Arachidonic acid-induced bronchomotor responses are partially mediated by release of sensory neuropeptides from capsaicin-sensitive structures.

Administration of arachidonic acid (AA) both in vitro and in vivo elicited prominent contractile responses in guinea-pig airways, which were markedly reduced after capsaicin desensitization. Furthermore, AA superfusion elicited a significant calcitonin gene-related peptide-like immunoreactivity release from isolated bronchi. It is suggested that at least part of the bronchomotor actions of AA rely upon stimulation of capsaicin-sensitive primary afferents.     

Prostacyclin activates tachykinin release from capsaicin-sensitive afferents in guinea-pig bronchi through a ruthenium red-sensitive pathway.

1. We have investigated the ability of prostacyclin (PGI2) to contract guinea-pig isolated bronchi and the possible involvement of capsaicin-sensitive primary afferents in the response to PGI2. 2. PGI2 (0.1-100 microM) produced concentration-dependent contractions of the guinea-pig isolated bronchi. In vitro capsaicin desensitization (10 microM for 30 min followed by washing) significantly reduced the PGI2-induced contraction at all concentrations tested. A capsaicin-resistant component of contraction (40-60% of the overall response) was also evident. 3. Ruthenium red (3 microM), an inorganic dye which acts as a selective functional antagonist of capsaicin, significantly decreased PGI2-induced contractions, without affecting the response to substance P, neurokinin A or acetylcholine. 4. MEN 10, 207, (Tyr5, D-Trp6,8,9, Arg10)-neurokinin A (4-10) (3 microM), a selective antagonist of NK2-tachykinin receptors, significantly decreased PGI2-induced contractions and neurokinin A-induced contractions, without affecting the response to acetylcholine. 5. The effect of ruthenium red and MEN 10,207 on the one hand, and that of ruthenium red and capsaicin on the other was non additive. 6. These results indicate that PGI2-induced contraction of the guinea-pig isolated bronchi involves two distinct mechanisms, one of which involves transmitter (tachykinins) release from peripheral endings of capsaicin-sensitive primary afferents. In as much as PGI2-activation of primary afferents is sensitive to ruthenium red, we suggest that PGI2 shares a common mechanism of tachykinin release with that activated by capsaicin.     

Mechanism of endothelin-induced contraction in guinea-pig trachea: comparison with rat aorta.

1. Endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of guinea-pig epithelium-containing (intact) trachea (EC50 = 30.9 nM). Endothelin was a less potent agonist than leukotriene D4 (LTD4; EC50 = 0.77 nM), but was more potent than carbachol (EC50 = 0.15 microM) or substance P (EC50 = 1.4 microM). Endothelin was a more potent contractile agent in rat endothelium-denuded aorta (EC50 = 2.1 nM) than in guinea-pig trachea. 2. Endothelin-induced contraction in guinea-pig trachea was unaffected by mepyramine (10 microM), atropine (1 microM), SK&F 104353 (10 microM), a leukotriene receptor antagonist, or SQ 29,548 (1 microM), a thromboxane receptor antagonist. The contraction produced by 0.3 microM endothelin was potentiated by cyclo-oxygenase inhibition with 5 microM indomethacin. 3. Nicardipine (0.01 or 0.1 microM) or incubation in calcium-free medium +0.1 mM EGTA for 30 min had a relatively minor or no effect on endothelin concentration-response curves in guinea-pig intact trachea, but markedly inhibited responses produced by endothelin in endothelium-denuded aorta of the rat. Increasing the EGTA concentration in calcium-free medium to 1 mM abolished endothelin-induced contraction in guinea-pig trachea. 4. In guinea-pig trachea, ryanodine (10 microM) produced a 2.1 fold shift to the right of endothelin concentration-response curves and reduced the maximum response elicited by 0.3 microM endothelin. 5. Staurosporine (0.01 microM and 0.1 microM), a protein kinase C inhibitor, was without effect on endothelin- or carbachol-induced contraction in guinea-pig trachea, but markedly inhibited the response produced by endothelin in rat aorta. 6. Endothelin (3 nM-0.3 microM) produced a concentration-dependent stimulation of phosphatidylinositol (PI) turnover in guinea-pig intact trachea, with an EC50 value of 45.9 nM. 7. Removal of the epithlium markedly potentiated endothelin-induced contraction in guinea-pig trachea, producing a 4.7 fold leftward shift in endothelin concentration-response curves and an increase in the contractile response elicited by 0.3 microM endothelin. 8. These data indicate that endothelin is a potent agonist in guinea-pig trachea whose response is markedly enhanced by removal of the airway epithelium. Endothelin-induced contraction is not mediated to a marked extent by calcium influx via dihydropyridine-sensitive calcium channels and does not involve the release of histamine, acetylcholine, leukotrienes or thromboxane. Rather, endothelin appears to produce contraction of guinea-pig trachea via a direct action which involves stimulation of PI turnover and utilization of calcium from intracellular stores and, also, calcium influx via a pathway that is not sensitive to dihydropyridine calcium channel inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Prejunctional modulatory action of neuropeptide Y on peripheral terminals of capsaicin-sensitive sensory nerves.

1. We have determined the effect of neuropeptide Y (NPY) on motor responses produced by activation of capsaicin-sensitive primary afferents in the guinea-pig isolated left atria (reserpine-pretreatment, atropine in the bath) and bronchi (atropine and indomethacin in the bath) using capsaicin itself and electrical field stimulation as stimuli. 2. In both preparations, NPY inhibited or suppressed the response produced by electrical field stimulation while leaving the response to a submaximal concentration of capsaicin unaffected. 3. NPY had no effect on motor responses produced by a submaximal concentration of calcitonin gene-related peptide (atria) or neurokinin A (bronchi), the putative endogenous mediators of the responses produced by activation of the 'efferent' function of sensory fibres in these preparations. 4. We conclude that NPY exerts a prejunctional inhibitory action on transmitter release from peripheral endings of capsaicin-sensitive nerves. Failure of NPY to modulate responses activated by capsaicin provides further evidence for the existence of two independent modes of activation of the 'efferent' function of capsaicin-sensitive sensory nerves.     

Endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea.

1. Exogenous and endogenous tachykinins facilitate cholinergic nerve-induced bronchoconstriction in guinea-pig. Using a vagally innervated guinea-pig tracheal tube preparation we have investigated the involvement of endogenous capsaicin-sensitive neuropeptides in both pre- and postganglionic cholinergic neurotransmission. The effects of the neutral endopeptidase inhibitor (NEP), phosphoramidon, were investigated in this preparation either alone or in conjunction with sensory neuropeptide depletion by capsaicin pretreatment. The subtype of neurokinin receptor mediating this facilitatory effect of tachykinins has also been examined, by the use of selective tachykinin receptor agonists and a selective NK1 receptor antagonist. 2. Cholinergic contractions of the sealed Krebs filled tracheal tube preparation were recorded as increases in intraluminal pressure and were induced either by (i) pre-ganglionic vagus nerve stimulation (PGS), (ii) stimulation of postganglionic intramural nerves via transmural stimulating electrodes (TMS) in the presence of ganglion-blocking concentrations of hexamethonium and (iii) application of exogenous acetylcholine (ACh). 3. The effect of phosphoramidon, which inhibits the breakdown of tachykinins, was investigated on ACh-, PGS- and TMS-induced contractions. Phosphoramidon (1-10 microM) facilitated contractions of the trachea induced by PGS, in a concentration-dependent manner, but had no effect on contractions of the trachea induced either by TMS or exogenous ACh. 4. The facilitatory effect of phosphoramidon (10 microM) on PGS-induced contractions was abolished by pretreating guinea-pigs with capsaicin 7 +/- 2 days before the in vitro experiments. Capsaicin pretreatment did not significantly alter responses to the spasmogens, ACh or substance P. Depletion of sensory neuropeptides, by capsaicin pretreatment was confirmed by the lack of response to capsaicin (1 microM) in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The positive inotropic and chronotropic effects of evodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Evodia rutaecarpa, on the guinea-pig isolated right atria: possible involvement of vanilloid receptors

Cardiotonic effects of evodiamine and rutaecarpine, constituents of the fruits of Evodia rutaecarpa Bentham Rutaceae, were evaluated on guinea pig isolated atria. Comparison with capsaicin, a vanilloid receptor agonist, revealed similar positive inotropic and chronotropic activity, as judged from antagonistic effects of the competitive vanilloid receptor (capsaicin receptor) antagonist capsazepine, the non-competitive vanilloid receptor antagonist ruthenium red, the calcitonin gene related peptide antagonist CGRP(8-37), the P2X purinoceptor antagonist PPADS, and various desensitization studies. Evodiamine and rutaecarpine produced transient positive inotropic and chronotropic effects on the guinea-pig isolated atria, followed by a desensitizing effect to additional administration. Dose-response relationships for evodiamine, rutaecarpine and capsaicin were obtained. All the compounds evoked positive inotropic and chronotropic effects in a concentration-dependent manner. Maximal contractions for evodiamine, rutaecarpine and capsaicin were observed at concentrations of 1 microM, 3 microM and 0.3 microM, respectively. The cardiotonic responses evoked by both evodiamine and rutaecarpine were shifted to the right by capsazepine, an established antagonist of vanilloid receptor (capsaicin-receptor). The effects of both evodiamine (1 microM) and rutaecarpine (3 microM) were abolished by pretreatment with a desensitizing dosage of capsaicin (1 microM), developing cross-tachyphylaxis between these compounds. The effects of evodiamine (1 microM), rutaecarpine (3 microM) and capsaicin (0.3 microM) were also significantly reduced by pretreatment with ruthenium red (10 microM) and CGRP (8-37) (10 microM). The effects of evodiamine, rutaecarpine and capsaicin were not affected by pretreatment with PPADS (100 microM), a highly selective P2X purinoceptor antagonist, and the possibility of the involvement of the P2X purinoceptor was excluded. These results suggest that the positive inotropic and chronotropic effects on the guinea-pig isolated right atria induced by both evodiamine and rutaecarpine could be attributed to their interaction with vanilloid receptors and the resultant release of CGRP, a cardiotonic neurotransmitter, from capsaicin-sensitive nerves as with capsaicin.     

Peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) activates capsaicin-sensitive primary afferent nerves in guinea-pig atria and urinary bladder.

1. We have investigated the ability of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) a synthetic analogue of a chemotactic peptide derived from a variety of bacteria, to activate capsaicin-sensitive primary afferents in the guinea-pig atria and urinary bladder. 2. In the isolated, electrically-driven left atria from reserpine-pretreated guinea-pigs (atropine in the bath), FMLP (3 nM-1 microM) produced a biphasic positive inotropic response. The late component of this response was selectively abolished by in vitro capsaicin pretreatment while both the early and late responses were abolished by indomethacin. 3. The inotropic response to FMLP in the guinea-pig atria was unaffected by ruthenium red. The late but not the early response was strongly inhibited or abolished by tetrodotoxin (TTX), omega-conotoxin (CTX) or by the C-terminal fragment (8-37) of human alpha-calcitonin gene-related peptide (hCGRP). hCGRP-(8-37) acts as competitive antagonist at CGRP receptors. 4. In the guinea-pig isolated bladder, FMLP (10 nM-10 microM) produced a concentration-dependent contraction which was unchanged by previous in vitro capsaicin, TTX or CTX pretreatment. The response to low concentrations of FMLP was suppressed by indomethacin, irrespective of the capsaicin pretreatment. 5. FMLP (10 microM) produced a significant increase in the outflow of CGRP-like immunoreactivity (CGRP-LI) from superfused guinea-pig atria or urinary bladder. CGRP-LI outflow induced by FMLP was blocked by indomethacin or in vitro capsaicin pretreatment. 6. These findings indicate that FMLP activates the 'efferent' function of capsaicin-sensitive primary afferents via prostanoid generation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory action of galanin on responses due to antidromic activation of peripheral terminals of capsaicin-sensitive sensory nerves

Galanin inhibited, in a concentration-dependent manner (EC50 7.2 nM), the positive inotropic response produced by field stimulation of isolated left atria from reserpine-pretreated guinea-pigs (in the presence of atropine). These responses were shown to involve antidromic activation of capsaicin-sensitive primary afferents. On the other hand, galanin did not affect the inotropic response to capsaicin or calcitonin gene-related peptide, the putative endogenous mediator released from sensory nerves. Galanin-(1-10) was at least 10,000 times less potent than the parent peptide, while galanin-(25-29) was ineffective. Likewise, galanin inhibited the non-cholinergic contraction produced by field stimulation of the isolated guinea-pig bronchus but not the contraction produced by exogenous neurokinin A. These findings indicate a prejunctional neuromodulatory action of galanin on the excitability of peripheral terminals of capsaicin-sensitive sensory nerves.     

Non-adrenergic, non-cholinergic contractions in the electrically field stimulated guinea-pig trachea.

1. The effects of drugs and altering stimulus parameters on neurogenic responses to electrical field stimulation (EFS) have been investigated in distal and proximal portions of the guinea-pig trachea. 2. In the presence of indomethacin (3 microM) and propranolol (1 microM) two contractile phases were evident in both the proximal and distal trachea. The first rapid phase was abolished by atropine (0.1 microM), whereas the prolonged, second phase was abolished by capsaicin (10 microM) pretreatment. Tetrodotoxin (3 microM) abolished the initial response and greatly inhibited the second phase. In proximal trachea this second phase was evident only in 9 of 22 preparations. The addition of the peptidase inhibitor thiorphan (10 microM) however, caused a second phase to be seen in all the proximal tissues examined. 3. The two phases of the contractions to EFS were differentially sensitive to the pulse duration applied. The initial, cholinergic contractions were evident at lower pulse durations than were the prolonged capsaicin-sensitive contractions, with the first phase being approximately 10 fold more sensitive than the second phase. 4. The magnitude of the capsaicin-sensitive contraction to EFS was significantly greater in the distal trachea than in the proximal trachea. This difference prevailed in the presence of thiorphan, an inhibitor of neutral endopeptidase. In contrast, concentration-response curves to capsaicin were similar in segments of proximal and distal trachea. 5. The non-adrenergic non-cholinergic (NANC) relaxant responses were studied in tissues in which excitatory neurogenic responses were pharmacologically abolished by capsaicin and atropine treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid receptors and prejunctional modulation of capsaicin-sensitive sensory nerves in guinea-pig left atrium

1. In the isolated electrically driven left atria from reserpine-pretreated guinea-pigs and in presence of 1 microM atropine, electrical field stimulation (EFS) at 10 Hz produces a delayed positive inotropic response (DPIR) involving activation of capsaicin-sensitive afferents. 2. Opioids inhibited the DPIR with the following order of potency: dermorphin greater than [D-Ala2,N-MePhe4, Gly5-ol]-enkephalin (DAGO) greater than or equal to [D-Ala2,D-Leu5]-enkephalin (DADLE) greater than morphine greater than dynorphin A (1-13) greater than [D-Pen2,D-Pen5]-enkephalin (DPDPE). U-50488 was ineffective up to 10 microM. 3. Opioids also inhibited resting inotropism (3 Hz) with the following rank order of potency: DADLE greater than DAGO greater than U-50488 = dynorphin A (1-13) = morphine = DPDPE. 4. Both inhibition of the DPIR and inhibition of resting inotropism were prevented by 10 microM naloxone. 5. Neither dermorphin (0.1 microM) nor DAGO (0.3 microM) or DADLE (1 microM) inhibit responses produced by capsaicin (30 nM) or calcitonin gene-related peptide (3 nM). 6. These findings indicate that capsaicin-sensitive nerves in the guinea-pig atrium are endowed with mu opioid receptors which inhibit transmitter release when sensory nerve terminals are activated by EFS but not by capsaicin.     

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.     

GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.

1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of bradykinin- and capsaicin-induced myocardial and coronary effects in isolated perfused heart of guinea-pig: involvement of substance P and calcitonin gene-related peptide release.

1.Bradykinin and capsaicin were compared for their ability to elicit functional effects and to release sensory neuropeptides from guinea-pig isolated perfused hearts. 2. Both bradykinin (10 microM) and capsaicin (1 microM) produced a marked increase in coronary flow, a large positive chronotropic effect and a significant reduction in contractile strength. These actions were associated with a marked release of substance P-like immunoreactivity (SP-LI) and calcitonin gene-related-like immunoreactivity (CGRP-LI). The percentage of the tissue content of SP-LI and CGRP-LI released by each agent was similar, although bradykinin was less effective than capsaicin. The ratio of SP-LI/CGRP-LI released by both agents was similar to that present in cardiac tissue. 3. Neuropeptide release could be evoked only once with capsaicin but at least four times with bradykinin. Also, functional responses to capsaicin underwent desensitization. After either in vitro or systemic capsaicin pretreatment, the release of SP-LI and CGRP-LI by bradykinin was reduced and the positive chronotropic effect of bradykinin was significantly reduced, while the increase in coronary flow and negative inotropic responses remained unchanged. 4. Pretreatment with indomethacin (10 microM) strongly antagonized the release of SP-LI and CGRP-LI by bradykinin and reduced the increase in heart rate. 5. These findings suggest that activation by bradykinin (probably through indirect mechanisms) of capsaicin-sensitive sensory nerves in the heart, leads to a local release of sensory neuropeptides. These neuropeptides, in turn, could participate in determining the complex functional effects of this kinin on cardiac performance.     

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.     

The antagonism induced by ruthenium red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies

Ruthenium Red, an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, reduced the capsaicin-induced release of substance P-like immunoreactivity from muscle strips of the guinea-pig urinary bladder in a concentration-dependent (30 nM - 3 microM) manner, and protected the sensory fibers from capsaicin-induced densensitization. A similar antagonism of the actions of capsaicin was observed in functional experiments (capsaicin-induced contraction of the isolated guinea-pig bladder or inhibition of twitches of the isolated rat vas deferens). In view of its established action on the depolarization-coupled entry of Ca into synaptosomes and the secretion of transmitter, we propose that Ruthenium Red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter secretion and preventing the establishment of desensitization.     

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.     

Ruthenium red antagonism of the effect of capsaicin on the motility of the isolated guinea-pig ileum

Ruthenium red (1 microM), an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, selectively reduced the capsaicin (1 microM)-induced contraction of the guinea-pig ileum and protected the sensory fibers from capsaicin-induced desensitization. The ruthenium red (0.5-1 microM) antagonism of capsaicin-induced inhibition of responses to mesenteric nerve stimulation or field stimulation in the isolated guinea-pig ileum was an example of a similar antagonism of the effect of capsaicin. In view of the known action of ruthenium red on the depolarization-coupled entry of Ca into synaptosomes and the release of transmitter, our results support the proposal that ruthenium red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter release and preventing the establishment of desensitization.     

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