Dr. Zaid Altany
The cross-talk of hydrogen sulfide and nitric oxide in vascular endothelial cells
SUPERVISOR: Dr. G. Yang (formerly adjunct in Biology)
ABSTRACT: Gasotransmitters, like hydrogen sulfide (H2S) and nitric oxide (NO), are small gaseous molecules that can be generated in different types of mammalian cells by enzymatic catalyzation. Cystathionine y-lyase (CSE) and endothelial NO synthase (eNOS) are responsible for the majority of endogenous production of H2S and NO in vascular endothelium, respectively. H2S and NO maintain different vascular functions. Here we show that H2S interacts with eNOS to increase NO release from endothelial cells (ECs). Two mechanisms are involved in this interaction. Firstly, H2S indirectly induces eNOS phosphorylation. Secondly, H2S directly modifies one cysteine residue of eNOS through S-sulfhydration. Stimulation of eNOS phosphorylation and S-sulfhydration by H2S subsequently increases NO release. The phosphorylation of eNOS by H2S is p38 and Akt-dependent. eNOS S-sulfhydration is partially affected by S-nitrosylation, but not by phosphorylation. We further found that knockdown of CSE gene by siRNA technique, or blockage of CSE enzyme activity by PPG (dl-propargylglycine), attenuates NO production. CSE overexpression or L-cysteine (a substrate of H2S) supplementation stimulates NO production. The level of eNOS S-sulfhydration in aortic tissue from CSE knockout (CSE-KO) mice was lower than that from wild type (WT) mice. L-cysteine treatment increases S-sulfhydration of eNOS in ECs isolated from WT mice, but not in ECs isolated from CSE-KO mice. GSNO (a NO donor) induces, but NaHS reduces, eNOS S-nitrosylation. However, GSNO does not alter eNOS S-sulfhydration whereas NaHS alters S-nitrosylation. Site-directed mutagenesis of one cysteine residue Cys-443 in eNOS (Cys-443-eNOS) completely eliminates eNOS S-sulfhydration and partially decreases eNOS S-nitrosylation. Although the mutation of serine 1179 (Ser-1179) completely abolishes eNOS phosphorylation, it does not affect eNOS S-sulfhydration. The dominant configuration of vascular eNOS proteins purified from WT mice is dimer, whereas in CSE-KO mice it is monomer. In the presence of GSNO, more monomers are found with WT-eNOS, which is reversed by a subsequent treatment with NaHS. Cys-443-eNOS manifests itself as monomers, which is not changed by either GSNO or NaHS treatments. The production of NO is decreased but superoxide is increased in CSE-KO ECs in comparison with WT ECs. H2S treatment increases EC proliferation, tube formation, angiogenesis, and accelerates wound healing. With an in vitro aortic ring angiogenesis assay, we found a reduction in the number of microvessels formed by culturing aortic rings from CSE-KO mice, even in the presence of VEGF (vascular endothelial growth factor). We further found that wound healing is faster in WT mice when compared with CSE-KO mice, and H2S promotes wound healing recovery. Blockade of NO production by eNOS-specific siRNA or L-NAME (L-NG-nitroarginine methyl ester) reverses, but eNOS overexpression potentiates the proliferative effect of H2S. In contrast, CSE knockdown attenuates the pro-proliferative effect of NO. Overall, our studies demonstrate that H2S increases NO release in ECs through phosphorylation and S-sulfhydration of eNOS. Thus, H2S and NO are required for the physiological control of angiogenesis and superoxide production. Mechanistic understanding of H2S-NO interaction in vascular endothelium will help advance novel therapeutic strategies for EC dysfunction related vascular diseases.
Dr. William Dew
The effects of copper, calcium, and nickel on the olfactory response of Fathead minnows: From neurophysiology to behaviour
SUPERVISOR: Dr. Greg Pyle (formerly in Biology)
ABSTRACT: The olfactory system is essential for a fish to be successful in an ecological context, and has been demonstrated to be sensitive to a variety of toxicants. The current biotic ligand model (BLM) for copper is an acute-toxicity model based on the gill that is used to predict site-specific safe copper concentrations. Recently, work has been done to develop a BLM based on a more sensitive tissue, namely the olfactory epithelium. The work presented in this dissertation determines that a number of the assumptions of the current acute-toxicity gill-based BLM do not hold for the olfactory epithelium. Two techniques were employed for the work contained within this dissertation, a neurophysiological measure of olfactory acuity, electro-olfactography (EOG), and measurement of behavioural responses. For all experiments, fathead minnows (Pimephales promelas) and yellow perch (Perca flavescens) were used as these species are ubiquitous in waterways across Canada. Fathead minnows were exposed to low, ecologically-relevant concentrations of copper for varying exposure durations in hard and soft water. While it was determined that there was a significant of inhibition of olfactory function as measured by EOG, there was recovery, at least partially, of EOG function with increased exposure duration. It was also determined that not only does calcium have no protective effect against copper-induced olfactory dysfunction at the olfactory epithelium as it does at the gill, but calcium induces its own response. The response to calcium in fathead minnows was further investigated. Fathead minnows had a strong olfactory-dependent avoidance response to calcium. The reduction in EOG response caused by calcium was demonstrated to be due to cross-adaptation with the odourant used, namely L-arginine. Different olfactory sensory neuron (OSN) classes within the olfactory epithelium respond specifically to different odourants. Exposures of fathead minnows or yellow perch to copper demonstrated that there was a specific impairment of ciliated OSNs, while exposure to nickel resulted in impairment of microvillous OSNs. Behavioural work with fathead minnows using an anti-predator cue demonstrated that copper impairs the response to an anti-predator cue, while nickel does not. These observations demonstrate that ciliated cells are responsible for mediating response to anti-predator cue, which is the first time response to a specific chemosensory cue has been directly connected with a specific OSN class. The work presented in this dissertation represents a significant advancement in our understanding in how copper impairs the olfactory system of fish, which will aid in the construction of models and regulations to protect fish populations.