Identification and characterization of leukotriene C 4 and D 4 receptors on a cultured smooth muscle cell line BC3H-1

Abstract

We studied the characteristics of the leukotriene (LT) C 4 and D 4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [ 3H]LTC 4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 ± 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 ± 2.0 nM (n=9). The association and dissociation of [ 3H]LTC 4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [ 3H]LTC 4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC 4 to compete for the specific binding of [ 3H]LTC 4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [ 3H]LTC 4 binding was LTC 4 ⪢ LTD 4 > LTE 4 > FPL-55712. The maximum number of binding sites (Bmax) of [ 3H]LTD 4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [ 3H]LTC 4. The calculated values of Kd and Bmax of [ 3H]LTD 4 binding were 9.3 ± 0.8 nM and 0.37 ± 0.04 pmol/mg protein, respectively ( n=3). The rank order of potency of the unlabeled competitors for competing specific [ 3H]LTD 4 binding was LTD 4 = LTE 4 > FPL-55712 ⪢ LTC 4. These findings demonstrate that BC3H-1 cell line possess both LTC 4 and LTD 4 receptors with a predominance of LTC 4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC 4 and LTD 4 receptors.