Human platelets are constantly regulated by activating and inhibitory effectors. Thrombin,the most potent platelet agonist, induces signaling through the protease-activated receptors(PARs) 1 and 4 which in turn convey their signal by coupling to G-proteins. Nitric oxide (NO)is a potent platelet inhibitor continuously formed by the endothelium exerting its effect byincreasing cGMP through activation of soluble guanylyl cyclase (sGC). The purpose of thiswork has been to investigate how NO would affect platelets already activated by PARagonists.To examine the different contributions of the PAR1- and PAR4-signals, the selectiveagonist peptides SFLLRN and AYPGKF-NH2 were utilized. Aggregation, Ca2+-mobilization andphosphorylation of threonine 696 in myosin phosphatase target subunit 1 (MYPT1) wereanalyzed. Intriguingly PAR1-, but not PAR4-, agonist provoked aggregation was rapidlyreversed upon NO exposure. PAR-agonist induced Ca2+-mobilization was markedly reducedafter exposure to NO, however this Ca2+-suppression did not cause the disaggregation ofPAR1-agonist evoked platelet aggregation. The reversal of aggregation was suspected to becaused by a cGMP-mediated inhibition of the Rho-kinase/ROCK-signaling pathway. This wassupported by Westen blot analysis where a marked decrease of MYPT1 phosphorylationcompared to basal levels could be observed. In conclusion, NO was found to reverse humanplatelet aggregation evoked by PAR1-activation by inhibition of a Rho-kinase/ROCK-signalingpathway.

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 lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.

Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent. / May 2009

Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.

In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA). The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.

In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA). The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.

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.

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.