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Mechanisms of cellular synchronization in the vascular wall. Mechanisms of vasomotion: Doctoral Dissertation

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  • Department of Physiology and Biophysics
Although the function of rhythmic contractions in the vascular wall - vasomotion - is still under debate, it has been suggested to play a significant role for tissue oxygen homeostasis and under pathological conditions where tissue perfusion is affected. Vasomotion has further been suggested to be important for blood pressure control and has been shown to be reduced in diabetes. Vasomotion is initiated by the coordinated activation of smooth muscle cells (SMCs) in the vascular wall leading to rhythmic contractions. We have suggested the model for generation of this rhythmic activity and have shown that vasomotion initiates via interaction between intracellular calcium released from the sarcoplasmic reticulum and changes in membrane potential. Rhythmic changes in intracellular calcium induce, under certain conditions (in the presence of sufficient concentration of cGMP), changes in membrane potential that lock the electrically-connected SMCs into phase. Synchronized depolarization induces synchronous calcium influx and thus produces rhythmic contraction of blood vessels. I have demonstrated and characterized a new chloride channel in vascular SMCs, which has properties necessary to coordinate SMCs in the vascular wall. Chloride channels have been investigated for many years but remained somewhat in the shadow of cation channels. We know now the molecular structures of some chloride channels, i.e. GABA receptors, "cystic fibrosis transmem-brane conductance regulator" (CFTR) and the ClC chloride channel family. There is one particular group of chloride channels, the calcium activated chloride channels (CaCCs), whose molecular structure is debated still. There are currently no pharmacological tools that activate or inhibit CaCCs with any significant selectivity. The existence of CaCCs in almost all cells in the body has been known for many years based on electrophysiological and other functional studies. CaCCs have been suggested to be important for regulation of membrane potential and cellular volume, as well as for body homeostasis. CaCCs are well characterized in vascular tissues but only at the functional level. The lack of their molecular structure makes it difficult to study the clinical significance of these channels. Based on patch clamp measurements of ion currents, I have previously characterized in SMCs a chloride current with unique properties. This chloride current activated by cGMP, has very high sensitivity to calcium and can be inhibited by low concentrations of zinc ions, while the traditional inhibitors of CaCCs affect this current only at very high concentrations. This cGMP-dependent, calcium-activated chloride current has a linear volt-age-dependence, which differs from previously characterized CaCCs, and it has characteristic anion permeability. This current has been detected in SMCs isolated from a number of different vascular beds but, importantly, it has not been detected in pulmonary arteries. Moreover, this current has been shown in SMCs isolated intestine indicating its broad distribution. Based on unique characteristics I have suggested that the cGMP-dependent calcium-activated chloride current can synchronize SMCs in the vascular wall and that bestrophin protein could be the molecular substrate for this current. Bestrophin has been characterized first as a gene in which mutations cause vitelliform macular dystrophy (VMD) or Best diseases. Based on heterologous expression it has been suggested that bestrophin is a chloride channel. This question is nevertheless controversial since caution should be taken in heterologous expression of calcium-activated chloride channel candidates. The presence of chloride channels in virtually all living cells is an essential problem as well as the dependence of ion channel properties on the complex interaction of many cellular proteins. I was the first who coupled the endogenous chloride current to one of four known bestrophin isoforms. PCR and Western blot studies on different blood vessels demonstrated the presence of bestrophin-3 protein with the exception of pulmonary arteries where the cGMP-dependent current is also absent). There was a strong indication that bestrophin-3 expression could be essential for the cGMP-dependent calcium-activated chloride current. To couple bestrophin-3 expression and this current I have used small interfering RNA (siRNA) technique to downregulate the expression of the candidate (bestrophin-3) and have studied the effect of this specific downregulation on chloride currents. I showed that bestrophin-3 expression is associated with the cGMP-dependent calcium-activated chloride current. This study does not tell us whether bestrophin-3 forms the channel or it is an essential subunit but the previous mutagenic experiments suggested the first possibility. Electrical communication between SMCs is essential for successful synchronization and depends on channels between the cells called gap junctions. The majority of cardiovascular diseases (e.g. hypertension and atherosclerosis) are associated with defects in intercellular communications or in gap junction regulation. The molecular mechanisms responsible for these defects are un-known because of lack of specific experimental tools. Our comprehensive study on the often used gap junction inhibitors heptanol and 18β-glycyrrhetinic acid demonstrated unspecific effects of these drugs at the concentrations where they have no or little gap junctions effects. Other drugs, e.g. 18α-glycyrrhetinic acid and connexin-mimetic peptides are better to inhibit gap junctions but also have demonstrated unspecific effects. Previous studies suggested that channels and transporters in the cell membrane do not function independently but interact as functional units in the spatially restricted areas of the cell. I have demonstrated a close functional interaction between gap junctions and Na+,K+-ATPase, Na+/Ca2+-exchanger and ATP-dependent K+ channels in the spatially restricted manner. I have shown that inhibition of the ouabain-sensitive Na+, K+-ATPase inhibits calcium efflux by the Na+/Ca2+-exchanger and this leads to the local elevation of intracellular calcium and inhibition of intercellular communications. This explains the inhibitory action of ouabain on vasomotion. I have also found that the ATP-dependent K+ channel is an important player in this functional unit and this interaction is reciprocal, since K+ channel supplies Na+, K+-ATPase with K+ ions while the ATP-dependent K+ channel current also regulates the Na+, K+-ATPase. This dissertation is based on nine scientific publications where I have suggested the model for generation of vasomotion and characterized the essential elements of this model.
Original languageEnglish
JournalDanish Medical Bulletin (Online)
Pages (from-to)1-50
Number of pages50
Publication statusPublished - 2010

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