Figure 1
RT-PCR, [3H]ICI198,615 binding and LTD 4 -induced [3H]thymidine incorporation. (A) Final RT-PCR products obtained using inner primers for human CysLT1-R (Upper Panel) and CysLT2-R (Lower Panel). The PCR mediated amplification of cDNA produced the expected 948 bp (CysLT1) and 1117 bp fragments (CysLT2), which were visualized upon agarose gel, by UV irradiation. Lane 1, purchased HASMC and lane 2, HASMC isolated in our lab; STD, standard. (B) Equilibrium binding curve of [3H]ICI198,615 in membranes from HASMC. Mixed type binding curves were performed using 0.1–1 nM [3H]ICI198,615 (saturation part of the curve) and 1 nM – 1 μM of unlabeled ICI198.615 (competition part of the curve). Binding is expressed as the ratio of bound ligand concentration over total ligand concentration, (B/T, dimensionless), vs. the logarithm of total ligand concentration. B (in M) is the sum of "hot", "cold", and non-specific binding; T (in M) is the sum of "hot" and "cold" ligand incubated. Data shown are representative of two independent experiments simultaneously analyzed with LIGAND. (C-D) Effect of LTD4, EGF, and CysLT1-R antagonists on [3H]thymidine incorporation in HASMC. Increase of [3H]thymidine incorporation induced by 1 μM LTD4 and 20 ng/ml EGF alone or in combination, in the absence and presence of 1 μM of the antagonists pranlukast (C) and zafirlukast (D) (30 min pretreatment). Control is represented by MEM additioned with 1% FBS. The results are presented as mean ± S.E.M. of at least three experiments performed in triplicate on two different cell lines. **P < 0.01 (one-way ANOVA).