Cyclic amp-dependent protein kinase : a potential target for actions of vanadium

Vanadium salts and organic complexes diminish or reverse many of the consequences of insulin deficiency and insulin resistance in vivo. It is widely assumed that the inhibition of protein tyrosine phosphatases can explain biological effects of vanadium; however, there is considerable evidence in vitro that vanadium can act significantly downstream from the protein tyrosine phosphorylation "level" of signal transduction. The aim of these studies was to focus on the ability of vanadium to inhibit hormone-sensitive triglyceride (TG) hydrolysis because this action is observed at rather low vanadium concentrations (typically 10-100 μM) and the mechanisms involved in the control of TG hydrolysis are well defined. Based on the balance of prior studies, this thesis focused on the possibility that cAMP-dependent protein kinase (PKA) might be a viable target for inhibition by vanadium. These studies confirmed that PKA could be potently inhibited by vanadium. Due to the complexity of interactions between vanadate, vanadyl and reagents used in assay mixtures, it was essential to define the experimental conditions carefully to allow unambiguous characterization of the effects of vanadium. Following initial optimization of enzyme activity, PKA was found to be inhibited by high concentrations of vanadyl sulphate (VS) (IC₅₀ > 400 μM). However, PKA inhibition was seen at dramatically lower VS concentration (IC₅₀< 25 μM) when sequestration of vanadyl ions was minimized. Under these conditions, the true concentration of vanadyl was lower than the threshold for detection by EPR spectroscopy (~ 15 μM). The derived kinetic constants (Κ[sub i] values < 20 μM) must still be considered "apparent" values and the true affinity constant of vanadyl for PKA is probably even lower. The effective PKA inhibitor species is likely to be vanadyl because a range of divalent cation chelators abolished PKA inhibition by VS. Vanadyl was both a weak cofactor and a strong inhibitor of PKA, perhaps replicating the dual roles hypothesized for magnesium. From the results of EPR and kinetic studies, it was concluded that the vanadyl EPR signal is enhanced in the presence of glutathione at physiological pH. Significantly, the combination of reduced and oxidized glutathione (GSH and GSSG) was more effective than either form in maintaining the vanadyl EPR signal at pH 7-9. The most effective combination of GSH and GSSG observed in these studies is similar to that expected within mammalian cells. In conclusion, these studies provide evidence that PKA could be an important target for vanadyl action in vivo, the vanadyl being produced and stabilized through the actions of GSH and GSSG.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat