Tesi sul tema "Nitric oxide – Physiological effect"

Three biologically-active aminothiols cysteamine (CA), DL-cysteine (CYSH) and DL-homocysteine, were studied in this thesis. These aminothiols react with nitrous acid (HNO2), prepared in situ, to produce S-nitrosothiols (RSNOs): S-nitrosocyteamine (CANO), S-nitrosocysteine (CYSNO) and S-nitrosohomocysteine (HCYSNO). They also react with S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) through a transnitrosation reaction to produce their corresponding RSNOs. A detailed kinetics and mechanistic study on the formation of these RSNOs and their subsequent decomposition to release nitric oxide (NO) were studied. For all three aminothiols the stoichiometry of their reaction with nitrous acid is strictly 1:1 with the formation of one mole of RSNO from one mole of HNO2. In all cases, the nitrosation reaction is first order in nitrous acid, thus implicating it as a nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating another nitrosating agent, the nitrosonium cation, NO+ ( which is produced from the protonation of nitrous acid) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Nitric oxide itself was not detected as a nitrosant. Bimolecular rate constants for the nitrosation of CA, CYSH and HCYSH were deduced to be 17.9, 6.4, 0.09 M-1 s-1 for the nitrosation by nitrous acid and 8.25 x 1010, 2.89 x 1010 and 6.57 x 1010 M-1 s-1 for the nitrosation by nitrosonium cation respectively. A linear correlation was obtained between the rate constants and the pKa of the sulfur center of the aminothiols for nitrosation by NO+. The stabilities of the three RSNOs were found to be affected by metal ions. They were unstable in the presence of metal ions, with half-lives of few seconds. However, in the presence of metal ion chelators, they were found to be relatively stable with half-lives of 10, 30 and 198 hours for CYSNO, CANO and HCYSNO respectively. The relative stability of HCYSNO may be an advantage in the prevention of its metabolic conversion to homocysteine thiolactone, the major culprit in HCYSH pathogenesis. This dissertation has thus revealed new potential therapeutic way for the modulation of HCYSH related cardiovascular diseases.

1. The free radical gas nitric oxide (NO) is now recognised as a ubiquitous and versatile signalling molecule and the investigation of its biological roles has involved a wide range of scientific disciplines in many different species. Yet despite this, its potential roles in the development of rhythmic motor activities in vertebrates have been largely ignored. 2. Physiological experiments recording extracellular ventral root output suggest that NO is playing an inhibitory role in the swimming system of Xenopus laevis larvae, shortening the duration of swim episodes and slowing swim frequency. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry labelled three populations of neurons in the brainstem, which putatively co-localise NO with the aminergic neuromodulators serotonin (5-HT) and noradranaline (NA), and the fast descending inhibitory neurotransmitter, y-aminobutyric acid (GABA). This suggests that the inhibitory role is supraspinal in origin. 3. Intracellular recordings from neurons presumed to be spinal motor neurons provide further evidence for the inhibitory influence of NO. My experiments suggest that NO potentiates both glycinergic and y-aminobutyric acid (GABA)-ergic inhibition onto spinal motor neurons. The facilitation of the release of these inhibitory transmitters is consistent with the observed effects on swim frequency and swim episode duration, respectively. Additionally, NO appears to affect membrane properties, causing a pronounced membrane potential depolarisation and a decrease in membrane conductance. This suggests that NO shuts off a resting membrane conductance. 4. NADPH-diaphorase histochemistry was subsequently applied to determine the four dimensional expression of putative nitrergic neurons in the central nervous system and related structures. The developmental sequence of staining identifies groups and subgroups of interconnected intemeurons, and provides further clues to their identity. NADPH-diaphorase labelling was also located in the eyes, skin and blood vessels, further confirming the validity of this staining technique for identirying nitric oxide synthase. 5. In the related anuran species, Rana temporaria nitric oxide donor drugs appear to have no affect on swimming, but instead reliably initiates a non-rhythmic "lashing" motor pattern similar to that elicited by dimming of the illumination. Interestingly the NADPH-diaphorase technique labelled three clusters of apparently homologous interneurons in the brainstem and additionally the inner layer of the skin was intensely stained, implicating a species-specific role for NO released from brainstem neurons.

 Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke.<br>published_or_final_version<br>Chinese Medicine<br>Master<br>Master of Philosophy

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009.<br>ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM). These conditions have for a long time been associated with infertility. Obesity is characterized by high levels of circulating leptin and cytokines as well as insulin resistance. Type I DM is associated with low or no insulin whereas, Type II DM is characterised by insulin resistance. As the prevalence of obesity and DM continues to rise, it is likely that the incidence of infertility associated with these pathological conditions will likewise increase. The effects of insulin and leptin on male reproductive function have been reported on the endocrine and spermatogenesis level, but their effects on cellular level of human ejaculated spermatozoa are yet to be elucidated. This study presents data on the role of insulin and leptin on human ejaculated spermatozoa and their interaction with cytokines and nitric oxide. In the first part of the study, we established the suitable concentrations of glucose, insulin and leptin that could be administered to human spermatozoa in vitro. Glucose concentration of 5.6 mM was chosen as the suitable concentration to be administered to human spermatozoa because it has previously been reported in the literature; furthermore, it is within the range of the physiological glucose levels found in the blood of fasting humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen respectively because they gave much improved sperm function and this was within the range of insulin and leptin levels previously measured in human ejaculated spermatozoa. This was followed by investigating the signalling pathway of insulin and its beneficial effects on human spermatozoa function. Endogenous insulin secretion from human ejaculated spermatozoa was blocked by nifedipine and its receptor tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol 3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous insulin administration significantly increased human sperm motility parameters as well as the sperm ability to acrosome react. The inhibition of endogenous insulin release from spermatozoa as well as the inhibition of the insulin receptor substrate (IRS) tyrosine phosphorylation significantly decreased motility parameters and the ability of spermatozoa to acrosome react. The study also investigated the effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin significantly increased sperm motility parameters, acrosome reaction and NO production. The NO production induced by insulin and leptin was via PI3K signalling as evidenced by a reduction in NO levels when PI3K activity was inhibited by wortmannin. To investigate whether insulin and leptin could improve motility parameters of asthernozoospermic and teratozoospermic spermatozoa, the spermatozoa were separated into two fractions by means of a double density gradient technique. The gradient system was able to separate spermatozoa into high morphologically abnormal and less motile spermatozoa similar to that of asthernozoospermic and teratozoospermic patients as well as a more motile fraction. Insulin and leptin significantly increased the motility parameters of spermatozoa from the immature and less motile fraction. The fourth part of the study was aimed at investigating the effects of the cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm motility, viability, acrosome reaction and NO production. The study shows that TNF-α and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4 and time-dependent manner. These cytokines were also shown to significantly increase NO production from human spermatozoa. The decreased motility parameters induced by these cytokines could be attributed to their ability to induce excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo the acrosome reaction. The fifth part of the study was to investigate the expression and localization of glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8 is constitutively expressed and located in the midpiece region of the human spermatozoa. The study also showed that stimulating spermatozoa with insulin led to an increase in GLUT8 expression as well as translocation to the acrosomal region. In the last part of the study we wanted to investigate why the increase in NO generation by spermatozoa due to insulin and leptin stimulation is accompanied with increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation is accompanied with decreased sperm function. We observed that TNF-α and IL-6 not only increased NO production but also ROS production. This study speculates that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6 were administered was due to the concomitant high increase in NO and ROS they induced. In conclusion, this study has established in vitro beneficial effects of insulin and leptin in normozoospermic and asthernozoospermic human sperm function. These hormones influence sperm function via the PI3K signalling pathway in two ways. Firstly, by increasing GLUT8 expression and translocation thereby possibly increasing glucose uptake and metabolism and secondly, by increasing NO production. The study has also established that TNF-α and IL-6 have detrimental effects on human spermatozoa in a dose and time dependent manner. These effects are mediated via their ability to stimulate both NO and ROS production in human spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of human spermatozoa and that insulin stimulation upgrades its expression and leads to its translocation to the acrosomal region.<br>AFRIKAANSE OPSOMMING: Oor die afgelope jare was daar `n toename in obesiteit en diabetes mellitus (DM). Hierdie toestande word reeds vir ’n geruime tyd geassosieer met onvrugbaarheid. Obesiteit word gekenmerk deur verhoogde sirkulerende vlakke van leptiene en sitokiene sowel as insulien weerstandigheid. Tipe I DM word geassosieer met lae of geen insulien terwyl Tipe II DM gekenmerk word deur insulien weerstandigheid. Soos wat die voorkoms van obesiteit en DM toeneem, is dit waarskynlik dat die insidensie van onvrugbaarheid wat met hierdie patologiese toestande geassosieer word, gevolglik ook sal toeneem. Die effek van insulien en leptien op die manlike voortplantingsfunksie is alreeds aangetoon op endokriene en spermatogenese vlak, maar hul effekte op sellulêre vlak van menslike geëjakuleerde spermatosoë is nog onduidelik. Die studie vertoon data oor die rol van insulien en leptien op die menslike geëjakuleerde spermatosoë en hul interaksie met sitokiene en stikstofoksied (NO). In die eerste gedeelte van die studie, het ons ’n toepaslike konsentrasie van insulien en leptien bepaal wat aan menslike spermatosoë in vitro toegedien kan word. Glukose konsentrasies van 5,6 mM is bepaal as die gepaste konsentrasie om aan menslike spermatosoë toe te dien, omdat dit beter resultate tot gevolg het; verder is dit vergelykbaar met fisiologiese glukose vlakke in die bloed van `n vastende persoon. Insulien en leptien konsentrasies is op 10 μIU en 10 nm onderskeidelik vasgestel, aangesien dit tot beter resultate gelei het, en omdat dit vergelykbaar was met insulien en leptien vlakke wat reeds voorheen in menslike geëjakuleerde spermatosoë gemeet is. Dit was gevolg deur `n ondersoek na die insulien seintransduksie pad en sy voordelige effekte op menslike spermatosoë funksie. Endogene insulien afskeiding deur menslike geëjakuleerde spermatosoë was deur nifedipien geïnhibeer en sy reseptor tirosien fosforilasie effekte was deur erbstatin geïnhibeer terwyl fosfatidielinositol 3-kinase (PI3K) fosforilasie deur wortmannin geïnhibeer is. Eksogene insulien toediening het menslike sperm-motiliteit parameters betekenisvol laat toeneem asook die vermoë van sperme om die akrosoomreaksie te ondergaan. Die inhibisie van endogene insulien afskeiding deur spermatosoë sowel as die inhibisie van die insulien reseptor substraat (IRS) tirosien fosforilasie het die motiliteit parameters en die akrosoomreaksievermoë van spermatosoë verlaag. Die studie het ook die effekte van insulien en leptien op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie nagevors. Beide insulien en leptien het sperm-motiliteit parameters, -akrosoomreaksie en -NO produksie betekenisvol verhoog. NO produksie is deur insulien en leptien via PI3K seintransduksie geïnduseer, soos bewys deur die verlaging waargeneem in NO vlakke toe PI3K aktiwiteit deur wortmannin geïnhibeer was. Om vas te stel of insulien en leptien die motiliteit parameters van asthenozoospermiese en teratozoospermiese spermatosoë kon verbeter, het ons spermatosoë in twee fraksies met ’n dubbel digtheid gradiënt geskei. Die gradiënt sisteem was daartoe instaat om die spermatosoë in ’n onvolwasse, (morfologies abnormaal en minder motiel - soortgelyk aan dié van asthenozoospermiese en teratozoospermiese pasiënte), sowel as ’n volwasse meer motiele fraksie te skei. Insulien en leptien het die motiliteit parameters van spermatosoë van die onvolwasse en minder motiele fraksie verhoog. Die vierde gedeelte van die studie was daarop gemik om die effekte van die sitokiene tumor nekrose faktor alfa (TNF-α) en interleukin-6 (IL-6) op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie, te ondersoek. Die studie het getoon dat TNF-α en IL-6 motiliteit parameters en akrosoomreaksie in ’n tyd- en dosis-afhanklike wyse betekenisvol verlaag het. Hierdie sitokiene was ook in staat om NO produksie in menslike spermatosoë te verhoog. Die verlaging in motiliteit parameters wat deur hierdie sitokiene geïnduseer is, kan toegeskryf word aan hul vermoë om die produksie van oormatige hoeveelhede NO te stimuleer. Dit is nog nie duidelik hoe hulle die akrosoomreaksie in spermatosoë kan inhibeer nie. Die vyfde gedeelte van die studie het dit ten doel gehad om die uitdrukking en lokalisering van die glukose transporter 8 (GLUT8) in menslike spermatosoë te ondersoek. Hierdie studie kon aantoon dat GLUT8 konstitutief uitgedruk is en in die middelstuk van die menslike spermatosoë voorkom. Die studie bewys ook dat stimulering van die spermatosoë met insulien tot `n toename in GLUT8 uitdrukking sowel as translokasie na die akrosomale area, lei. In die finale gedeelte van die studie wou ons ondersoek waarom die toename in NO produksie in spermatosoë (as gevolg van insulien en leptien stimulasie) deur `n toename in spermfunksie gekenmerk word, terwyl die toename in NO produksie (as gevolg van TNF-α en IL-6 stimulasie) deur ’n afname in spermfunksie gekenmerk word. Ons het waargeneem dat TNF-α en IL-6 nie alleen NO produksie nie, maar ook reaktiewe suurstof spesies (ROS) produksie verhoog het. Ons vermoed dat die afname in sperm motiliteit en akrosoomreaksie met TNF-α en IL-6 toediening, die gevolg van die gelyktydige verhoging in NO en ROS was. In gevolgtrekking kan ons sê dat hierdie studie die voordelige in vitro effekte van insulien en leptien op asthenozoospermiese en teratozoospermiese menslike spermfunksie aangetoon het. Hierdie hormone beïnvloed spermfunksie via die PI3K seintransduksie pad op twee maniere. Eerstens, deur `n toename in GLUT8 uitdrukking en translokasie, met die gevolg dat glukose opname en metabolisme moontlik verhoog is, en tweedens, deur die toename in NO produksie. Die studie het ook vasgestel dat TNF-α en IL-6 nadelige effekte op menslike spermatosoë in `n dosis- en tyd-afhanklike wyse het. Hierdie effekte vind plaas a.g.v. hul vermoë om beide NO en ROS produksie in menslike spermatosoë te induseer. Die studie toon aan dat GLUT8 uitdrukking in die middelstuk van die menslike spermatosoon voorkom en dat insulien stimulasie GLUT8 uitdrukking opreguleer en tot translokasie na die akrosomale area lei.

Cardiovascular disease is the leading cause of death in the western world. Hypertension is a major risk factor for cardiovascular diseases and blood pressure is in turn markedly influenced by large artery stiffness. Recently a number of studies have reported that apparently healthy normotensive individuals who exhibit an exaggerated systolic blood pressure response on exercise are at increased risk of developing subsequent sustained hypertension and cardiovascular disease. It is likely that an exaggerated systolic blood pressure response on exercise represents an abnormal response of the large artery during dynamic exercise. In this thesis the normal responses of large arteries to different types and intensities of exercise was investigated in healthy normotensive subjects. Whilst distensibility of limb conduit arteries was measured for up to 15 minutes following exercise aortic distensibility did not change. An exaggerated systolic blood pressure response on exercise was not observed in healthy subjects without other conventional cardiovascular risk factors, but was observed frequently in the presence of such risk factors. Subjects with an exaggerated systolic blood pressure response on exercise did not show increased limb conduit artery distensibility immediately following exercise (and by implication during exercise). Blockade of NO synthesis prevented the increase in limb conduit artery distensibility seen in the first few minutes following exercise, but did not abrogate the more sustained increase in arterial distensibility following exercise. Systemic blockade of NO synthesis caused marked changes in systemic haemodynamics at risk, but these were markedly attenuated during exercise. The impact of mental stress on arterial function was also assessed. Whilst peripheral microvessels vasodilated, large arteries stiffened and this largely accounted for the observed increase in blood pressure.

Nitric Oxide (NO) has been detected in the expiratory air of normal animals and human subjects. Recent experiments revealed that expiratory NO production rises during exercise and correlates well with O$ sb2$ consumption and heart rate. Whether or not physical conditioning influences expiratory NO output production remains unclear. In this study, NO concentration in expired gas was measured in 18 healthy male volunteers subdivided into 3 groups (sedentary, intermediate, athletes) based on their state of physical conditioning. Measurements were taken at rest and during two steady-state exercise bouts on a bicycle ergometer designed to elicit VO$ sb2$ of 1 and 2 1/min with the athletes performing an additional bout at VO$ sb2$ of 4 1/min. In the sedentary and intermediate groups, expired NO concentrations declined significantly with increasing VO$ sb2.$ In contrast, expired NO levels declined only slightly with increasing VO$ sb2$ in athletes. At a VO$ sb2$ of 2 1/min, expired NO concentrations were significantly higher in athletes compared with the other groups. When correlated with V$ rm sb{E},$ expired NO concentrations declined linearly with the increase in $ rm V sb{E}$ in sedentary and intermediate groups but not in the athletes. Only the athletes had a significant linear increase in NO output (expired NO x V$ rm sb{E})$ with increasing VO$ sb2$ (p $<$ 0.001). These results support the notion that physical conditioning increases expiratory NO output during exercise. We speculate that the rise in expiratory NO output in athletes might be due to increased vascular and/or epithelial production of NO. Enhanced vascular NO production may be the result of increased shear stress and/or upregulation of endothelial NO synthase gene expression.

A study of the reactions of various S-nitrosothiols, particularly S-nitroso-N- acetylpenicillamine (SNAP), was undertaken. These compounds were known to produce nitric oxide (NO) when decomposing, which has important and diverse biological roles. An example of their use in physiological research was demonstrated. The Griess method was used to determine the stoichiometry of nitrite production from S-nitrosothiol decomposition in various buffer solutions. In all cases the production was found to be almost quantitative. The kinetic measurement of SNAP decomposition in a variety of buffers and pH was undertaken. The results were complex and often erratic, conforming to first order but also half order kinetics in many cases. There was some indication that decomposition products and light could affect the reaction. The presence of disulphide (dimer) as a major reaction product was confirmed in the case of SNAP. Free-radical traps were used to probe the decomposition mechanism, as were hemin and haemoglobin as NO detectors to determine decomposition kinetics. The true agent of S-nitrosothiol decomposition was found to be intrinsic copper in the water supply and buffer salts. S-nitrosothiols were found to be stoichiometrically decomposed by Hg(^2+) ions, but catalytically decomposed by Cu(^2+) ions. Kinetic measurement confirmed the complex nature of the catalysis. The importance of SNO and NH(_2), and SNO and COO- as binding sites was demonstrated. Some explanation was found for the differing structure/reactivity relationships observed. It was shown that transnitrosation of a thiol could occur, involving thiolate anion attack upon the S-nitrosothiol. However, the reaction appeared to be very slow at physiological pH. The nitrosation of N-methylaniline by S-nitrosothiols was found to occur only in the presence of oxygen - direct transfer of NO did not occur, nitrosation being mediated after SNAP decomposition.

Thesis (Ph. D.)--West Virginia University, 1999.<br>Title from document title page. Document formatted into pages; contains x, 265 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

The thesis examines the regulation of neuronal nitric oxide synthase (nNOS) mRNA expression in magnocellular neurosecretory neurones under physiological circumstances including pregnancy, hyperosmotic challenges and prolonged stimulation of the axons of the magnocellular neurones, and the functional role of nitric oxide (NO) in regulating oxytocin and vasopressin neuronal activities, and the release of oxytocin. The studies have included measurement of mRNA expression by <I>in situ</I> hybridization, <I>in vivo</I> electrophysiological studies combined with intrasupraoptic nucleus retrodialysis, Fos immunocytochemistry studies, and functional studies involving measurement of oxytocin secretion. Expression of nNOS mRNA in the magnocellular neurones in the supraoptic (SON) and paraventricular nuclei (PVN) has been confirmed to be regulated in response to chronic osmotic stimuli, suggesting an important involvement of NO in this situation. It has been demonstrated that an induction of this gene is not a rapid response since an increase of NOS gene expression in the magnocellular neurones in the SON and PVN has been found at 6 h after acute hypertonic saline intraperitoneal injection, but not at 4 h after injection. Neuronal activity of oxytocin and continuous firing vasopressin neurones in the SON was inhibited by local application of NO donor, and was increased by local administration of NOS inhibitor. Nitric oxide donor inhibited neuronal activity of phasic vasopressin neurones, while NOS inhibitor did not alter neuronal activities of these neurones. Blocking the central production of NOS by NOS inhibitor enhanced the expression of Fos in the magnocellular neurones in the SON and PVN following high doses, but not low doses of hypertonic saline. In addition, inhibition of NOS potentiated the release of oxytocin evoked by electrical stimulation of the axons of the magnocellular neurones. Furthermore, prolonged electrical stimulation of the axons of the magnocellular neurones for 2 h produced a down-regulation nNOS mRNA expression in the magnocellular neurones in the SON. Thus NO, generated in an activity-dependent manner, appears to act as a feedback inhibitor of oxytocin release at the cell bodies and the terminals of oxytocin neurones. However, although NOS expression is up-regulated in conditions of chronic demand of oxytocin, the present experiments indicate that this does not reflect a coupling of spike activity to increased NOS mRNA expression. In experiments in virgin rats, central administration of NO donor attenuated the release of oxytocin in response to acute intraperitoneal hypertonic saline and systemic administration of NOS inhibitor potentiated oxytocin release induced by acute intraperitoneal hypertonic saline but not cholecystokinin.

Nitric oxide or nitrogen monoxide (NO) is a tiny free radical gas. The discovery of this intriguing molecule has revolutionized physiology and pharmacology research during the last 20 years. Currently, it is known that NO is endogenously synthesized by several molecules and tissues via two pathways: the synthase-dependent pathway and the synthase-independent pathway. In the first, the amino acid L-arginine is the main donnor of NO synthesis. In the second, inorganic nitrate is the main substrate for the synthesis of this molecule. Interestingly, both pathways are directly connected. While the synthase-dependent pathway is oxygen dependent, the synthase-independent metabolic route is greatly facilitated under hypoxia conditions. Thus, these mechanisms of NO production regulate levels of NO in the tissues. An adequate production of NO is important because it plays an essential role in mechanisms related with vasodilatation and blood flow distribution. Additionally, NO modulates other important functions in the human body such as mitochondrial respiration and immune mechanisms. For all these reasons, the interest for dietary NO donors have increased during the last years. It has been suggested that the consumption of food rich in L-arginine or in inorganic nitrate may enhance NO availability in the human body. This hypothesis has not been unnoticed in exercise physiology. In fact, it has been suggested that supplementation with NO donors may improve the cardio-respiratory response, as well as the tolerance to endurance exercise in humans. However, there is a lack of studies analyzing this issue. Therefore, the aim of this doctoral thesis was to assess the effect of L-arginine and inorganic nitrate in the cardio-respiratory and metabolic response of healthy humans. To develop this aim, three studies and one review were carried out. In the first, it was found that L-arginine supplementation during three days at several doses, between 5.5 and 20.5 g/day was not effective to increase plasma markers of NO, as well as the cardio-respiratory and metabolic response during endurance test. In the second study we found that acute dose of inorganic nitrate supplementation (10 mg/kg of body mass) raised significantly plasma levels of nitrate and nitrite. However, this effect did not report an improvement in the cardio-respiratory response at low-to-moderate intensities of exercise. However, at maximal work loads of exercise dietary nitrate induced significantly reduction of oxygen consumption (VO2peak) compared with placebo. Other cardio-respiratory parameters, as well as blood lactate concentration did not differ between nitrate and placebo. In addition, exercise performance measured as time to exhaustion during an incremental test did not increase compared with placebo. All these findings together suggested that at higher intensities of exercise energy production became more efficient after inorganic nitrate ingestion. Accordingly, in the third study it was analyzed the effect of dietary inorganic nitrate ingestion for three days during endurance exercise in a cycle ergometer at high intensity (time-trial of 40-min). Results of this study showed that nitrate supplementation did not increase significantly plasma levels of nitrite, as well as enhance performance in healthy subjects. Interestingly, a significant, negative correlation was found between change in nitrite and endurance capacity measured as VO2peak during the exercise test. These results indicated that the effect of dietary nitrate ingestion was lower in subjects with high cardiovascular capacity compared with subjects with poor tolerance capacity to endurance exercise. This fact is very important, since it is known that endurance training increase values of VO2peak in sedentary population and this fact is correlated with lower incidence of cardiovascular diseases. These and other important conclusions of these studies are included in the last work of this thesis which was a review article.<br>L’òxid nítric (NO) es un radical lliure alliberat per diverses molècules i teixits en l’organisme humà. El descobriment d’aquesta intrigant molècula ha revolucionat la recerca en el camp de la fisiologia i la farmacologia durant els últims 20 anys. Actualment, es coneix que la alliberació de NO per part de les cèl•lules endotelials estimula el procés de vasodilatació. A més, també es coneix que aquesta molècula es un important regulador de la respiració mitocondrial i del sistema immunològic. Totes aquestes funcions han generat un gran interès per els precursors nutricionals de NO. En l’àmbit de la fisiologia de l’exercici físic s’ha suggerit que la suplementació amb alguna d’aquestes substancies (L-arginina o nitrat inorgànic) pot millorar la tolerància a l’exercici físic de resistència. No obstant, hi ha molta controvèrsia en els resultats dels estudis que han analitzat aquesta hipòtesi. Per tant, l’objectiu principal d’aquesta tesi doctoral va ser analitzar els efectes dels principals precursors de NO, L-arginina i nitrat inorgànic, en la resposta cardiorrespiratòria i metabòlica durant l’exercici físic de caràcter aeròbic en humans. Per dur a terme aquest objectiu es van realitzar 3 estudis i una revisió bibliogràfica. Els principals resultats d’aquests estudis van mostrar que la suplementació de L-arginina en diferents dosis no va ser efectiva per augmentar el marcadors plasmàtics de NO, així com, la resposta cardiorrespiratòria i metabòlica durant un exercici físic aeròbic en intensitats moderades. En referència als nitrat inorgànic, es va observar que la suplementació augmenta els nivells d’aquests compostos en plasma. No obstant, aquest fet no es va correlacionar amb una millora de la tolerància a l’exercici físic de resistència. A més, es va observar una correlación negativa i significativa entre l’augment dels nitrits plasmàtics i la potència aeròbica màxima (VO2max). Tots aquests resultats van ser àmpliament tractats en l’últim treball (revisió bibliogràfica) d’aquesta tesi. En resum, l’ingesta nutricional de L-arginina i/o nitrat inorgànic no resulta efectiva per millorar la resposta cardiorrespiratòria i la tolerància a l’exercici físic de resistència en humans sans i entrenats físicament.

Neuropeptide Y (NPY) is widely expressed in both the central and peripheral nervous system and has an important role in the regulation of adult hippocampal neurogenesis by mediating the proliferation of neural precursor cells in both health and disease. The mechanisms underlying this neuroproliferative effect of NPY, however, are unknown. The aim of this project was to investigate these cellular pathways and the possible involvement of nitric oxide (NO) in NPY-mediated neuroproliferation using postnatal rat hippocampal cultures in vitro. NPY was found to have a purely proliferative effect on hippocampal neural precursor cells. The role of NO was explored by inhibiting the NO synthesising enzyme, nitric oxide synthase (NOS), which abolished the proliferative effect of NPY and supported the involvement of NO in NPY-mediated proliferation. Pharmacological analyses using subtype-selective inhibitors suggested that the neuronal isoform of NOS is the sole NOS subtype involved, which was expressed by both nestin+ precursors and class III β-tubulin+ neurons, the cell types previously shown to be responsive to NPY. The involvement of NO was further verified through loading hippocampal cells with an NO indicator, diaminofluorescein diacetate, where an increase in NO/N2O3 production was observed in nestin+ precursors and class III β-tubulin+ neurons in response to NPY treatment. The downstream signalling pathways coupling NPY-mediated NO synthesis to cell proliferation were identified, through the use of selective pharmacological agonists and antagonists, as soluble guanylate cyclase, cGMP-dependent protein kinase (PKG) and the extracellular signal-regulated kinases (ERK) 1/2. By assessing levels of NPY-mediated ERK 1/2 phosphorylation in response to NOS inhibition, it was found that ERK 1/2 activation was mediated only via NOS/NO mechanisms. This proliferative cGMP-PKG-ERK 1/2 signalling cascade appears to be mediated by intracellularly released NO, while on the other hand, the addition of extracellular NO through the application of NO donors exerted an inhibitory effect on neural precursor cell proliferation. In addition to demonstrating the dual nature of NO, this is the first time that the signalling mechanisms underlying the proliferative effect of NPY on neural precursor cells have been described. Understanding the mechanisms underlying the proliferation of neural precursor cells will ultimately be beneficial by allowing the development of novel therapeutic interventions for promoting hippocampal neurogenesis. To analyse the role of NO in the NPY-mediated neuroproliferation of hippocampal cells in three-dimensional (3D) cultures, Laponite, a novel synthetic silica hydrogel, was used. Culture medium-based Laponite hydrogels were developed before cell viability within the hydrogels were assessed by culturing hippocampal monolayers under gel cover. Hydrogel cover, however, resulted in cell behaviour reminiscent of preservation/fixation as monolayers showed no spatial or morphological changes over time, with one possible explanation being the high gel osmolarity. Although attempts at cell seeding showed more positive results, with cells adhering to a low heavy metal content variation of the hydrogel, determination of cell viability remained a problem due to prominent dye-gel binding. Although the rheological properties of Laponite make its use attractive, the biocompatibility of the hydrogels with hippocampal cells still require further optimisation if they are to be used as cell culture matrices.

Nitric oxide (NO) and reactive oxygen species are two key components in the induction of the hypersensitive response (HR) during plant defense against pathogen infection. In animal cells, the production of NO is catalyzed by nitric oxide synthase (NOS). Although NOS activity has been documented in plants, the process of NO synthesis in plants is not well understood. Isolation of the NOS protein and/or cloning of the corresponding gene will greatly facilitate the understanding of NO synthesis and its role in plant defense. The objectives of this research were to analyze the physiological and biochemical properties of a NOS-like protein (peaNOS) of pea (Pisum sativum L.), to purify and characterize peaNOS, and to clone the gene(s) encoding peaNOS and relate its expression to NOS activity in pea-bacteria interactions. The application of abiotic agents that induce systemic defense in plants [copper chloride, ActiguardÒ, Triton-X100 and salicylic acid (SA)] to pea leaves did not induce NOS activity and verified reports that NO and NOS function upstream of SA in the signaling pathway of defense responses. Maximum (two-fold) NOS activity was detected three hours before the onset of HR in pea leaves infiltrated with incompatible bacteria (Ralstonia solanacearum), which is consistent with the effect of NO in the activation of HR after interaction with H2O2. The compatible bacteria (Pseudomonas syringae pv pisi) induced NOS activity significantly, suggesting that NO generation may also be a general response to biotic stress in plants. Antibodies raised against mammalian NOS did not have apparent specificity and utility for isolating peaNOS and should be used with caution in non-mammalian systems. The peaNOS protein was most efficiently extracted under alkaline conditions (pH 8.5 and 9.0) as compared to the neutral conditions (pH 7.4-7.5) in animal systems. Precipitation of the peaNOS protein with various concentrations of ammonium sulfate, sodium citrate and sodium chloride caused rapid loss of NOS activity. The peaNOS protein did not bind to 2',5'-ADP-Sepharose and calmodulin (CaM)-agarose indicating that the protein lacks binding sites for NADPH and CaM. Cloning of a peaNOS gene based on mammalian NOS was unsuccessful suggesting that the structure of peaNOS gene may be significantly different from mammalian NOS. Analysis of the Arabidopsis thaliana genome database identified two gene sequences related to animal NOS, i.e., accessions At4g09680 (similar to NOS of Rattus norvegicus) and At3g47450 (similar to NOS of Helix pomatia). Gene At4g09680 is probably not expressed since attempts to clone cDNA of this gene using reverse transcription-polymerase chain reaction (RT-PCR) consistently failed, even when RNA of Arabidopsis was used as a template. A potential expressed peaNOS gene was successfully cloned using RNA template of pea HR tissues in RT-PCR. The 784-bp peaNOS cDNA sequence had 50% nucleotide identity to the At3g47450 coding sequence and had no other significant match in the database. The correlation of the gene expression of P protein of glycine decarboxylase complex (GDC) of pea (peaP) and NOS activity during HR in pea was not demonstrated here but peaP gene was highly expressed concomitant with NOS activity during disease development. The NOS-like protein involved in NO production during HR in pea appears to be more related to At3g47450 sequence, and is possibly encoded partially by the cloned 784-bp pea cDNA.

Air pollution in the home has been associated with asthma and asthma-like symptoms in children. To understand how they contribute to the initiation and/or exacerbation of asthma, the toxic effects of these pollutants on the human respiratory system have been studied under controlled laboratory conditions. In these clinical studies exposure is short and concentrations are generally higher than those encountered on a daily basis. Domestic levels of air pollutants are quite low and little is known about how chronic exposure to these low level pollutants can affect asthma prevalence and severity. Inflammatory changes in the airways are important for both the development of asthma and triggering of symptoms. The main aim of this thesis was to investigate the effects of selected indoor pollutants on exhaled nitric oxide, a marker of asthmatic airway inflammation, in healthy children. The primary objective was to improve our knowledge regarding a possible mechanism by which these pollutants can contribute to asthma in children. The research was divided into two parts. In the first part methodological and physiological factors that can affect measurements of exhaled nitric oxide in a healthy paediatric population were investigated. One hundred and fifty seven children aged between 6 and 13 years were recruited for this study. Children were recruited from local primary schools via a respiratory health questionnaire and only those children without current respiratory disease or a history of respiratory illness were included. The children underwent a respiratory assessment at the Respiratory Medicine Department of Princess Margaret Hospital for Children (Perth, Western Australia). This included spirometry, a skin prick test and measurement of exhaled nitric oxide. Methodological issues that were investigated included a suitable expiratory flow for children for eNO measurements and the repeatability of eNO measurements. Physiological factors studied included skin prick reactivity (atopy), age, height and spirometric variables (forced vital capacity (FVC) and forced expiratory volume in one second (FEV 1)). Exhaled NO levels are flow dependent and if the flow is too high differences in eNO levels between subjects is reduced. Three expiratory flows were used (50, 75 and 100 mis'1) for measuring eNO levels in this study. A flow of 75ml.s'1 seemed to be the most comfortable for the children, while, at this flow, there remained good discrimination of eNO levels between subjects. The twenty-four hour repeatability of eNO levels for each expiratory flow was measured in 10 children. The coefficient of repeatability of measurements were 8.3 ppb, 5.2 ppb and 6.3 ppb, respectively, which represents 9.9%, 6.9% and 10.0% of the range of eNO levels at each flow. Two physiological factors were positively associated with eNO levels in healthy children. These were age (p < 0.05) and atopy (p < 0.001). These findings had not been reported previously and the mechanisms require further investigation. A possible mechanism for the relationship between atopy and raised eNO levels is subclinical inflammation caused by exposure to allergens. To investigate this house dust mite allergen levels were collected from the mattress of those children who had a positive skin prick reaction to house dust mite. Exhaled NO measurements were repeated in these children during the period allergen levels were collected. There was no association between current allergen exposure, as determined by mattress levels, and eNO in these children. The main part of this thesis was a cross sectional study to investigate the effect of indoor air pollution on eNO levels in healthy children. This study involved 224 children (6 to 13 years old) who were free of respiratory disease. The homes of the children were monitored for formaldehyde and nitrogen dioxide using passive sampling techniques, while their exposure to environmental tobacco smoke was determined by questionnaire. These three pollutants - formaldehyde, nitrogen dioxide and environmental tobacco smoke - were chosen for the study due to reported associations with asthma and evidence that they can induce inflammatory responses in human airways. During the week their homes where monitored the children underwent a respiratory assessment (as above). Neither measured N02 concentrations nor reported smoking in the home were associated with changes in eNO levels in the children. However, children living in homes with formaldehyde levels that were greater than or equal to 50 parts per billion had significantly higher levels of eNO than children exposed to levels less than 50 ppb in the home (15.5 ppb v 8.7 ppb, p < 0.002). The results of this study suggest that exposure to low levels of air pollution in the home can be associated with inflammatory changes in the airways. These results need to be confirmed and extended in future research. Future studies should include an investigation of how these indoor pollutants affect the developing lungs and immune system in infants, thereby improving our understanding of their role in the initiation of asthma.

The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.

Three bovine casein peptides, LLY, PGPIPN, and TTMPLW, have been reported to stimulate phagocytosis of sheep red blood cells by murine macrophages. TTMPLW also protected mice against Klebsiella pneumoniae infection. The purpose of this study was to investigate the effects of these three peptides on cytokine (TNF-$ alpha$ and IL-6) production and nitric oxide (NO) release by bone marrow macrophages (BMM). The peptides alone were incapable of stimulating cytokine production or NO release in naive or IFN-$ gamma$-primed BMM. However, when BMM were coincubated with the peptides (1.0 $ mu$M) and LPS (100 ng/ml), an augmentative effect on TNF-$ alpha,$ IL-6 and NO production was observed. The peptides increased the response of BMM to stimulation with LPS in a dose- and time-dependent manner. Of the three peptides, TTMPLW (0.01, or 1.0 $ mu$M) had the greatest augmentative effect on NO production by LPS-stimulated BMM. Tumor necrosis factor-$ alpha$ production peaked after 4 hr stimulation, and decreased rapidly thereafter. Among three peptides, TTMPLW induced the highest amount of TNF-$ alpha$ production at a concentration of 1.0 $ mu$M. When used at a concentration as low as 0.01 $ mu$M, TTMPLW and PGPIPN, but not LLY, potentiated TNF-$ alpha$ production. All the peptides (1.0 $ mu$M) stimulated IL-6 production by BMM, which plateaued after 12 hr. The auto/paracrine TNF-$ alpha$ produced by LPS-stimulated BMM was partially responsible for release of NO. After all the TNF-$ alpha$ was neutralized, release of NO was reduced by about 21% (P $<$ 0.01). However, neutralization of IL-1$ beta$ and IL-6 did not have any effect on NO production by LPS-stimulated BMM. These results demonstrate that bovine casein peptides can costimulate naive macrophages with LPS for proinflammatory cytokine production and NO release and may play a role in host defense against pathogens.

Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), alone or in combination with estrogen treatment, would potentiate the beneficial effects of EPCs in the context of restenosis.We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (hCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of hCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with hCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced Bcl-2/Bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in hCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, hCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs. These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate hCASMC migration through paracrine effects.<br>La resténose artérielle, qui se produit dans presque 20% des patients ayant subi une angioplastie, est caractérisée par une prolifération excessive des cellules musculaires lisses vasculaires résultant de la disparition du revêtement des cellules endothéliales. Les progéniteurs endothéliaux circulants (EPC) ont la capacité de recoloniser et réparer l'endothélium vasculaire endommagé, ce qui réduit la resténose. L'oxyde nitrique (NO) contribue à la mobilisation et l'activité fonctionnelle des EPCs, et l'oestrogène a été montré pour augmenter le taux circulant des EPCs et d'accélérer le processus de réendothélialisation par les EPCs chez la souris. En outre, le NO est important pour la transduction de la signalisation oestrogène-dépendante et la réendothélialisation. Nous avons proposé que la surexpression d'oxyde nitrique synthase endothéliale (eNOS), seule ou en combinaison avec un traitement à l'oestrogène, potentialiserait les effets bénéfiques des EPCs dans le cadre de la resténose.Nous avons constaté que les EPC humains natifs (hEPCs) n'ont pas d'effet sur la prolifération et la migration in vitro des cellules musculaires lisses de l'artère coronaire humaine (hCASMC), évaluées par l'incorporation de BrdU et le l'essai « wound-scratch » respectivement. En revanche, le donneur de NO SNAP a diminué de façon significative la prolifération et la migration des hCASMCs. Par la suite, des hEPCs ont été soit transfectés avec un plasmide eNOS humain ou stimulés par du 17β-estradiol (E2) avant d'être co-cultivés avec des hCASMCs. Les niveaux d'eNOS totale et phosphorylée évalués par western blot ont été augmentés de 3 - à 3,5 fois dans les hEPCs transfectés avec eNOS ou stimulés avec E2. Cela a été associé à une augmentation de 3 fois de la production de NO, mesuré par immunofluorescence avec DAF-FM diacétate (p <0,05). Des hEPCs surexprimant eNOS ont montré une augmentation du rapport Bcl-2/Bax et le marquage d'annexine V/iodure de propidium a été réduit indiquant une augmentation de la survie. Fait intéressant, nous avons observé une diminution significative (p <0,05) de la migration des hCASMC de 23% pendant la co-culture avec des hEPCs surexprimant eNOS, et de 56% avec des hEPCs stimulés avec E2. Cependant, la prolifération des hCASMC n'a été affectée ni par des hEPCs surexprimant eNOS, ni par les hEPCs stimulés avec E2. Ces résultats suggèrent que la surexpression de eNOS dans des EPCs humains augmente leur survie et améliore leur capacité à moduler la migration des hCASMC via des effets paracrines.

Background: Vascular dysfunction due to hyperglycemia in individuals with diabetes is a factor contributing to distal symmetric polyneuropathy (DSP). Reactive oxygen species (ROS) reduce the bioavailability of nitric oxide (NO), a powerful vasodilator, resulting in reduced circulation and nerve ischemia. Increases in blood NO concentrations and circulation have been attributed to whole body vibration (WBV). The purpose of this study was to the determine the effects of low frequency, low amplitude WBV on whole blood NO concentration and skin blood flow (SBF) in individuals with symptoms of DSP. Research Design and Methods: Ten subjects with diabetes and impaired sensory perception in the lower limbs participated in this cross-over study. Each submitted to two treatment conditions, WBV and sham, with a one week washout period between. Blood draws for NO analysis and Doppler laser image scans of SBF were performed before, immediately after and following a 5 minute recovery of each the treatments. Results: Low frequency, low amplitude WBV vibration significantly increased skin blood flow compared to the sham condition (p=0.0115). Whole blood nitric oxide concentrations did not differ between the WBV and sham condition immediately or 5 minutes post-treatment ( p=0.1813) Conclusions: These findings demonstrate that subjects with diabetes respond to whole body vibration with increased skin blood flow compared to sham condition. The implication is that WBV is a potential non-pharmacological therapy for neurovascular complications of diabetes.

A wealth of physiological and pathophysiological roles have been ascribed to nitric oxide (NO) in the nervous system. Many of these seem diverse and unrelated. However, the widespread role NO has to play in modulation of the nervous system suggests an underlying common and fundamental role. The ongoing popularity of NO research has lead to a mass of literature on the subject yet great controversy still prevails as to the true physiological function of NO in the nervous system. In this thesis, the existing literature has been critically evaluated. Histological, autoradiographic and electrophysiological research approaches have been used to investigate further the physiological role for NO in the nervous system in the hope of explaining some of the controversy that still prevails. Due to an unforseen move, two different models have been used during the investigations, the brain and the pituitary gland of the ring dove (Streptopelia risoria) and the superior cervical ganglion (SCG) of the rat. The histological NADPH-diaphorase (NDP) technique was used to show the distribution of the NO synthesising enzyme nitric oxide synthase (NOS) for the first time in the ring dove brain. Previously, the NDP distribution has been mapped in the brain of other species and found to be distinct from all other known neurotransmitters. NADPH-diaphorase is a known marker of the mammalian pontine cholinergic reticular formation. However, findings presented here strongly suggest that the distribution of NOS is far more widespread in structures comprising the avian reticular formation. The distribution of the NO synthesising enzyme and the freely diffusible property of NO make it a highly attractive messenger molecule that might coordinate the spatially distinct structures of the reticular formation enabling them to act as one functional unit. Nitric oxide is released following NMDA receptor activation. Both the presence and the distribution of this component of the NO-cGMP pathway have been shown here for the first time in the ring dove brain. This has been achieved using receptor autoradiography of the [3H]-MK-801, a ligand that binds to the NMDA receptor channel. The ring dove is a popular model for the study of behaviour changes and the hormonal control of the avian breeding cycle. Here, a study was set up to compare regional [3H]-MK-801 ligand binding in the brains of non breeding birds (naive) and first time breeders. Quantitative receptor autoradiographic analysis of the ligand binding has shown that significant regional binding changes occur during the breeding cycle of the ring dove. This result implies that the NO-cGMP pathway might have a physiological role in the breeding cycle of the dove and that further investigation of this matter would be worth undertaking. Here, histological and electrophysiological approaches using the rat SCG model have been employed to investigate the role of NO-cGMP pathway in modulation of synaptic transmission. Histological mapping of the pathway components has reconfirmed that NOS is present in the presynaptic fibres and terminals, but it also suggests that it is restricted to a subpopulation of the principal neurones in the ganglion. Immunocytochemistry of the NO-donor induced cGMP accumulations shows that the satellite cells surrounding the principal neurones are major target sites for NO. This finding is consistent with that seen in the dorsal root ganglion (DRG), and supports a glial target for NO in this tissue. Although, NO-donors depolarise the ganglion and enhance synaptic transmission when extracellularly recording the compound action potential (CAP), intracellular recording did not reveal such clear responses. This difference was attributed to the fact that a subpopulation of the principal neurones receives a NO input. These findings suggest that the SCG is not a particularly efficient model for intracellular investigation of the NO­cGMP pathway. Both intracellular and extracellular investigations into involvement of NO-cGMP pathway in ganglionic L TP suggest that it does not have a role to play in long-term potentiation (L TP) of synaptic efficacy in this tissue. Here, intracellular and extracellular approaches agreed. Further to this, extracellular recording ofLTP of the CAP supported the involvement of the pathway in a short term enhanced synaptic efficacy by a mechanism distinct from L TP. Moreover, it has been shown here that L TP of the epsp can be reliably produced in the SCG. It is therefore suggested that the SCG is a suitable preparation for both intracellular and extracellular investigation of LTP at the peripheral synapse.