The mechanisms underlying the regulation of the expression of endothelin-1 (ET-1) in vascular endothelial cells (EC) are not well established. ET_B receptors, the sole ET-1 receptor subtype expressed in EC, are generally perceived as being responsible for the clearance of circulating ET-1. As such, these receptors are well positioned to regulate endothelial ET-1 production. We hypothesised that ET-1-dependent stimulation of ET_B regulates the level of preproET-1 mRNA in passage 1 primary cultures of porcine aorta EC.

Quiescent EC (in serum-free medium) were exposed to ET-1 (0.1 µM) alone or combined with antagonists. To quantify preproET-1 mRNA, competitive reverse transcription (RT) PCR was performed with a mimic RNA containing a 460 bp fragment of rabbit cardiac -actin and gene-specific primer pairs (forward and reverse) for the preproET-1 mRNA at both ends. For RT, a series of serial 5 fold dilutions of the RNA mimic were prepared, combined with 1 µg of sample EC RNA. cDNA was prepared from total RNA by RT reaction and amplified by PCR using oligonucleotide primers specific for porcine preproET-1 mRNA (sense; 5'-ATGGATTATTTCCCCATGATTATCG-3', nucleotides 75-99: antisense; 5'TCAGTGTGTTCGGTTATGGGTCA-3', nucleotides 690-714). These two primers define a specific 640 bp fragment within the preproET-1 mRNA. The products of each PCR reaction were separated, stained and visualised under U.V. light. The mass of each amplified mimic and preproET-1 mRNA fragment DNA was calculated from their fluorescence intensity using the DNA mass ladder. Activation of the p38 MAPK cascade was assessed by measuring the activity of MK-2. Phospho-ERK1/2 was determined using anti-phosphoprotein antibodies. Data are represented as the mean±SEM. P<0.05 was considered as statistically significant (ANOVA, Scheffé's F test).

In EC, exogenous ET-1 markedly reduced preproET-1 mRNA levels, with a maximal effect at 4 hrs (2 amol of preproET-1 mRNA / µg of total RNA compared to 15 fmol/µg in control EC, P<0.05). The negative effect of ET-1 was conserved in the presence of actinomycin D (1µg/ml). Addition of an ET_B selective antagonist, BQ788 (1µM), increased preproET-1 mRNA (0.7 pmol/µg, P<0.05). ET-1 increased (P<0.05) p38 MAPK activity, which peaked at 30 min and returned to control levels within 90 min. In contrast, ET-1 induced a slow increase in phosphoERK1/2, which remained elevated after 4 hrs. Inhibition of the p38 MAPK pathway (SB202190; 1 µM) prevented the decrease in mRNA stability by ET-1, whereas ERK 1/2 inhibition (PD98059, 1 µM), like BQ788, reversed the down-regulatory effect of ET-1. Similarly, inhibition of receptor internalisation with dansylcadaverine (DAN, 50 µM) increased mRNA expression in the presence and absence of ET-1. The effect of DAN was blocked by SB202190.

In EC, ET-1 reduces preproET-1 mRNA stability via ET_B receptors. ET-1 activates p38 leading to internalisation of the ET-1/ET_B complex. ERK1/2 activation follows internalisation and preproET-1 mRNA destabilisation.