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.     

Vesico-inhibitory responses and capsaicin-sensitive afferents in rats

Summary (1) The effect of perineal pinching and distension of a balloon inserted into the colon on motility of the urinary bladder has been investigated in adult urethane-anesthetized rats pretreated with capsaicin (50 mg/kg s.c.) or its vehicle 4 days before the experiments. (2) At bladder volumes which were sufficient to elicit reflex micturition, perineal pinching or colonic distension transiently inhibited the ongoing bladder voiding contraction. The somato-vesical inhibitory response was markedly reduced or even abolished by division of pudendal nerves. Neither the somato-vesical nor the colovesical inhibitory response were modified by desensitization with systemically administered capsaicin. (3) Intraurethral administration of capsaicin produced a transient inhibition of the reflexly-activated bladder contractions. A second administration of the drug was less effective, indicating desensitization. Intravenously administered capsaicin had a similar inhibitory effect on bladder motility. (4) The vesico-inhibitory response produced by intraurethral administration of capsaicin was not affected by phentolamine, propranolol, guanethidine, picrotoxin or naloxone, while it was greatly reduced or even abolished by bilateral section of the pudendal nerves. (5) These findings provide evidence that capsaicin-sensitive chemoreceptors in the rat urethra are involved in generating a vesico-inhibitory response via pudendal nerves. On the other hand, no evidence was found for the participation of capsaicin-sensitive nerves in the generation of the somato- or colo-vesical inhibitory response. Send offprint requests to C. A. Maggi at the above address     

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     

Effects of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin.

Piperine (1 microM), a congener of capsaicin, produced an initial contraction blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 microM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 microM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1 microM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross-tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10 microM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 microM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P and CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle.     

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.     

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.     

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.     

Cadmium chloride induces contractions of the rat isolated urinary bladder by activation of capsaicin-sensitive sensory nerves

Cadmium chloride (CdCl2)(30 microM-1 mM) produced a concentration-related contraction of the rat isolated urinary bladder which was abolished by tetrodotoxin (1 microM) or extrinsic bladder denervation (72 h before). Complete cross-desensitization was observed between the contractile response to Cd and capsaicin, indicating that, at the peripheral level, this inorganic calcium channel blocker can activate the 'efferent' function of capsaicin-sensitive sensory nerves.     

Capsaicin-induced relaxation in the rat isolated external urethral sphincter: characterization of the vanilloid receptor and mediation by CGRP.

1. The potential role of capsaicin-sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2. Capsaicin (1 microM) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mM). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin-pretreated rats. 3. Capsaicin-induced relaxation of NA-precontracted REUS was mimicked by calcitonin gene-related peptide (CGRP, 0.3-10 microM), but not by substance P (1 microM), vasoactive intestinal polypeptide (VIP, 1 microM), alpha-beta methylene ATP (10 microM), gamma-aminobutyric acid (GABA, 3 mM) or galanin (1 microM). A cross-tachyphylaxis between capsaicin (1 microM) and CGRP (1 microM) was observed. Both capsaicin and CGRP-induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8-37) (10 microM). 4. Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 microM) and CGRP (0.01-1 microM) produced an almost complete inhibition of EFS-induced tonic contraction. A cross-tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8-37) (10 microM). 5. [3H]-resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pM and a Bmax of 40 fmol mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of capsaicin-sensitive sensory nerves to xylene-induced visceral pain in conscious,freely moving rats

1. Intravesical instillation of xylene (10-100%, dissolved in silicone oil) through a catheter implanted into the bladder of conscious, freely-moving rats produced behavioural effects (licking of lower abdomen or perineal region) suggestive of intense visceral pain, not mimicked by topical application of the irritant on the urethral outlet. 2. The xylene-induced visceral pain was prevented, to the same extent, by systemic desensitization to capsaicin (50 mg/kg s.c.) performed in either adult or newborn rats, as well as by extrinsic bladder denervation (pelvic ganglionectomy), thus indicating the involvement of primary afferents in the bladder wall. 3. Other behavioural responses induced by xylene instillation into the bladder (hind limb hyperextension, grooming) were not affected by systemic capsaicin desensitization in either adult or newborn rats, but were abolished by bladder denervation. 4. Systemic capsaicin desensitization produced an almost complete depletion of substance P-, neurokinin A-like and calcitonin gene-related peptide-like immunoreactivity in the rat urinary bladder. 5. These findings indicate that, in addition to their role in activating reflex micturition, the neuropeptides-containing capsaicin-sensitive sensory nerves of the rat bladder are involved in chemogenic visceral pain.     

The role of sensory neuropeptides in motor innervation of the hamster isolated urinary bladder

In this study we have characterized the role of sensory fibers and of the sensory peptides, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), on the contractile responses evoked by single pulse electrical field stimulation (EFS) in the hamster urinary bladder. EFS of the hamster isolated urinary bladder produced twitch contractions which were unaffected by atropine but abolished by tetrodotoxin. The P2 purinoreceptor antagonist PPADS (30 microM) inhibited twitches by 66+/-4% on its own and by 78+/-3% in the presence of atropine. The selective tachykinin NK2 receptor antagonist nepadutant produced a slight but consistent reduction of twitch amplitude (-21+/-3%) at 1 microM. Addition of nepadutant to atropine and PPADS did not further increase their inhibitory effect. The application of hCGRP (10-300 nM) produced a concentration-dependent inhibition of twitches (Emax -38+/-3%, EC50=12 nM) and a small reduction of tone (0.5+/-0.09 mN). Similar effects were obtained with capsaicin (0.1-10 microM) which inhibited EFS-evoked contractions with an EC50 of 100.0 nM and a maximal effect of 34+/-4% inhibition at 1 microM. Under submaximal parameters of stimulation NKA (10 nM) increased the amplitude of twitches by 45+/-6% and produced a concentration-dependent tonic contraction (EC50=55.9 nM). The CGRP1 receptor subtype antagonist, hCGRP(8-37), increased by 29+/-8% the EFS-evoked contractions and significantly reduced the response to 0.1 microM CGRP. Capsaicin (10 microM) increased both CGRP-LI and NKA-LI release from superfused slices of hamster urinary bladder by about sixfold and by about 70%, over baseline, respectively. A second application of capsaicin was ineffective, indicating a complete desensitization of sensory nerve efferent function. In the hamster urinary bladder the sensory neuropeptides NKA and CGRP are co-released by sensory fibers after stimulation either by EFS or capsaicin. However, the role of CGRP appears functionally predominant.     

Several neuropeptides determine the visceromotor response to capsaicin in the guinea-pig isolated ileal longitudinal muscle

Capsaicin (1 microM) produced, after an initial contraction, a depression of the field stimulation-induced contraction of the guinea-pig isolated ileal longitudinal muscle. Both effects exhibited prompt desensitization, indicating the involvement of a specific action on sensory nerves. The initial contraction was inhibited by [D-Pro4,D-Trp7,9,Phe11]SP-(4-11), a substance P (SP) antagonist, which did not affect the inhibitory component of the response. Incubation of the strips with antiCGRP (CGRP = calcitonin gene-related peptide) serum did not modify the amplitude of the capsaicin-induced contraction but inhibited the twitch depression induced by capsaicin. AntiCGRP serum blocked the effects of exogenous CGRP but not the inhibitory response induced by baclofen. These findings provide evidence that the release of several neuropeptides from sensory nerves determines the visceromotor response to capsaicin in this preparation. In particular, a CGRP-like peptide could be responsible for the inhibitory phase which follows the initial contraction which is due to release of SP and/or related peptides.     

Use and limitations of three TRPV-1 receptor antagonists on smooth muscles of animals and man: a vote for BCTC

Specificity of the effect is a crucial factor in using antagonists for detecting the physiological/pathophysiological roles of receptors. Here we examined the capsaicin receptor antagonist effects of three commercially-available substances, capsazepine, iodo-resiniferatoxin (I-RTX) and BCTC, on isolated smooth muscle preparations, including the human intestine. Care was taken to observe possible non-specific effects, to find out safe and effective concentrations. Capsazepine appeared to have a low margin of safety. I-RTX (up to 1μM) specifically inhibited capsaicin-induced contractions in the guinea-pig ileum and urinary bladder. I-RTX showed agonist activity on the rat urinary bladder. BCTC (1μM) abolished the contractile effects of capsaicin (1 or 2μM) on all preparations tested (guinea-pig ileum, bladder, trachea, as well as rat and mouse bladder), and on the guinea-pig renal pelvis, where it failed to influence capsaicin-sensitive, sensory neuron-mediated positive inotropy in response to field stimulation. On human intestinal preparations BCTC prevented the relaxant effect of capsaicin. It is concluded that of the three antagonists tested BCTC seems the safest one for inhibiting TRPV-1 receptors. The effect of capsazepine may be complicated by non-specific inhibition of smooth muscle contractility and that of I-RTX by agonist activity. The "local efferent" function of capsaicin-sensitive sensory neurons is not influenced by BCTC, as shown by the results obtained in the renal pelvis. In conclusion, of the TRPV-1 receptor antagonists studied, BCTC (1μM) seems the most reliable in isolated organ experiments. This substance is also effective in the human intestine.     

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.     

Evidence that toluene diisocyanate activates the efferent function of capsaicin-sensitive primary afferents

Isocyanates are an important cause of occupational asthma. The mechanism of isocyanate-induced asthma is still unknown. To determine whether toluene diisocyanate stimulates the 'efferent' function of peripheral endings of capsaicin-sensitive sensory nerves, we investigated the effect of toluene diisocyanate in the rat isolated urinary bladder, a preparation in which the action of capsaicin has been well characterized. Toluene diisocyanate (0.03-3 mM) produced a concentration-dependent contraction of the bladder strips. Its maximal effect was about 50% of the response to capsaicin (1 microM). Previous exposure of the strips to capsaicin followed by washing out produced complete unresponsiveness, both to the first exposure to toluene diisocyanate and to a second exposure of capsaicin. Further, the response to both toluene diisocyanate and capsaicin was completely prevented by extrinsic bladder denervation, achieved by bilateral removal of pelvic ganglia (72 h before). Repeated exposure of the rat bladder to toluene diisocyanate reduced the capsaicin-evoked release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI), taken as biochemical marker of activation of these sensory nerves. These experiments provide the first evidence that toluene diisocyanate activates directly or indirectly the efferent function of capsaicin-sensitive primary sensory nerves.     

The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig and rat bladder

1. Capsaicin produced a prompt release of substance P-like immunoreactivity (SP-LI) from superfused mucosa-free muscle strips excised from the guinea-pig urinary bladder. A second application of capsaicin had no further effect, indicating desensitization. 2. Neither tetrodotoxin (1 microM) or nifedipine (10 microM) had any inhibitory effect on SP-LI release by capsaicin nor influenced the establishment of the desensitized state. Nifedipine produced per se some SP-LI release. 3. SP-LI release by capsaicin was abolished by incubation in a Calcium(Ca)-free medium containing EDTA (1.0 mM) which also afforded a partial protection toward desensitization. A lower EDTA concentration (0.1 mM) did not suppress SP-LI release by capsaicin but still inhibited desensitization. 4. When the concentration of CaCl2 in the medium was lowered to 1/10-1/100 of that present in normal Krebs solution, capsaicin still evoked a marked SP-LI release and desensitization occurred. In a nominally Ca free medium (maximal Ca concentration due to impurities was 6.7 microM) SP-LI release was still observed and desensitization was incomplete. 5. In a nominally Ca free medium, removal of Mg ions enhanced the SP-LI release induced by capsaicin and enhanced desensitization. 6. In functional studies, nifedipine greatly reduced or abolished the capsaicin- or SP-induced contraction of the rat or guinea-pig isolated bladder but did not prevent desensitization. Likewise, SP-LI depletion in the rat bladder following systemic capsaicin desensitization was not prevented by nifedipine pretreatment. On the other hand, the protective action of Ca free media (containing EDTA) was confirmed in organ bath studies (guinea-pig bladder). 7. These findings indicate that: (a) the requirements of extracellular calcium for activation of neuropeptide release from sensory nerves by capsaicin are very low; (b) both excitation of sensory fibers (SP-LI release) and desensitization are dependent upon the presence of extracellular calcium and (c) L-type voltage-sensitive Ca channels are not likely to be involved in the actions of capsaicin on sensory nerve terminals.     

Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of capsaicin pretreatment on neuropeptides and salivary secretion of rat parotid glands.

1. 'Atropine-resistant' secretion of saliva in response to parasympathetic stimulation may reflect antidromic activation of sensory nerve fibres. In this investigation, the effect of pretreatment in the rat with capsaicin (total dose of 125 mg kg-1, s.c.), was determined. 2. In the parotid glands substance P (SP)/calcitonin gene-related peptide (CGRP)-containing nerve fibres around ducts and blood vessels disappeared after capsaicin, while periacinar SP-containing fibres (devoid of CGRP) and CGRP-containing fibres (devoid of SP) remained. Vasoactive intestinal peptide (VIP)-containing nerve fibres seemed to be unaffected. The parotid content of SP and CGRP was reduced by 11 and 36% respectively, while that of VIP remained unchanged. 3. The weights of the parotid glands and their sensitivity to the secretagogues methacholine and SP, injected intravenously, were unchanged as was the response to stimulation of the auriculo-temporal nerve in the presence and absence of atropine. 4. In contrast to capsaicin pretreatment, parasympathetic denervation of the parotid gland reduced the weight of the gland and produced an increase in the response to methacholine and SP. 5. For comparison, the effectiveness of the capsaicin treatment on neuropeptide content was determined in the urinary bladder. The bladder of capsaicin-pretreated rats increased in weight (21%) and in VIP content (31%), while the content of SP and CGRP was reduced by 86 and 94%, respectively. SP- and CGRP-containing nerve fibres were virtually eliminated, while VIP-containing nerve fibres seemed unaffected. 6. In conclusion, antidromic activation of primary afferent (capsaicin-sensitive) C-fibres does not contribute significantly to the 'atropine-resistant' secretory response of the parotid gland to stimulation of the parasympathetic nerve.     

Involvement of capsaicin-sensitive nerves in the bronchomotor effects of arachidonic acid and melittin: a possible role for lipoxin A4.

1. Functional studies have been performed to evaluate the potential involvement of capsaicin-sensitive nerves in the bronchomotor responses evoked by lipid mediators produced from the metabolic breakdown of arachidonic acid (AA) in the guinea-pig bronchus. 2. In the presence of indomethacin, the exogenous administration of AA (0.01-1 mM) produced a concentration-dependent contractile response in guinea-pig isolated bronchial rings. AA-induced contractions were augmented by epithelium-removal and by thiorphan (10 microM), an inhibitor of tachykinin breakdown. A sustained downward and rightward displacement of the complete concentration-response curve to AA was observed after in vitro capsaicin desensitization. 3. BWA4C (1 microM), a selective inhibitor of 5-lipoxygenase, shifted the AA concentration-response curve to the right. In the presence of this inhibitor, capsaicin desensitization did not have any further inhibitory action. 4. A potent, concentration-dependent and capsaicin-sensitive bronchoconstrictor effect was also observed with the polypeptide, melittin (10 nM-1 microM), an activator of phospholipase A2, which therefore should generate endogenous AA. 5. In vitro capsaicin-desensitization produced a significant reduction of the bronchomotor responses evoked by lipoxin A4 (1-6 microM), but not of those elicited by other lipoxygenases products such as leukotriene D4 (1-100 nM) or by 15-hydroxyeicosatetraenoic acid (15-HETE, 1-6 microM). 6. These findings indicate that lipoxin A4 but not leukotriene D4 or 15-HETE, might be one of the lipoxygenase mediators of excitatory effects of AA on capsaicin-sensitive sensory nerves.     

Endogenous calcitonin gene-related peptide suppresses vasoconstriction mediated by adrenergic nerves in rat mesenteric resistance blood vessels

The role of perivascular calcitonin gene-related peptide (CGRP)-containing nerves in the modulation of adrenergic nerve-mediated vasoconstrictions was studied in the rat perfused mesenteric vascular bed. A frequency-dependent vasoconstriction induced by periarterial nerve stimulation (1-6 Hz) of the bed was significantly potentiated by perfusion of 1 microM CGRP-(8-37) (CGRP receptor antagonist) or to a similar extent after treatment with 500 nM capsaicin. In the preparations treated with capsaicin, CGRP-(8-37) caused a small potentiation of periarterial nerve stimulation-induced vasoconstriction. Exogenous CGRP (0.1-1 nM) concentration-dependently attenuated the augmented vasoconstriction in response to periarterial nerve stimulation after treatment with capsaicin. However, exogenous CGRP (1 nM) did not attenuate the periarterial nerve stimulation-induced vasoconstriction in the bed untreated with capsaicin. These results suggest that endogenous CGRP, which is released from CGRP-containing nerves, suppresses the adrenergic nerve function involved in mechanisms regulating the tone of resistant blood vessels.