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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