Figure 2. Induction of AR by ET-1 is specific to GPCR pathway.

LNCaP cells cultured in medium containing 5% CS were pre-treated with recombinant neutral endopeptidase (NEP) (50 ug/ml) which degrades ET-1 (A), ET-1 receptor antagonists BQ123 (1 uM) and BQ788 (1 uM) (B), and then treated with 50 nM ET-1 for 18 hrs. Western blots based on these cell lysates were conducted and probed with AR and actin antibodies. (C) Real time PCR analysis of the up-regulation of androgen receptor mRNAs by ET-1. Message RNAs prepared from LNCaP cells cultured in medium containing 5% CS and treated with 50 nM of ET-1 for 18 hours following pre-exposure to BQ123, an ET_A receptor antagonist, or neutral endopeptidase (NEP) were subjected to real time PCR analysis with a pair of primers specific to AR and normalized with 18s rRNA. The treatment of 10 nM DHT was used as positive control. Relative levels of androgen receptor mRNAs were calculated by taking control (no treatment) as 100. Experiments were repeated at least twice with similar results. All data represent the average of one experiment and are presented as mean ± SEM, ** p < 0.01 represent statistical significance compared to the value of control. All experiments were repeated at least two times with three independent preparations of cell lysates with similar results.