Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.
Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-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 2'-deoxyadenosine 3',5'-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-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](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 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-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) 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(2+)](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.
Dixon
C Jane
CJ
The Cell Signaling Laboratory, Leicester School of Pharmacy, The Hawthorn Building, De Montfort University, Leicester LE1 9BH, United Kingdom.
Hall
John F
JF
Webb
Tania E
TE
Boarder
Michael R
MR
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D024981:Glycogen Phosphorylase / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D058472:Receptors, Purinergic P2Y1; D014544:Uridine Triphosphate / Q000378:metabolism
C117445:AR C67085MX; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C546384:P2ry1 protein, rat; D020033:Protein Isoforms; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D024981:Glycogen Phosphorylase; D014544:Uridine Triphosphate