Modulation of affinity and density of LTB4 receptors on guinea pig lung membranes by divalent cations and guanine nucleotides.
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+ greater than Mn2+) stimulated, whereas EDTA inhibited (IC50 = 0.31 +/- 0.08 mM) binding of [3H]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 less than 0.01, for control and EDTA-treated respectively), but no significant change in receptor affinity (KD = 0.67 +/- 0.16 nM and 1.01 +/- 0.19 nM, P greater than 0.05). Competition experiments with LTB4 and the low-affinity (Ki = 165 nM) competitive LTB4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6-fold, P less than 0.05 and P less than 0.01, respectively), in affinity to both ligands. In the same experiments, the 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Falcone
R C
RC
Department of Pharmacology, ICI Pharmaceuticals Group, Wilmington, DE 19897.
Aharony
D
D
D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005233:Fatty Alcohols / Q000378:metabolism; D006018:Glycols / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D018102:Receptors, Leukotriene B4
D002413:Cations, Divalent; D005233:Fatty Alcohols; D006018:Glycols; D006150:Guanine Nucleotides; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; C062137:U 75302; D007975:Leukotriene B4; D006165:Guanylyl Imidodiphosphate; D004492:Edetic Acid