TRANSITION OF AFFINITY STATES FOR LEUKOTRIENE-B4 RECEPTORS IN SHEEP LUNG MEMBRANES

Leukotriene B4 (LTB4) is a pro-inflammatory arachidonate metabolite. We have characterized the LTB4 receptors in sheep lung membranes and have assessed the contribution of the guanine-nucleotide-binding (G) protein in the regulation of receptor affinity states. Saturation isotherms have demonstrated a single class of LTB4 receptor with a K(d) of 0.18 ± 0.03 nM and a density (B(max.)) of 410 ± 84 fmol/mg of protein in sheep lung membranes. The effect of the G-protein on receptor affinity was assessed in the presence of non-hydrolysable GTP analogues (e.g. GTP[S]) and in membranes following alkali treatment (pH 12.1) to remove the G-protein. Saturation isotherms produced either in the presence of GTP[S] (K(d.GTP[S]) = 0.51 ± 0.02 nM) or with alkali-treated membranes (K(d.alk.) = 0.52 ± 0.02 nM) demonstrated a 3-fold shift in receptor affinity for [3H]LTB4 binding. In competition experiments, the rank order of affinity of LTB4 analogues was LTB4 > 20-OH-LTB4 > trans-homo-LTB4 > 6-trans-LTB4 > 20-COOH-LTB4 using either untreated or alkali-treated membranes, both in the presence and absence of GTP[S]). These findings demonstrate that, in sheep lung membranes, there is only one class of LTB4 receptor. Removal of the G-protein or uncoupling of the receptor from the G-protein shifted the agonist-binding affinity of the receptor by 3-4-fold, without affecting the specificity of the LTB4 receptor in either the high- or the low-affinity state.