Cell Calcium 37 (2005) 87–96 Nitroxyl triggers Ca 2+ release from skeletal and cardiac sarcoplasmic reticulum by oxidizing ryanodine receptors Eunji Cheong a , Vassil Tumbev a , Jon Abramson b , Guy Salama a , Detcho A. Stoyanovsky c,∗ a Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA b Department of Physics, Portland State University, Portland, OR 97207, USA c Department of Surgery, University of Pittsburgh, 3459 Fifth Ave, MUH-NW 654, Pittsburgh, PA 15213, USA Received 18 March 2004; received in revised form 8 July 2004; accepted 13 July 2004 Abstract The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli’s salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca 2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca 2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056 ± 0.026 to 0.270 ± 0.102 (P < 0.005, n = 4) then DTT (3 mM) reduced Po to 0.096 ± 0.040 (P < 0.01, n = 4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca 2+ release indicating that picomolar concentrations of HNO triggered SR Ca 2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca 2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca 2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO’s activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure. © 2004 Elsevier Ltd. All rights reserved. Keywords: NO; Ca 2+ release; Ryanodine receptors; Nitroxyl 1. Introduction The sarcoplasmic reticulum (SR) is the major storage compartment of intracellular Ca 2+ in striated muscle that controls cytosolic free Ca 2+ and force generation by se- questering and releasing Ca 2+ during each contraction. The release of Ca 2+ from skeletal and cardiac SR is mediated by a high conductance Ca 2+ release channel or ryanodine receptor (RyR). Studies aimed at the identification, cloning and characterization of the RyRs have shown that this protein exists in three highly homologous isoforms [1]. Extensive experiments on intact muscle, isolated SR vesicles ∗ Corresponding author. Tel.: +1 412 647 6087; fax: +1 412 647 5959. E-mail address: stoyanovskyd@upmc.edu (D.A. Stoyanovsky). and RyRs channel proteins reconstituted in planar bilayers have identified numerous potentially physiological signaling processes and pharmacological agents as activators and/or inhibitors of RyRs [2]. However, the molecular mechanisms that link the electrical excitation of skeletal and cardiac muscle plasma membranes to the opening of RyRs to release Ca 2+ from the SR and the process responsible for the closure of RyRs remain incomplete. Among the various activators and inhibitors of RyRs, the oxidation and reduction of RyRs was shown to be one of the most potent mechanisms that can reversibly open and close the channel [3,4]. The hypothesis that redox modifi- cation of critical or hyper reactive thiols on the RyR is a mechanism involved in the regulation of this class of ion channels has become increasingly persuasive. Initial studies 0143-4160/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2004.07.001