[³H]leukotriene B₄ binding to the guinea-pig spleen membrane preparation: A rich tissue source for a high-affinity leukotriene B₄ receptor site

Intact human granulocytes contain a leukotriene (LT) B₄ receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [³H]LTB₄ to guinea-pig spleen membranes and we have determined the ability of LTB₄ to compte with [³H]LTB₄ for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl₂ and CaCl₂ enhanced [³H]LTB₄ binding, but NaCl and KCl decreased it. Spleen [³H] LTB₄ binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25°C was 0.47 nM^-1 and 0.099 min^-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg protein. Moreover, the LTB₄/[³H]LTB₄ competition study performed at 4 or 25° C revealed the inhibitory constant (K(i)) of LTB₄ to be in the nanomolar range. The rank order of agents competing for spleen [³H]LTB₄ binding was: LTB₄ (K(i) = 2.8 nM) > 20-hydroxyl-LTB₄ (23 nM) > LTA₄ (48 nM) > LTA₄ methyl ester (0.13 μM) > 20-carboxyl-LTB₄ (>6.6 μM) ≥ arachidonic acid (0.15 mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihydropyridine Ca⁺⁺ channel blocker, exhibited micromolar inhibition of spleen [³H]LTB₄ binding. Comparison of the ability of LTB₄ to inhibit [³H]LTB₄ binding among various tissue homogenates demonstrated the following tissue rank for its effect: spleen > thymus gland > lung ≥ uterus = urinary bladder = brain = adrenal gland = small intestine = liver = kidney = heart. The low-affinity binding of [³H]LTB₄ to the guinea-pig granulcoyte argues against the the concept that binding to this cell accounts for the [³H]LTB₄ high-affinity binding site in the spleen. We conclude that guinea-pig spleen contains a rich tissue source of [³H]LTB₄ receptor high-affinity binding sites and suggest that these binding sites may play a role in immunoregulation.