Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium.
[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees 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 degrees 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 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hogaboom
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Mong
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Stadel
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Crooke
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000032:analysis* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis*; D018077:Receptors, Leukotriene; D011982:Receptors, Prostaglandin / Q000032:analysis*; D013189:SRS-A / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014316:Tritium
D011956:Receptors, Cell Surface; D018077:Receptors, Leukotriene; D011982:Receptors, Prostaglandin; D013189:SRS-A; D013439:Sulfhydryl Reagents; D014316:Tritium