Characterization of guinea pig myocardial leukotriene C4 binding sites. Regulation by cations and sulfhydryl-directed reagents

GK Hogaboom, S Mong, JM Stadel and ST Crooke

Using [3H]leukotriene C4 (LTC4) and radioligand-binding techniques, specific leukotriene C4 binding sites have been identified in membranes derived from guinea pig ventricular myocardium. High performance liquid chromatography analyses indicated that, in the presence of the gamma- glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 2% of membrane-bound [3H]LTC4 was converted at 20 degrees to [3H]leukotriene D4 or [3H]leukotriene E4. The specific binding of 4 nM [3H]LTC4, in the presence of 80 mM L-serine-borate, reached a stable steady state within 15 min at 20 degrees (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded a dissociation constant (Kd) of 27.5 +/- 6.0 nM and a maximum number of binding sites (Bmax) of 19.9 +/- 5.2 pmol/mg of membrane protein. Competition binding studies of [3H]LTC4 with synthetic leukotriene C4, leukotriene D4, and leukotriene E4 and the putative peptidoleukotriene antagonists FPL 55712, SKF 88046, and 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid revealed an order of potency of leukotriene C4 much greater than 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid greater than SKF 88046 greater than LTE4 greater than LTD4 greater than FPL 55712. The specific [3H]LTC4 binding was stimulated by the divalent cations Ca2+, Mg2+, and Mn2+ and to a lesser degree by the monovalent cations Na+, K+, Li+, and NH4+. CaCl2 (3 mM) and NaCl (150 mM) stimulated the LTC4 binding by increasing the Bmax to 42.6 +/- 5.9 and 35.0 +/- 2.0 pmol/mg, respectively, but had minimal effects on Kd. Pretreatment of the heart membranes with the sulfhydryl reagent N-ethylmaleimide decreased the specific [3H]LTC4 binding in a concentration-dependent manner. The N-ethylmaleimide-induced inactivation of [3H]LTC4 binding sites was protected by occupation of the binding site with the agonist leukotriene C4, but no protection was observed with the antagonist SKF 88046. Scatchard analyses of saturation isotherms indicated that 30 microM N-ethylmaleimide pretreatment reduced the Bmax of the [3H]LTC4 binding to 8.2 +/- 3.1 pmol/mg with minimal effects on Kd. The data provide direct biochemical evidence for specific [3H]LTC4 binding sites in the guinea pig heart membranes. The [3H]LTC4 binding sites appear to be modulated by divalent and monovalent cations and free sulfhydryl group(s) may be associated with the agonist-binding site. The results suggest that the physiological effects of the leukotrienes on the guinea pig heart may be mediated through membrane-bound receptors.

Volume 27, Issue 2, pp. 236-245, 02/01/1985
Copyright © 1985 by American Society for Pharmacology and Experimental Therapeutics

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