The Effect Of Nitric Oxide Donors On Heart Rate PDF Download

Nitric oxide (NO) may modulate mitochondrial O2 consumption in the heart, and provide another level of respiratory control under normal or hypoxic conditions. NO and NO donors have also been proposed to have both beneficial and deleterious effects during ischaemia-reperfusion (IR) injury, and both of these maybe mediated by interactions of NO with mitochondria. The aims of the research in this thesis were to investigate (i) whether there is a specific form of mitochondrial nitric oxide synthase (NOS), (ii) which NO donors release NO spontaneously and which require bioactivation, (iii) whether different NO donors are protective or deleterious during IR injury, (iv) the effect of NO donors on mitochondrial membrane permeability transition pore (mPTP) opening. In this study, using 3 different techniques (Western blotting, immunoprecipitation and DAF fluorescence), no evidence was found to suggest the existence mitochondrial NOS in highly purified heart and liver mitochondria. However, it was possible to detect all forms of NOS in crude heart and liver mitochondria, and the same applied for the ryanodine receptor and caveolin, in "mitochondrial" fractions. Despite the fact that NO donors are commonly used in medicine and in research, in many cases it is still unclear as to how NO is released from the NO donors; whether it is spontaneously released or whether bioactivation is required. Using the NO- sensitive fluorescent dyes DAF-2 and DAF-2 DA, the results of this study showed that the NO donors DEA and SIN-l spontaneously released NO. SNAP, a donor that has been assumed to be spontaneous, showed no release of NO. Sodium nitroprusside did show significant spontaneous release of NO, but only at 5 and 10 mM. The novel NO donor NCX2057 was the only NO donor that showed release of NO by mitochondrial metabolism. The nitrates BDMN and ISMN both released NO in post-mitochondrial supernatant, indicating the involvement of cytosolic enzymes in NO donor metabolism. Previous studies have shown that NO can have both protective and deleterious effects in whole heart IR injury. In this study it was shown that the administration of the NO donors, SNAP and DEA before the onset of global ischaemia and throughout reperfusion, resulted in a concentration-dependent protection against IR injury. Beyond a concentration threshold level (40 ~ SNAP and 2 ~ DEA), the protection was lost. Since IR injury is critically regulated by the opening and closing of the mPTP during reperfusion, the effects of DEA on mPTP opening, mitochondrial Ca2+ uptake and membrane potential were investigated using isolated mitochondria. The lower concentrations of DEA (2-25 flM) caused partial mitochondrial membrane depolarisation, sensitised the mPTP to Ca2+ and promoted mPTP opening. By contrast, the higher concentrations of DEA depolarised the mitochondrial membrane potential, prevented Ca2+ uptake and inhibited mPTP opening. This biphasic effect observed could be attributed to the inhibition of cytochrome c oxidase, as potassium cyanide had similar effects on membrane potential, Ca2+ uptake and mPTP opening as DEA. DEA also inhibited mPTP opening under de-energised conditions, indicating that in addition to modulating mPTP opening via inhibition of cytochrome c oxidase, DEA can also directly interact and modify mPTP components, possibly via nitration.