A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate.
Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005934:Glucagon / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D025481:Phosphofructokinase-2; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism; D013873:Thionucleotides / Q000494:pharmacology*
D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D005934:Glucagon; D000242:Cyclic AMP; D010770:Phosphotransferases; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D011770:Pyruvate Kinase; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium