Regulation of Haem Oxygenase-1 by Nitrosative Stress in Cardiac Cells

The reactive nitrogen species (RNS) nitric oxide (NO), nitroxyl anion (N0‘) and \ nitrosonium cation (NO+), modulate a myriad of biological processes. The microsomal haem oxygenases (HO-1, HO-2 and HO-3) oxidatively catabolise haem to bilirubin, carbon monoxide (CO) and ferrous iron (Fe2+). Sensitivity of the inducible isoform (HO-1) to a variety of inducers has identified HO-1 as an effective endogenous cytoprotectant against oxidative stress. Although nitrosative stimuli can enhance HO-1 expression, little is known about the biochemistry and mechanisms of this response. This Thesis examines a number of aspects related to HO-1 and nitrosative stimuli in cardiac cells, including: 1. induction by NO'; 2. the biochemistry of NO'/NO-mediated induction of HO-1; 3. identification of a possible mechanism for the activation of HO-1 by NO congeners; 4. the antinitrosative potential of bilirubin; and 5. the potential of glyceryl trinitrate (GTN), a clinically used NO donor, to activate the haem oxygenase pathway. These different aspects of HO-1 were addressed using biochemical, molecular biology and cell culture techniques. The results indicate that NO', in analogy with other RNS, is a potent inducer of haem oxygenase activity and HO-1 mRNA and protein expression. A proposed mechanism for this response is modulation of thiol groups within redoxsensitive transcription factors. An antinitrosative and HO-1 inducing capacity was identified for bilirubin and GTN, respectively. Collectively, these findings suggest that the haem oxygenase pathway can act both as a sensor to, and target of, redox based mechanisms involving RNS, and extend our knowledge on the biological function of HO-1 in response to nitrosative stress. LIST OF ABBREVIATIONS pl microlitre gM micromolar AS Angeli’s salt ' BR bilirubin BVR biliverdin reductase + CO O calcium CaM calmodulin C-PTIO 2-(4-carboxyphenyl)-4I4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (potassium salt) CO carbon monoxide cGMP cyclic guanidine monophosphate DeaNO diethylamine NONOate DetaNO diethylenetriamine NONOate DMEM Dulbecco’s modified eagle’s medium DPBS Dulbecco’s phosphate buffered saline Fe3+ ferric iron Fe2+ ferrous iron GTN glyceryl trinitrate g gram(s) h hour(s) HO haem oxygenase HO-1 ,-2 and -3 haem oxygenase-1, -2 and -3 I litre(s) min minute(s) ml mililitre mM milimolar MW molecular weight n number of replicates per group NADH reduced nicotinamide adenine dinucleotide NADP oxidised-nicotinamide adenine dinucleotide phosphate NADPH reduced nicotinamide adenine dinucleotide phosphate NO nitric oxide NOS nitric oxide synthase N03‘ nitrate N02‘ nitrite NO+ nitrosonium cation NO' nitroxyl anion OD optical density P probability RNS reactive nitrogen species ROS reactive oxygen species Sec seconds S.E.M. standard error of the mean sGC soluble guanylate cyclase vs. versus xfif times gravity iii