• Increased S-nitrosylation Impairs Contraction and Relaxation in Mouse Aorta

      Choi, Hyehun; Department of Physiology (2011-06)
      S-Nitrosylation is a ubiquitous protein modification in redox-based signaling. This modification uses nitric oxide (NO) to forms S-nitrosothiol (SNO) on cysteine residues. Thioredoxin (Trx) and Trx reductase (TrxR) play a role in limiting Snitrosylation. We hypothesized overall that S-nitrosylation of intracellular signaling molecules impairs contraction and relaxation of vascular smooth muscle cells. Aortic rings from C57BL/6 mice were used to measure vascular contraction and relaxation. The rings were treated with TrxR inhibitors, auranofin or 1-chloro-2,4-dinitrobenzene (DNCB), and/or NO donors, propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO), to increase Snitrosylation. Contractile responses of aortic rings to phorbol-12,13-dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. PKCa S-nitrosylation was increased by a TrxR inhibitor and CysNO; concomitantly, PKCa activity and downstream signaling were inhibited as compared to control protein. Vascular relaxation in aortic rings from normotensive (Sham) and angiotensin II (Angll)-induced hypertensive mice was measured after contraction with phenylephrine in the presence or absence of DNCB. DNCB reduced relaxation to acetylcholine (ACh) compared to vehicle, but the antioxidants, apocynin and tempol, normalized DNCB-induced impaired relaxation to ACh in sham aorta. Soluble guanylyl cyclase (sGC) S-nitrosylation was increased by DNCB, and sGC activity (cyclic GMP assay) was reduced in sham aorta. In aortic rings from Angll-treated mice, DNCB did not change relaxation to ACh compared to vehicle. DNCB decreased relaxation to sodium nitroprusside (SNP) in aortic rings from both sham and Angll mice. Total protein S-nitrosylation was enhanced in Angll aorta compared to sham, and TrxR activity was inhibited in Angll aorta compared to sham. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits contractile responses to PDBu. TrxR inhibition reduces vascular relaxation via increasing oxidative stress and sGC S-nitrosylation. In Angll-induced hypertensive mice, augmented S-nitrosylation is associated with impaired vasodilation. Thus, TrxR and Snitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.