Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their ability to undergo desensitization. For this, PAR1- and PAR4- activating peptides (APs) (SFLLRN and AYPGKF, respectively) were used. Different aspects of platelet activities were studied: aggregation and the rise in intracellular Ca2+ concentrations ([Ca2+]i). Aggregation was analyzed with lumiaggregometry, and [Ca2+]i were studied using the fura-2 method. PKC substrate phosphorylation and the expression of PAR1 surface receptors were also analyzed, using Western blot and flow cytometry, respectively. The results from this study showed that NO exerted similar inhibitory effects on the two thrombin receptors. However, PAR1 and PAR4 differed in their ability to undergo desensitization. In cumulative dose-response studies, a low concentration of PAR1-AP induced desensitization of platelets towards higher PAR1-AP concentrations. This was not the case when studying PAR4-AP. The mechanism behind the desensitization of PAR1 to some part involved PKC, at least when studying the mobilization of intracellular Ca2+. PAR1 desensitization did not seem to involve receptor internalization and neither did it affect the activity of PAR4. This thus suggests that PAR4 might be a more suitable therapeutic target in the future management of thrombosis.

The increase of calcium in the cell can induce cellular functions such as fertilization, cell division and cell communication. Calcium (Ca2+) carries out these processes through proteins called calcium sensors. An important calcium modulator is calmodulin. Calmodulin has four possible Ca2+ binding sites that have the characteristic helix-loop-helix (EF hand) motif. When the EF hands bind to Ca2+, methionine rich hydrophobic patches are exposed allowing for CaM to interact with target proteins. However, there are proteins that can interact with CaM at low levels of Ca2+ or in the absence of Ca2+. An enzyme that is activated by CaM is nitric oxide synthase (NOS), which converts L-arginine to L-citrulline and nitric oxide (•NO), where •NO is used to carry out important cellular functions. There are three isoforms of the enzyme; endothelial, neuronal and inducible NOS. The first two isoforms are activated by Ca2+-bound CaM when there is an influx of Ca2+ and are therefore Ca2+-dependent whereas inducible NOS (iNOS) is activated and binds tightly to CaM regardless of the Ca2+ concentration and is therefore Ca2+-independent. Of particular interest is the iNOS enzyme, since no three-dimensional structures of the reductase domain or the CaM-binding region have been solved. All three isoforms of NOS exist as homodimers, where each monomer consisting of a reductase domain and an oxygenase domain separated by a CaM-binding region. The reductase domain contains binding sites for NADPH and the flavins, FAD and FMN, which facilitate electron transfer from the NADPH to the catalytic heme in the oxygenase domain of the opposite monomer. The transfer of electrons from the FAD to the heme is carried out by the FMN domain which is proposed to swing between the two docking points since the distance between the two points is too large for electron transfer. This electron transfer point is under the control of CaM, which is essential for NOS activation. This dynamic process and the direct role of CaM have yet to be observed structurally. A method to monitor dynamics structurally is through the use of nuclear magnetic resonance (NMR) spectroscopy. Therefore as the first step to determine the NMR structure of the FMN domain with the CaM-binding region, the structure of the iNOS CaM-binding region bound to CaM will be determined. The structure will allow for further characterization and identification of important interactions between the iNOS CaM-binding region and CaM which contribute to the unique properties of iNOS.

Glutathione (GSH), a 3-amino acid compound is ubiquitously expressed in eukaryotic cells and is the most abundant low molecular weight thiol. The importance of GSH is highlighted by its multitude of effects. Within the vascular wall GSH plays a crucial role as an intracellular antioxidant and it possess the ability to act as a signalling intermediate and store for nitric oxide (NO). The importance of NO and its role in vascular wall homeostasis is well recognized. Within the coronary circulation, NO is the primary dilator of many of the large arteries and the smaller arterioles. In addition to controlling coronary vascular tone, the importance of NO is highlighted by its antithrombotic, antihypertrophic, and antriproliferative effects. During instances of cardiovascular disease and normal aging, increases in the production of reactive oxygen species occur. A portion of the deleterious vascular effects of reactive oxygen species are believed to be due to reduction in NO bioavailability as a result of increased ROS-mediated destruction of NO. Altered GSH production in humans has been demonstrated to reduce endothelial function. Conversely, supplementation with GSH augments endothelium-dependent dilation. The mechanisms by which these alterations in GSH influence vasomotor function have not been resolved. The purpose of the studies within this thesis was to examine the impact of chronic and acute GSH modulations on coronary vascular resistance (CVR) and endothelium dependent dilation. In all experiments vascular reactivity was assessed in the isolated perfused rat heart. The advantage of this technique is that it allows the global coronary vasomotor functioning to be examined. Hearts were allowed to stabilize for 30 minutes to allow for the development of spontaneous coronary vascular resistance, followed by a bradykinin (BK) dose-response curve to assess endothelium-dependent dilation. The coronary circulation was then maximally dilated using an endothelium-independent agonist. In all cases BK-mediated dilation is expressed as a percentage of the endothelium-independent dilation. Chapter 2 of this document examines the chronic nature of GSH depletion and examines whether GSH depletion augments the influence of natural aging. Animals (mean age 33 and 65 weeks) were randomized to receive L-Buthionine-(S,R)-sulphoximine (BSO) in the tap water in order to inhibit GSH synthesis, or regular tap water (normal controls). Following 10 days of BSO treatment, ventricular GSH content was reduced in the BSO group compared to the control (0.182±0.021 vs 2.022±0.084 nmol/mg wet weight, p<0.05) and there was increased ventricular H2O2 content (1.345±0.176 vs 0.877±0.123 pmol/µg PRO, p<0.05). Baseline CVR was significantly reduced in the older animals compared to the adult animals (3.92±0.34 vs 4.76±0.20 and 3.67±0.24 vs 5.12±0.37 mmHg/ml×min-1 in the control and BSO treated groups, p<0.05). Conversely, in the presence of LNAME there was a significant increase in CVR in the adult BSO group (14.15±0.99, p<0.05) compared to all other groups. In the absence of LNAME, maximal dilation (percent endothelium-independent response) was reduced in the older animals compared to the adult animals (77±10.3% vs 95.0±1.0% for older and adult control and 92.7±4.5% vs 98.6±0.6% for the older and adult BSO, main effect of age). In the presence of LNAME the adult BSO group had a significantly reduced sensitivity (EC50) compared to all other groups (-7.39±0.09 Log M, p<0.05). Additionally, adult BSO treated animals had an increase in eNOS protein content. These results demonstrate that chronic thiol depletion resulted in an increased reliance on NO in the adult BSO group only. In chapter 3 the beneficial effects of GSH supplementation on BK mediated dilation were examined. Acute GSH was administered in the perfusate at either 0 (control) or with 10 µM for 2 reasons, 1) this concentration does not reduce basal coronary vascular resistance, allowing for a similar baseline CVR across conditions and 2) the 10 µM concentration is a physiologically relevant concentration of plasma/extracellular fluid GSH. The sensitivity to the endothelial agonist bradykinin was enhanced in the presence of GSH (-8.70±0.16 vs -7.94±0.06 LogM, p<0.01). The GSH effect was not dependent on NO production or utilization by soluble guanylate cyclase (sGC) as the enhanced dilation in the GSH group was maintained despite NOS (LNAME) and/or sGC inhibition. When the hearts were supplemented with a ROS scavenger TEMPOL, enhanced dilation was seen in the control group, but was not further enhanced in the GSH group. The requirement for ROS was best demonstrated when both the CON and GSH groups were supplemented with both TEMPOL and LNAME. This condition resulted in similar sensitivity (-7.76±0.19 vs -7.75±0.17 LogM, p>0.05) and area under the curve (182.33±12.70 vs 170±13.86, p>0.05) between GSH and CON. Thus, it was concluded that the effects of GSH administration requires the presence of ROS and exerts its effect in the microvasculature. The study presented in chapter 4 examined the effects of acute thiol modulation (depletion) on CVR and endothelium-dependent dilation. Previous reports have suggested that a reduction in intracellular GSH causes impaired NO production, and functional data support this contention. However, a majority of the data regarding the effects of thiol manipulation are from endothelial-removed vessels. The following agents were used to reduce GSH: the glutathione reductase inhibitor, BCNU; the thiol oxidizing agent, diamide; the thiol conjugating agent, ethacrynic acid (EA); and a thioredoxin inhibitor (CDNB). Preliminary data revealed that only CDNB (11.46±0.71 mmHg/ml×min-1) and EA (8.61±0.36 mmHg/ml×min-1) caused an elevation in CVR compared to the control (6.73±0.24 mmHg/ml×min-1). Conversely, Diamide and BCNU did not significantly affect baseline CVR, or the BK mediated responses. In the presence of EA, there was an overall blunting of the BK-response curve as observed by reduced EC50 (-7.85±0.07 Log M) and maximal dilation (90.8±1.8 %, percent endothelium-independent dilation) compared to the control group (-8.42±0.08 Log M and 97.7±1.6%). In the presence of CDNB the maximal dilation was 74.4±1.9% and the EC50 was -8.83±0.28 Log M. In addition to altering BK mediated responses, acute thiol depletion with all agents resulted in an increased minimal CVR with significant increases observed in the presence of CDNB and EA. There was a significant correlation with GSH:GSSG ratio and baseline (-0.547, p<0.05) and minimal CVR (r=-0.581, p<0.05). This study demonstrates that modulation of the GSH:GSSG ratio using a variety of agents with diverse mechanisms elicits differential responses within the vasculature. Specifically conjugation of GSH and inhibition of thioredoxin significantly alters BK mediated response, where as BCNU and dimaide did not. These results suggest that a modulation in the GSH:GSSG ratio impairs endothelium-dependent dilation and alters total dilatory capacity (baseline-minimal CVR) and thus may have implications for adequate tissue perfusion. Across all studies there was significant correlation between GSH and GSSG with both baseline and minimal CVR. Therefore it is likely that changes in overall glutathione content plays a role in determining baseline and minimal coronary vascular resistance. These results demonstrate the complexity that manipulations of GSH have on both CVR and endothelium-dependent dilation, and provide mechanistic insight into how changes in GSH alter coronary vascular resistance and endothelium-dependent dilation.

ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are unequivocally useful for lowering cholesterol levels in patients with dyslipidemias. In addition to cholesterol lowering properties, statins exert a number of pleiotropic, vascular-protective effects include improvement of endothelial function, increased nitric oxide (NO) bioavailability, antioxidant properties. Since endothelial dysfunction and reactive oxygen species (ROS) are important pathophysiological determinants of essential hypertension, these actions of statins raise the possibility that statin therapy may be useful for simultaneously clinical hypertension management. However, the signaling mechanisms of statins that improve hypertension remain unclear. Our previous study showed, in the NTS, insulin may decrease blood pressure and heart rates through PI3K-Akt-eNOS pathway, and NTS integrates convergent information from peripheral baroreceptors and central cardiovascular regulatory center. Statins also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, members of the Ras and Rac1 GTPase family are major substrates for posttranslational modification by isoprenylation and may be important targets for inhibition by statins. Statins could inhibit Rac1 isoprenylation and Rac1-mediated nicotinamide adenine dinucleotide phosphate oxidase activity attenuates reactive oxygen species production. The aim of this study was to investigate the possible signaling pathways involved in simvastatin-mediated blood pressure regulation in the nucleus tractus solitarii (NTS). Male 20-week-old spontaneously hypertensive rats (SHR) were divided into two groups: control group and intracerebroventricular injection with simvastatin group for three days. We found that systolic blood pressure measured with tail-cuff method of the simvastatin-treated rats decreased significantly, and the NO level in the NTS was significantly increased. In addition, we observed that simvastatin could lower the ROS level and increase Ras GTPase activity in the NTS. Immunoblotting and immunohistochemistry analysis further showed that simvastatin increased the phosphorylation ratio of ERK1/2, Akt, and endothelial nitric oxide synthase (eNOS) in the NTS. Taken together, these results suggest that eNOS signaling in the NTS may play an important role in simvastatin-induced blood pressure lowering effects. Keywords: statins, nucleus tractus solitarii, nitric oxide, oxidative stress, central cardiovascular regulatory, isoprenylation

Recent studies demonstrated that fructose intake can increase blood pressure in experimental animals. Oxidative stress has emerged as an important pathogenic factor in the development of hypertension. It has been reported that increased superoxide production in fructose-fed rat mediated through nicotinamide adenine dinucleotide phosphate NAD(P)H oxidase. Superoxide dismutase (SOD) is one of the most important enzymes against oxidative stress. However, the signaling mechanisms of fructose which induce hypertension through superoxide remain unclear. Nucleus tractus solitarii (NTS) is the integrative center for baroreflex. Our previous study had revealed that accumulation of superoxide in the NTS can induce hypertension. As an important antioxidant in red wine, resveratrol is likely to contribute to the potential of red wine to prevent cardiovascular disease. At pharmacological doses, resveratrol increases vascular nitric oxide (NO) levels and improves NO bioavailability in animal models. Resveratrol is a potent activator of AMPK in neuronal cell lines, primary neurons, and the brain. Recent reports have indicated that metformin targets AMPK which activates nNOS and eNOS. Therefore, we hypothesized that resveratrol causes blood pressure decrease through regulating nitric oxide and superoxide production in the NTS of fructose-fed rats. There were three specific aims: 1. To investigate whether fructose induce superoxide production and causes hypertension in the NTS. 2. To investigate which signaling pathway is involved in fructose-induced hypertension. 3. To investigate which signaling pathway is involved in resveratrol modulates blood pressure. Male Wistar Kyoto rats (WKY) were divided into two groups: control group and fed with 10% fructose water group for 1 week. After one-week treatment, the systolic blood pressure and superoxide production increased significantly and the nitrate level in the NTS was significantly decreased. Immunoblotting showed that administration of fructose significantly increased NADPH oxidase subunits p22-phox, p67-phox activity, RAGE activity and reduce SOD2 activity in the NTS. Based on our previous studies, male Wistar-Kyoto rats (WKY) were divided into five groups: Group I: Control group; Group II: fructose-fed rats (FFR) fed with 10% fructose for 4 weeks; Group III: Control + resveratrol (R) rats received a gavage of resveratrol; Group IV: FFR+ resveratrol (FR) fed with 10% fructose and resveratrol ; Group V: FFR + 2weeks resveratrol (F2R) fed with 10% fructose and received a gavage of resveratrol 2 weeks. We found that systolic blood pressure measured by tail-cuff method in F group rats and F2R group rats revealed a significantly increased than C group rats continuously through week 0 to week 2 but R group rats and FR group rats were no difference with C group. However, received a gavage of resveratrol (10 mg/kg/d) 2 weeks, F2R group revealed a significantly decrease in SBP than the F group continuously through week 2 to week 4. Fructose-induced hypertension increased NADPH oxidase activity and SOD2 activity related to inhibit the production of NO in the regulation of blood pressure. These results suggest that in the NTS, intake of fructose induces NADPH oxidase activity and reduces SOD2 activity to increase blood pressure. Resveratrol can not only reverse fructose-induced hypertension but also prevent fructose-induced hypertension.

We hypothesized that the interaction between tumor necrosis factor alpha(TNF)/nuclear factor-kappaB (NFkB) via activation of IKK may amplify one anotherresulting in the evolution of vascular disease and insulin resistance associated withdiabetes. The interaction between TNFa and monocyte chemoattractant protein-1 (MCP-1) may contribute to the evolution of vascular inflammation and endothelial dysfunctionin coronary arterioles in type 2 diabetes. To test this hypothesis, endothelium-dependent(ACh) and –independent (SNP) vasodilation of isolated, pressurized coronary arterioles(40-100 μm) from mLeprdb (heterozygote, normal), Leprdb (homozygote, diabetic) andLeprdb mice null for TNF (dbTNF-/dbTNF-) were examined. Although dilation of vesselsto SNP was not different between Leprdb and mLeprdb mice, dilation to ACh was reducedin Leprdb mice. The NFkB antagonist, MG-132, IKK inhibitor, sodium salicylate(NaSal), or Anti-MCP-1 partially restored endothelium-dependent coronary arteriolardilation in Leprdb mice. Protein expression of IKK and IKK were higher in Leprdb thanin mLeprdb mice. The expression of IKK, but not the expression of IKK was increasedin dbTNF-/dbTNF- mice. Leprdb mice showed increased insulin resistance, but NaSal improved insulin sensitivity. Protein expression of TNFa, NFkB, phosphorylation ofIKK and JNK were greater in Leprdb mice, but NaSal attenuated protein expression ofthem in Leprdb mice. The ratio of phosphorylated IRS-1 at Ser307 (pIRS-1)/IRS-1protein expression was elevated in Leprdb mice; both NaSal and JNK inhibitor SP600125reduced pIRS-1/IRS-1 in Leprdb mice. MG-132 or neutralization of TNF reducedsuperoxide production in Leprdb mice. Anti-MCP-1 attenuated superoxide productionand protein expression of nitrotyrosine (N-Tyr), which is an indicator of peroxynitriteproduction, in isolated coronary arterioles of Leprdb mice. Immunostaining resultsshowed that expression of MCP-1 and vascular cellular adhesion molecule-1 (VCAM) isco-localized with endothelial cells and macrophages. Anti-TNFa or anti-MCP-1markedly reduced macrophage infiltration and the number of MCP-1 positive cells.Neutralization of TNFa or anti-MCP-1 reduced the expression of adhesion molecules. Inconclusion, our results indicate that the interaction between NFkB and TNFa signalinginduces activation of IKKb. In addition, TNFa and TNFa-related signaling, includingthe expression of MCP-1 and adhesion molecules, further exacerbates oxidative stressleading to endothelial dysfunction in type 2 diabetes.

As global focus shifts towards the health and conservation of the planet, greater importance is placed upon the hazardous emissions of our fossil fuels, as well as their finite supply. These two areas remain intense topics of research in order to reduce green house gas emissions and increase the fuel efficiency of our vehicles. A particular solution to this problem is the diesel engine, with its inherently fuel-lean combustion, which gives rise to low CO2 production and higher efficiencies than its gasoline counterpart. Diesel engines, however, typically exhibit higher nitrogen oxides (NOx [NOx = NO NO2, where NO is nitric oxide and NO2 is nitrogen dioxide]) and soot. There exists the possibility to simultaneously reduce both emissions with the application of low temperature diesel combustion (LTC). While exhibiting great characteristics in simultaneous reductions in nitrogen oxides and soot, LTC faces challenges with higher carbon monoxide (CO) and hydrocarbon (HC) emissions, as well as penalties in fuel efficiency. The following study examines the characteristics of LTC which contribute to the differences in emissions and efficiency compared to typical conventional diesel combustion. More specifically, key engine parameters which are used to enable LTC, such as EGR and fuel pressure are swept through a full range to determine their effects on each combustion regime. Analysis will focus on comparing both combustion regimes to determine how exhaust gas recirculation (EGR) and fuel pressure relate to lowering NO and smoke concentrations, and how these relate to a penalty in fuel efficiency. This study finds that the application of LTC is able to realize a 99 percent reduction in NO while simultaneously reducing smoke by 17 percent compared to the conventional combustion counterpart. Through a sweep increasing EGR, LTC is able to defeat the typical soot – NO tradeoff; however, brake fuel conversion efficiency decreases 6.8 percent for LTC, while conventional combustion realizes a 4 percent increase in efficiency. The sweep of increasing fuel pressure confirms typical increases in NO and decreases in smoke for both LTC and conventional combustion; however, brake fuel conversion efficiency increases 2.3 percent for LTC and drops 4 percent for conventional combustion.

Interest in biodiesel has piqued with advent of stringent emissions regulations. Biodiesel is a viable substitute for petroleum diesel because biodiesel produces significantly lower particulate and soot emissions relative to petroleum diesel. Higher nitric oxide (NO) emissions for biodiesel, however, are of primary concern in biodiesel-fueled engines. Search for an in-cylinder technique to reduce NO emissions for biodiesel has motivated studies to gain an improved understanding of fundamental factors that drive increase in NO emissions with biodiesel. Potential factors include fuel-bound oxygen, fuel-bound nitrogen and post-flame gas temperature. The role of fuel-bound oxygen however is debated in the literature. The research objective of this study is to computationally determine if biodiesel and petroleum diesel yield equivalent concentrations of NO with the same oxygen equivalence ratio in a 0-D homogeneous reactor, to explain the role of fuel-bound oxygen in biodiesel on increases in NO emissions with biodiesel. The results from this study indicate that the biodiesel surrogate yields higher NO emissions than the n-heptane because of its lower oxygen consumption efficiency. The lower oxygen consumption efficiency for biodiesel is likely because of the slower decomposition of the individual components and the blending ratios of the biodiesel surrogate blend. The relative differences in combustion efficiency of individual components of the biodiesel blend suggest this conclusion. The more efficient burning of the methyl esters relative to the n-heptane in biodiesel surrogate perhaps indicates the favorable role of fuel-bound oxygen in the fuel’s combustion. The low utilization of oxygen by the biodiesel surrogate could not be explained in this study. The dominance of NO2 H ↔ NO OH and N NO ↔ N2 O mechanisms during biodiesel combustion however explain the high NO emissions for the biodiesel surrogate relative to the n-heptane. The biodiesel may yield lower NO emissions than the petroleum diesel if the blending ratios for the biodiesel are adjusted such that combustion efficiency of biodiesel and petroleum diesel is same or the NO2 H ↔ NO OH and N NO ↔ N2 O mechanisms are suppressed during biodiesel combustion.

Environmental factors such as ambient temperature and oxygen availability are variation in different altitude. Individuals within a species, living in variable environments often display phenotypic plasticity by changing morphology, behavior, reproduction, and physiology to meet the individual¡¦s ability to survive demanding conditions. This study was aimed to investigate the expression of angiotensin receptor and nitric oxide synthase genes of individuals resided at differential altitude, in an attempt to find the role of these molecules in cardiovascular adaptation to altitude. Spiny rats (Niviventer coxingi) are widely elevational distributed in Taiwan. They were studied under more natural conditions to provide an ecological context data on physiological plasticity between the different altitudes. I examined the body weight, blood pressure, heart rate and the expression of angiotensin type 1 or type 2 (ATI or ATII) receptor and nitric oxide synthase (NOS) genes in tissues (cortex, hypothalamus, medulla, lung, heart, aorta, adrenal gland and kidney) of spiny rats resided at differential altitude and during the domesticated period. The results of the study showed that spiny rats resided at higher altitudes were lighter than that at lower altitudes (750 m: 178.6¡Ó35.8 g and 1600 m: 122.3¡Ó29.3 g). Spiny rats resided at 1600 m did not change their body weight during the domesticated period, but rats resided at 750 m gradually reduced their body weight. Blood pressure and heart rate were similar between rats resided at different altitudes, and did not change during the domesticated period. ATI receptor, endothelelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) mRNA expression in these tissues were similar between rats resided at different altitudes. ATII receptor mRNA expressed in these tissues under our detection limit. Rats resided at 750 m declined the level of nNOS in heart, when they were domesticated at 100 m. ATI receptor in kidney reduced at first, but subsequently increase to same level like native. Moreover, rats resided at 1600 m declined the level of iNOS in heart, when they were domesticated at 100 m. Together, these results indicate that heart rate, blood pressure, ATI receptor, eNOS, iNOS and nNOS mRNA expressions in these tissues were similar between rats resided at different altitudes. If there was no other compensatory mechanism, individuals resided at higher altitude were limited in low available oxygen. A reduced body weight could help in adaptation to high-altitude.