Effects of superoxide on signaling pathways in smooth muscle cells from rats

Abstract

The effects of hypoxanthine and xanthine oxidase-induced superoxide anion were evaluated on various signal transduction pathways in aortic smooth muscle cells (SMCs) from spontaneously hypertensive rats (SHR) and Wistar- Kyoto rats (WKY). Superoxide increased inositol 1,4,5-tris-phosphate (IP3) formation in a concentration- and time-dependent manner in both strains but more markedly in SMCs from SHR. Various antioxidants significantly decreased the superoxide-induced 1P3 formation in both strains. In addition, tyrosine kinase inhibitors, genistein and tyrphostin A25, inhibited the superoxide- induced IP3 formation more markedly in SHR than in WKY. Moreover, superoxide decreased the basal level of cGMP to a greater extent in SHR and also suppressed the rise in cGMP induced by S-nitroso-N-acetylpenicillamine. In addition, the superoxide-induced increase in IP3 formation was significantly inhibited by guanylyl cyclase stimulator S-nitroso-N-acetylpenicillamine but was potentiated by ODQ (a guanylyl cyclase inhibitor, 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and KT5823 (a cGMP-dependent protein kinase inhibitor), with a greater effect in SHR. Finally, the superoxide-enhanced IP3 formation was not accompanied by simultaneous changes in cAMP levels, and inhibition of the adenylyl cyclase pathway did not modify the superoxide-induced IP3 formation. Our results thus demonstrate a stimulatory effect of superoxide on 1P3 formation, mediated by the tyrosine kinase-coupled phospholipase C(γ) activity, and an inhibitory effect of superoxide on cGMP formation in vascular SMCs. The increased reactivity of the phospholipase C pathway and the decreased cross inhibition of the IP3 pathway by cGMP in the presence of superoxide may underlie the altered functions of vascular SMCs in SHR.