EFFECTS OF NICOTINE AND COTININE ON THE SYNTHESIS AND RELEASE OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS

Abstract

While cigarette smoking has beenimplicated in the development of cardiovascular diseases, it has been reported to increase fibrinolytic activity in vivo. However, no data is available regarding the underlying mechanism of action. The present investigation was carried out to evaluate the effects of nicotine and its major metabolite, cotinine, on the seretion of plasminogen activator (PA) and PA inhibitor (PAI) by cultured bovine aortic endothelial cells. PA activity was determined by the fibrin plate method, and individual molecular species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both nicotine and cotinine increased PA secretion in a time- and dose-dependent manner. A maximum stimulation in PA secretion after 24-hour incubation was observed for nicotine at 10-8 M and for cotinine at 10-7> M, which corresponded to 2.5- and 2.7-foldincreases over control, respectively. These concentrations are in the range observed after cigarette smoking. The pharmacological stimulation of PA activity required both RNA and protein synthesis, as evidencedby its inhibition by cycloheximide and actinomycin D. Both control cells and cells treated with nicotine and cotinine produced multiple forms of PA and a single form of PAI. The PAI was mainly of the latent form as no quenching effect was observed on standard tissue plasminogen activator (tPA) and urokinase (UK) after they were mixed with the conditioned culture medium. The PAs werefound to consist of both tPA and UK,and the corresponding complexes with PAI, however, the UK bands were wider than the tPA bands. Both species were enhanced by nicotine and cotinine. Although activities of all species of PA were enhanced by nicotine and cotinine, these compounds had no apparent quantitative or qualitative effects on the release ofPAI. These results thus suggest that the mechanism underlying the enhanced fibrinolytic activity after cigarette smoking may be due to nicotine- and cotinine-induced stimulation of PA synthesis and subsequent release.