Dynamic denitrosylation via S -nitrosoglutathione reductase regulates cardiovascular function

Abstract

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.excitation-contraction coupling | nitroso-redox imbalance G uanosine 3′,5′-cyclic monophosphate (cGMP)-dependent and -independent signaling by nitric oxide (NO) has been described in many organ systems, including the cardiovascular (CV) system (1, 2). Accumulating evidence indicates that the principal non-cGMP signal is effected by the covalent attachment of NO to the thiol group of cysteine (Cys) residues (Snitrosylation) (3) and that this posttranslational modification may influence cardiac contractility (4) and peripheral vascular resistance (5) through effects on ion channels (6) and adrenergic receptors (7). Because deletion or inhibition of NO synthase (NOS) diminishes all forms of NO bioactivity and thus impairs both cGMP and S-nitrosylation signaling, it has been difficult to elucidate the exact roles of S-nitrosylation vs. cGMP in CV regulation.Investigation of the role of S-nitrosylation in cellular signaling has been aided by discovery of enzymes that metabolize Snitrosothiols (SNOs) without affecting NOS activity or levels of NO itself (mammalian enzymes that directly metabolize NO have not been identified) (8-10). In particular, S-nitrosoglutathione reductase (GSNOR), an enzyme involved in the removal of NO groups from Cys thiols in proteins (SNO-proteins) through metabolism of S-nitrosoglutathione (GSNO, which is in equilibrium with SNO-proteins), has been ascribed an indispensable role in regulating S-nitrosylation in the CV system (9). Although GSNOR does not affect baseline blood pressure, it mitigates hypotension induced by anesthetics and infectious agents (5) and plays an essential role in regulating both β-adrenergic receptor expression and responsiveness in the heart (7). These studies suggest that SNOs may exert physiological roles in the control of systemic hemodynamics and cardiac contractility.In the CV system, endothelial NOS (NOS3, eNOS) and neuronal NOS (NOS1, nNOS) subserve endothe...

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

“…Surprisingly, but consistent with our results, NTG increased responsiveness to Ca 2ϩ in NOS1 Ϫ/Ϫ (Fig. 6, B and D), as shown by a leftward shift (EC 50 (Fig. 6E).…”

“…This effect would be consis-tent with a cGMP-dependent regulation of L-type Ca 2ϩ channels and/or restoration of RyR2 S-nitrosylation (33,50) and occurs without affecting cardiac contractility, which correlates with the observed improvement in myofilament sensitivity induced by NTG in NOS1 Ϫ/Ϫ CMs. This improvement may also be attributed to the restoration of the S-nitrosylation/normal redox state of redox-sensitive sites on sarcomeric proteins.…”

“…Amplex Red XOR activity was inhibited by allopurinol (Ͼ0.01 mM, IC 50 0.19 Ϯ 0.06 mM, Fig. 5D; n ϭ 3) and HYD (IC 50 Ϫ/Ϫ in a concentration-dependent manner toward normal levels as exhibited by WT CMs. NTG alone had no significant effects.…”

“…4F shows the drug concentration-SR Ca 2ϩ leak response relationship. The IC 50 for HYD was 0.0705 Ϯ 0.0473 M (at Fig. 4G; p ϭ 0.0186).…”

Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia

“…Mechanistically, hemoglobin allostery mediates both the sensing of O 2 concentration and the transduction of O 2 -coupled SNO-based signaling. Our observation that Cysβ93-derived NO bioactivity participates in both hypoxia-and flow-coupled responses, ascribed previously to endothelium-derived NO, provides a unifying explanation for the recent findings that GSNO and other SNOs in equilibrium with SNO-Hb are exported from RBCs under hypoxia (4,10,15,17) and that, at least in part, EDRF is identified in vivo with GSNO (by strict genetic criteria) (45,46). Thus, RBC-endothelium interactions that regulate microvascular blood flow and tissue oxygenation, and thereby maintain organ function, are best understood in terms of coupled equilibria that govern SNO-based bioactivity in blood and vasculature viewed as an integrated system.…”