Abstract
In human bronchial muscle preparations, nifedipine (3 μM) significantly inhibited the histamine, ACh and KCl contractions. However, the dihydropyridine did not modify the contractile responses induced by either leukotriene D4 (LTD4) or anti-human IgE (a-IgE). In human airways, SK&F 96365 (30 μM and 100 μM) markedly reduced the KCl and, at the higher concentration, LTD4 maximal contractions. In addition, when preparations were treated with nifedipine (3 μM), SK&F 96365 (100 μM) significantly blocked responses to both LTD4 and a-IgE. The calcium chelating agent ethylene glycol-bis (β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (4 mM) also inhibited the a-IgE-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; Ahmedet 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 Ca2+ 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 LTD4- and 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 ± 4 years (mean ± 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.
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 μM; 2.28 ± 0.28 g; N = 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 re-established. 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 μM–100 μM). Cumulative concentration-effect 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 μM). 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 μM) or nifedipine (3μM)/SK&F 96365 (100 μM). 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 ε 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 min).
Calculations.
The changes in force produced by the different agonists were determined from recordings and expressed as percent of ACh (100 μM) contractions. Because the calcium antagonists blocked the agonist maximal contractile response, no attempt was made to interpolate the effective concentration (EC50) values. Results were evaluated using the ANOVA followed by Student’st test for the individual data points.
Drugs.
The drugs and their sources were as follows: acetylcholine chloride, atropine sulfate, chlorpheniramine maleate, indomethacin, histamine dihydrochloride and EGTA (Sigma Chemical Co., St. Louis, MO), sheep antiserum to human IgE (anti-IgE; Nordic Immunological Laboratories, Tilberg, Netherlands). LTD4 was synthesized by Bayer plc (Stoke Court, UK). Nifedipine and SK&F 96365 were a gift from Dr. P.J. Gardiner (Bayer plc). The receptor antagonists and indomethacin were dissolved in dimethylsulfoxide, and subsequent dilutions were made in Tyrode’s solution. The leukotrienes were shipped on dry ice from Bayer plc in vials containing a stock solution of LTD4 in 20% ethanol and physiological salt solution at pH 7.2.
Results
The data presented in figure 1demonstrate the effects of nifedipine on contractions induced by several agonists in human airways. Although nifedipine (3 μM and 10 μM) significantly reduced the histamine, ACh and KCl responses, nifedipine (3 μM) did not alter the LTD4 contractions. At a higher concentration (nifedipine, 10 μM), no effect on the maximal LTD4 contractions was observed (control 142 ± 16% and nifedipine 147 ± 44%, N = 3).
Effects of nifedipine on contractile agonists in human isolated airways. Control (•) and nifedipine 0.01 μM (▴), 0.1 μM (○), 1 μM (▿), 3 μM (□) and 10 μM (▵). Results are presented as percent of ACh (100 μM) precontraction. Values are means ± S.E.M., and results are derived from 3 to 17 different lung samples. *Values significantly different from control data (P < .05).
SK&F 96365 (30 μM and 100 μM) significantly reduced the maximal contractions induced by KCl and, at the higher concentration, by LTD4 in human bronchial preparations (fig.2). However, the maximal histamine contractions were not significantly altered by SK&F 96365 at 100 μM (control 137 ± 25% and SK&F 96365 96 ± 16%,N = 3). In contrast, the maximal ACh contractions (148 ± 10%) were significantly reduced (92 ± 23%;N = 5; P < .05) by SK&F 96365 (100 μM). In bronchial preparations treated with nifedipine (3 μM), SK&F 96365 (30 μM and 100 μM) also blocked the LTD4 contractions in human airways (fig. 3).
Effects of SK&F 96365 on KCl and LTD4contractions in human airways. Control (•) and SK&F 96365 30 μM (○) and 100 μM (□). Results are presented as percent of ACh (100 μM) precontraction. Values are means ± S.E.M., and results are derived from 5 to 7 different lung samples. *Values significantly different from control data (P < .05).
Effects of SK&F 96365 on LTD4contractions in human airways. Protocols were performed in the presence of nifedipine (3 μM). Control (•) and SK&F 96365 30 μM (○) and 100 μM (□). Results are presented as a percent of ACh (100 μM) precontraction. Values are means ± S.E.M., and results are derived from three different lung samples. *Values significantly different from control data (P < .05).
When human airways were pretreated with AIC, the contractions produced by a-IgE were not significantly increased compared with the response in control tissues (table 1). However, a significant reduction in the anti-IgE-induced contraction of human airways was observed in tissues treated for 5 min with EGTA (4 mM). This reduced response after EGTA treatment was observed in the absence and presence of AIC (table 1).
Effects of EGTA on anti-IgE-induced contractions in human airways in absence or presence of AIC
Nifedipine (3 μM) in AIC-treated tissues did not alter the anti-IgE-induced contraction. However, SK&F 96365 (100 μM) in the presence of nifedipine (3 μM) significantly reduced responses to anti-IgE in human airways in the presence of AIC (fig.4).
Effects of SK&F 96365 on anti-IgE-induced contractions in human airways. Protocols were performed in tissues treated with the drug combination AIC for 30 min. Results are presented as a percent of the ACh (100 μM) precontraction. Left panel: anti-IgE (dilution 1/1000)-stimulated bronchial preparations (control, •) and in presence of nifedipine (3 μM; ○). Right panel: anti-IgE challenge in the presence of nifedipine (3 μM; •) or nifedipine (3 μM) and SK&F 96365 (100 μM; □). Values are means ± S.E.M. obtained from 4 to 5 lung samples. *Values different from appropriate controls (P < .05).
Discussion
In human airways (present report), nifedipine (3 μM), which significantly reduced the histamine, ACh and KCl contractions, failed to block the LTD4 and anti-IgE-induced responses. However, treatment of human airways with SK&F 96365 reduced the maximal response obtained with LTD4 and a-IgE. Because contractions produced by a-IgE in human airways (Bjorck and Dahlén, 1993; Gorenneet al., 1994) are due to the effects of endogenously released leukotrienes, these results suggest that stimulation of the CysLT1 receptor may be associated with the mobilization of calcium via a ROC in human airways.
In guinea pig tracheal preparations, verapamil and nitrendipine markedly inhibited the contractions induced by barium chloride, whereas the responses to other agonists (histamine, carbachol) were not affected (Duncan and Douglas, 1985). Recently, Cuthbert and co-workers (1994) demonstrated that in physiological medium (calcium present), nifedipine (1 μM and 10 μM) significantly inhibited the LTD4 contractions and did not alter the ACh responses. These latter results are similar to those results previously reported in guinea pig tracheal preparations for LTD4 (Raeburn and Roger, 1984). In addition, Raeburn and Roger (1984) clearly showed that LTD4 did not stimulate calcium uptake in guinea pig airway smooth muscle preparations and suggested that the mobilization of Ca2+ during LTD4 contraction may involve internal Ca2+ pools.
The LTD4 contractions of human airways have also been reported to rely preferentially on intracellular calcium, whereas methacholine contractions rely on the extracellular source of this ion (Roberts et al., 1986). However, these latter results in human airways are not consistent with an earlier observation byHenderson and co-workers (1983), who demonstrated that histamine, ACh and antigen-induced contractions were reduced by nifedipine, which suggests that a voltage-dependent Ca2+ channel entry mechanism may be involved. Other investigators (Black et al., 1986; Roberts et al., 1986; Raeburn et al., 1986) using human airways in calcium-depleted medium reported a significant reduced response to a variety of contractile agonists. The data (present report) support these latter observations; nifedipine (3 μM) significantly inhibited ACh, histamine and KCl contractions in human airways but did not block LTD4 and anti-IgE-induced responses.
Although the presence of VOC in human airway smooth muscle cells (Marthan et al., 1989) has been demonstrated, an exploration of the ROC in airway muscle function has received less attention, because appropriate antagonists have not been available. Merritt and co-workers (1990) provided the initial observations on a putative inhibitor (SK&F 96365) for the calcium entry during agonist-receptor interactions. This compound has recently been reported to block effectively the nifedipine-resistant contractions of ACh and LTD4 in guinea pig airways (Cuthbert et al., 1994). These results from guinea pig airways, together with those derived from human airways (present report), suggest that certain agonists and their specific receptors may be more closely associated than others with a ROC type of calcium mobilization. The observation that SK&F 96365 (100 μM) had no significant effect on histamine responses although it significantly inhibited KCl, ACh and LTD4 contractions suggests that this compound may have both VOC and ROC inhibitory activity. However, the compound is less effective than nifedipine as a VOC antagonist. The observation that SK&F 96365 (100 μM) caused a marked reduction in LTD4contractions, as compared with the data obtained with histamine, provides further indirect evidence that certain receptors may be intimately linked to a ROC mechanism in human airways.
Henderson and co-workers (1983) showed that human airways, after passive sensitization, contracted when challenged with a specific allergen. This contraction was reduced, though only marginally, by treatment of the tissues with nifedipine (100 μM). Previous reports have shown that under specific experimental conditions, the major mediators involved in antigen-induced contraction in human airways are cysteinyl-leukotrienes (Bjorck and Dahlén, 1993; Gorenne et al., 1994). In human airways (present report), the contractions induced by LTD4 and a-IgE are apparently independent of any mobilization of calcium via a VOC, because nifedipine did not affect either response. These results support the work ofBourdillat and co-workers (1987), who showed that LTD4contractions in human bronchial preparations were resistent to treatment with the VOC inhibitor diltiazem. In the presence of both antagonists (nifedipine and SK&F 96365), the contractions induced by LTD4 and a-IgE were markedly inhibited. These results suggest that LTD4 and a-IgE induced contraction by activating an SK&F 96365-sensitive/nifidepine-insensitive calcium-entry mechanism. Whether this calcium mobilization system that is activated by LTD4 is identical to a receptor-mediated calcium channel entry mechanism in human airways is presently not known. However, these observations are similar to data reported by Soergel and co-workers (1992), who demonstrated that maitotoxin activates phosphoinositide breakdown in C6 glioma cells and is inhibited by SK&F 96365 but not by nifedipine. In contrast, these authors also showed that in RIN cells, the effects of endothelin, which stimulated a specific receptor and activated phosphoinositide breakdown, were not inhibited by SK&F 96365. Such observations lend support to the initial studies in human platelets (Merritt et al., 1990), rat pancreatic acini (Busik et al., 1993) and PC12 cells (Fasolato et al., 1990), which suggested that SK&F 96365 was an inhibitor of a receptor-mediated calcium-entry channel.
The inhibition of LTD4 and a-IgE contractions by SK&F 96365 provides indirect evidence that a ROC mechanism may be present and functional in human airway muscle. Because human airways contain a single cysteinyl-leukotriene receptor responsible for contraction (CysLT1; Buckner et al., 1986; Labat et al., 1992), these data suggest that the CysLT1receptor may be intimately linked to ROC and that LTD4 may be a potent activator of this channel.
Footnotes
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Send reprint requests to: Dr. Charles Brink, CNRS ERS 566, Centre Chirurgical Marie Lannelongue, 133 av de la Résistance, 92350 Le Plessis-Robinson, France.
- Abbreviations:
- LTD4
- leukotriene D4
- a-IgE
- anti-human IgE
- EGTA
- ethylene glycol-bis (β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid
- CysLT1
- cysteinyl-leukotriene receptor
- VOC
- voltage-operated channel
- ROC
- receptor-operated channel
- AIC
- (atropine, 1 μM
- indomethacin
- 3 μM and chlorpheniramine, 1 μM)
- Received May 27, 1997.
- Accepted October 17, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
