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.
|Number of pages||7|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|Publication status||Published - 1986|
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