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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: Abstract  We aimed at studying the mechanism(s) of the inhibitory effect exerted by calcitonin gene-related peptide (CGRP) on the spontaneous activity of the guinea-pig isolated renal pelvis. In organ bath experiments, CGRP (1–100 nM) produced a concentration-dependent (EC50 8 nM) partial inhibition (Emax about 35% inhibition of motility index) of spontaneous contractions. The potassium (K) channel opener, cromakalim (3–10 μM) promptly suppressed the spontaneous contractions in a glibenclamide- (10 μM) sensitive manner. Glibenclamide (10 μM) did not affect the inhibitory action of CGRP. The calcium (Ca) channel agonist, Bay K 8644 (1 μM), markedly enhanced the spontaneous activity of the renal pelvis and reduced the inhibitory effect of CGRP. The protein kinase A inhibitors Rp-cAMPS (300 μM), H8 (100 μM) and H89 (10 μM), and the blockers of intracellular Ca handling by sarcoplasmic reticulum, ryanodine (100 μM) and thapsigargin (1 μM) did not affect the response to CGRP. The response to CGRP was likewise unaffected by the nitric oxide synthase inhibitor, L-nitroarginine (30 μM) and by the protein kinase G inhibitor, KT5823 (3 μM). Furthermore, the inhibitory action of CGRP was not modified by lowering the extracellular concentration of K (from 5.9 to 1.2 mM) nor by increasing (from 2.5 to 3.75 mM) or decreasing (from 2.5 to 0.25 mM) the extracellular Ca concentration. Replacement of 80% glucose with 2-deoxyglucose (2-DOG) reduced the amplitude of spontaneous contractions, both in the absence and presence of 10 μM glibenclamide. In the presence of 2-DOG, the inhibitory action of CGRP was enhanced at a similar extent, either in the absence or presence of glibenclamide. In sucrose gap, the effect of CGRP (0.1 μM for 5 min) was separately analyzed in the proximal (close to the kidney) and distal (close to the ureter) regions of the renal pelvis. Both preparations discharged spontaneous (pacemaker) action potentials having different shape, duration and frequency. CGRP had no effect on pacemaker potentials in the proximal renal pelvis while producing about 30% reduction of the frequency of pacemaker potentials and motility index in the distal renal pelvis. Cromakalim (3 μM) abolished pacemaker potentials in both regions of the renal pelvis. In conjunction with the results of previous studies in the guinea-pig ureter, the present findings document the existence of remarkable regional differences in the effector mechanisms initiated by CGRP receptor occupancy in the guinea-pig pyeloureteral tract. CGRP appears to be inherently unable to activate glibenclamide-sensitive K channels in the guinea-pig renal pelvis, a mechanism which is central for its ability to suppress latent pacemakers in the ureter. Within the renal pelvis, the sensitivity to the inhibitory effect of CGRP appears in the more distal region, from which an ‘ureter-like’ action potential is recorded.

Notes: Abstract The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 μmol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2–0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 μmol/kg i.v.) or hexamethonium (28 μmol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 μmol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4–5 mmHg by further distension of the balloon (0.4–0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 μmol/h), to provide a suitable baseline (amplitude of contractions was 7–8 mmHg) for studying the effect of tachykinin receptor antagonists. I.v. administration of the selective tachykinin NK2 receptor antagonists, MEN 10,627 (10–100 nmol/kg) and SR 48968 (100–300 nmol/kg) or of the selective NK1 antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 µmol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR 48965 (300 nmol/kg), the enantiomer of SR 48968 devoid of NK2 receptor blocking activity, was without effect. I.v. administration of the selective NKi receptor agonists [Sar9] substance P sulfone and septide or of the NK2 receptor selective agonist, βAla8] neurokinin A(4–10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar9] substance P sulfone and septide without affecting those produced by [βAla8] neurokinin A(4–10). On the other hand, MEN 10,627 (30–100 nmol/kg i.v.) and SR 48968 (100–300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK2 and NK1 receptor agonists. We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK1 and NK2 receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK1 receptor agonists, [Sar9] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK2 receptors.

Notes: Abstract. The aim of this study was to compare the stimulus-response characteristics of the cholinergic and tachykininergic excitatory transmission to the circular muscle of the guinea-pig proximal colon and their susceptibility to inhibition by the N-type calcium channel blocker ω-conotoxin (CTX). All experiments were performed in the presence of guanethidine (3 μM), indomethacin (10 μM), L-nitroarginine (L-NOARG, 30 μM) and apamin (0.1 μM). In the presence of the tachykinin receptor antagonists, FK 888 (10 μM) and GR 94800 (3 μM), to block NK1 and NK2 receptors, respectively, electrical field stimulation (EFS) produced frequency-dependent atropine- (1 μM) sensitive contractions. In the presence of atropine (1 μM), EFS produced tachykininergic contractions which were abolished by the combined administration of FK 888 (10 μM) and GR 94800 (3 μM). The maximal responses produced by cholinergic and tachykininergic neurotransmission ranged between 80 and 100% of the maximal contractile response to 80 mM KCl. The frequency of stimulation, pulse width and voltage required to produce 50% of the maximal cholinergic and tachykininergic contraction were not different from each other, although cholinergic transmission appeared more efficient in producing twitch contractions in response to single pulse EFS. Furthermore, cholinergic transmission was more efficient than tachykininergic transmission in producing contraction in response to short periods of EFS.   CTX (0.1 μM for 30 min) produced a large and comparable rightward shift of the cholinergic and tachykininergic frequency-response curve (19 and 17 fold increase in the frequency of stimulation producing 50% of the maximal response, respectively) and markedly depressed (51 and 43% inhibition, respectively) the maximal concentrations response. CTX failed to affect the contraction of the colon produced by submaximally effective concentrations of the muscarinic receptor agonist, methacholine (0.1–0.3 μM) and those produced by the tachykinin NK1 and NK2 receptor selective agonists [Sar9] substance P sulfone and [βAla8] neurokinin A (4–10) (1–3 nM).   The present findings demonstrate that the cholinergic and tachykininergic components of the excitatory transmission to the circular muscle of the guinea-pig colon are activated at comparable intensities of nerve stimulation and are both inhibited, in a qualitatively and quantitatively comparable manner, by CTX at the prejunctional level. These findings are consistent with the idea that acetylcholine and tachykinins are co-released from the same population of enteric motoneurones which innervate the circular muscle of the colon.

Notes: Abstract We have characterized the contractile responses produced by stimulation of the tachykinin NK2 receptor in the hamster urinary bladder in vitro and in vivo. In isolated bladder strips, neurokinin A (NKA, pD 2 7.40, Emax 71% of the response to 80 mM KCl) and the synthetic tachykinin NK2 receptor selective agonist [βAla8]NKA(4–10) (pD 2 7.48, Emax 77% of the response to KCl) both induced a concentration-dependent contraction, whereas the tachykinin NK1 and NK3 receptor selective agonists, [Sar9]substance P sulfone and senktide, respectively, produced a negligible contractile effect. The bicyclic peptide antagonists MEN 11420 and MEN 10627 behaved as competitive antagonists of the response to [βAla8]NKA(4–10) with apparent pK B values of 9.3 and 9.7, respectively. Comparable apparent pK B values were estimated against NKA (pK B 9.2 and 9.4 for MEN 11420 and MEN 10627, respectively). Under isovolumetric recording of the intravesical pressure, the nicotinic receptor agonist DMPP (0.6 µmol/kg i.v.) produced a phasic contraction of the hamster bladder in vivo that was abolished by hexamethonium (110 µmol/kg i.v.) or by surgical ablation of pelvic ganglia. In vivo [βAla8]NKA(4–10) (10 nmol/kg i.v.) induced a tonic-type sustained bladder contraction with superimposed high frequency and small amplitude (〈12 mmHg) phasic contractions and, in about 70% of cases examined, a few high amplitude (〉20 mmHg) phasic contractions. Hexamethonium abolished the high amplitude phasic contractions, indicating their reflex origin. In animals subjected to the ablation of pelvic ganglia, the urinary bladder response to [βAla8]NKA(4–10) was comparable to that observed after administration of hexamethonium. Moreover, hexamethonium did not affect the contractile responses to [βAla8]NKA(4–10) in ganglionectomized animals. MEN 10627 and MEN 11420 produced a dose-dependent and long-lasting inhibition of the contractile response to [βAla8]NKA(4–10): the least effective doses of the two antagonists were 30 and 3 nmol/kg i.v. for MEN 10627 and MEN 11420, respectively. An almost complete and long-lasting inhibition of the response to the agonist was produced at doses of 10 and 100 nmol/kg i.v. of MEN 11420 and MEN 10627. In urethane-anaesthetized hamsters the non-stop intravesical infusion of saline (50 µl/min) produced repetitive micturition cycles which were abolished by hexamethonium (110 µmol/kg i.v.) or by surgical removal of the pelvic ganglia. MEN 11420 (100 nmol/kg) had no significant effect on the volume-evoked micturition reflex in anaesthetized hamsters. In conclusion, the hamster urinary bladder is a suitable preparation for studying the action of tachykinin NK2 receptor antagonists in vivo: in this species, the stimulation of tachykinin NK2 receptors induces bladder contractions. Blockade of tachykinin NK2 receptors does not appreciably modify the volume-evoked micturition reflex in this species.