Oxidative stress and calcium signalling : Implications for diabetes and cardiac glycosides

Abstract: The signal transduction processes by which an individual cell is able to recognize and respond to its external environment are critical to determining its survival as well as that of the entire organism. In the current study we have investigated the intracellular signalling cascades activated downstream of two separate stimuli: the diabetic milieu and the cardiac glycoside, ouabain. The development and progression of diabetic nephropathy is dependent on the maintenance of adequate glucose homeostasis. Based on studies using diabetic rats, we found that increased oxidative stress was implicated in the cellular activation of the PKC/TGF-b1 pathway and consequent kidney dysfunction. Diabetic rats fed a diet containing the antioxidant, nitecapone, were protected from the development of diabetic nephropathy. Using a renal mesangial cell model of diabetes, we present evidence that AGE, a class of molecules typically present in the diabetic milieu, initiate a cascade of signalling events including activation of PKC, TGF-b1, and NF-kB. Increased oxidative stress was involved in the regulation of these signalling molecules by AGE since antioxidants were able to normalize their activation. The endogenous ligand of Na,K-ATPase, ouabain, is a newly recognized steroid hormone, the effect of which on signal transduction is largely unknown. Using renal epithelial cells, we show that ouabain induces slow oscillations in cytosolic calcium. The generation of this unique response was facilitated by a number of calcium transport proteins that regulated both intra- and extra-cellular calcium compartments. Initiation of the calcium response and release from intracellular stores was dependent on a novel molecular interaction between the NH2-terminus of Na,K-ATPase and IP3R. The physical interaction between Na,K-ATPase and IP3R and the induction of calcium oscillations was fundamental to the ability of ouabain to activate NF-kB. Regulation of gene expression following short term ouabain exposure was investigated by DNA microarray analysis. Inhibition of Na,K-ATPase was the main factor establishing the observed gene changes. In conclusion, this thesis presents new insight into the signal transduction cascades activated by the diabetic milieu and by ouabain. Oxidative stress has a role in the stimulation of key signalling molecules implicated in the pathogenesis of the diabetic condition. Binding to Na,K-ATPase by its ligand, ouabain, reveals a signal transducing function of the pump and the generation of calcium oscillations via protein-protein interaction between Na,K-ATPase and IP3R.