Biomedical subjects

Lefteris C Zacharia

Publications and source records attributed to Lefteris C Zacharia.

18 recordsLinked to original sources

PubMed2006

Adenosine-mediated inhibition of the cytotoxic activity and cytokine production by activated natural killer cells.

Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-gamma, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI(14-22) peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells.

2-Chloroadenosine↗

PubMed2006

Adenosine receptor expression and function in bladder uroepithelium.

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A1, A2a, A2b, and A3) are expressed in the uroepithelium. A1 receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A2a, A2b, and A3 receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 microF approximately 1 cm(2) tissue area) of approximately 30% or approximately 24%, respectively, after 5 h. Although A1, A2a, and A3 selective agonists all stimulated membrane capacitance after being administrated serosally, only the A1 agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells.

Animals↗

PubMed2006

cAMP-adenosine pathway in the proximal tubule.

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.

Adenosine↗

PubMed2005

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells.

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.

2-Chloroadenosine↗

PubMed2004

Cytochromes 1A1/1B1- and catechol-O-methyltransferase-derived metabolites mediate estradiol-induced antimitogenesis in human cardiac fibroblast.

We investigated the role of specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) in the growth inhibitory effects of estradiol in cardiac fibroblasts (CFs) expressing functional estrogen receptors. 3-Methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and beta-naphthoflavone (CYP1A1/1A2 inducer) augmented, and 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor) blocked, the inhibitory effects of estradiol on serum-induced CF growth (DNA synthesis, cell number, and collagen synthesis). Neither ketoconazole (3A4 inhibitor) nor furafylline (selective 1A2 inhibitor) altered the antimitogenic effects of estradiol on CF growth. In contrast, ellipticine (selective 1A1 inhibitor), pyrene (selective 1B1 inhibitor), and alpha-naphthoflavone (1A1>1A2 inhibitor) abrogated the antimitogenic effects of estradiol on CF growth. OR486 (COMT inhibitor) also blocked the antimitogenic effects of estradiol in both the presence and absence of the CYP450 inducers. ICI182780 (estrogen receptor antagonist) attenuated the growth inhibitory effects of estradiol, but only at concentrations that inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). CFs expressed CYP1A1 and CYP1B1, isozymes that convert estradiol to hydroxyestradiols. Moreover, CFs metabolized estradiol to hydroxyestradiol, and 2-hydroxyestradiol to 2-methoxyestradiol. OR486 and quercetin (COMT inhibitor) blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol in CFs. We conclude that the antimitogenic effects of estradiol on CF growth are mediated in part by conversion to hydroxyestradiols via CYP1A1 and CYP1B1, followed by metabolism of hydroxyestradiols to methoxyestradiols by COMT.

Aryl Hydrocarbon Hydroxylases↗

PubMed2004

Hormone replacement therapy and cardiovascular disease: what went wrong and where do we go from here?

Observational studies in humans and experimental studies in animals and isolated cells supported the widely held belief that hormone replacement therapy protects the cardiovascular system from disease. To nearly everyone's astonishment, the Women's Health Initiative Study and the Heart and Estrogen/Progestin Replacement Study overturned the conclusion that hormone replacement therapy protects the cardiovascular system and, in fact, supported the opposite view that such therapy may actually increase the risk of cardiovascular disease. This review addresses 2 questions: what went wrong and where do we go from here?

Animals↗

PubMed2004

2-hydroxyestradiol is a prodrug of 2-methoxyestradiol.

Previous in vivo studies indicate that 2-hydroxyestradiol (2OHE) attenuates cardiovascular and renal diseases. In vitro studies suggest that the biological effects of 2OHE are mediated by 2-methoxyestradiol (2MEOE) after methylation of 2OHE by catechol-O-methyltransferase (COMT). This study tested the hypothesis that in vivo 2OHE is a prodrug of 2MEOE. We administered to male rats i.v. boluses of either 2OHE or 2MEOE and measured plasma levels of 2OHE and 2MEOE by gas chromatography-mass spectrometry at various time points after drug administration. After administration of 2OHE, plasma levels of 2OHE declined extremely rapidly [t(1/2(1)) = 0.94 min and t(1/2(2)) = 10.2 min] becoming undetectable after 45 min. Concomitant with the disappearance of 2OHE, 2MEOE occurred and then declined [t(1/2(1)) = 7.9 min and t(1/2(2)) = 24.9 min]. The peak concentration and total exposure (area under the curve) for 2OHE were much lower than for 2MEOE. 2OHE had a much higher plasma clearance (CL) and volume of distribution (V(d)) compared with 2MEOE (2OHE: CL = 1215 ml min(-1) kg(-1) and V(d) = 17,875 ml/kg; 2MEOE: CL = 50 ml min(-1) kg(-1) and V(d) = 1760 ml/kg). After administration of 2MEOE, plasma levels of 2MEOE declined [t(1/2(1)) = 2.5 min and t(1/2(2)) = 20.2 min] with a plasma CL of 50 ml min(-1) kg(-1) and a V(d) of 1500 ml/kg. We could not detect 2OHE in plasma from rats receiving 2MEOE. We conclude that the conversion of 2OHE to 2MEOE is so efficient that in terms of 2MEOE exposure, administration of 2OHE is bioequivalent to administration of 2MEOE itself.

2-Methoxyestradiol↗

PubMed2004

A gas chromatography/mass spectrometry assay to measure estradiol, catecholestradiols, and methoxyestradiols in plasma.

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.

Animals↗

PubMed2004

Catecholamines block the antimitogenic effect of estradiol on human coronary artery smooth muscle cells.

Sequential conversion of estradiol to catecholestradiols and methoxyestradiols by cytochrome-P(450) (CYP450) and catechol-O-methyltransferase (COMT), respectively, contributes to the antimitogenic effects of estradiol on vascular smooth muscle cell (SMC) growth via estrogen receptor-independent mechanisms. Because catecholamines are also substrates for COMT, we hypothesize that catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic/inhibitory effects of estradiol on human coronary artery SMC growth (cell number, DNA synthesis, collagen synthesis, and SMC migration) and ERK1/2 phosphorylation in the presence and absence of catecholamines. Norepinephrine, epinephrine, isoproterenol, and OR486 (COMT inhibitor) abrogated the inhibitory effects of estradiol on SMC growth and ERK1/2 phosphorylation. The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, alpha- and beta-adrenoceptor antagonists, respectively. The antimitogenic effects of 2-hydroxy-estradiol, but not 2-methoxyestradiol, were abrogated by epinephrine, isoproterenol, and OR486. Catecholamines inhibited the conversion of both estradiol and 2-hydroxy-estradiol to 2-methoxyestradiol, and SMCs expressed CYP1A1 and CYP1B1. Our findings suggest that catecholamines within the coronary arteries may abrogate the antivasoocclusive effects of estradiol by blocking the conversion of catecholestradiols to methoxyestradiols. The interaction between catecholamines and estradiol metabolism may importantly define the cardiovascular effects of estradiol therapy in postmenopausal women.

2-Methoxyestradiol↗

PubMed2003

Methoxyestradiols mediate the antimitogenic effects of 17beta-estradiol: direct evidence from catechol-O-methyltransferase-knockout mice.

BACKGROUND: Studies using pharmacological agents suggest but do not prove that the antimitogenic effects of estradiol are caused by conversion of estradiol to hydroxyestradiols (mediated by CYP450s) followed by methylation of hydroxyestradiols to methoxyestradiols (mediated by catechol-O-methyltransferase, COMT). METHODS AND RESULTS: To test this hypothesis more rigorously, we used aortic smooth muscle cells (SMCs) from mice lacking COMT (COMT-KO). Wild-type (WT) but not COMT-KO SMCs efficiently converted 2-hydroxyestradiol to 2-methoxyestradiol. Both WT and COMT-KO SMCs expressed estrogen receptors. Estradiol and 2-hydroxyestradiol concentration-dependently inhibited serum-induced DNA synthesis, cell numbers, and collagen synthesis in WT but not COMT-KO SMCs. 2-Methoxyestradiol inhibited DNA synthesis, cell numbers, and collagen synthesis in both WT and COMT-KO SMCs. CONCLUSIONS: These data provide strong evidence that the vascular antimitogenic effects of estradiol are estrogen receptor-independent and involve the sequential conversion of estradiol to hydroxyestradiols and then to methoxyestradiols.

2-Methoxyestradiol↗

PubMed2003

Catecholamines block the antimitogenic effect of estradiol on human glomerular mesangial cells.

Local sequential conversion of estradiol to hydroxyestradiols and methoxyestradiols by CYP450 and catechol-O-methyltransferase, respectively, contributes to the antimitogenic effects of estradiol on glomerular mesangial cell growth via estrogen receptor-independent mechanisms. Catecholamines are also substrates for catechol-O-methyltransferase and therefore, might abrogate the renoprotective effects of estradiol by inhibiting formation of methoxyestradiols. To test this hypothesis, we investigated the antimitogenic effects of estradiol on human glomerular mesangial cell proliferation and collagen synthesis in the presence and absence of catecholamines. Norepinephrine, epinephrine, and isoproterenol abrogated the inhibitory effects of estradiol on cell number, DNA synthesis, and collagen synthesis. For example, serum-induced DNA synthesis was inhibited from 100% to 62+/-1.9% by 0.1 micromol/L estradiol, and these inhibitory effects were reversed to 91+/-1.9% by 1 micromol/L epinephrine, 90.7+/-3.3% by 1 micromol/L isoproterenol, 87.5+/-2.8% by 10 micromol/L norepinephrine, and 92+/-1% by 10 micromol/L OR486 (catechol-O-methyltransferase inhibitor). The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, alpha- and beta-adrenoceptor antagonists, respectively. Similar to estradiol, the antimitogenic effects of 2-hydroxyestradiol were abrogated by epinephrine, isoproterenol, and OR486. In contrast to estradiol and 2-hydroxyestradiol, the antimitogenic effects of 2-methoxyestradiol were not attenuated by epinephrine, isoproterenol, or OR486. Norepinephrine, epinephrine, and isoproterenol inhibited the conversion of both estradiol and 2-hydroxyestradiol to 2-methoxyestradiol. Our findings suggest that catecholamines within the glomeruli might abrogate the antimitogenic effects of estradiol by blocking the conversion of 2-hydroxyestradiol to 2-methoxyestradiol.

2-Methoxyestradiol↗

PubMed2003

Methylation of 2-hydroxyestradiol in isolated organs.

Vascular smooth muscle and glomerular mesangial cells in culture express a biochemical pathway that methylates 2-hydroxyestradiol (17beta-estradiol metabolite) to produce 2-methoxyestradiol, a cell growth inhibitor that may mediate the cardiorenal protective effects of 17beta-estradiol. Whether this pathway exists in intact organ systems is currently unclear. Accordingly, the purpose of the present investigation was to characterize the methylation of 2-hydroxestradiol in intact organs from both male and female rats. No significant differences were detected in the ability of male and female tissues to methylate 2-hydroxyestradiol. In isolated hearts, kidneys, and mesenteries perfused with Tyrode's solution, Km values for 2-hydroxyestradiol methylation were 0.175+/-0.021, 0.387+/-0.054, and 0.495+/-0.089 micromol/L, respectively, and Vmax values were 21.0+/-1.58, 24.9+/-1.49, and 1.01+/-0.148 pmol 2-methoxyestradiol x min(-1) x ml(-1) per gram, respectively. The catalytic efficiency (Vmax/Km) was greatest in the heart compared with the kidney and mesentery (132+/-14.3, 78.4+/-15.1, and 2.30+/-0.263 pmol 2-methoxyestradiol x min(-1) x mL(-1) x micromol/L(-1) per gram, respectively). In the kidney, the catechol-O-methyltransferase inhibitor quercetin and norepinephrine (10 micromol/L) reduced methylation of 2-hydroxyestradiol by approximately 90% and 41%, respectively. Importantly, methylation in the kidney was inhibited by an average of 16.6+/-1.80% by endogenous norepinephrine released by renal artery nerve stimulation. Our results indicate that a robust 2-hydroxyestradiol methylation pathway exists in the kidney and heart, but not in the mesentery, and that this pathway is mediated by catechol-O-methyltransferase. Our findings also suggest that catecholamines may interfere with 2-hydroxyestradiol methylation and thereby attenuate the cardiorenal protective effects of 17beta-estradiol.

2-Methoxyestradiol↗

PubMed2002

CYP450- and COMT-derived estradiol metabolites inhibit activity of human coronary artery SMCs.

The purpose of this study is to test the hypothesis that the inhibitory effects of estradiol in human coronary vascular smooth muscle cells are mediated via local conversion to methoxyestradiols via specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT). The inhibitory effects of estradiol on serum-induced cell activity (DNA synthesis, cell number, collagen synthesis, and cell migration) were enhanced by 3-methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and beta-naphthoflavone (CYP1A1/1A2 inducer) and were blocked by 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor). Ellipticine, alpha-naphthoflavone (selective CYP1A1 inhibitors), and pyrene (selective CYP1B1 inhibitor), but not ketoconazole (selective CYP3A4 inhibitor) or furafylline (selective CYP1A2 inhibitor), abrogated the inhibitor effects of estradiol on cell activity, a profile consistent with a CYP1A1/CYP1B1-mediated mechanism. The inhibitory effects of estradiol were blocked by the COMT inhibitors OR486 and quercetin. The estrogen receptor antagonist ICI 182,780 blocked the inhibitory effects of estradiol, but only at concentrations that also blocked the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). Western blot analysis revealed that coronary smooth muscle cells expressed CYP1A1 and CYP1B1. Moreover, these cells metabolized estradiol to hydroxyestradiols and methoxyestradiols, and the conversion of 2-hydroxyestradiol to 2-methoxyestradiol was blocked by OR486 and quercetin. These findings provide evidence that the inhibitory effects of estradiol on coronary smooth muscle cells are largely mediated via CYP1A1- and CYP1B1-derived hydroxyestradiols that are converted to methoxyestradiols by COMT.

2-Methoxyestradiol↗

PubMed2002

Methoxyestradiols mediate estradiol-induced antimitogenesis in human aortic SMCs.

Estrogen receptors (ERs) are considered to mediate the ability of 17beta-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-alpha or ER-beta challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis (3[H]-thymidine incorporation), collagen synthesis (3[H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (>50 micromol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.

2-Methoxyestradiol↗

PubMed2002

Catecholamines block 2-hydroxyestradiol-induced antimitogenesis in mesangial cells.

Methylation of 2-hydroxyestradiol to 2-methoxyestradiol by catechol-O-methyl transferase (COMT) mediates the antimitogenic effects of 2-hydroxyestradiol on vascular smooth muscle cells. Moreover, 2-hydroxyestradiol inhibits growth of glomerular mesangial cells (GMCs). Because catecholamines are substrates for COMT, which is expressed in GMCs, we hypothesize that catecholamines may abrogate the antimitogenic effects of 2-hydroxyestradiol on GMCs by competing for COMT and inhibiting 2-methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic effects of 2-hydroxyestradiol on rat GMCs in the presence and absence of catecholamines. The capability of GMCs to methylate 2-hydroxyestradiol in the presence and absence of catecholamines was also evaluated. GMCs metabolized 2-hydoxyestradiol in a concentration-dependent manner with a V(max) of 12.03+/-0.32 pmol/10(6) cells/min and an apparent K(m) of 0.23+/-0.04 micromol/L. Norepinephrine (10 micromol/L) and epinephrine (10 micromol/L) significantly inhibited methylation of 0.25 micromol/L 2-hydroxyestradiol. Norepinephrine concentration-dependently abrogated the ability of 2-hydroxyestradiol to inhibit 3H-thymidine incorporation (index of DNA synthesis). In the presence of 5, 10, and 40 micromol/L norepinephrine, the inhibitory effect of 0.1 micromol/L 2-hydroxyestradiol on 3H-thymidine incorporation was reduced from 51+/-0.7% to 46+/-0.4%, 39+/-0.3%, and 25+/-0.7%, respectively. Similar to DNA synthesis, the inhibitory effects of 2-hydroxyestradiol on cell number and 3H-proline incorporation (index of collagen synthesis) on GMCs were abrogated by catecholamines. Our findings provide evidence that methylation of 2-hydroxyestradiol inhibits GMC proliferation and extracellular matrix synthesis and may in part protect against renal proliferative diseases. Moreover, catecholamines may abrogate the renoprotective effects of 2-hydroxyestradiol in the glomeruli by inhibiting COMT and 2-methoxyestradiol formation.

2-Methoxyestradiol↗

PubMed2002

Methoxyestradiols mediate the antimitogenic effects of locally applied estradiol on cardiac fibroblast growth.

Estradiol inhibits cardiac fibroblast growth and may protect against cardiac remodeling associated with heart disease. However, the mechanisms by which estradiol attenuates cardiac fibroblast growth remain unclear. Because cardiac fibroblasts express cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) capable of converting estradiol to hydroxyestradiols and methoxyestradiols, respectively, and because hydroxyestradiols and methoxyestradiols (estradiol metabolites with little affinity for estrogen receptors) are potent inhibitors of cardiac fibroblast growth, we hypothesized that the antimitogenic effects of estradiol are mediated via hydroxyestradiols and/or methoxyestradiols. The inhibitory effects of estradiol (1 to 100 nmol/L) on serum-stimulated (3)H-thymidine incorporation (DNA synthesis), (3)H-proline incorporation (collagen synthesis), and cell number (proliferation) were enhanced (P<0.005) by CYP450 inducers 3-methylcholanthrene (10 micromol/L) and phenobarbital (10 micromol/L). Moreover, the inhibitory effects of estradiol were blocked by the CYP450 inhibitor 1-aminobenzotriazole (10 micromol/L) and the COMT inhibitors quercetin (10 micromol/L) and OR486 (10 micromol/L). In contrast to estradiol, the modulators of CYP450 and COMT were poor ligands for estrogen receptors (binding affinity less-than-or-equal 0.0001% versus estradiol). In cardiac fibroblasts, both quercetin and OR486 inhibited the metabolism of hydroxyestradiol to methoxyestradiol and blocked the inhibitory effects of hydroxyestradiol on cardiac fibroblast proliferation and DNA and collagen synthesis. The abrogating effects of quercetin and OR486 on the metabolism and antimitogenic effects of 2-hydroxyestradiol were mimicked by 20 micromol/L norepinephrine and isoproterenol, substrates for COMT. Our findings provide evidence that estradiol can inhibit cardiac fibroblast growth via an estrogen receptor--independent pathway that involves the local metabolism of estradiol to methoxyestradiols.

Animals↗

PubMed2002

Role of methoxyestradiols in the growth inhibitory effects of estradiol on human glomerular mesangial cells.

Metabolism of locally applied 17beta-estradiol (estradiol) to methoxyestradiols contributes to the growth inhibiting effects of estradiol on vascular smooth muscle cells via an estrogen receptor (ER)-independent mechanism. Because vascular smooth muscle cells are phenotypically similar to glomerular mesangial cells, it is feasible that estradiol inhibits glomerular mesangial cell growth via a similar mechanism, and this possibility was investigated. In human glomerular mesangail cells, estradiol concentration dependently (1 to 100 nmol/L) inhibited serum-induced proliferation (cell number) and DNA ((3)[H]-thymidine incorporation) and collagen ((3)[H]-proline incorporation) synthesis. The inhibitory effects of estradiol were mimicked by 2-hydroxyestradiol and 2-methoxyestradiol, metabolites of estradiol with little affinity for ERs. 2-Hydroxyestradiol and 2-methoxyestradiol were more potent growth inhibitors than estradiol. The inhibitory effects of estradiol were enhanced by CYP450 inducers 3-methylcholanthrene (10 micromol/L) and phenobarbital (10 micromol/L) and blocked by the CYP450 inhibitor 1-aminobenzotriazole (10 micromol/L). The growth inhibitory effects of estradiol were also blocked by quercetin (10 micromol/L) and OR 486 (10 micromol/L) inhibitors of catechol-O-methyltransferase (converts catecholestradiols to methoxyestradiols). ICI182780 (ER antagonist with ER binding affinity similar to estradiol) blocked the growth inhibitory effects of estradiol (1 to 100 nmol/L) only at concentrations (>50 micromol/L) that inhibited estradiol metabolism to catecholestradiols. The growth inhibitory effects of 2-hydroxyestradiol were abrogated by quercetin and OR486 (two structurally dissimilar catechol-O-methyltransferase inhibitors), but not by ICI182780. However, the growth inhibitory effects of 2-methoxyestradiol were unaltered by catechol-O-methyltransferase inhibitors and ICI182780. In conclusion, our findings provide the first evidence that methoxyestradiols mediate the growth inhibitory effects of locally applied estradiol on glomerular mesangial cell growth via an ER-independent mechanism.

Catechol O-Methyltransferase Inhibitors↗

PubMedDate not supplied

Conversion of tibolone to 7alpha-methyl-ethinyl estradiol using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry: interpretation and clinical implications.

OBJECTIVE: Tibolone, a hormone therapy drug, is used to treat climacteric symptoms. This drug is rapidly metabolized into three major metabolites (3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta4-tibolone). One clinical study provided evidence of conversion of tibolone to another estrogenic metabolite, 7alpha-methyl-ethinyl estradiol (MEE). However, no evidence of MEE formation was found in another study using the human aromatase enzyme. Because MEE was analyzed by gas chromatography-mass spectrometry (GC-MS), which requires derivatization, together with the fact that derivatization of some steroids may lead to aromatization, it is feasible that the MEE detected resulted from an artifact generated during the derivatization process. Hence, our objective was to assess whether tibolone is converted to MEE. DESIGN: We assayed MEE formation in a nonbiological system using GC-MS after derivatization and by analyzing MEE formation using liquid chromatography-mass spectrometry (LC-MS) in nonderivatized samples. RESULTS: MEE formation was evident in tibolone samples derivatized with either pentafluoropropionic anhydride or trimethylsilyl and analyzed by GC-MS. The amount of MEE formed increased with increasing amounts of tibolone (0.5, 1, 2.5, and 5 microg) derivatized; however, relative to tibolone, the percentage of MEE formed remained constant and ranged between 0.22% and 0.29% of tibolone. In contrast to GC-MS, no MEE formation was seen when tibolone was analyzed by liquid chromatography-mass spectrometry without derivatization. CONCLUSIONS: Our findings prove that conversion of tibolone to MEE is an artifact that is generated in a GC-MS system and is largely due to the intense heating step involved in GC-MS. Caution should be exercised to extrapolate clinical implications from existing data on MEE formation using a GC-MS system.

Chromatography, Liquid↗