Aim: 

Endothelin-1 is a strong vasoconstrictor and growth factor expressed mainly in the endothelium. Under pathological conditions, the peptide can be produced in vascular smooth muscle cells (VSMC). Regulation of preproendothelin-1 (pET-1) expression in VSMC has not been extensively studied. Our aim was to study vasopressin-induced expression of pET-1 in a rat aortic smooth muscle cell line, A7r5.

Methods: 

Subconfluent serum-deprived cells were used. Gene expression of pET-1 was quantitated using radioactive RT-PCR followed by phosphorimager analysis. Ribosomal Protein Large-32 (RPL) was used as a housekeeping gene.

Results: 

A7r5 cells expressed pET-1 mRNA under basal conditions. Vasopressin concentration-dependently increased the expression of pET-1 with a maximum about 3-fold of the basal value and EC50 of 5.5nM. Transcription blockade with actinomycin D suppressed induction of pET-1 by vasopressin. However, actinomycin D chase experiments showed that vasopressin also stabilized pET-1 mRNA, increasing its half-life from approx. 20-50min. Cycloheximide, an inhibitor of protein synthesis, caused an induction of pET-1 and a superinduction of pET-1 by vasopressin. Removal of extracellular calcium had no effect on pET-1 induction, while chelation of intracellular calcium with BAPTA decreased basal and vasopressin-stimulated pET-1 expression. Thapsigargin stimulated pET-1 expression, which was blocked by BAPTA.

Conclusions: 

A7r5 cells express pET-1 mRNA and this expression is increased by vasopressin. Transcriptional and posttransctriptional mechanisms are involved in the regulation of pET-1 biosynthesis in these cells. Release of calcium from intracellular stores, rather than calcium influx from the extracellular space, is important for pET-1 expression in these cells.