Regulation of Rat Hepatocyte Function by P2Y Receptors: Focus on Control of Glycogen Phosphorylase and Cyclic AMP by 2-Methylthioadenosine 5′-Diphosphate

Abstract

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5Ј-diphosphate (2-MeSADP), an agonist at P2Y 1 , P2Y 12 , and P2Y 13 receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N 6 -methyl 2Ј-deoxyadenosine 3Ј,5Ј-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y 1 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 P2Y 1 antagonists, adenosine-3Ј-phosphate-5Ј-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 M, indicating that it was not mediated by P2Y 1 receptors. This contrasts with the increase in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] 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-Me-SADP 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 P2Y 12 and P2Y 13 receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y 12 and P2Y 13 receptor antagonist, 2-propylthio-␤,␥-dichloromethylene-D-ATP (AR-C67085) (10 nM to 300 M) 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 P2Y 1 receptors coupled to raised [Ca 2ϩ ] c , and by inhibiting cyclic AMP levels by an unknown G i -coupled receptor subtype, distinct from P2Y 1 , P2Y 12 , or P2Y 13 receptors.Glycogen phosphorylase, the rate-controlling enzyme in hepatic glycogenolysis, is activated by increases in both cyclic AMP and cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] c ), resulting in a net output of glucose to supply extrahepatic tissues, crucially the brain, when blood glucose levels drop. Extracellular nucleotides play a well established role in the regulation of this key function in rat hepatocytes through the activation of P2Y receptors (Okajima et al., 1987;Keppens et al., 1992Keppens et al., , 1993Keppens, 1993), a family of G-protein-coupled receptors responding to the native nucleotides ADP, ATP, UDP, UTP, and UDP-glucose (Boarder and Hourani, 1998;Abbracchio et al., 2003). Curiously, stimulation of P2Y receptors on rat hepatocytes leads to both increases in [Ca 2ϩ ] c and inhibition of adenylyl cyclase; these responses will stimulate and limit, respectively, activation of glycogen phosphorylase (Okajima et al., 1987;Dixon et al., 1990Dixon et al., , 1995Dixon et al., , 2000Keppens et al., 1992;1993;Keppens, 1993;Edgecombe et al., 1999). The mechanism underlying the regulation of glycogen phosphorylase by cyclic AMP is well established, with ...

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

“…Statistical analysis was performed by the Student's t-test, and results were considered statistically different at P Ͻ 0.05. 12 and P2Y 13 in rat cholangiocytes and cilia. RT-PCR and Western blotting show that both P2Y 12 and P2Y 13 are expressed in rat cholangiocytes (Fig.…”

“…1, A and B). P2Y 12 and P2Y 13 are also expressed in rat hepatocytes and brain, which we used as a positive control (Fig. 1, A and B).…”

“…To test the expression of P2Y 12 and P2Y 13 in cholangiocyte cilia, paraffin sections of rat liver were stained with antibodies to a ciliary marker, acetylated ␣-tubulin, and to either P2Y 12 or P2Y 13 . Figure 1C shows the IBD with cilia (stained in red) extending from the cholangiocyte apical plasma membrane into the ductal lumen.…”

“…In contrast, P2Y 11 is associated with both [Ca 2ϩ ] i and cAMP signaling (1,10,56). Among four P2Y receptors (i.e., P2Y [11][12][13][14] ) associated with the cAMP signaling pathway, P2Y 12 and P2Y 13 are most suitable for a chemosensory function of cholangiocyte cilia in rodents. This conclusion is based on three critical observations.…”

“…A family of P2Y receptors consists of eight members (i.e., P2Y 1,2,4,6,11,12,13, and 14 13 , and P2Y 14 are associated with cAMP signaling. In contrast, P2Y 11 is associated with both [Ca 2ϩ ] i and cAMP signaling (1,10,56).…”