Cloning of bovine preproadrenomedullin and inhibition of its basal expression in vascular endothelial cells by staurosporine

Abstract

The cDNA encoding preproadrenomedullin (preproAM) was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends from total RNA from bovine aortic endothelial cells (BAEC). Bovine preproAM cDNA shows high sequence homology with human, porcine and rat preproAM. Bovine-specific primers derived from this sequence were used in RT-PCR to study regulation of this gene. Treatment of BAEC or a human endothelial cell line (Ea.hy 926) with the non-selective protein kinase inhibitor staurosporine resulted in significantly reduced preproAM mRNA levels. The reduction in preproAM mRNA appeared to be absolute when Ea.hy 926 cells were exposed to 100 nM staurosporine for 2 h. However, this dramatic reduction could not be reproduced by treatment with the protein kinase A (PKA) inhibitor H-89, or the protein kinase C (PKC) inhibitors chelerythrine chloride and bisindolylmaleimide I. These observations suggest that activation of a novel staurosporine-sensitive protein kinase is necessary for basal expression of the preproAM gene in these cells.