Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

8-Bromo-7-hydroxyquinoline as a photoremovable protecting group for physiological use: mechanism and scope.

Two-photon excitation (2PE) of "caged" biomolecules represents a powerful method to investigate the temporal and spatial relevance of physiological function in real time and on living tissue, because the excitation volume can be restricted to 1 fL. Additionally, low-energy IR light is used, which minimizes tissue destruction and enables deeper penetration into tissue preparations. Exploitation of this technology for studying cell physiology requires the further development of photoremovable protecting groups with sufficient sensitivity to 2PE for use in "caged" compounds. 8-Bromo-7-hydroxyquinoline (BHQ) is efficiently photolyzed by classic 1PE (365 nm) and 2PE (740 nm) under simulated physiological conditions (aqueous buffer of high ionic strength, pH 7.2) to release carboxylates, phosphates, and diols-functional groups commonly found on bioactive molecules such as neurotransmitters, nucleic acids, and drugs. It is stable in the dark, soluble in water, and exhibits low levels of fluorescence, which will enable use in conjunction with fluorescent indicators of biological function. BHQ-protected effectors are synthetically accessible. Stern-Volmer quenching, time-resolved infrared (TRIR), and (18)O-labeling experiments suggest that the photolysis occurs through a solvent-assisted photoheterolysis (S(N)1) reaction mechanism on the sub-microsecond time scale. BHQ has the requisite photochemical and photophysical properties as a photoremovable protecting group to regulate the action of biological effectors in cell and tissue culture with light, especially 2PE.

Photosensitive precursors to nitric oxide.

The photochemistry and use of recently developed photosensitive precursors to nitric oxide (NO) are reviewed. As "caged NO" donors these precursors are able to deliver NO in a spatially and temporally controllable manner. These properties have made such precursors useful in applications in biology and medicine, especially in elucidating neurophysiological roles of NO and in new cancer therapies.

Controlled photochemical release of nitric oxide from O2-substituted diazeniumdiolates.

Diazeniumdiolates are a well-established class of nitric oxide (NO) donors that have been employed in a wide variety of biochemical and pharmacological investigations. To provide a means of targeting NO release, photosensitive precursors to diazeniumdiolates have been developed and are reviewed here. After a brief description of diazeniumdiolate chemistry and the potential uses of photosensitive precursors to NO, three different classes of phototriggered diazeniumdiolates are discussed: 2-nitrobenzyl derivatives, meta-substituted benzyl derivatives, and naphthylmethyl and naphthylallyl derivatives. In addition, the photochemistry of diazeniumdiolate salts themselves is covered.

Photochemistry of 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate: an experimental and computational investigation.

The aqueous photochemistry of the sodium salt of 1-(N,N-diethylamino)-diazen-1-ium-1,2-diolate (3) has been investigated by both experimental and computational methods. Photolysis results in the formation of the N-nitrosodiethylamine radical anion (5) and nitric oxide (NO) via a triplet excited state. The nitrosamine radical anion either undergoes electron transfer with NO before cage escape to form triplet NO(-) and nitrosamine (minor process) or rapidly dissociates to form an additional molecule of NO and ultimately amine (major process). The production of nitrosamine radical anion 5 upon photolysis of diazeniumdiolate 3 is confirmed by low-temperature EPR spectroscopy. The calculated energetics for the ground and excited states of the parent diazeniumdiolate ion at the CIS and B3LYP levels of theory as well as B3LYP calculations on the fragmentation processes were very effective in rationalizing the observed photodissociation processes.

Controlled photochemical release of nitric oxide from O(2)-naphthylmethyl- and O(2)-naphthylallyl-substituted diazeniumdiolates.

The photochemistry of O2-naphthylmethyl- and O2-naphthylallyl-substituted diazeniumdiolates has been investigated. Electron-donating methoxy group substitution is shown to have a significant effect on the observed photochemistry, with the appropriate substitution pattern resulting in efficient diazeniumdiolate photorelease. Observed nitric oxide release rates from these photoprecursors are consistent with those expected for normal thermal dissociation of the diazeniumdiolate in aqueous solutions and show the same pH dependence.

Controlled photochemical release of nitric oxide from O2-benzyl-substituted diazeniumdiolates.

An investigation of potential photosensitive protecting groups for diazeniumdiolates (R2N-N(O)=NO-) has been initiated, and here the effect of meta electron-donating groups on the photochemistry of O2-benzyl-substituted diazeniumdiolates (R2N-N(O)=NOCH2Ar) is reported. Photolysis of the parent benzyl derivative (Ar = Ph) results almost exclusively in undesired photochemistry-the formation of nitrosamine and an oxynitrene intermediate with very little, if any, photorelease of the diazeniumdiolate. We have been able to use meta substitution to tune the photochemistry of these benzylic systems. The desired diazeniumdiolate photorelease has been shown to become more substantial with stronger pi-donating meta substituents. This effect has been verified by direct observation of the photoreleased diazeniumdiolate with 1H NMR spectroscopy and by NO quantification measurements conducted in high- and low-pH solutions. In addition, the observed rates of NO release are consistent with that expected for normal thermal decomposition of the diazeniumdiolate in aqueous solutions and also show the same pH dependence.