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Notes: Summary 1. Capsaicin produces a concentration-related relaxation of the longitudinal muscle of the rat isolated duodenum in the presence of atropine (3 μM) plus guanethidine (3 μM). This effect of capsaicin is partly (about 40%) antagonized by tetrodotoxin (1.0 μM) suggesting the involvement of intramural non-adrenergic non-cholinergic (NANC) neurons. 2. The capsaicin-induced relaxations are unaffected by previous bilateral vagotomy or removal of the inferior mesenteric ganglion but are completely prevented by removal of the coeliac ganglia plus the superior mesenteric ganglion (72 h before). Acute duodenal denervation did not modify the response to capsaicin. 3. Unlike various neuropeptides (substance P, kassinin, neurokinin A, cholecystokinin octapeptide, somatostatin, vasoactive intestinal polypeptide) only the calcitonin gene-related peptide (CGRP) closely mimicked, both qualitatively and quantitatively, the capsaicin-induced relaxations. The CGRP-induced relaxations were unaffected by hexamethonium and partly reduced (about 40%) by tetrodotoxin. 4. In preparations desensitized to adenosine-triphosphate (ATP) a putative NANC inhibitory neurotransmitter of the rat duodenum, the effects of CGRP were reduced (about 30%) as compared to controls. After ATP-desensitization tetrodotoxin did not produce any further reduction of the CGRP-induced relaxations suggesting the involvement of endogenous ATP in the neuronal (tetrodotoxin-sensitive) component of the CGRP-induced relaxations. 5. Either ATP-or CGRP-desensitization reduced (about 50 and 65% respectively) the amplitude of the capsaicin-induced relaxations. In the presence of both ATP and CGRP capsaicin did not affect motor activity of the rat isolated duodenum indicating a complete desensitization of the effect of endogenously released substance(s). 6. These findings indicate that the capsaicin-induced relaxations of the rat isolated duodenum are due to activation of a neural mechanism of extrinsic origin, presumably primary afferent fibers. CGRP is a likely candidate for the role of neuropeptide released by capsaicin in this preparation. Endogenous CGRP may produce NANC relaxation both directly and by releasing the endogenous NANC neurotransmitter(s).

Notes: Summary 1. The effect of capsaicin on bladder motility in vivo (urethane anaesthesia) and in vitro, plasma extravasation (Evans blue leakage technique) and content of substance P-like immunoreactivity (SP-LI) of the urinary bladder was investigated in various mammalian species. 2. Systemic capsaicin desensitization (rat and hamster, 50 mg/kg s.c. 4 days before; guinea-pig 55 mg/kg s. c. 4–7 days before) increased bladder capacity in rats and guinea-pigs and reduced voiding efficiency in guinea-pigs. All other urodynamic parameters were unaffected in both rats, guinea-pigs and hamsters. 3. Reflex bladder voiding was abolished by spinal cord transection in anaesthetized rats and hamsters. On the other hand, hexamethonium-(20 mg/kg i.v.)sensitive voiding contractions were obtained in response to saline filling 45 min from cord transection in guinea-pigs, indicating a profound interspecies variation in the basic organization of micturition. 4. Exposure to capsaicin (1 μM) produced a contraction of the isolated bladder from rats, guinea-pigs (dome) and mice. Capsaicin produced only a slight contractile response in the guinea-pig bladder base. The motor response to capsaicin of the rat, guinea-pig and mouse bladder exhibited marked desensitization, suggesting a specific effect on sensory nerves. On the other hand, capsaicin (1 μM) produced a slight relaxation of the hamster isolated bladder but this effect was reproducible at 1–2 h intervals, suggesting an unspecific effect. Capsaicin (1–10 μM) did not affect motility of strips from the dome or the base of the rabbit bladder. 5. Intravenously administered capsaicin produced a marked plasma extravasation (Evans blue leakage) in the lower urinary tract of rats, mice and guinea pigs. In rats but not guinea-pigs the reaction in the bladder base was greater than in the dome. In hamsters intravenous capsaicin failed to induce any significant Evans blue leakage in the lower urinary tract. 6. SP-LI was detected in the lower urinary tract of rats, guinea-pigs, rabbits and mice but not hamsters. Bladder SP-LI was depleted by systemic capsaicin desensitization in rats, guinea-pigs and mice. Reverse phase HPLC indicated that all the immunoreactive material co-eluted with authentic substance P or its oxidized form. 7. These findings indicate that noticeable species-related differences exist with regard to the functions mediated by the Capsaicin-sensitive neurons in the urinary bladder.

Notes: Summary In the superfused isolated rat urinary bladder, capsaicin as well as electrical field stimulation evoked the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-IR). Carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP, threshold 2 μM) reduced both, the capsaicin- and the electrical field stimulation-evoked release of CGRP-IR while a low concentration of Ruthenium Red (RR, 0.6 μM and 2 μM) selectively attenuated the capsaicin-evoked release of CGRP-IR but did not influence the effect of electrical field stimulation. 20 μM RR nearly abolished the capsaicin-evoked release, but also attenuated the effect of electrical field stimulation. In the isolated guinea-pig bronchus, electrical field stimulation and capsaicin induced non-cholinergic contractions which are known to be caused by tachykinin release from afferent nerve terminals. CCCP (0.6 μM) only reduced the response to field stimulation; a ten-fold higher concentration of CCCP attenuated field stimulation as well as capsaicin-induced contractions. This is in contrast to the reported selective inhibition of capsaic-ininduced contractions by RR. The present data demonstrate that CCCP generally inhibits evoked neuropeptide release, regardless of the kind of stimulation used while low concentrations of RR preferentially inhibit capsaicin-evoked neuropeptide release.

Notes: Abstract We have investigated the effect of the dihydropyridine calcium channel agonist, Bay K 8644, and of the plant alkaloid blocker of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, ryanodine, on the refractory period, action potential and mechanical response of the guinea-pig isolated ureter to electrical stimulation. All experiments were performed in ureters pre-exposed to 10 μM capsaicin to eliminate the inhibitory influence exerted by local release of sensory neuropeptides on ureteral excitability and contraction. In organ bath experiments, electrical field stimulation with parameters which produce direct excitation of ureteral smooth muscle (train of pulses at 10 Hz, 5 ms pulse width, 60 V for 1 s) produced tetrodotoxin- (1 μM) resistant phasic contractions. The response to EFS was abolished by nifedipine (1 nM-3 μM) and was enhanced by Bay K 8644 (1 nM-3 μM). In the presence of Bay K 8644 (1 μM), nifedipine (30 μM) abolished the evoked contractions. Ryanodine (10–100 μM) had no significant effect on the amplitude of evoked contraction. The response of the guinea-pig ureter to direct electrical stimulation of smooth muscle is characterized by a refractory period: at least 40 s interstimulus interval was required to produce a second response in all preparations tested. Bay K 8644 (1 μM) markedly reduced the refractory period of the ureter and a similar effect was observed with ryanodine (100 μM). To further analyze the effect of Bay K 8644 and ryanodine on the refractory period, the response of the ureter was investigated over a 10 s period of stimulation (other parameters as above). In control ureters, continuous stimulation for 10 s produced only one phasic contraction just after the beginning of the train of stimuli. In the presence of Bay K 8644 or ryanodine, more than one phasic contraction developed during a 10 s stimulation, i.e. the refractory period became shorter than the train duration. When both Bay K 8644 and ryanodine were tested on the same preparations, an additive excitatory effect was observed on the mechanical response to electrical stimulation. A slight elevation of KCI concentration (5–10 mM) reduced the refractory period of the ureter as observed with ryanodine or Bay K 8644. Application of KCI (80 mM) produced a biphasic contractile response of the ureter: a series of phasic contractions occurred first, which were then replaced by a slowly developing tonic response. Bay K 8644 (1 μM) enhanced both components of the response to KCI. Ryanodine (10 and 100 μM) markedly prolonged the duration of phasic contractions evoked by KCI and, at 100 μM, slightly (about 25%) reduced the amplitude of tonic contraction. In sucrose gap experiments, electrical stimulation (single pulse, 40–130 V, 1–3 ms pulse duration) evoked an action potential and accompanying phasic contraction which were abolished by 1 μM, nifedipine. Bay K 8644 (1 μM) produced a marked prolongation of action potential duration, increased the number of spikes and enhanced contraction amplitude and duration. Ryanodine (100 μM) depolarized the membrane, reduced the delay between stimulus application and onset of the action potential, shortened the action potential at 50% of repolarization and increased afterhyperpolarization, without producing marked effects on the accompanying mechanical response. KCI (5 mM) likewise produced a slight membrane depolarization and decreased latency between stimulus application and onset of the action potential but did not affect action potential duration. The combined administration of ryanodine and Bay K 8644 produced additive effects on action potential and contractions: furthermore, the contractile phase of the overall contraction-relaxation cycle was significantly prolonged by the combined administration of the two agents, an effect not observed with either drug alone. In the presence of both Bay K 8644 and ryanodine, multiple action potentials and contractions were observed during a train of pulses delivered at a frequency of 1 Hz for 12 s: when a second action potential was triggered before relaxation of the preceding contraction, a summation of the contractile response was observed. These findings demonstrate that availability of voltage-dependent L-type calcium channels is a major mechanism in determining the refractory period of the guinea-pig ureter and, consequently, can be considered as a limiting step in regulating the maximal frequency of ureteral peristalsis. Furthermore, a ryanodine-sensitive mechanism regulates the excitability and contraction-relaxation cycle of ureteral smooth muscle. The increased electrical excitability of the ureter observed in the presence of ryanodine may involve blockade of transient outward currents triggered by spontaneous calcium release from the store and consequent membrane depolarization.

Notes: 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 μM) 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.

Notes: Summary 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.

Notes: Summary In urethane-anesthetized rats with an intact spinal cord, application of capsaicin on the outer surface of the urinary bladder produced a transient bradycardia, hypotension and negative cardiac inotropism which were neither prevented by i. v. atropine (0.5 mg/kg) nor by cervical vagotomy. In acute spinal rats (C2-C3) application of capsaicin (0.2 and 2 pg in 25 pl) on the urinary bladder induced a transient hypertension, tachycardia and positive cardiac inotropism. A second application (30 min later) induced minor cardiovascular effects, expecially with the higher dose, indicating desensitization. All cardiovascular responses to topical capsaicin were abolished by systemic capsaicin desensitization (50 mg/kg s. c., 4 days before). The excitatory cardiovascular response to capsaicin in acute spinal rats was markedly reduced by bilateral section of pelvic but not hypogastric nerves. Further, it was abolished by pretreatment with hexamethonium (20 mg/kg i.v.) or reserpine (5 mg/kg i. p., 2 days before) and reduced, at various extent for the different components, by phentolamine (0.5 mg/kg i. v.) or propranolol (1 mg/kg). In rats with pelvic and hypogastric nerves intact, section of the cord at a level (T12-L1), just above the medullary segments which receive primary afferent input from the bladder (L6-S1), abolished the excitatory cardiovascular response to application of capsaicin on the bladder. In spinal rats (C2-C3) rapid distension of the urinary bladder with saline produced transient tachycardia, hypertension and positive cardiac inotropism similar to that evoked by capsaicin. These responses were not observed in rats systemically pretreated with capsaicin. These findings indicate that certain bladder afferents which are susceptible to capsaicin desensitization in adult rats activate a spinal reflex having excitatory influence on cardiovascular function. This response is apparently mediated by spinal centers located above the site of entry of bladder pelvic afferents into the cord and most likely involves excitation of preganglionic sympathetic neurons in the spinal cord.

Notes: Summary 1. The effect of omega-conotoxin (CTX) GVIA, a peptide which blocks neuronal calcium channels, were investigated on nerve-mediated motor responses in a variety of isolated smooth muscle preparations from rats and guinea-pigs. 2. In the rat or guinea-pig isolated vas deferens CTX (1 nM − 1 μM) produced a concentration and time-related inhibition of the response to field stimulation, while the responses to KCI, noradrenaline or adenosine triphosphate were unaffected. In the presence of CTX a series of tetrodotoxin-resistant contractions could be elicited by field stimulation by increasing pulse width and/or voltage. 3. In the rat or guinea-pig isolated urinary bladder, CTX produced a concentration and time-dependent inhibition of twitch responses to field stimulation without affecting the response to exogenous acetylcholine. In the rat bladder the maximal effect did not exceed 25% inhibition while a much larger fraction of the response (about 70%) was inhibited in the guinea-pig bladder. The CTX-resistant response was abolished, in both tissues, by tetrodotoxin. 4. The effects of CTX in the rat bladder were also studied with a whole range of frequencies of field stimulation (0.1–50 Hz). Maximal inhibition was observed toward contractions elicited at frequencies of 2–5 Hz. At low frequencies the inhibitory effects of CTX and atropine were almost additive while at high frequencies of stimulation a large component of the atropine-sensitive response was CTX-resistant. 5. In the rat isolated proximal duodenum, field stimulation in the- presence of atropine and guanethidine produced a primary relaxation followed by a rebound contraction. Both responses were abolished by tetrodotoxin, indicating the activation of intramural nonadrenergic noncholinergic nerves. The primary relaxation was totally CTX resistant while the rebound contraction was slightly inhibited. 6. The motor responses produced by capsaicin (1 μM) in the rat or guinea-pig bladder (contraction) and in the rat proximal duodenum (relaxation) were unaffected by CTX. Likewise, the release of substance P-like immunoreactivity from sensory nerves of the guinea-pig bladder muscle was unaffected by CTX. 7. These findings indicate that CTX-sensitive calcium channels modulate transmitter release in autonomic nerve terminals of mammals, but noticeable species and organ related variations exist in sensitivity to this peptide, possibly reflecting the existence of a heterogenous population of voltage-sensitive calcium channels. CTX-sensitive calcium channels are apparently not involved in the excitatory action of capsaicin on sensory nerve terminals.

Notes: Summary 1. Capsaicin applied on the serosal surface of the urinary bladder in urethane-anaesthetized rats produces two distinct types of motor effects: a) a tetrodotoxin-, hexamethonium- and lidocaine-insensitive ‘tonic’ contraction and b) a series of tetrodotoxin-, hexamethonium- and lidocaine-sensitive rhythmic contractions. 2. 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. 3. The motor effects of topical capsaicin are unaffected by pretreatment with indomethacin or diphenhydramine plus cimetidine. 4. 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 capsaicindesensitization. 5. 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. 6. 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.

Notes: Summary (1) Topical administration of Ruthenium Red (10–100 μM in saline) to the serosal surface of the urinary bladder in urethane-anesthetized rats prevented the motor response of the urinary bladder to topical administration of capsaicin and protected the sensory fibers from capsaicin desensitization, but had no effect on the volume-evoked contractions (micturition reflex). At 1 mM increased bladder capacity and decreased amplitude of micturition contraction were observed. (2) At 100 μM, topical Ruthenium Red prevented the blood pressure rise produced by topical administration of capsaicin onto the bladder but did not affect the blood pressure rise produced by sudden bladder distension in spinal rats. (3) After intrathecal administration, Ruthenium Red (80–800 ng/rat) produced a long lasting inhibition of the micturition reflex in urethane-anesthetized rats, this effect being evident in both vehicleor capsaicin- (50 mg/kg s. c. 4 days before) pretreated rats. At 800 ng/rat, intrathecal Ruthenium Red did not affect the blood pressure rise produced by topical administration of capsaicin onto the rat bladder nor that produced by bladder distension. (4) These findings provide further evidence that Ruthenium Red acts quite selectively as a “capsaicin antagonist” preventing both reflex and “efferent” responses activated by peripherally administered capsaicin. By contrast, sensory impulse generation by a natural stimulus such as bladder distension is apparently unaffected by Ruthenium Red. The marked inhibition of the micturition reflex observed after intrathecal administration of Ruthenium Red does probably not involve an interaction with primary afferents in the spinal cord.