We have recently characterized a cGMP-dependent Ca2+-activated Cl- current (ICl,cGMP) with unique characteristics in smooth muscle cells (SMCs) and suggested that this could be important for vasomotion. In SMCs this current co-exists with the "classical" Ca2+-activated Cl- current. We hypothesized that a bestrophin could be responsible for ICl,cGMP based on similarities between the membrane current produced by heterologous expression of bestrophin and ICl,cGMP and similarities in the distribution pattern of ICl,cGMP and bestrophin-4 protein expression in different SMCs (see also abstract Bouzinova et. al). In this study we tested the hypotheses that ICl,cGMP is mediated by bestrophin-4 and that bestrophin-4 is important for vasomotion. Bestrophin-4 was downregulated with small interference (siRNA) in A7r5 cells and in rat mesenteric small arteries in vivo. siRNAs targeting different exons of bestrophin-4 were used. Non-related siRNA and bestrophin-4 siRNA with 2 changed nucleotides were used as the controls for non-specific siRNA action. Downregulation induced by siRNA was evident by reduced expression of bestrophin-4 mRNA and protein (by 82±6 % and by 54±4 % (n=3 and 6), respectively, for cell culture; in vivo transfection showed similar results). Transfection with siRNAs did not affect other bestrophin isoforms. SMCs (both cultured and in vivo) downregulated for bestrophin-4 demonstrated a significant reduction of ICl,cGMP, while the "classical" Ca2+-activated Cl- current was unaffected. Control transfections had no effect. Surprisingly, arteries with reduced bestrophin-4 were more sensitive to noradrenaline than control vessels. The downregulation of bestrophin-4 significantly reduced the amplitude of arterial vasomotion (by 75±10 % n=6) without significant effect on frequency. We provide evidence that at least one bestrophin family member - bestrophin-4 - is essential for ICl,cGMP and for generation of vasomotion in rat mesenteric small arteries.
Journal of Vascular Research, 2008
Main Research Area:
9TH INTERNATIONAL SYMPOSIUM ON RESISTANCE ARTERIES (ISRA), 2008