The goals of these studies were to determine if maturational changes occurred in the responsiveness of airway and vascular porcine smooth muscle in vitro. Pulmonary arteries from immature pigs were less responsive than those from adult pigs to adrenoreceptor mediated vasoconstriction. An alpha-2 adrenoreceptor mediated release of endothelium-derived relaxing factor was demonstrated in pulmonary arteries from adult pigs but not in those from immature pigs. Arteries from immature pigs were less responsive to relaxation induced by nitric oxide and hypoxia but not by sodium nitroprusside. Bronchial rings from immature pigs produced greater force (normalised to wet weight) in response to acetylcholine or potassium chloride than did rings from adult pigs, although the concentration of acetycholine required to produced half-maximal response (EC50) was similar. Rings from newborn pigs were more sensitive to the bronchodilator ketamine than those from adult pigs, as manifested by a shift in EC50. The relaxant action of ketamine on tracheal smooth muscle was shown to be due to an inhibitory effect on excitation of the postsynaptic nicotinic receptors of the intramural parasympathetic ganglion and a direct effect on the smooth muscle cell. Nonadrenergic noncholinergic innervation was shown to be present from birth. Hypoxic bronchodilation was seen in bronchial rings from all ages. In conclusion, this study demonstrates age-related changes in the in vitro responsiveness of both pulmonary vascular and airway smooth muscle to contractile and relaxant agonists, responses depending not only upon the agonist used to elicit contraction or relaxation, but also upon the age of the animal studied.

HCO3- secretion plays a vital role in regulating the pH and mucus viscosity of airway surface liquid to facilitate airway mucociliary clearance of inhaled pathogen. In cystic fibrosis (CF), reduced HCO3- secretion contributes to defective mucociliary clearance which predisposes the lungs to bacterial infection. Calu-3 cells are used as a model of human submucosal gland serous cells which are involved in CFTR (cystic fibrosis transmembrane conductance regulator)-dependent HCO3- secretion, a process that appears to involve functional interactions with both apical and basolateral Cl-/HCO3- anion exchangers (AE), but through regulatory pathways that are not well understood. The aim of this thesis was to investigate the signalling mechanisms that regulate CFTR-dependent AE activity in Calu-3 cells. Under resting conditions, Calu-3 cells showed a DIDS-sensitive Cl- and HCO3--dependent basolateral anion exchange activity consistent with AE2 (SLC4A2) expression. However, apical AE activity was not detected. Increasing cytosolic Ca2+, or removal of extracellular Ca2+, had no effect on basolateral AE activity. In contrast, lowering cytosolic Ca2+ with BAPTA-AM, or inhibiting calmodulin (CaM), reduced basolateral AE activity. Furthermore, an intact actin cytoskeleton, as well as active dynamin, were essential for maintaining basolateral AE activity, possibly via supply of new proteins to the basolateral membrane. Inhibiting CK2 or protein phosphatase 1 (PP1) abolished basolateral AE activity, and CK2 inhibition was linked to CaM. This suggests that AE activity was maintained through a novel CaM-dependent mechanism involving phosphorylation/dephosphorylation by CK2/PP1. In support of this, transient transfection of HEK293 cells with mouse AE2, with and without CK2 co-transfection, clearly demonstrated CK2-dependent AE2 activity. Stimulation of Calu-3 cells with cAMP agonists both activated an apical anion exchanger via a PKA and Epac-dependent mechanism, and inhibited the basolateral anion exchanger, but through a PKA and Epac-independent mechanism. Blocking CFTR with GlyH-101 caused an apparent inhibition of apical AE activity, but addition of basolateral DIDS restored apical activity, suggesting that a basolateral HCO3- transporter was activated when CFTR was inhibited. Removal of extracellular Ca2+ partially reduced the cAMP-induced inhibition of the basolateral AE activity, but had no effect on cAMP-stimulated apical AE activity. Moreover, increasing cytosolic Ca2+, or lowering cytosolic Ca2+ with BAPTA-AM, markedly reduced cAMP-stimulated apical AE activity, but it had no effect on cAMP-induced inhibition of the basolateral anion exchanger. Actin-cytoskeleton disruption had no effect on apical AE activity but dynamin inhibition caused a significant decrease. A similar decrease in apical AE activity was observed when CK2 was inhibited, but in contrast to the basolateral anion exchanger, this appeared to be via a CaM-independent mechanism. Inhibiting CK2, however, had no effect on the cAMP-induced inhibition of the basolateral AE activity, suggesting that CK2 regulation of Calu-3 anion exchangers is through cAMP-independent mechanisms. These findings provide new insights into the signalling pathways that regulate both the apical and basolateral anion exchangers in Calu-3 cells and help define their respective roles in airway HCO3- secretion. The results could potentially open up new avenues for modulating AE activity which could be beneficial in HCO3- secretory diseases such as CF.

No description available.

No description available.

No description available.

The aim of this project was to construct equations to predict the mechanism of threshold values of sensory irritation (nose and eye irritation) caused by Volatile Organic Compounds (VOCs). Mathematical models were established to predict Nasal pungency thresholds (NPTs) and odour detection thresholds (ODTs). Firstly, the Abraham Solvation Equation was used to determine the octanol/water partition coefficient (log Poct) for fluorescein. A similar equation was used to characterize several Gas-Liquid Chromatography (GLC) stationary phases. Abraham descriptors were determined for several VOC compounds using data obtained from experiments using GLC and data on water-solvent partition coefficients. The QSAR analysis on NPTs and ODTs revealed that "selective" processes (i.e., chemically-broad, transfer driven effects) overwhelmingly dominate chemesthetic detection, whereas both selective and "specific" (i.e., chemically-narrow, ligand-receptor interactions) processes control olfactory potency. To understand further the nature of the chemical factors that influence ODT values, the possibility was explored that NPT values could be used to estimate "selective" effects in ODTs. In the present study, concentration-detection, i.e., detectability, functions for the human olfactory detection of some chemical vapours of diverse chemical structures were analyzed. The functions cover the complete peri-threshold range from chance to almost perfect detection and provide much more information than can be conveyed by a single "threshold value". These odour functions have been modeled with a uniform approach: a simplified sigmoid equation, y = 1 / (1 + e*"0), and have been correlated with the set of up to five physicochemical descriptors taken from the Abraham solvation equation that has quite successfully described the simpler "odour threshold" and "nasal pungency" psychophysical outcome. Previous studies of ocular and nasal chemesthetic thresholds along and across homologous chemical series have suggested the existence of a "cut-off, i.e., a point where a homolog fails to stimulate even at vapor saturation. All larger homologs fail as well. The present study sought, first, to identify the particular cut-off member along homologous alkylbenzenes and 2-ketones and, second, to probe the likely basis for the effect.

Cough is an important protective reflex. Sensory nerve activity mediating cough may be enhanced during disease. Current antitussives possess little clinical efficacy, therefore research is needed to investigate the modulation of airway sensory nerves in order to understand the mechanisms driving chronic cough. A conscious guinea-pig cough model using different tussive stimuli, known to elicit cough in man, was established. Corresponding in vitro effects of these tussive agents on sensory nerve activation were characterised. These agents were also characterised into sub-types as they produced distinct patterns of response. Cigarette smoke (CS) exposure elicited distinct effects on the cough reflex in conscious guinea-pigs and on sensory nerve activity measured in vitro; CS enhanced responses to citric acid (CA), capsaicin (CAPS) and bradykinin, inhibited a prostaglandin E2 (PGE2)-induced response and had no effect on the response to hypertonic saline. The CS model demonstrated neutrophilia and increased mucus in the airways. Focussing on mechanisms driving enhanced cough, revealed that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), may play a role in cough reflex sensitisation. An in vitro bioassay confirmed that NGF and BDNF exhibited bioactivity and the anticipated species cross-reactivity. CS exposure augmented the expression of transient receptor potential vanilloid 1 (TRPV1) in vagal sensory nerve ganglion. Pharmacological modulation of guinea-pig and human vagus nerve activation showed that TRPV1 antagonists, capsazepine and SB366791, abolished CAPS-induced depolarisation, partially inhibited low pH, bradykinin and PGE2-induced responses and had no effect on hypertonic saline. Despite positive in vitro and pharmacokinetic analyses, TRPV1 antagonists did not inhibit CAPS-induced cough in vivo. Further work is required to expand upon these findings. This thesis contains an assessment of the effects of sensory nerve modulation in guinea-pig models of cough. Understanding the neural mechanisms associated with this research may help to guide the development of novel antitussive therapies.

No description available.

No description available.

Previous studies have implicated oxygen (02)-dependent substances in the vasodilator response to muscle contraction. This thesis explores the effect of breathing supplementary O2, (40% O2: hyperoxia) on the increase in forearm blood flow (FBF) and vascular conductance (functional hyperaemia) evoked by forearm contractions in healthy young male subjects. Hyperoxia given throughout the protocol did not affect resting vascular conductance, yet reduced the hyperaemia evoked by sub-maximal static and rhythmic contractions and by maximal voluntary effort contractions (100% MVE). Hyperoxia given only during two successive periods of 100% MVE to exhaustion reduced the postcontraction hyperaemia but had no affect on the shortened time to voluntary exhaustion (TVE) of Contraction 2. By contrast, hyperoxia given only during recovery did not affect the hyperaemia, but lengthened the TVE of Contraction 2. Hyperoxia given during 100% MVE also reduced forearm venous concentrations of lactate and H+ but not of K+ and the cyclooxygenase (COX) product, prostaglandin h (PGh). Given the concomitant decrease in FBF this suggests reduced efflux of lactate, H+ and K+ and PGh. Further, COX inhibition with aspirin attenuated the hyperaemia but, in the presence of aspirin, hyperoxia given during recovery had no further effect on the hyperaemia: despite the reduced FBF, the TVE of Contraction 2 was stil1 improved. It is proposed that hyperoxia and the consequent increase in arterial O2tension (Pa02) can improve O2 delivery to forearm muscle even during 100% MVE so limiting anaerobic metabolism and possibly muscle and/or endothelial efflux of PGh and K+. These results are considered in relation to the influence of Pa02 in determining the role of PGs and other metabolites in functional hyperaemia and recovery from static contraction.