We further note that application of similar high concentrations of GSNO to skinned fibres has been previously demonstrated to indeed produce S-nitrosylation of key contractile proteins (including fast troponin I, TnI f), shown both by using antibodies to CysNO and by the biotin switch method ( Nogueira et al. Our aim was to induce the final state and not to try to emulate the unknown reactions actually occurring in intact fibres in vivo. NO itself, or even more likely that it occurred via transnitrosation ( Foster et al., 2003 Halliwell & Gutteridge, 2007).We fully agree that the S-nitrosylation of the cysteine residues was probably brought about by higher oxides of In our study we used various agents including GSNO to produce both S-nitrosylation and S-glutathionylation of proteins in the skinned fibres. This suggests that the major acute effects reported here probably do generally reflect those happening in intact fibres …’ We point out, however, that the findings here and in our earlier skinned fibre studies ( Lamb & Posterino, 2003 Posterino et al., 2003 Murphy et al., 2008) do seem in generally good accord with those found when applying ROS and RNS in intact fibres ( Andrade et al., 1998 a, b), such as in regard to the increases or decreases in contractile sensitivity occurring without change in maximum force, and the lack of major changes in fibre excitability and Ca 2+ release. Consequently, it is possible that ROS and RNS generated within, or applied to, an intact muscle fibre might exert different or additional effects to those found here. In regard to the above points, we note that we wrote the following in our paper in the section Consideration of experimental limitations: ‘Firstly, the results here were all obtained in skinned fibres, where various diffusible intracellular constituents present in vivo would have been lost from the fibre, possibly including redox-sensitive kinases or phosphatases or other co-factors.
myoglobin, GSH, etc.) in order to determine the effects of each individually, which is not possible in intact cells where the relevant reactions, and identities and concentrations of the myriad reactive entities and intermediates, are unknown and effectively not determinable. Indeed, this is one of the very strengths of this type of skinned fibre examination, as it makes it possible to sequentially add back in various normal constituents (e.g.
Furthermore, we need to point out that in these skinned fibre experiments all the diffusible cytoplasmic constituents have been entirely lost from the preparation, and so the additional pathways to which Dr Tsikas refers were absent in these experiments. 2008), the experiments involved applying saturating concentrations of the agents sufficient to induce maximal activation of the particular processes under examination. Just as in virtually all biochemical studies of isolated proteins and systems, and as in our previous skinned fibre studies using oxidants such as H 2O 2 and dithiodipyridine (DTDP) ( Lamb & Posterino, 2003 Murphy et al. Firstly, although not explicitly stated in the work in question, we in no way meant to imply that the concentrations of agents applied to the skinned fibre preparation were at all comparable with those pertaining within muscle fibres or other cells. We do nevertheless wish to make some particular points about our experiments. We agree with points Dr Tsikas makes about the chemistry of GSNO, indeed specifically having made some of them ourselves. We thank Dr Tsikas for his interest in our work ( Dutka et al.
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