Abstract 4884: Identification of Hetero-multimeric P2X Receptor Which Regulates Vascular Tone in Rat Arterial Smooth Muscle
Background: ATP is one of important vasoactive substances and is released from sympathetic nerve terminal, endothelial cells and other cells surrounding smooth muscle cells. Ionotropic P2X channel is one of the representative receptors, and only homomeric P2X1 has been thought to be the functional molecular architecture in vascular smooth muscle cells for a long time. However, some inconsistencies exist between native P2X1-like and cloned P2X1 channels.
Methods: Male rats aged 10 –11w were used. P2X channel current was measured in smooth muscle cells isolated from aorta, cerebral or mesenteric artery using patch-clamp technique. RT-PCR and Western blot analyses were employed for detecting their messenger RNAs and proteins, and their interaction in combination with an immunoprecipitation method.
Results: Application of ATP (≥100nM) elicited dual phase, fast and slow, desensitizing inward current by whole cell recording. The ATP-induced current also showed rebound current, which is a typical feature specific to cloned P2X1/5 channel. I-V relationship of the current was that of inward-rectifying, and reversal potential was around 0mV, close to the equivalent potential of non-selective cation channel in spite of various intracellular chloride ion concentration. Sensitivity of several ATP analogs is as follows; 2-methylthio ATP>BzATP>ATP>α, β-methylene ATP>ATPγS. The channel conductance was 10.5±0.1pS by a single channel recording. The current was blocked by several P2X and P2 receptor antagonists, TNP-ATP, suramin and PPADS, and their 50% inhibitory concentrations (IC50) were similar to those of cloned P2X1/5 channel (1.0, 0.5 and 0.6μM, respectively; n=3~5). RT-PCR and Western blot analysis revealed highly expression of both P2X1 and P2X5 channels in extracts from arteries examined, and both channel proteins were found to be complexed as assessed by an immunoprecipitation method.
Conclusion: Our results indicate that hetero-multimeric P2X1/5 channel, but not only P2X1, is functionally expressed in arterial smooth muscle, and may play an important role in vascular tone regulation.