P38: Mechanisms for cross-membrane vasodilatory signaling of nitrosothiols in isolated sheep femoral arteries

Abstract

Nitrosothiols (SNOs) are proposed to play an important role in nitric oxide (NO) mediated vasodilation. Vasoactivity of SNOs rely, at least in part, on transfer of NO equivalent from extracellular SNOs into smooth muscle cells. There are several possible pathways for this cross-membrane vasodilatory signaling. First, SNOs may release free NO (NO ) outside the cell membrane followed by diffusion of the NO into cell to activate guanylyl cyclase. Second, SNOs may be taken up into cell via l -type amino acid transporter (LAT) before releasing NO within cell. A third possibility is that SNOs may transnitrosate thiol groups on cell membrane to initiate trans-membrane signaling events. These experiments test for the role of these three possibilities by using selective blockers of each pathway, and by comparison of vasodilating effects of SNOs which have distinct stereo conformation and membrane permeability. Endothelium-denuded sheep femoral arterial segments were mounted on a wire myograph, pre-contracted with phenylephrine (10 μmol/L), and then subjected to dose–responses to various SNOs. To test the extent to which SNO-mediated dilation occurs by NO activation of guanylyl cyclase, experiments were carried out in the presence of the competitive guanylyl cyclase inhibitor ODQ (10 μmol/L). To test whether SNO-mediated dilation involves the release of NO outside the cell, experiments were performed in the presence of CPTIO (200 μmol/L), a membrane impermeable NO scavenger. To test for a role for the stereo-selective LAT, experiments were performed with S -nitroso- l -cysteine ( l -cysNO) which is a substrate of LAT, compared to its stereoisomer S -nitroso- d -cysteine ( d -cysNO). In addition, we measured responses to the membrane impermeable low and high molecular weight SNOs, S -nitroso-glutathione (GSNO) and S -nitroso-albumin (AlbSNO). Experiments were also performed in the presence of threonine (10 mmol/L) and BCH (10 mmol/L), inhibitors of the LAT. To test for the possibility of transnitrosation of membrane thiols, experiments were carried out after incubation of vessels with impermeable thiol modifier HMBA (10 μmol/L; extensively washed out before application of SNOs to artery). The vasodilation of l -cysNO was significantly attenuated by competitive guanylyl cyclase inhibitor ODQ and partially attenuated by impermeable NO scavenger CPTIO. Surprisingly, vasodilation was significantly potentiated by the LAT inhibitors threonine and BCH. Vasodilation was not affected by blocking extracellular thiols with HMBA. The vasoactivities of d -cysNO, GSNO and AlbSNO were similar ( p > 0.05 for EC 50 s) to that of l -cysNO. In addition, the effects of ODQ, CPTIO, threonine, BCH and HMBA on d -cysNO were all similar ( p > 0.05 for EC 50 s) to that on l -cysNO. Our results suggest that the SNO-derived vasodilating NO equivalent is not NO released outside the cell membrane. Besides, uptake of SNO as a whole is not a prerequisite for its vasodilation, but weakens vasodilation instead. Further more, membrane thiol was not involved in the cross-membrane vasodilatory signaling of SNOs. Nothing to disclose.