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 Gi-coupled receptor subtype, distinct from P2Y1, P2Y12, or P2Y13 receptors.
Footnotes
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Financial support was provided by The Wellcome Trust.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.067744.
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ABBREVIATIONS: RT-PCR, reverse transcriptase-polymerase chain reaction; A3P5P, adenosine-3′-phosphate-5′-phosphate; AR-C67085, 2-propylthio-β,γ-dichloromethylene-d-ATP; bp, base pair(s); FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high-pressure liquid chromatography; IBMX, 3-isobutyl-1-methylxanthine; 2-MeSADP, 2-methylthioadenosine 5′-diphosphate; MRS 2179, N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate.
- Received March 4, 2004.
- Accepted May 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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