Naunyn-Schmiedeberg’s Arch Pharmacol (2001) 364 : 242–248 DOI 10.1007/s002100100447
O R I G I N A L A RT I C L E
Sandro Giuliani · Paolo Santicioli · Annalisa Lippi · Alessandro Lecci · Manuela Tramontana · Carlo Alberto Maggi
The role of sensory neuropeptides in motor innervation of the hamster isolated urinary bladder Received: 26 March 2001 / Accepted: 4 May 2001 / Published online: 5 July 2001 © Springer-Verlag 2001
Abstract 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 µM) 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 µM. 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 µM) which inhibited EFS-evoked contractions with an EC50 of 100.0 nM and a maximal effect of 34±4% inhibition at 1 µM. 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 µM CGRP. Capsaicin (10 µM) 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 inef-
S. Giuliani (✉) · P. Santicioli · A. Lippi · A. Lecci · M. Tramontana · C.A. Maggi Pharmacology Department, Menarini Ricerche S.p.A., via Rismondo 12A, 50131 Florence, Italy e-mail:
[email protected], Tel.: +39-055-5680750, Fax: +39-055-5680419
fective, 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. Keywords Neurokinin A · CGRP release · Hamster urinary bladder · Capsaicin-sensitive sensory fibers
Introduction Capsaicin-sensitive sensory fibers containing CGRP and tachykinins are widely distributed in mammalian tissues (Maggi 1995, for a review) and their role in the urinary bladder of several species has been extensively characterized (Maggi et al. 1987; Maggi 1991, for a review). In contrast to rat, guinea-pig and mouse bladder (Maggi et al. 1987), the application of capsaicin on the hamster isolated urinary bladder produces a small relaxation. Apparently no excitatory substance is released by stimulation of the sensory nerve endings in this species: substance P-like immunoreactivity (SP-LI) has not been detected by radioimmunoassay in hamster urinary bladder (Maggi et al. 1987). Notwithstanding, tachykinin NK2 receptor agonists produce a potent contractile response (Watson et al. 1983; Dion et al. 1987; Tramontana et al. 1998) and, in agreement with functional data, tachykinin NK2 receptors have been demonstrated by binding studies (Van Giersbergen et al. 1992; Guard et al. 1993) and are abundantly expressed in the smooth muscle layer (Burcher and Buck 1986). The hamster isolated urinary bladder has been considered as a monoreceptorial organ for studying tachykinin NK2 receptors (Dion et al. 1987), and stimulation of the tachykinin NK1 and NK3 receptors with selective agonists did not produce contractile responses (Tramontana et al. 1998). The stimulation of sensory fibers in the hamster urinary bladder with capsaicin has been described to produce a facilitatory effect on the micturition reflex (Lecci et al.
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1999) which may be ascribable to the sensitizing effect on sensory fibers produced by tachykinins (Ishizuka et al. 1994; Kibble and Morrison 1996; Lecci et al. 1997) and in particular by neurokinin A through the stimulation of the tachykinin NK2 receptor (Tramontana et al. 1998). Little or no information is available about the effect of CGRP which apparently does not produce intrinsic effects on the bladder from hibernating hamster (Pinna et al. 1998), but it could be responsible for the relaxation produced by capsaicin administration (Maggi et al. 1987). Specific binding to vanilloid receptor has been detected in hamster urinary bladder (Szallasi et al. 1993), although in a relatively small amount, being approximately one-fifth of the amount found in the rat bladder. In preliminary experiments we have observed that CGRP, capsaicin and the selective tachykinin NK2 receptor antagonist nepadutant all inhibit the twitches produced by single pulse electrical field stimulation (EFS) in isolated hamster urinary bladder and these effects were reduced after in vitro capsaicin desensitization. The aim of this study was to characterize the effect of the sensory neuropeptides, neurokinin A and CGRP, on the contractile responses evoked by single pulse EFS of the hamster isolated urinary bladder and to check if activation of capsaicin-sensitive sensory fibers may produce release of these peptides.
Materials and methods In vitro experiments. Male Golden Syrian hamsters, weighing 100– 150 g (Charles River, Calco, Italy), were sacrificed by cervical dislocation. The whole urinary bladder was rapidly excised, divided into two longitudinal strips and placed in a 5-ml organ bath containing oxygenated (96% O2 and 4% CO2, pH 7.4 at 37°C) standard Krebs solution of the following composition (mM): NaCl 119, KH2PO4 1.2, MgSO4 1.5, KCl 4.7, CaCl2 2.5, NaHCO3 25 and glucose 11. A resting load of 10 mN was applied and tension was recorded by means of an isometric force transducer connected to a Basile 7050 Unirecord. All recordings were stored on a Power Macintosh 6100/66PC and analyzed using Mac Lab/8s (ADInstruments, Hastings, UK). During the stabilization period the tone of the preparation faded to approximately 5 mN. After 60–90 min equilibration period the urinary bladder strips were electrically field stimulated with single pulses with a frequency of 0.05 Hz, 0.5 ms pulse width and maximal voltage by means of two platinum wire electrodes, placed at the top and bottom of the organ bath and connected to a GRASS S88 stimulator. In these conditions, regular and stable contractile responses (twitches) were obtained. Concentration-response curves to human calcitonin gene-related peptide (hCGRP), neurokinin A or capsaicin were obtained in a cumulative manner, the next concentration being added to the bath when the effect of the preceding one had reached the steady state, usually every 5–10 min. For the experiments with neurokinin A also submaximal conditions of stimulation were used (0.18± 0.01 ms pulse width). In some experiments the response produced by 0.3 µM hCGRP was further investigated. In vitro capsaicin desensitization was obtained by exposure of the preparations to 10 µM capsaicin for 15 min followed by thorough washing out. In each experiment the twitch height was measured as an increase from the current tension of the preparation. Neurokinin A-LI and CGRP-LI release induced by capsaicin. Male Golden Syrian hamsters weighing 100–150 g were sacrificed by
cervical dislocation and the urinary bladder was rapidly removed and placed in oxygenated (96% O2 and 4% CO2) Krebs solution at 37°C. Tissues taken from 6–8 animals were sliced (thickness 0.4 mm) using a MacIlwain tissue chopper (Campden Instruments, Leicester, UK). The pooled slices (160±6.5 mg) were divided into four thermostated (37°C) 2-ml Perspex perfusion chambers and superfused, at a flow rate of 0.4 ml/min, with oxygenated Krebs solution containing 0.1% bovine serum albumine (BSA) and 1 µM thiorphan. After a 60-min stabilization period, 5-min fractions (2 ml) were collected before (two fractions), during (three fractions) and after (one fraction) exposure to 10 µM capsaicin into polypropylene tubes containing acetic acid (final concentration 2 N). In all experiments the same protocol was repeated after 60 min washing out with Krebs solution, in order to verify if capsaicin-induced sensory peptides release undergoes desensitization. At the end of the experiments the tissue was blotted two to three times with filter paper and weighed. Superfusates were freeze-dried and stored at –80°C until assay. Tissue release of neurokinin A-like immunoreactivity (NKALI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was determined in reconstituted superfusate by the enzyme immunoassay (EIA) method. The sensitivity of the test was 3.5 pg/well and 2.5 pg/well for NKA-LI and CGRP-LI, respectively. Statistical analysis. All data in the text and figures are means ± standard error of the mean (SEM). Statistical analysis was performed by means of one-way analysis of variance (ANOVA) followed by the Dunnett test for multiple comparisons and by means of the Student’s t-test for unpaired data. Values of P
