Stimulation of Anchorage-independent Cell Growth by Endothelin in NRK 49F Cells¹

Endothelin (ET) is a vasoconstrictor peptide originally isolated from vascular endothelial cells. Recent studies have revealed that ET has many biological functions including growth factor-like activity. The present study aims to clarify whether ET-1 possesses the ability to stimulate anchorage-independent cellular growth, an indicator of factors with transforming activity.

We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled ¹²⁵I-ET-1 binding was displaced by unlabeled ET-2 in a similar dose response, but in the case of ET-3, 100-fold more was required. Specific ¹²⁵I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are ET-1/ET-2 selective.

NRK 49F is a cell line which is most commonly used to assay for anchorage-independent cellular growth. Therefore, we explored whether ETs promote anchorage-independent cellular growth in this cell line. ET-1 and ET-2 stimulated NRK colony formation dose dependently in the presence of 1 nm epidermal growth factor (EGF). In contrast, ET-3 did not have colony-stimulating ability. In the presence of EGF, the maximal effect of ET-1 was approximately 90% of that of transforming growth factor-β. Moreover, in the presence of maximal stimulating concentrations of EGF and transforming growth factor-β, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess transforming growth factor-like activity for NRK 49F cells. Since ET-1 and -2 increased intracellular calcium levels, this ion may participate in signal transduction pathways by which ET-1 and -2 promote colony formation.