7-(3-(3-hydroxy-4-(4--iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic-acid and Leukemia--Erythroblastic--Acute

Previous studies have demonstrated an increased thromboxane A2 (TXA2) receptor expression in human erythroleukemia (HEL) cells and rat aortic smooth muscle (RASM) cells in response to testosterone treatment. HEL cells have served as a model for megakaryocytes, the progenitor cell for platelets. Platelets have previously been shown to convert androstenedione to testosterone. This study investigated the effects of androstenedione on the TXA2 receptor density in HEL and cultured RASM cells. Both cell lines were incubated with vehicle, 150 nM testosterone or 250, 500 or 750nM androstenedione for 48 hours. Co-incubation with testosterone or androstenedione significantly (p<0.05) increased the maximum number of TXA2 binding sites (Bmax) in HEL cells compared to controls. There was no significant change in Kd values. In a separate series of experiments, HEL cells were incubated with the androgen receptor antagonist hydroxyflutamide (2.5mM). Treatment with androstenedione (500nM) significantly (p<0.05) increased the Bmax value by 35% compared to control and hydroxyflutamide completely antagonized this effect of androstenedione. Incubation with hydroxyflutamide alone had no effect on the Bmax values compared to control. RASM cells also showed an increase in Bmax values by 25% and 23% over control (95+/-6.6, 118+/-7.2 and 117+/-5.1 fmoles/mg protein, control, testosterone and androstenedione, n=3). Both cell lines converted androstenedione to testosterone. The results raise the possibility that the adrenal androgen, androstenedione can regulate the expression of TXA2 receptors either on its own or via conversion to testosterone and through an androgen receptor.

Topics: Androgen Antagonists; Androstenedione; Animals; Aorta; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cells, Cultured; Drug Interactions; Fatty Acids, Unsaturated; Flutamide; Humans; Indomethacin; Iodine Radioisotopes; Kinetics; Leukemia, Erythroblastic, Acute; Muscle, Smooth, Vascular; Radioligand Assay; Rats; Receptors, Thromboxane; Testosterone; Time Factors; Tumor Cells, Cultured

Thromboxane A2 (TXA2) induces activation of platelets and vascular smooth muscle contraction via cell surface receptors. A platelet type TXA2 receptor from the megakaryocyte-like HEL cell was cloned with a deduced amino acid sequenced identical to that previously reported for the human placental TXA2 receptor. Transient expression of the HEL cell TXA2 receptor cDNA and radioligand binding studies with the agonist 125I-BOP showed a single class of binding sites with an affinity comparable to a low affinity platelet TXA2 receptor. Using a series of 13-azapinane TXA2 analogs, which discriminate between TXA2 receptor subtypes in platelets and vascular smooth muscle, we found that the cloned HEL cell TXA2 receptor is characteristic of a platelet type TXA2 receptor and that its binding characteristics are different from those of vascular smooth muscle cells. The affinity of the HEL cell TXA2 receptor for 125I-BOP was significantly (P < .05) increased upon co-transfection with G alpha 13 alone, or with G alpha q alone and with G alpha 13 and G alpha 12 together (n = 4-6). GTP gamma S significantly (P < .05) decreased the affinity of the receptor for 125I-BOP in COS-7 cell membranes coexpressing HEL-TXR and G alpha 13 to a value comparable to HEL-TXA2 receptor alone. We conclude that 1) the cloned HEL cell TXA2 receptor has pharmacological characteristics of a low affinity platelet type receptor and 2) that the affinity state of this receptor may be influenced by interaction with G alpha 13 and G alpha q.

Topics: Base Sequence; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Fatty Acids, Unsaturated; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Thromboxane; Transfection; Tumor Cells, Cultured