Background Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are both important relaxing factors. Their synthesis, release and downstream signaling pathways are controlled by a number of proteins, such that alteration in the activity of these proteins may disturb vascular tone. Aim This study was aimed to investigate the role of some of the regulatory proteins in NO- and EDHF-mediated relaxations. The regulatory proteins that were examined include: 1) calcium-calmodulin dependent protein kinase II (CaMK II), 2) mitogen-activated protein kinase (MAPK), 3) adenosine monophosphate-activated protein kinase (AMPK), 4) phosphoinositide 3-kinase (PI3K) / protein kinase B (Akt) and 5) phosphoprotein phosphatase. Experimental approach Organ chamber system was used for measuring isometric tension of porcine coronary arteries. The role of the regulatory proteins was investigated by using their activators or inhibitors. In the contraction study, arterial rings without endothelium were contracted with U46619 (0.1 nM to 10 μM) or phorbol 12,13-dibutyrate (PDBu, 0.1 nM to 1 μM). In the relaxation study, arterial rings with and without endothelium were contracted with U46619 (30 or 100 nM). They were incubated with indomethacin (cyclooxygenase inhibitor, 10 μM) and TRAM-34 plus UCL1684 (intermediate- and small-conductance calcium-activated potassium channel blockers, respectively; 1 μM each) or L-NAME (NO synthase inhibitor, 30 μM) for the study of NO and EDHF components of bradykinin (0.1 nM to 10 μM)-induced relaxations. Moreover, endothelium-independent relaxations by sodium nitroprusside (SNP, exogenous NO donor, 0.1 nM to 100 μM) and diazoxide (ATP-sensitive potassium channel activator, 1 nM to 1 mM) were examined in arteries without endothelium. Key findings 1. NO and EDHF are both involved in endothelium-dependent relaxation in porcine coronary arteries, in which NO is the dominant relaxing factor. 2. KN-93 (CaMK II inhibitor, 30 μM) significantly reduced contractions to U46619 and PDBu. On the other hand, CaMK II partly involved in EDHF signaling but not in the NO-mediated relaxations. 3. Calyculin A (phosphoprotein phosphatase inhibitor, 30 nM) greatly inhibited both endothelium-dependent and –independent relaxations. 4. PD98059 (MAPK inhibitor, 30 μM) significantly potentiated bradykinin-induced relaxation that was mediated by EDHF but not that mediated by NO. On the other hand, it potentiated SNP-induced but not diazoxide-induced endothelium-independent relaxations. 5. AMPK and Akt do not play a role in regulating vascular tone as compound C (AMPK inhibitor, 30 μM), AICAR (AMPK activator, 1 mM) and wortmannin (PI3K inhibitor, 100 nM) did not affect contractions to U46619 and PDBu, and relaxations to bradykinin, SNP and diazoxide in porcine coronary arteries. Conclusions and implications Different regulatory proteins (CaMKII, MAPK, AMPK, Akt, phosphoprotein phosphatase) have different effects on the regulation of vascular tone. While the present study has the limitation of using pharmacological agents at only one concentration to examine the role of these proteins, it still produces scientific information for the development of therapeutic agents. In considering CaMK II, MAPK and phosphoprotein phosphatase as potential therapeutic targets, the vascular effects (which can be therapeutic or adverse) of the compounds acting on these proteins should be taken into account. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

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published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

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Dysfunction of the vascular endothelium can initiate atherosclerosis. Mechanical forces, particularly wall shear stress (WSS) are believed to cause endothelial dysfunction. Present in vitro cell culture models are often simplified and thus, ignore the wall shear stress spatial gradients inherent in complex geometries. The aim of this project was to study endothelial cell response in an anatomically correct right coronary artery model (RCA) under more physiologically realistic flow conditions. / Human Abdominal Aortic Endothelial Cells (HAAECs) were seeded in the lumen of a pre-treated faithful RCA and a straight tubular model. The cells were subjected to steady or non-reversing oscillatory flow (Re=196, alpha=1.82) at a mean physiological flow rate of 20 dynes/cm2 for 8, 12 and 24 hours of flow. The results show that under all flow conditions, the cells became progressively more elongated and aligned. Moreover, differences in endothelial morphology in the inner (myocardial) and outer (pericardial) walls were seen in the inlet region. The morphologic adaptation to steady and oscillatory flow was similar. The results suggest that spatial, not temporal gradients in shear in the inlet region are responsible for the differential endothelial cell response.

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