We have recently characterized a cGMP-dependent Ca²⁺-activated Cl^- current (I_Cl,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 I_Cl,cGMP based on similarities between the membrane current produced by heterologous expression of bestrophin and I_Cl,cGMP and similarities in the distribution pattern of I_Cl,cGMP and bestrophin-4 protein expression in different SMCs (see also abstract Bouzinova et. al). In this study we tested the hypotheses that I_Cl,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 I_Cl,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 I_Cl,cGMP and for generation of vasomotion in rat mesenteric small arteries.