Nitric oxide (NO ^• ) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S -nitrosates thiol proteins. We developed mitochondria-targeted S -nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S -nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO ^• and S -nitrosated thiol proteins. MitoSNO1-induced NO ^• production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO ^• generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S -nitrosation. These results support the idea that selectively targeting NO ^• donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO^–) and carbonate radical anion ( ) using stopped-flow and pulse radiolysis, respectively. Rate constants for the reactions of Mn(III) porphyrins with ONOO^– ranged from 1 × 10⁵ to 3.4 × 10⁷ m^–1 s^–1 and correlated well with previously reported kinetic and thermodynamic data that reflect the resonance and inductive effects of the substituents on the porphyrin ring. Rate constants for the reactions of Mn(III) porphyrins with ranged from 2 × 10⁸ to 1.2 × 10⁹ m^–1s^–1 at pH ≤ 8.5 and increased with pH as a consequence of the ionization of the complexes. Mn(II) porphyrins reacted with with rate constants ranging from 1 × 10⁹ to 5 × 10⁹ m^–1s^–1 at pH 10.4. Hence, fast scavenging of ONOO^– and by manganese porphyrins could occur in vivo because of the catalytic reduction at the expense of a number of cellular reductants. Additionally, we determined the pK_a of the axial water molecules of the Mn(III) complexes at pH 7.5–13.2 by spectrophotometric titration. Results were consistent with two acid-base equilibria for most of the complexes studied. The pK_a values also correlated with the resonance and inductive effects of the substituents. The correlations of E½ with the rate constants with ONOO^– and with the pK_a values display a deviation from linearity when N-alkylpyridinium substituents included N-alkyl moieties longer than ethyl, which is interpreted in terms of a decrease in the local dielectric constant.

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca²⁺-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca²⁺-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO₂⁻)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO₂-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO₂-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO₂-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO₂⁻ under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO₂-cLA) and nitro-oleic acid (NO₂-OA). The presence of free and protein-adducted NO₂-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO₂-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.