Extracellular ATP, via specific G protein-coupled P2Y receptors, modulates important hepatic functions such as, gluconeogenesis, protein synthesis and bile secretion. However, some observations suggest that additional P2 receptor types could also contribute to the hepatocyte response to ATP. We thus examined rat hepatocytes for the functional expression of P2X receptors, the ATP gated channels.

Hepatocytes were prepared from Wistar rats. Expression of mRNA were investigated by RT-PCR. Proteins were detected by western blotting and localisation was achieved by confocal microscopy. Pore formation induced by activation of P2X receptors was detected using YO-PRO-1, a low molecular weight (629 Da) fluorescent dye.

RT-PCR showed that P2X₄ and P2X₇ receptor mRNA were expressed in rat hepatocytes, even when resident liver macrophages were selectively eliminated before hepatocytes isolation. Indeed it has been shown that macrophages express these receptors. In hepatocyte lysates, P2X₄ and P2X₇ proteins were readily detected. This was confirmed by immunocytochemistry. Confocal analysis showed a patchy distribution of the P2X₇ receptor at the hepatocyte plasma membrane level, whereas P2X₄ was localised at the hepatocyte plasma membrane and throughout the cytoplasm. Interestingly, in hepatocyte doublets, P2X₄ receptors were preferentially localised around the bile canaliculus. High concentration of ATP and BzATP, the P2X₇ preferring agonist, induced membrane blebbing and significant uptake of YO-PRO-1, which was inhibited in the presence of oxidised ATP, a blocker of P2X₇ receptors. Our results show that P2X₄ and P2X₇ receptors are expressed in rat hepatocytes and provide evidence for a functional participation of P2X receptors in the purinergic signalling complex in these cells.