Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL.
The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.
Turner
J T
JT
Department of Pharmacology, School of Medicine, University of Missouri-Columbia.
Camden
J M
JM
Kansra
S
S
Shelton-James
D
D
Wu
H
H
Halenda
S P
SP
D000262:Adenylyl Cyclases / Q000378:metabolism; D000527:Alprostadil / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D000242:Cyclic AMP / Q000096:biosynthesis*; D004357:Drug Synergism; D011464:Epoprostenol / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
C026678:carboprostacyclin; D011464:Epoprostenol; D000242:Cyclic AMP; D011493:Protein Kinase C; D013917:Thrombin; D000262:Adenylyl Cyclases; D000527:Alprostadil; D002118:Calcium