ABSTRACT
This study was designed to compare the effects of beta-adrenoceptor agonists formoterol and BRL 37344 on spontaneous contractions and the levels of cAMP and cGMP of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from term-pregnant Wistar albino rats (n=12), mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. We evaluated the effect of increasing concentrations of formoterol and BRL 37344 on oxytocin-induced myometrial contractions and on contractions of myometrial smooth muscle pretreated with metoprolol, ICI 118.551 and SR 59230A (beta1, beta2, beta3-adrenoceptor antagonist, respectively, 10(-6) M). Effects of formoterol and BRL 37344 on cAMP and cGMP levels in isolated myometrial strips (n=6) were evaluated by radioimmunoassay kits. Formoterol (10(-12)-10(-8) M) and BRL 37344 (10(-11)-10(-5) M) concentration-dependently decreased the amplitude of oxytocin-induced contractions. E(max) value (100%) of formoterol was increased significantly more than E(max) value (70.6%) of BRL 37344 (P<0.05), with no change in pD(2) value (9.54+/-0.12 and 9.12+/-0.12, respectively). The inhibition of the amplitude of oxytocin-induced contractions by formoterol was antagonized with ICI 118.551 (10(-6) M), but they were not changed by metoprolol (10(-6) M) or SR 59230A (10(-6) M). The inhibition of the amplitude of oxytocin-induced contractions by BRL 37344 were antagonized with SR 59230A (10(-6) M), but they were not changed by metoprolol (10(-6) M) or ICI 118.551 (10(-6) M). Formoterol and BRL 37344 increased cAMP levels. BRL 37344 increased cGMP levels in BRL 37344 group more than control group, but this increase is less significant than cAMP levels (P>0.05). Formoterol and BRL 37344 decreased amplitude of myometrial contractions with similar potency, but efficacy of formoterol was better than BRL 37344.
Subject(s)
Adrenergic beta-Agonists/pharmacology , Ethanolamines/pharmacology , Myometrium/drug effects , Uterine Contraction/drug effects , Adrenergic beta-2 Receptor Agonists , Adrenergic beta-3 Receptor Agonists , Adrenergic beta-Antagonists/pharmacology , Animals , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Female , Formoterol Fumarate , In Vitro Techniques , Myometrium/metabolism , Myometrium/physiology , Oxytocin , Pregnancy , Propanolamines/pharmacology , Rats , Rats, WistarABSTRACT
The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.
