Abstract 15714: Metabolic Modulation Of Vascular Distinctive P2X Receptors By Intracellular ATP In Rat Arterial Smooth Muscle

Introduction: It is broadly accepted that extracellular nucleotide sensitive P2 receptors are composed of two different groups, namely ionotropic P2X and metabotropic P2Y receptors. Therefore, it seems to be considered in general that P2X receptor lacks metabolic regulation site. However, we found that P2X1 and 5 receptors, which we identified as P2X receptors in vascular smooth muscle ( Circulation , 120: S1012 - S1013, 2009), are equipped with intracellular ATP binding site and its activation kinetics are modulated by cytosolic ATP. Hypothesis: Vascular smooth muscle type P2X1 and 5 subunits could be controlled by not only extracellular, but also intracellular ATP. Methods: We used male rats aged 10-11w, and recorded P2X receptor current from myocytes isolated from rat cerebral and mesenteric arteries, or aorta using whole-cell patch-clamp technique. We also performed tension recording by use of rat aorta ring preparation, western blot, immunoprecipitation and immunofluorescent analyses to detect the receptors in the smooth muscle. Results: We found that desensitization of α,β-methylene ATP(10μ M)-induced P2X receptor current is greatly suppressed by increasing intracellular ATP concentration through patch pipette solution (Initial desensitization rates were 52.4±6.0, 45.7±5.4 and 33.5±3.0% at 0, 2 and 5mM ATP, respectively: n=5-13). In contrast, ADP and AMP did not modulate the desensitization of the current at all. Similar effects were observed in rat cerebral and mesenteric artery myocytes. In addition, α,β-methylene ATP (10μ M)-induced contraction also showed desensitization similar to the results of patch-clamp experiment with 5mM ATP (Initial desensitization rate was 30.7±6.7%: n=5). Therefore, we searched ATP-binding motifs in P2X receptors, and found a putative ATP-binding motif, G-X-G-X-X-G, in intracellular domains of human P2X1 and 5 receptors. Expression of P2X1 and 5 receptor proteins and their co-localization were confirmed in each arterial tissue. Conclusions: Vascular distinctive P2X receptors, namely P2X1 and 5, are controlled by extracellular and intracellular ATP bi-directionally. These results suggest that P2X1 and 5 receptors may play important role under the pathophysiological condition including hypoxia.