or greater. 5 (+/-)-Propranolol (5 x 10(-6)M), guanethidine (1 x 10(-6)M), phentolamine (5 x 10(-6)M) and clonidine (3 x 10(-8)M) each enhanced the contractile response to field stimulation. 6 Contractile responses obtained in the presence of atropine (4 x 10(-7) M) and guanethidine (1 x 10(-6) M) increased with time and were inhibited 60 to 80% by TTX (5 x 10(-7)M. 7 It is concluded that the cholinergic nervous system contributes, in part, to electrically-induced excitatory contractions of the isolated urinary bladder of the guinea-pig. Concomitant sympathetic stimulation appears to serve an inhibitory role. In addition, a major portion of the contractile response appears to be due to a non-cholinergic non-adrenergic, as yet unidentified, substance.     

A study of the atropine-resistant component of the neurogenic response of the rabbit urinary bladder

Rabbit bladder body was stimulated to contract by a number of agonists, of which bradykinin was the most potent, and ATP one of the least potent substances tested. The atropine-resistant component of the neurogenic response was unaffected by 2 X 10(-5) M chlorpheniramine or 10(-6) M methysergide, doses which suppressed responses to histamine or 5HT. Indomethacin 10(-5) M, or 10(-5) M capsaicin both reduced the atropine-resistant component. Following treatment with 10(-6) M atropine and 10(-5) M prazosin, 10(-4) M ANAPP3 produced a further suppression of the response, but did not antagonize the response to ATP. In the bladder body, the transmitter(s) responsible for the neurogenic response may be acetylcholine and prostaglandins and possibly ATP and substance P.