Abstract

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 compete 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 degrees C was 0.47 nM-1 min-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 of protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25 degrees C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy-LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding.(ABSTRACT TRUNCATED AT 250 WORDS)