Le couplage étroit entre l'activité neuronale et l'augmentation du flux sanguin, appelé couplage neurovasculaire (CNV), est essentiel aux fonctions cérébrales. Ce processus est à la base de l'imagerie médicale cérébrale non invasive utilisée pour déterminer l'activité neuronale chez l'individu sain ou malade. Cependant, les mécanismes cellulaires et moléculaires du CNV restent encore débattus. La compréhension de ce processus permettra non seulement une interprétation plus fine des signaux d'imagerie cérébrale mais également un meilleur diagnostic des maladies neurologiques.De nombreux messagers vasoactifs sont impliqués dans le CNV du cortex cérébral. Les prostanoïdes, notamment libérés lors de l'activation des récepteurs NMDA, sont impliqués dans l'augmentation du flux sanguin cérébral. Cependant, l'origine cellulaire, moléculaire, la nature ainsi que les cibles de ces messagers lipidiques restent incertaines.La prostaglandine E2 (PGE2) et la prostacycline (PGI2), produits par les cyclo-oxygénases de type 1 ou 2 (COX-1 ou COX-2) et des enzymes terminales, sont les deux principaux prostanoïdes vasodilatateurs du cortex cérébral. Ce travail a montré que les vasodilatations induites par le NMDA dépendent de la COX-2 et nécessitent également l'activation des récepteurs EP2 et EP4 de la PGE2 et IP de la PGI2et que les neurones pyramidaux sont les principales cellules du cortex cérébral équipées pour la biosynthèse de la PGE2 et de la PGI2.L'ensemble de ces travaux démontre que les neurones pyramidaux jouent donc un rôle clé dans le CNV cortical via la libération de la PGE2. Produite par la COX-2, la PGE2 agit sur les récepteurs EP2 et EP4 et induit des vasodilatations. / The tight coupling between neuronal activity and cerebral blood flow, known as neurovascular coupling (NVC), is essential for brain functions. It is also the physiological basis of cerebral imaging, widely used to map neuronal activity in health and disease. Despite this importance, its cellular and molecular mechanisms are poorly understood. A better understanding of NVC will not only permit an accurate interpretation of cerebral imaging but also a better diagnosis of neurological diseases. In the cerebral cortex, numerous messengers are involved in NVC. Prostanoids, released during NMDA receptors activation, play a key role in NVC. However, the cellular and molecular origins, as well as the nature and the targets of this lipid messengers remain elusive. Prostaglandin E2 (PGE2) and prostacyclin (PGI2), produced by the rate limiting cyclo-oxygenases 1 or 2 (COX-1 or COX-2) and specific terminal enzymes, are the main cortical vasodilatory prostanoid. This work shows that NMDA-induced vasodilations are COX-2 dependent and require the activation of EP2 and EP4 receptors of PGE2 and IP receptors of PGI2. Furthermore, pyramidal cells are the main cell type equipped for the biosynthesis of PGE2 and PGI2 derived from COX-2 activity. In summary, these observations demonstrate that pyramidal cells play a key role in NVC by releasing PGE2 produced via COX-2 and acting on the vasodilatory EP2 and EP4 receptors.

Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function. / Allergan Inc

No / There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory cytokines (e.g. IL-1β or TNFα), as well as regulating the basal levels of this prostanoid. Furthermore, cPLA2γ, cPLA2δ, and iPLA2 were found to also to regulate the basal levels of PGE2 production. On the other hand, cPLA2β was not involved in prostanoid synthesis in A549 cells either in the presence or absence of inflammatory cytokines. Thus, our studies show that cPLA2α plays the pivotal role in the production of PGE2 in response to inflammatory cytokines, and suggests that cPLA2α may be a possible drug target in diseases such as asthma, inflammation, and cancer.

No description available.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.