Neurons in the marginal layer (lamina I) of the dorsal horn of the spinal cord play a major role in the transmission and integration of pain-related sensory information that is relayed to the brain. Alteration in excitability of these cells greatly influences pain perception. Among these, alterations of the substance P (SP) system play a major role because of its known involvement in spinal cord nociceptive mechanisms. In this thesis, our main focus was on the characterization of the cell populations in lamina I of the spinal cord which express the SP receptor (NK-1r) and project to the parabrachial nucleus in normal rats and in an animal model of localized polyarthritis. Lamina I projection neurons can be classified into three morphological types, the fusiform, the multipolar (flattened) and the pyramidal. Our combined tract-tracing and immunocytochemical studies demonstrated that in control animals the fusiform and multipolar neurons project abundantly to the parabrachial nuclei and express the NK-1r, whereas pyramidal neurons, although projecting in almost identical proportion to the same target, express little, if any, NK-1r. In rats treated with a single, unilateral low dose subcutaneous injection of complete Freund's adjuvant (CFA) in the thick skin of the hind paw, we demonstrated an ipsilateral de novo NK-1r expression on the normally non-nociceptive spinoparabrachial lamina I pyramidal cell type starting at 15 days post-injection. We also observed, as from 15 days post-CFA, an innervation of pyramidal neurons by SP-immunoreactive (IR) boutons. It should be pointed out that pyramidal neurons are normally not innervated by SP, which would confirm their non-nocic / Dans la moelle épinière, les neurones de la couche marginale (couche 1) de la corne dorsale jouent un rôle majeur dans l'intégration et la transmission de l'information sensorielle vers le cerveau. Des changements au niveau de l'excitabilité de ces cellules influencent grandement la perception de la douleur. Parmi ceux-ci, un changement au niveau du système de la substance P (SP) peut avoir un grand impact en raison de son implication dans les mécanismes nociceptifs. Dans cette thèse, notre but premier était de caractériser la population neuronale de la couche 1 qui exprime le récepteur de la substance P (NK-1 et qui projette au noyau parabrachial. Les observations se sont faites chez les rats normaux et dans un modèle animal de polyarthrite locale. Les cellules de projection de la couche 1 peuvent être classifiées en trois types morphologiques; les fusiformes, les multipolaires (aplaties) et les pyramidales. Notre approche combinant le traçage rétrograde et l'immunocytochimie a démontré que, chez les animaux contrôles, les neurones fusiformes, multipolaires et pyramidaux projettent abondamment au noyau parabrachial. Les deux premiers types de cellules expriment le récepteur NK-1, alors que les cellules pyramidales l'expriment très peu, sinon pas. Chez les rats traités avec une seule dose unilatérale d'adjuvant complet de Freund (ACF), administrée sous-cutanée dans la plante de la patte, nous avons démontré une nouvelle expression du récepteur NK-1. Les cellules pyramidales qui sont habituellement non nociceptives sont imunoréactives pour le récepteur NK-1 à partir de 15 jours après l'injection. Nous avons aussi observé, à 15 jours apr

Numerous premature infants subjected to oxidant stress are afflicted with retinopathy of prematurity (ROP). Reactive oxygen species (ROS) and increased products of peroxidation with vasoconstrictor and cytotoxic properties ultimately lead to the degeneration of retinal microvessels. However, synthesis and activity of the vasodilator, nitric oxide (NO), are increased in the newborn. Furthermore, NO has been reported to protect cells, including endothelial cells, against injury associated with excess production of ROS. We thus postulated that NO plays a protective role in the development of oxygen-induced retinopathy by limiting the extent of microvascular obliteration and tested this hypothesis in a rat model of ROP. / Rat pups receiving inhibitors with differential selectivity for the NO synthase (NOS) isoforms were exposed to 80% O2 from postnatal days 6 to 12 and retinas were compared for extent of capillary obliteration. Endothelial cells being particularly susceptible to oxidative injury, we tested the effect of NO directly on microvascular endothelial cell cultures under oxidant stress. (Abstract shortened by UMI.)

In this thesis, we addressed different issues related to drug exposure as it may bear on the estimates of risk in the context of hypertension treatment. A cohort of 19,501 subjects initiating therapy for uncomplicated hypertension was identified from Saskatchewan Health databases. In a first study aimed at documenting the equivalence of the angiotensin-converting-enzyme (ACE) inhibitors, we found that medical visits and hospitalizations following treatment initiation were lower among patients initially dispensed enalapril and lisinopril relative to captopril. Baseline characteristics could not be ruled out as possible explanations but variability in the outcomes suggest that ACE inhibitors may not be equivalent in all respects. Due to concerns about the appropriateness of using initial treatment as the exposure, patterns of use of antihypertensive were examined longitudinally in the second manuscript using the same cohort. ACE inhibitors, followed by calcium antagonists and beta-blockers, were the most commonly prescribed agents to initiate therapy for hypertension. Compliance with therapy was found to decrease over time with only 28% of patients still being compliant after seven years. In addition, 89% of patients underwent at least one modification to therapy, interrupted treatment being the most frequently encountered. Important differences were also found across agents with regard to compliance, type and timing of treatment modifications. The third manuscript reports on a case-control study assessing the association between antihypertensive drug use and the risk of myocardial infarction (MI). Overall, 812 cases of MI were identified using hospital discharge data and death certificates. Four controls were matched to each case on entry date and time at risk of an event. Compared with beta-blockers, current use of calcium antagonists was associated with an increased risk of MI (RR = 2.3; 95% Cl = 1.7--3.1). The risk ratio for ACE inhibitors was 1.3 (95% Cl = 1.0

The contribution of use-dependence to the actions of two novel antiepileptic drugs acting upon the GABA neurotransmitter system was investigated using the rat hippocampal slices preparation. The first study of this thesis demonstrated that tiagabine, a GABA uptake blocker, causes a more pronounced increase of GABAergic responses evoked by high-frequency stimulation (HFS) than of those elicited by a single stimulus. The predominant effect of tiagabine on HFS-evoked responses was to facilitate the generation of GABAA receptor-mediated depolarizations (DRs) which were capable of triggering bursts of action potentials when evoked from the stratum radiatum. / Since these results suggested that tiagabine may paradoxically promote rather than inhibit epileptiform discharges, the second study investigated the functional consequences of tiagabine-augmented DRs. Consistent with our observation of enhanced inhibition following HFS, the amplitude of evoked EPSPs superimposed upon tiagabine-augmented DRs were markedly reduced and could no longer trigger action potentials. A similar inhibitory effect on evoked EPSCs was also observed. The large conductance increase associated with the occurrence of DRs suggests that tiagabine can reduce the depolarizing influence of excitatory transmission by increasing the effectiveness of GABA-mediated shunting inhibition. / In the third study of this thesis, the effects of gamma-vinyl GABA (GVG, vigabatrin) were investigated in slices prepared from animals pretreated with either an anticonvulsant dose of GVG or saline. GVG pretreatment produced a frequency-dependent strengthening of GABA-mediated transmission such that repetitive stimulation at low-frequencies (2.5--10 Hz) was no longer associated with a depression of inhibition. A reduced sensitivity of evoked IPSCs to the actions of baclofen, a GABAB receptor agonist, suggests that GVG may have produced its frequency-dependent effects by depressing the efficacy of the GABA release regulating negative feedback mechanisms through the presynaptic GABAB autoreceptors. / In the final study of this thesis, the acute effects of bath applied GVG on GABAergic inhibition were examined. GVG (100--500 muM) caused a concentration-dependent disinhibitory effect which did not appear to be mediated through an antagonism of postsynaptic GABAA receptors. These results suggest that to acute actions of GVG, distinct from those mediated through is inactivation of GABA-T, do not contribute to the anticonvulsant properties of this drug. / In conclusion, to results presented suggest that both tiagabine and GVG produce use-dependent increases in inhibition, an effect which may importantly contribute to their anticonvulsant properties.

In this thesis, the innervation patterns by substance P- (SP), dopamine-beta-hydroxylase- (DbetaH) and the vesicular acetylcholine transporter- (VAChT) immunoreactive (IR) fibres were investigated in the skin of the rat lower lip, in order to provide a morphological basis for the possible interactions between sensory and autonomic fibres in the periphery. Our immunocytochemical approach revealed that SP-IR sensory fibres were widely distributed throughout the epidermis and dermis. In the dermis, they were associated with blood vessels, hair follicles, sebaceous glands and mast cells. Electron microscopical quantification revealed that SP-IR fibres equally innervated arterioles, capillaries and venules, all of which were shown to possess SP receptor (NK-1r) immunoreactivity within their walls. DbetaH- and VAChT-IR fibres were present around blood vessels in the lower dermis, but not in the upper dermis, indicating that these fibres are not as widely distributed as the SP-IR fibres. SP-DbetaH, SP-VAChT and DbetaH-VAChT fibre combinations were observed around lower dermal blood vessels. The absence of single labeled blood vessels suggested that blood vessels in the lower dermis are innervated by all three fibre-types. Quantification at the ultrastructural level revealed that DbetaH- and VAChT-IR terminals lie closer to their targets than SP-IR terminals. Bilateral lesions of the mental nerve, a purely sensory nerve, led to the total loss of SP-IR fibres from the lower lip skin, and to the migration of DbetaH-IR fibres into the upper dermis, a territory from which they are normally absent. These sprouted fibres remained in their new territory despite the reinnervation of the lower lip by SP-IR fibres. The bilateral removal of the superior cervical ganglia (SCG) caused the complete degeneration of DbetaH-IR fibres and a transient SP-IR fibre sprouting and NK-1r up-regulation. The number of SP-IR fibres and the content of SP within these fibres was increased both in th / Therefore we can conclude that SP-, DbetaH- and VAChT-IR fibres all innervate the same blood vessels in the lower dermis of the rat lower lip skin. Furthermore, the plastic changes observed in the association between SP- and DbetaH-IR fibres following trauma to peripheral nerve injuries, may lead to painful sensations and, possibly, abnormal blood flow in the affected region.

The delayed rectifier current, IK, responsible for terminating the plateau phase of the cardiac action potential, is composed of two components; the slowly activating component, IKs, and the rapidly activating component, IKr. IKr is the primary target of Class III antiarrhythmic agents, inhibition of which prolongs cardiac action potential and reduces the probability of reentrant arrhythmia induction. However, Class III antiarrhythmic agents can also excessively prolong the QT interval and precipitate a potentially lethal arrhythmia called torsade de pointes (TdP). Furthermore, mutations at the gene coding for IKr can cause congenital long QT syndrome and TdP. Heterologous expression of the human ether-a-go-go -related gene (HERG) elicits currents that closely resemble native IKr. The work presented in this thesis explores in detail heterologously expressed HERG channels, in particular their biophysical and pharmacological properties. We first probed HERG channels with the divalent cation Ba 2+ and the potent Class III antiarrhythmic agent dofetilide to examine the voltage-dependent pore accessibility. Our experimental and mathematical modeling data indicate that Ba2+ blocked wild-type HERG channels in their closed and open states but channel inactivation relieved Ba 2+ block. In contrast to previous reports indicating that dofetilide only blocked the open state of HERG channels, our data clearly show that dofetilide interacted with both the open and inactivated states. We then examined the need for association between the recently cloned MiRP1 (minK-related peptide 1) subunit in imparting properties similar to that of native IKr to HERG. We demonstrated that the pharmacological characteristics of HERG channels expressed in CHO cells is very similar to IKr recorded from guinea pig myocytes. The minor discrepancies between the cloned and native currents (i.e. deactivation kinetics and voltage dependence of activation) were not rectified by MiRP1 co-expression. Thu / In summary, our findings demonstrate that HERG channels are accessible in a state-dependent manner from the extracellular side by Ba2+ ions and the intracellular side by dofetilide. The characteristics of HERG channels that govern its sensitivity to drug blockers do not appear to require an association with its putative MiRP1 beta-subunit. The latter finding has important implications for screening of novel drugs for an early determination of their potential to prolong the QT interval, in particular those agents that are not meant to possess IKr inhibitory properties. Inhibition of HERG channels expressed in a mammalian cell line will provide a very close approximation of the sensitivity of native IKr to these agents.

I investigated the desensitization mechanisms of the serotonin 1A (5-HT$ sb{ rm 1A}$) and 5-HT$ sb{ rm 1B}$ receptors at the molecular level. Receptor phosphorylation by second messenger kinases and G-protein coupled receptor kinases (GRK) is an initial step in the functional desensitization of G-protein coupled receptor receptors (GPCRs). Evidence suggested that PKC preactivation desensitizes the 5-HT$ sb{ rm 1A}$ receptor. In Ltk-cells, PKC preactivation is pathway-selective, blocks receptor-mediated calcium mobilization but not cAMP inhibition. My first objective was to identify if the prime phosphorylation target of PKC was the receptor itself. Four PKC phosphorylation sites on the 5-HT1A receptor were eliminated using site-directed mutagenesis. The signalling profile of single mutants, and third loop double and triple mutants were examined in Ltk-cells. I determined that the prime target of PKC was the receptor since multiple PKC phosphorylation sites in the third loop mediated pathway-selective uncoupling of the receptor from the calcium mobilization pathway and not the adenylyl cyclase. The PKC site in the second loop of the 5-HT$ sb{ rm 1A}$ receptor was shown to be a critical G-protein-contact site in both neuroendocrine and mesenchymal cells. / My second objective was to investigate if GRK-mediated phosphorylation could enhance homologous desensitization of the endogenously expressed 5-H$ sb{ rm 1B}$ receptor in the opossum kidney (OK) cell line. To define the role of endogenous GRK in desensitization, I cloned the OK-GRK$ sb2$ from the OK cell Line and generated a kinase inactive mutant. The GRK$ sb2$ was shown to phosphorylate an epitope tagged 5-HT$ sb{ rm 1B}$ receptor in vitro. However, in intact cells the OK-GRK$ sb2$ did not enhance agonist-induced desensitization of the 5-HT1B receptor, but enhanced the desensitization of the $ alpha$2C receptor. The kinase-inactive mutant or reduction in OK-GRK2 protein levels using antisense GRK2 cDNA both attenuated the desensitization of the $ alpha$2C receptor but not the 5-HT$ sb{ rm 1B}$ receptor. These results suggest that phosphorylation mediated by GRKs in vitro may not mimic in vivo receptor desensitization. Processes other than those mediated by GRKs may be more important for the desensitization of the 5-HT$ sb{ rm 1B}$ receptor in the OK cells. / In conclusion, I have identified two possible mechanisms by which two related receptors, 5-HT$ sb{ rm 1A}$ and 5-HT$ sb{ rm 1B}$ receptors, are regulated by protein kinases: receptor phosphorylation by PKC and GRK. My results suggest that receptor phosphorylation by PKC plays a role in pathway selective desensitization of the 5-HT$ sb{ rm 1A}$ receptor, while phosphorylation the 5-HT$ sb{ rm 1B}$ receptor by GRK, observed in vitro, does not play an important role in the homologous desensitization of the 5-HT$ sb{ rm 1B}$ receptor.

Mono-N-dealkyldisopyramide (MND), the major metabolite of disopyramide, reaches significant concentrations in patients; however, little is known about its electrophysiological effects. We therefore assessed its activity in canine ventricular tissue superfused in vitro. MND produced a concentration and rate dependent increase in the magnitude of V$ sb{ rm max}$ depression. MND shortened all phases of repolarization in Purkinje fibers but prolonged action potential duration and effective refractory period in ventricular muscle. / We then assessed the effects of the combination of disopyramide and MND at clinically relevant concentrations. The combination produced additive effects on depression of V$ sb{ rm max}.$ MND accentuated the shortening of action potential duration in Purkinje fibers and the prolongation of refractory periods in both Purkinje fibers and ventricular muscle produced by disopyramide at normal heart rates. / In contrast, at slow stimulation rates and under redisposing electrolyte conditions, disopyramide produced a reverse use-dependent prolongation of action potential duration which led to the development of early afterdepolarizations and triggered activity in Purkinje fibers. Mexiletine and pacing abolished triggered activity, while MND shifted the incidence of triggered activity to longer cycle lengths.

The present study was done to determine whether the muscle nicotinic acetylcholine receptor (nAChR) function and expression could be regulated by two different factors: (a) thymopoietin (TPO)$ sp *$, a nicotinic antagonist and (b) MyoD, a myogenic transcription factor. / Exposure of rat neonatal muscle cells in culture to TPO on a long-term (days) and short-term (minutes) basis resulted in the inhibition of $ sp{125}$I-$ alpha$-bungarotoxin (BGT) binding. Short-term pretreatment with TPO also led to a decrease in carbachol-stimulated $ sp{22}$Na uptake; however, long-term exposure resulted in an enhanced carbachol-stimulated uptake. Chronic treatment also resulted in greater muscle cell morphological development. These results suggest that TPO regulates the nAChR and exerts trophic effects on myotube morphology. / As another approach to study factors that affect nAChR expression, non-muscle cells were transfected with MyoD cDNA. After transfection, saturable, high affinity $ sp{125}$I-$ alpha$-BGT binding was readily detectable, as was carbachol-stimulated $ sp{22}$Na uptake. Both these parameters developed in parallel over time and were inhibited by nicotinic antagonists. These results suggest that the transfection of a non-muscle cell line with MyoD cDNA results in the expression, at the cell surface, of a functional muscle-type nAChR. / This work shows that the nAChR function and expression can be regulated through (a) the chronic interaction of TPO at the nAChR at the cell surface and (b) the action of MyoD at the gene level. ftn$ sp *$ As stated in the addendum to this thesis, recent work by Quik et al. 1993a has shown that the preparation presumed to be TPO, contained $ alpha$-cobratoxin; the effects observed in the present thesis must therefore now be attributed to the presence of $ alpha$-cobratoxin contaminant.

Maximal inotropic response of electrically driven (0.5, 1 or 2 Hz) rat ventricular tissues in vitro to $ alpha sb1$-adrenoceptor agonist, methoxamine, was comparable to that of $ beta sb1$-adrenoceptor agonist isoprenaline up to 2 weeks of age. The inotropic effect of methoxamine decreased to adult levels at 3 weeks so that in tissues from rats 3 weeks and older, its inotropic effect was significantly less than that of isoprenaline. The inotropic effect of isoprenaline was not age-dependent. The inotropic effect of methoxamine was antagonized by prazosin and that of isoprenaline by propranolol. Treatment of pups at birth with dexamethasone, triiodothyronine or 6-hydroxydopamine did not influence that inotropic effect of methoxamine. Radioligand binding studies using $ sp3$(H) -prazosin and $ sp3$(H) -dihydroalprenolol as ligands and phentolamine and propranolol as displacers, respectively, revealed no age-dependent differences in myocardial $ alpha sb1$- and $ beta sp1$-adrenoceptor density or affinity. As well the increase in the second messenger inositol triphosphate (IP$ sb3$) after methoxamine was less in neonatal than in adult hearts. It is concluded that a greater inotropic effect of $ alpha sb1$-adrenoceptor agonists in neonatal rats may be due to unusual receptor-response coupling, which remains to be identified.