We investigated the mechanism by which guanine nucleotides and divalent cations modulate the affinity and apparent density of high-affinity receptors for Leukotriene B4 (LTB4) on guinea pig lung membranes (GPLM). Divalent cations (Mg2+ = Ca2+ > Mn2+) stimulated, whereas EDTA inhibited (IC50 = 0.31 +/- 0.08 mM) binding of [H-3]LTB4. Saturation analysis demonstrated that omission of divalent cations caused a two-fold reduction in apparent site density, (B(max) = 297 +/- 24 fmol/mg protein vs. 149 +/-21 fmol/mg protein, P < 0.01, for control and EDTA-treated respectively), but no significant change in receptor affinity (K(D) = 0.67 +/- 0.16 nM and 1.01 +/- 0.19 nM, P > 0.05). Competition experiments with LTB4 and the low-affinity (K(i) = 165 nM) competitive LTB4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6 fold, P < 0.05 and P < 0.01, respectively), in affinity to both ligands. In the same experiments, the guanine nucleotide analog GppNHp also reduced the affinity for LTB4 and U75302, similar to that observed with EDTA, suggesting that removal (Mg2+), or addition (GppNHp), of allosteric modulators of G-protein(s), causes reduction in receptor affinity. Saturation experiments also demonstrated that GppNHp, or GTP(gamma-S), caused a significant reduction (40-50%) in receptor density. A larger reduction in affinity for U75302 (3- to 3.6-fold) than for LTB4 (1.7-fold) was induced by EDTA as well as GTP analogs. The data suggest that LTB4 receptors on GPLM can undergo a transition between a G-protein coupled, high-affinity state, to an uncoupled state with fewer number of receptors, that possess lower affinity for ligands.
