Mevinphos (Mev) is an organophosphate insecticide that acts on the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone, to induce cardiovascular responses. This study investigated the role of Trk (tropomyosin-related kinase) (receptor form) or Src (non-receptor form) tyrosine kinase at the RVLM in Mev-induced cardiovascular responses. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Western blot assay showed that whereas p-Trk490 was increased during Phase I, p-Src416 was increased only during Phase II Mev intoxication. Interestingly, application of a Trk specific inhibitor (K252a; 1 pmol) or Src specific inhibitor (SU6656; 100 pmol) into the bilateral RVLM blunted the Mev-elicited sympathoexcitatory or sympathoinhibitory effect, respectively. Besides, K252a was limited to block NOS I protein expression in the RVLM during Mev intoxication, SU6656 only inhibited NOS II protein expression in the RVLM during Mev intoxication. We conclude that Trk tyrosine kinase (p-Trk490) in the RVLM participates in the Phase I cardiovascular responses during Mev intoxication, Src tyrosine kinase (p-Src416) in the RVLM participates in the Phase II cardiovascular responses associated with Mev intoxication.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xv, 190 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Diss. (sammanfattning) Linköping : Univ., 2004. / I publikationen felaktig serie: Linköping studies in health sciences. Härtill 4 uppsatser.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 217-246.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most important constitutively expressed enzymatic source of nitric oxide (NO), a major cardiac signalling molecule. On the whole, NO is regarded as a cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is controversial; however, it is generally accepted that ischaemia / hypoxia results in increased cardiac NO production. Most studies focus either on the whole heart or isolated cell models. As yet, no study has compared findings with regard to NO metabolism in these two distinct models, in a single study. We hypothesise that observations in a whole heart model with regard to increased NO production and eNOS involvement in ischaemia are the result of events on cellular level and that the increase in NO production observed during hypoxia in cardiomyocytes and endothelial cells is at least in part due to the increase in expression and / or activation of eNOS. Furthermore, we hypothesize that these effects are mediated via the PI3-K / PKB pathway. We aimed to measure and compare NO-production and eNOS expression and activation in the whole heart and isolated cardiac cells and measure PKB expression and activation in the cells under normoxic and ischaemic / hypoxic conditions. We also aimed to determine the effects of PI3-K / PKB pathway inhibition on NO production and eNOS expression and activation in isolated cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were perfused and global ischaemia induced for 15 and 20 min. Tissue homogenates of perfused hearts were used for the measurement of nitrites and determination of expression and activation of eNOS. Expression of eNOS in the heart was also determined by immunohistochemical (IHC) analysis. Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion, and adult rat cardiac microvascular endothelial cells (CMEC) purchased commercially. In the cells, hypoxia was induced by covering cell pellets with mineral oil for 60 min. Cell viability was determined by trypan blue and propidium iodide (PI) staining and intracellular NO production measured by FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite production increased by 12 % after 15 min ischaemia and 7 % after 20 min ischaemia. Total eNOS expression remained unchanged (Western Blot and IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15 min ischaemia and decreased by 43% after 20 min ischaemia. In the cells, both viability techniques verified that the hypoxia-protocol induced significant damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by DAF-2/DA fluorescence), total eNOS expression increased 2-fold and activated eNOS increased 1.8-fold over control. In CMECs, NO-production increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8- fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB investigations, results showed an increase of 84 % and 88 % in expression vii and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes, respectively. In hypoxic CMECs, there was no change in PKB expression but there was a 69 % increase in phosphorylated PKB. NO production in wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared to untreated hypoxic cells. In treated hypoxic CMECs, NO production decreased by 58 % as compared to untreated hypoxic cells. Treatment with wortmannin did not change the expression of eNOS protein in the cardiomyocytes, however, activated eNOS decreased by 41 % and 23 % under baseline and hypoxic conditions in treated cells respectively. There was a significant increase in NO production after exposure to O2 deficient conditions in all models investigated, a trend similar to what previous studies in literature found. However, the source of this NO is not fully understood although it has been discovered that NOS plays a role. Our data reveals similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the cells; however, the trends observed at 20 min ischaemia are in conflict with our cell data (i.e. decrease in activated eNOS). This may be due to the severity of the ischaemic insult in whole hearts and/or the presence of other cell types and paracrine factors in the whole heart. Hypoxia increased the activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB pathway reduced NO production and hypoxia-induced eNOS activation in cardiomyocytes. In conclusion, we have, for the first time, demonstrated that the increase in NO production during hypoxia is due (at least in part) to an increase in eNOS phosphorylation at Ser1177 and that this is mediated via the PI3-K / PKB pathway.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xxiii, 251 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 8 uppsatser.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis.

How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.