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  • Calcium Colorimetric Assay Kit br Methods br Results br Disc

    2019-10-11


    Methods
    Results
    Discussion MEN91507 is a potent and selective CysLT1 receptor antagonist. Guinea-pig lung membranes have been widely used for the detection and characterization of CysLT1 receptor antagonists: in this assay the high affinity (subnanomolar) binding of [3H[leukotriene D4 or [3H]leukotriene E4 was potently displaced (subnanomolar affinity) by MEN91507, Montelukast (Jones et al., 1995) as well as by other well-characterized CysLT1 antagonists (e.g., Aharony et al., 1989). In contrast, neither MEN91507, nor Montelukast (or other selective CysLT1 antagonists) displaced specific [3H]leukotriene C4 binding in guinea-pig membranes, in agreement with studies showing that [3H]leukotriene D4 and [3H]leukotriene C4 Calcium Colorimetric Assay Kit are distinct in terms of distribution, function, and pharmacology Norman et al., 1987, Carstairs et al., 1988, Jones et al., 1995. A similar situation is found in DMSO-differentiated human cell line dU937, where [3H[leukotriene D4 but not [3H]leukotriene C4 binding was potently displaced (subnanomolar affinity) by both MEN91507, Montelukast, and other CysLT1 receptor antagonists (Frey et al., 1993). In this cell line, most of the [3H]leukotriene C4 binding has been previously shown to correspond to the microsomal γ-glutathione-S-transferase rather than an actual CysLT receptor (Metters et al., 1994), and the lack of effect of MEN91507 and Montelukast on [3H]leukotriene C4 binding would exclude the interaction of these compounds with γ-glutathione-S-transferase. These findings indicated that affinity of MEN91507 vs. [3H[leukotriene D4 binding at CysLT1 receptors in both guinea-pig and human tissues is similar to that previously reported for Montelukast or other CysLT1 receptor antagonists Krell et al., 1990, Obata et al., 1992, Jones et al., 1995. Both MEN91507 and Montelukast antagonized in a concentration-related manner leukotriene D4-induced Ca2+ transients in dU937 cells and the apparent affinity was higher for MEN91507 (pKB 10.3) as compared to Montelukast (pKB 9.4). Interestingly, both compounds behaved as insurmountable antagonists in dU937 cells, despite the fact that in binding studies in this system both Montelukast and MEN91507 were competitive ligands. Ca2+ transients induced by leukotrienes in dU937 cells are exclusively mediated by the stimulation of CysLT1 receptors, as judged by agonists potency and blockade by selective antagonists (Wetmore et al., 1991). Furthermore, no other CysLT receptor was found in this system (Nothacker et al., 2000). Therefore, the insurmountable antagonism of leukotriene D4-induced Ca2+ transients by both MEN91507 and Montelukast could be putatively attributed to the kinetic of the interactions between leukotriene D4, CysLT1 receptor antagonists, and their receptors to determine the Ca2+ response in dU937 cells. In this contest, it is important to remind that leukotriene D4-induced response develops in about 10 s, whereas 60 min incubations were performed in binding studies on dU937 cell membranes: we speculate that a long incubation time, as performed in binding experiments may be required to leukotriene D4 to completely displace the antagonists from CysLT1 receptors, and this may explain the insurmountable antagonism of both MEN91507 and Montelukast observed toward leukotriene D4-induced Ca2+ transients in dU937 cells. MEN91507, Montelukast, Zafirlukast, and Pranlukast antagonized leukotriene D4-induced bronchoconstriction in guinea-pigs, following both intravenous and oral administration. When administered intravenously, MEN91507 was the most potent amongst the CysLT1 receptor antagonists tested, since the ED50s for Montelukast, Pranlukast, and Zafirlukast were about 2, 10 and 20 times greater than that of MEN91507. Following oral administration Montelukast was slightly (but non-significantly) more potent of MEN91507, whereas the ED50s of Pranlukast and Zafirlukast were 8 and 11 fold greater than that of MEN91507. The protection afforded by oral administration of MEN91507, Montelukast, and Pranlukast against leukotriene D4-induced bronchoconstriction was long-lasting, since after 8 h this response was still substantially antagonized. Therefore, although following the oral route of administration the absolute potency of MEN91507 and Montelukast was greater than that of Pranlukast, the duration of the anti-bronchoconstrictor effect of the latter compound was relatively longer than that of both MEN91507 and Montelukast.