Crucial Role of Type 2 Inositol 1,4,5-Trisphosphate Receptors for Acetylcholine-Induced Ca 2+ Oscillations in Vascular Myocytes

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Abstract
Objective— The aim of this study was to correlate the expression of InsP 3 R subtypes in native vascular and visceral myocytes with specific Ca 2+ -signaling patterns. Methods and Results— By Western blot and immunostaining, we showed that rat portal vein expressed InsP 3 R1 and InsP 3 R2 but not InsP 3 R3, whereas rat ureter expressed InsP 3 R1 and InsP 3 R3 but not InsP 3 R2. Acetylcholine induced single Ca 2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca 2+ oscillations. By correlating Ca 2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP 3 R1 and InsP 3 R2 whereas nonoscillating vascular cells expressed only InsP 3 R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca 2+ -ATPases, Ca 2+ influx, and mitochondrial Ca 2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP 3 R subtypes, we showed that acetylcholine-induced Ca 2+ oscillations were specifically blocked by the anti-InsP 3 R antibody. These data were supported by antisense oligonucleotides targeting InsP 3 R2, which selectively inhibited Ca 2+ oscillations. Conclusions— Our results suggest that in native smooth muscle cells, a differential expression of InsP 3 R subtypes encodes specific InsP 3 -mediated Ca 2+ responses and that the presence of the InsP 3 R2 subtype is required for acetylcholine-induced Ca 2+ oscillations in vascular myocytes.