CHARACTERIZATION OF [H-3] LEUKOTRIENE D4 BINDING-SITES IN GUINEA-PIG VENTRICULAR MYOCARDIUM

  • 1 January 1985
    • journal article
    • research article
    • Vol. 233  (3) , 686-693
Abstract
[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High performance liquid chramotography analyses indicated that, in the presence of the .gamma.-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), < 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30.degree. C. The specific [3H]LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30.degree. C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 .+-. 2.1 nM and a maximum number of binding sites of 850 .+-. 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S,6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 > LTE4 > LTC4 > 5R,6S-LTD4 .mchgt. 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 .+-. 61 fmol/mg of protein with minimal effects on the apparent Kd. Competition binding analyses with [3H]LTD4 indicated that 0.3 mM dithiothreitol pretreatment of heart membranes had minimal effects on Ki values for LTD4 and 2-nor-LTD1. The dithiothreitol-dependent decrease in specific [3H]LTD4 binding was not protected by prior occupation of the binding sites with either the agonist LTD4 or the antagonist 2-nor-LTD1. Specific [3H]LTD4 binding sites in guinea-pig heart membranes are supported. One or more essential disulfide bonds may be required for the interaction of leukotriene D4 with its binding site and LTD4 binding sites in guinea-pig heart may be distinct from LTD4 binding sites in guinea pig-lung.