Leukotriene D4 Contractions in Human Airways are Blocked by SK&F 96365, an Inhibitor of Receptor-Mediated Calcium Entry ISABELLE GORENNE, CARLOS LABAT, JEAN-PIERRE GASCARD, XAVIER NOREL, NABILA NASHASHIBI and CHARLES BRINK

In human bronchial muscle preparations, nifedipine (3 mM) significantly inhibited the histamine, ACh and KCl contractions. However, the dihydropyridine did not modify the contractile responses induced by either leukotriene D4 (LTD4) or antihuman IgE (a-IgE). In human airways, SK&F 96365 (30 mM and 100 mM) markedly reduced the KCl and, at the higher concentration, LTD4 maximal contractions. In addition, when preparations were treated with nifedipine (3 mM), SK&F 96365 (100 mM) significantly blocked responses to both LTD4 and a-IgE. The calcium chelating agent ethylene glycol-bis (b-amino-ethyl ether) N,N,N9,N9-tetraacetic acid (4 mM) also inhibited the aIgE-induced contractions. These data demonstrate that the nifedipine-resistant component of the LTD4 and a-IgE contractions was inhibited by SK&F 96365 and suggest that the cysteinyl-leukotriene receptor in human airways may be intimately linked with a receptor-operated calcium-entry mechanism. In guinea pig airways, a differential sensitivity to the inhibitory effects of calcium antagonists has been reported for a variety of contractile agonists (Foster et al., 1984; Ahmed et al., 1984; Ahmed et al., 1985; Cuthbert et al., 1994). These results suggested that there was a source of calcium that was not readily affected by blocking the availability of extracellular calcium via a VOC. The mobilization of calcium from internal stores by contractile agonists was one suggestion for explaining the considerable resistance of contractions induced by some agonists to calcium antagonists such as verapamil and nifedipine (Foster et al., 1984; Ahmed et al., 1985). However, another explanation—that a ROC entry mechanism was involved— (Benham and Tsien, 1987) has also been proposed. Data from electrophysiological studies have provided some evidence for the mobilization of calcium entry via a ROC in platelets (Merritt et al., 1990) and airway smooth muscle (Murray and Kotlikoff, 1991). Initial reports in guinea pig airways have shown that the contractions induced by LTD4 exhibited little or no dependence on extracellular calcium when compared with data from a number of other contractile agonists (Weichman et al., 1983; Raeburn and Rodger, 1984). Cuthbert and co-workers (1994) have recently shown that in guinea pig tracheal preparations, the contractile response to LTD4 was dependent to some extent on the extracellular calcium because nifedipine significantly reduced these responses. Thus results from functional studies in this species suggested that LTD4 may mobilize calcium via a VOC. However, the LTD4 contractions were to some extent resistant to high concentrations of the dihydropyridines, which suggested that the contribution of the VOC in the airways of the guinea pig may represent only one possible route for Ca mobilization during the LTD4 response. Unfortunately, the little information that is available on the mechanisms of calcium entry in isolated human airways (Jones et al., 1982; Drazen et al., 1983; Roberts et al., 1986; Kohrogi et al., 1985) is conflicting. Therefore, the aim of this investigation was to evaluate the LTD4and a-IgE-induced contractions in human airways in the presence of nifedipine and the putative VOC/ROC antagonist, SK&F 96365 (Merritt et al., 1990). Materials and Methods Isolated preparations. Human lung samples were obtained from 17 male and two female patients who had undergone surgery for lung carcinoma. The age was 59 6 4 years (mean 6 S.E.M.). After resection, the bronchus was dissected free from parenchymal lung tissue and placed in Tyrode’s solution at 4°C for 12 h. Experiments were performed on 180 subsegmental bronchial preparations derived from 19 lung samples (N). The bronchial ring preparations were 2 to 5 mm in internal diameter. Received for publication May 27, 1997. ABBREVIATIONS: LTD4, leukotriene D4; a-IgE, anti-human IgE; EGTA, ethylene glycol-bis (b-amino-ethyl ether) N,N,N9,N9-tetraacetic acid; CysLT1, cysteinyl-leukotriene receptor; VOC, voltage-operated channel; ROC, receptor-operated channel; AIC, (atropine, 1 mM; indomethacin, 3 mM and chlorpheniramine, 1 mM). 0022-3565/98/2842-0549$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 284:549–552, 1998 549 at A PE T Jornals on A ril 5, 2017 jpet.asjournals.org D ow nladed from Agonist contractions. Bronchial ring preparations were set up in 10-ml organ baths containing Tyrode’s solution of the following composition (mM): NaCl, 139.2; KCl, 2.7; CaCl2, 1.8; MgCl2, 0.49; NaHCO3, 11.9; NaH2PO4, 0.4 and glucose, 5.5; pH 7.4. The rings were mounted under initial loads of 2 to 3 g, maintained at 37°C and gassed with 5% CO2/95% O2. Changes in force were monitored on Linseis recorders using isometric force-displacement transducers (Narco F-60). The preparations were allowed to equilibrate for 90 min, and the medium was replaced every 15 min with fresh Tyrode’s solution. After the 90-min equilibration period, bronchial rings were contracted with ACh (100 mM; 2.28 6 0.28 g; N 5 19 lung samples). When the response reached a plateau, the tissues were washed every 10 min by exchanging the medium until the basal tone was reestablished. The bronchial tissues were then incubated for 30 min with Tyrode’s solution or Tyrode’s solution containing the different drugs (nifedipine, SK&F 96365 or nifedipine/SK&F 96365) at different concentrations (0.01 mM–100 mM). Cumulative concentrationeffect curves were then produced with ACh, histamine, KCl and LTD4. a-IgE-induced contractions. After the equilibration period, the bronchial tissues were contracted with ACh (100 mM). When the ACh response reached a plateau, the rings were washed until the basal tone was re-established. The preparations were then incubated for 30 min with Tyrode’s solution (control), with Tyrode’s solution containing AIC (30 min) or with Tyrode’s solution containing this drug combination and either nifedipine (3 mM) or nifedipine (3mM)/SK&F 96365 (100 mM). At the end of the incubation period, the tissues were challenged with a-IgE (1:1000). In protocols involving EGTA (4 mM), the tissues, which had been incubated with AIC, were subsequently exposed to this chelator for 5 min before a-IgE stimulation. The tissues used were not passively sensitized because the a-IgE serum was directed against the Fc fraction of the IgE (that is, the constant domains on the e chain) found on the membranes of mast cell and basophils. In one experimental protocol, the a-IgE response was monitored every 6 min until the contraction reached a plateau (30