Abstract

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5′-diphosphate (2-MeSADP), an agonist at P2Y₁, P2Y₁₂, and P2Y₁₃ receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N⁶-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y₁ receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y₁ antagonists, adenosine-3′-phosphate-5′-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 μM, indicating that it was not mediated by P2Y₁ receptors. This contrasts with the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y₁₂ and P2Y₁₃ receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y₁₂ and P2Y₁₃ receptor antagonist, 2-propylthio-β,γ-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 μM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y₁ receptors coupled to raised [Ca²⁺]_c, and by inhibiting cyclic AMP levels by an unknown G_i-coupled receptor subtype, distinct from P2Y₁, P2Y₁₂, or P2Y₁₃ receptors.