Crucial role of type 2 inositol 1,4,5-trisphosphate receptors for acetylcholine-induced Ca2+ oscillations in vascular myocytes

Abstract

Objective - The aim of this study was to correlate the expression of InsP3R subtypes in native vascular and visceral myocytes with specific Ca2+-signaling patterns. Methods and Results - By Western blot and immunostaining, we showed that rat portal vein expressed InsP 3R1 and InsP3R2 but not InsP3R3, whereas rat ureter expressed InsP3R1 and InsP3R3 but not InsP 3R2. Acetylcholine induced single Ca2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca2+ oscillations. By correlating Ca2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP3R1 and InsP3R2 whereas nonoscillating vascular cells expressed only InsP3R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca2+-ATPases, Ca2+ influx, and mitochondrial Ca2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP 3R subtypes, we showed that acetylcholine-induced Ca2+ oscillations were specifically blocked by the anti-InsP3R antibody. These data were supported by antisense oligonucleotides targeting InsP 3R2, which selectively inhibited Ca2+ oscillations. Conclusions - Our results suggest that in native smooth muscle cells, a differential expression of InsP3R subtypes encodes specific InsP 3-mediated Ca2+ responses and that the presence of the InsP3R2 subtype is required for acetylcholine-induced Ca 2+ oscillations in vascular myocytes.