Purinergic P2Y1 and P2Y2/4 receptors elicit distinct Ca2+ signaling patterns in hepatocytes via differential feedback regulation by Protein Kinase C

Extracellular nucleotides are key regulators of liver physiology. In primary rat hepatocytes, P2Y1 receptor (P2Y1R) activation by ADP generates cytosolic calcium ([Ca2+]c) oscillations with narrow spikes, whereas P2Y2/4R activation by UTP led to more complex broad [Ca2+]c oscillations. Both [Ca2+]c oscillation signatures were observed with the common agonist ATP. Inhibition of Gαq signaling with YM-254890 abolished ATP-induced [Ca2+]c oscillations, indicating that they depend on inositol 1,4,5-trisphosphate (IP3), and are not mediated by P2X receptors. The narrow P2Y1-linked [Ca2+]c spikes and the broad P2Y2/4-linked [Ca2+]c spikes are shaped by differential and complex PKC-mediated feedback mechanisms. Downregulation of PKC broadened both ADP- and UTP-induced [Ca2+]c oscillations, with a more pronounced effect on the former. PKC downregulation also selectively elicited a more robust response to ADP stimulation, enhancing oscillatory and sustained [Ca2+]c responses. Acute PKC modulation confirmed the importance of the negative PKC feedback regulation of P2Y1R-linked [Ca2+]c signals; such that PKC activation decreased [Ca2+]c oscillation frequency and PKC inhibition increased [Ca2+]c spike width. However, both PKC activation and inhibition decreased the spike width of P2Y2/4R-induced [Ca2+]c oscillations, suggesting that multiple opposing PKC feedback mechanisms shape P2Y2/4R responses. Significantly, plasma membrane Ca2+ entry was required for negative PKC feedback on P2Y1R-linked [Ca2+]c oscillations, whereas P2Y2/4R-linked [Ca2+]c oscillations were less sensitive to negative regulation by PKC and independent of Ca2+ influx. Thus, differential feedback regulation by PKC gives rise to receptor-specific [Ca2+]c oscillation profiles, which can encode the diverse physiological and pathophysiological responses to distinct agonists that all act through the IP3 signaling cascade.