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 Kd of 0.18 +/- 0.03 nM and a density (Bmax.) 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] (Kd.GTP[S] = 0.51 +/- 0.02 nM) or with alkali-treated membranes (Kd.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 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than 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.
Votta
B
B
Department of Immunology, Smith Kline & French Laboratories, King of Prussia, PA 19406-0939.
Mong
S
S
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000494:pharmacology; D007975:Leukotriene B4 / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D011869:Radioligand Assay; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D018102:Receptors, Leukotriene B4; D012756:Sheep
D006150:Guanine Nucleotides; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4; D000246:Adenosine Diphosphate Ribose