Endothelin-1 enhances plasminogen activator inhibitor-1 production by human brain endothelial cells via protein kinase C-dependent pathway

The effects of endothelin-l (ET-1) on the production of plasminogen activat or inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) by human bra in-derived endothelial cells in culture were studied. At 100 nmol/L, ET-1 i ncreased PAI-1 production by 88+/-6% within 72 hours, and increased PAI-1 m RNB expression within 1 hour of stimulation; there was no significant effec t on t-PA production. PAI-1 activity was also examined and found to increas e with ET-1 treatment. Suboptimal concentrations of ET-1 and tumor necrosis factor-alpha (TNF-alpha) acted synergistically to increase PAI-1 productio n. ET-1 activated protein kinase C and cAMP-dependent protein kinase pathwa ys within 3 to 5 minutes of treatment, with the peak at 10 minutes. Activat ion of protein kinase C by phorbol-12-myristate-13-acetate (PMA) resulted i n increased PAI-1 production, whereas activation of the cAMP-dependent prot ein kinase by forskolin or dibutyryl cAMP (dBu-cAMP) significantly decrease d PAI-1 production. However, simultaneous activation of protein kinase C by PMA and cAMP-dependent protein kinase by dBu-cAMP only slightly attenuated PMA-induced PAI-1 increase. Inhibition of protein kinase C by GF-109213X a bolished the effects of ET-1. These results demonstrate that ET-1 and TNF-a lpha function synergistically to induce procoagulant activity of brain endo thelial cells in a process that involves a protein kinase C-dependent pathw ay.