COMPETITION OF LEUKOTRIENES AND ICI-198,615 FOR [H-3] LTD4 BINDING-SITES IN GUINEA-PIG LUNG MEMBRANES SUGGESTS THE INVOLVEMENT OF 2 LTD4 RECEPTOR SUBTYPES

Pharmacological analysis of the effects of leukotriene D4 (LTD4) antagonists on contraction of guinea pig airway smooth muscle to leukotrienes reveals the presence of two subtypes of the LTD4 receptor. This finding is, however, inconsistent with [H-3]LTD4 equilibrium binding results, which show no evidence of a heterogeneity of pulmonary [H-3]LTD4 binding sites. It is possible that LTD4 binds to the two receptor subsets with equal affinity, and the pharmacological difference between them lies in the relative ability of leukotriene (LT) agonists and antagonists to interact at the receptor sites. This study was, therefore, undertaken to determine the rank order of potency of LTs and ICI-198,615 in competing with [H-3]LTD4 for their respective binding sites in guinea pig lung membranes. To determine precisely the inhibitory constant (K(i)) of LTC4, we used the irreversible gamma-glutamyl transpeptidase inhibitor, acivicin (AT-125), to prevent LTC4 metabolism. Incubation of lung membranes with 5 mM AT-125 for 120 min at 25-degrees-C resulted in >98% recovery of LTC4. Unlike L-serineborate complex, AT-125 failed to inhibit pulmonary [H-3]LTD4 binding. These results suggest that AT-125 can be used in this study. Nonlinear least squares analysis of the results of LTD4/[H-3]LTD4 or ICI-198,615/[H-3]LTD4 competitive binding reveals that either LTD4 (K(i) = 0.49 nM) or ICI-198,615 (K(i) = 6.89 nM) interacts at a single homogenous population of [H-3]LTD4 binding sites. However, the data of competitive binding results of LTC4 (in the presence of AT-125) or LTE4 are best fitted for its interaction with high- and low-affinity [H-3]LTD4 binding sites, designated as LTD4-alpha and LTD4-beta sites, respectively. The rank order of potency of these agents for the LTD4-alpha site is LTD4 greater-than-or-equal-to LTE4 greater-than-or-equal-to LTC4 greater-than-or-equal-to ICI-198,615, an order which differs significantly from that for the LTD4-beta site (LTD4 >> ICI-198,615 > LTE4 greater-than-or-equal-to LTC4). Although elimination of 10 mM Mg++ in the assay increased the K(i) value of LTD4, the agonists' order of potency for the LTD4-alpha and LTD4-beta site remains unchanged. Interestingly, these two types of rank orders of agonists' potency can be demonstrated in several biological systems where the relative ability of LTs to elicit responses was examined. Based on the relative potency of LTs determined from [H-3]LTD4 competitive binding studies, we conclude that there are likely to be two subtypes of [H-3]LTD4 binding sites (LTD4-alpha and LTD4-beta sites) and that LTD4 and ICI-198,615 interact at these two sites with equal affinity, whereas LTC4 and LTE4 are more potent in acting on LTD4-alpha than LTD4-beta sites.