Apocynin exerts rapid inhibition and slow stimulation of I Ca,L associated with changes of reactive oxygen species in vascular smooth muscle cells

Regulation of Ca 2+ current by endogenous reactive oxygen species (ROS) has been little clarified. Apocynin (APO), a methoxy‐substituted catechol with NADPH oxidase inhibitor and anti‐ and pro‐oxidant activities, reversibly inhibits I Ca,L . We recorded I Ca,L and dichlorofluorescein fluorescence (DCFF), a signal of ROS, continuously, before, during and after application of APO (6 mM, 5min) in A7r5 rat aortic smooth muscle cells. APO inhibited I Ca,L by ~50% and significantly decreased DCFF. Washout of APO (WO) rapidly increased I Ca,L and DCFF to above control. N‐Acetylcysteine (NAC, 10 mM) little affected the basal and WO‐induced DCFF increase but decreased APO‐induced decrease of I Ca,L . Ebselen (Ebs, 1 μM), a H 2 O 2 scavenger, markedly and irreversibly inhibited I Ca,L and abolished WO‐induced increases of I Ca,L and DCFF. NAC prevented the Ebs‐induced decrease of I Ca,L and in presence of NAC, Ebs (10 μM) significantly decreased WO‐induced increases of I Ca,L and DCFF. APO rapidly decreased GSH/GSSG, which was compromised by NAC. APO decreased ROS (H 2 O 2 ) and GSH to inhibit I Ca,L apparently by glutathione peroxidase action. Additional slow pro‐oxidant action of APO stimulated generation of ROS, which was manifested as WO‐induced increase of ROS and I Ca,L . We conclude that H 2 O 2 and GSH are intimately involved in the regulation of I Ca,L in vascular smooth muscles. This study is supported by NIH grant #R01HL85352