Doctor of Medical Science Danish Medical Bulletin

1. PREFACE This thesis is based on the work carried in the Vascular Smooth Muscle group at the Institute of Physiology and Biophysics, Aarhus University. This thesis is focused on a mechanistic understanding of cellular synchronization in the small resistance arteries. I have started this work because of my general interest in vasomotion, a phenomenon of synchronized activity in the vascular wall which has been known for more than 150 years. In spite of the long history and suggestions that vasomotion is important for pathological states the studies of vasomotion have been mostly descriptive. Development of new experimental techniques such as small artery myography, intracellular Ca imaging and electrophysiological approaches brought new possibilities to the studies of cellular mechanisms of vascular synchronization. I have used these advanced methods to characterize vasomotion in detail and have suggested and tested a model for generation of vasomotion in the rat mesenteric artery. The suggested model is one of several models of vasomotion but it has strong experimental support and is supplemented by the mathematical modeling published by our group. Two key elements for the synchronized oscillation in the mesenteric small arteries a cGMP-dependent Ca-activated Cl current and the electrical intercellular communication were further explored in my research. I have characterized the cGMP-dependent Ca-activated Cl current suggested by our model for vasomotion and demonstrated this current in different vascular beds. Using a novel siRNA approach I have then shown the association between this current and bestrophin-3 protein expression in vivo and in vitro. Based on these results I suggested the molecular identity of this current and its significance for smooth muscle cell synchronization by a membrane potential-dependent mechanism. The studies of intercellular communication in the vascular wall are lacking specific and effective tools to manipulate these intercellular contacts. I have performed comprehensive studies to analyze the action of the most commonly used gap junction blockers and demonstrated that vasomotion can be used as a “readout” for intercellular communication. Using this approach I demonstrated that inhibition of the ouabain-sensitive Na/K-ATPase uncouples smooth muscle cells in the vascular wall and suggested the mechanism responsible for this electrical uncoupling. In my studies on the role of the ouabain-sensitive Na/K-ATPase for vascular function I suggested the presence of Na/K-ATPase-based signalosome which also includes the Na/Ca-exchanger, gap junctions and the ATPdependent K channels. These studies provide a useful tool for manipulations intercellular communication in the small arteries.