Br.
J.
Pharmacol.
(1990), 100, 886-888 (1990),
100,
886-888
(--', MacmiHan Press Ltd, 1990
Pharmacological modulation of the contractile response to toluene diisocyanate in the rat isolated urinary bladder 'Cristina E. Mapp, Pasquale Chitano, Leonardo M. Fabbri, *Riccardo Patacchini & *Carlo A. Maggi Institute of Occupational Medicine, University of Padua, Padua, Italy, and *Pharmacology Department, Research Laboratories, A Menarini Pharmaceuticals, Florence, Italy 1 Toluene diisocyanate produced concentration-dependent contractions of the rat isolated urinary bladder. 2 The contractions were tetrodotoxin-resistant and were abolished by previous exposure of the strips to capsaicin.
3 Indomethacin (5,UM) and ruthenium red (30,pM) inhibited toluene diisocyanate-induced contractions. Responses expressed as a percentage of the response obtained with substance P, 30nm, were respectively 141.6 + 24.8% and 20.1 + 5.1% in control and indomethacin-treated strips (P < 0.005); 123.0 + 30.2% and 14.0 + 6.5% in control and ruthenium red-treated strips (0.01 < P < 0.05). 4 These results suggest that toluene diisocyanate-induced contractions of the rat isolated bladder are the result of the release of cyclo-oxygenase products which may act by activating the capsaicin receptor.
Introduction Toluene diisocyanate is a major causative agent in occupational asthma (Mapp et al., 1988). The mechanism of sensitization is unknown, even though studies performed in man and in animal models have shown the importance of airway inflammation (Boschetto et al., 1987; Fabbri et al., 1987; Thompson et al., 1987). It is known that capsaicin-sensitive primary afferents release several transmitters, including tachykinins and calcitonin gene-related peptide (CGRP) in the periphery and produce motor and inflammatory responses (Szolcsany, 1984; Maggi & Meli, 1988). Since the specific aspects of the acute inflammatory process related to asthma and airway hyperresponsiveness consequent to the exposure to toluene diisocyanate are unknown, we speculated that toluene diisocyanate might activate capsaicin-sensitive afferents and cause transmitter release which, in turn, could play a role in the inflammatory process. Recently, we have shown that toluene diisocyanate possesses a capsaicin-like activity, i.e. that toluene diisocyanate activates the 'efferent' function of these primary afferents in the rat isolated urinary bladder (Mapp et al., 1990), an experimental test object in which the action of capsaicin has been well characterized (Santicioli et al., 1986). The aim of this study was the pharmacological characterization of the response to toluene diisocyanate in the rat urinary bladder.
Methods Male albino rats of Wistar-Morini strain weighing 250-400g were killed by cervical dislocation and exsanguinated. The urinary bladder was rapidly removed, and two small strips of detrusor muscle were dissected out and placed in a 15ml organ bath (at 370C) containing a standard Krebs-Henseleit solution (composition in mM: NaCI 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, CaCl22.5 and D-( + )glucose 11.1) gassed with 96% 02 plus 4% C02, as described previously (Santicioli et al., 1986). A resting load of IOmN was applied and the preparations were allowed to equilibrate for 90 min before beginning the experiment. During the equili-
' Author for correspondence at: Istituto di Medicina del Lavoro, Universita degli Studi di Padova, via J Facciolati 71, 35127 Padova, Italy.
bration period, the strips were washed every 20 min. Tension was recorded by means of an isometric strain gauge (Grass FT03) connected to a Battaglia Rangoni Recorder (Mod. K 380, Bologna, Italy). Contractions were normalized as a percentage of the active tension obtained in response to substance P, 30nm (mean of three reproducible responses). Each dose was delivered at 1215 min intervals and the duration of application was 5 min. To determine the effect of possible inhibitors on toluene diisocyanate-induced contractions in the bladder strips, we studied the effect of the inhibitors on smooth muscle contraction produced by toluene diisocyanate, 0.3 mm. This concentration, in preliminary experiments produced the maximum effect on the bladder strips (Mapp et al., 1990). Two strips were excised from the same bladder and studied in parallel. After the equilibration period, control substance P responses were obtained. At this stage the test substance (tetrodotoxin (TTX) 1fM contact time 15 min; ruthenium red (RR), 30,UM contact time 30min; indomethacin, 5,UM, contact time 90min; methylprednisolone, 5,uM, contact time 90 min; atropine, 1 pM, contact time 30 min) was administered to one strip while the other served as control. In some experiments, one strip was exposed to capsaicin (1 pM) to inactivate the sensory nerves and then after 4560 min the response to toluene diisocyanate was investigated in both strips. Drugs included capsaicin, atropine sulphate, tetrodotoxin, indomethacin were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Substance P was obtained from Peninsula Labs. (Belmont, CA, U.S.A.). Methylprednisolone was obtained from Hoechst (Frankfurt, F.R.G.) and ruthenium red from Serva (Heidelberg, F.R.G.). Toluene diisocyanate consisted of an 80:20 mixture of the 2,4 and 2,6 isomers, and was obtained from Montedison (Porto Marghera, Italy). Capsaicin was dissolved in 100% ethanol to give a stock solution 10mM that was stored at 4°C and diluted in Krebs solution as necessary for the experiment. Tetrodotoxin was prepared in deionized water to give a stock solution -f 0.3 mm, that was stored at 4°C and diluted in Krebs solution as necessary for the experiment. Indomethacin was prepared in 100% ethanol, and ruthenium red was prepared in deionized water, both on the day of the experiment. Other drugs were prepared in Krebs-Henseleit solution on the day of the experiment. Toluene diisocyanate was dissolved in dimethyl sulphoxide (DMSO) and it was prepared freshly before each administration. The maximal final concentration of DMSO in the
TOLUENE DIISOCYANATE AND 'EFFERENT' SENSORY NERVES
organ bath was 3%. Addition of the vehicles used to dissolve the drugs had no effect on resting tension. Data analysis All the data are mean + s.e.mean of 5-6 experiments. The effect of the inhibitors on toluene diisocyanate-induced contractions was compared by two-tailed Student's t test for paired data. P < 0.05 was considered significant.
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response to substance P. Thus, in the presence of methylprednisolone, there was a small but significant inhibitory effect on toluene diisocyanate-induced contractions (0.01
0.05; Figure 1).
Results
Discussion
Toluene diisocyanate caused a concentration-dependent contraction of the bladder strips. The effect was maximum at a concentration of 0.3 mM, and was 103.6 ± 20.2%, whereas capsaicin (1 IM) induced a contraction which averaged 158.8 + 27.5% of the response to substance P (30nM). In the strips pretreated with capsaicin, toluene diisocyanate was ineffective (n = 5) (Figure 1). TTX (1pM) did not produce inhibition of the responses to toluene diisocyanate or to capsaicin. In control strips, toluene diisocyanate and capsaicin produced a contraction which averaged respectively 91 + 13.8% and 147 + 20.3% of the response to substance P (30 nm), and in strips pretreated with TTX, toluene diisocyanate and capsaicin-induced contractions were respectively 88.4 + 13.7% and 146.8 + 13.7% (P > 0.05, n = 6; Figure 1). Ruthenium red (30uM) produced a significant inhibition of toluene diisocyanate-induced contractions which was decreased from 123 + 30.2% to 14 + 6.5% of the substance P response (0.01 < P < 0.05, n = 6; Figure 1). We have previously shown that ruthenium red abolishes the contractile response to capsaicin (1 pM), apparently due to a prejunctional action on sensory nerves (Maggi et al., 1988). Ruthenium red (30pM) did not inhibit the response to exogenous substance P (30nM). In fact, in strips pretreated with ruthenium red and in control strips the contractions were respectively 158 + 24.5% and 156 + 17% (n = 6). Indomethacin (5pM) had a significant inhibitory effect on toluene diisocyanate-induced contractions. In the presence of indomethacin, the toluene diisocyanate-induced response was 20 + 5.3% whereas in control strips it was 141 + 24.8% of the response to substance P (P < 0.005, n = 6; Figure 1). Indomethacin did not decrease significantly capsaicin-induced contractions which were 251 + 26% in control strips and 185 + 25% in the strips pretreated with indomethacin (n = 6, P > 0.05). In the presence of methylprednisolone (5M), the toluene diisocyanate-induced contraction was 121 + 17.5%, whereas in control strips, the contraction was 165 + 18.5% of the
We have demonstrated previously that toluene diisocyanate produces a contractile response of the rat isolated urinary bladder by activating the efferent function of the primary afferents and the present experiments were designed to investigate the mechanisms involved (Mapp et al., 1990). The response to toluene diisocyanate, like that to capsaicin, was totally TTX-resistant while the effect of both agents was abolished by chronic extrinsic bladder denervation (Mapp et al., 1990). Therefore both agents activate the 'sensory receptorcoupled efferent' function of the sensory nerves (Maggi & Meli, 1988) without any significant contribution of a propagated action potential (axon reflex). A further common feature of the response to capsaicin and toluene diisocyanate in the rat bladder is their sensitivity to ruthenium red. Ruthenium red has recently been characterized as a selective capsaicin antagonist in a variety of preparations in which it blocked prejunctionally peptide release from sensory nerves when the latter are activated by capsaicin but not by other stimuli, such as bradykinin (Amman et al., 1989a,b; Maggi et al., 1989ab). Apparently, ruthenium red blocks, in some way, a nonselective cation channel which is opened by activation of the capsaicin 'receptor' (Amman et al., 1989b). In view of the above possibilities, it is interesting to assess whether toluene diisocyanate acts directly or indirectly on capsaicin-sensitive afferents. Experiments with indomethacin and the slight but significant inhibitory effect of methylprednisolone indicate that the action of toluene diisocyanate on sensory nerves is chiefly indirect and that toluene diisocyanate activates the generation of a cyclo-oxygenase product which in turn activates the capsaicin-sensitive afferents via a ruthenium red-sensitive mechanism. In view of the high selectivity of ruthenium red for the capsaicin-induced mode of activation of sensory nerves, the present findings raise the attractive hypothesis that a cyclo-oxygenase product activates these sensory nerves via a mechanism common to that of capsaicin. Recently, evidence was presented for the existence of a capsaicin 'receptor' on primary afferent neurones (James et al., 1988; Szallasi & Blumberg, 1989) which, in turn poses the problem of the possible existence of an endogenous ligand for this receptor. The present findings might therefore be interpreted as a preliminary indication that some cyclo-oxygenase product(s) act as endogenous ligands for the capsaicin receptor on primary afferents in the rat urinary bladder. Interestingly, previous in vivo studies have shown that the action of indomethacin and capsaicin pretreatment on bladder capacity are non-additive, indicating a common site of action at this level (Maggi et al., 1988). In conclusion, toluene diisocyanate, a chemical able to cause occupational asthma, causes smooth muscle contraction in the rat bladder through the activation of the efferent function of capsaicin-sensory nerves, and its seems likely that this activation is indirect. Since the same mechanism may occur in the airways, research in this area must proceed to answer this relevant question.
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Figure I Effect of various pretreatments on the contractile response to toluene diisocyanate (TDI, 0.3 mM) (open column = control strips; closed columns = pretreated strips). Capsaicin (1 pM) inhibited almost completely TDI-induced contractions, ruthenium red (RR, 30OM), indomethacin (5pM), and methylprednisolone (5juM) decrease significantly TDI-induced contractions. 0.01 < *P < 0.05; **P < 0.005. 1TX = tetrodotoxin; steroids = methylprednisolone.
Supported in part by Italian Research Council grants No. 89.02961.04, 89.02664.04, by the Italian Ministry of Education and by a grant of Regione Veneto.
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