Role of Bioactives in Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) affect millions worldwide, with the two most prevalent being Alzheimer's and Parkinson disease [...].

Systematic Review of Naturally Derived Substances That Act as Inhibitors of the Nicotine Metabolizing Enzyme Cytochrome P450 2A6.

Tobacco smoking has been highlighted as a major health challenge in modern societies. Despite not causing death directly, smoking has been associated with several health issues, such as cardiovascular diseases, respiratory disorders, and several cancer types. Moreover, exposure to nicotine during pregnancy has been associated with adverse neurological disorders in babies. Nicotine Replacement Therapy (NRT) is the most common strategy employed for smoking cessation, but despite its widespread use, NRT presents with low success and adherence rates. This is attributed partially to the rate of nicotine metabolism by cytochrome P450 2A6 (CYP2A6) in each individual. Nicotine addiction is correlated with the high rate of its metabolism, and thus, novel strategies need to be implemented in NRT protocols. Naturally derived products are a cost-efficient and rich source for potential inhibitors, with the main advantages being their abundance and ease of isolation. This systematic review aims to summarize the natural products that have been identified as CYP2A6 inhibitors, validated through in vitro and/or in vivo assays, and could be implemented as nicotine metabolism inhibitors. The scope is to present the different compounds and highlight their possible implementation in NRT strategies. Additionally, this information would provide valuable insight regarding CYP2A6 inhibitors, that can be utilized in drug development via the use of in silico methodologies and machine-learning models to identify new potential lead compounds for optimization and implementation in NRT regimes.

Fascin-1 in Cancer Cell Metastasis: Old Target-New Insights.

As metastasis is responsible for most cancer-related deaths, understanding the cellular and molecular events that lead to cancer cell migration and invasion will certainly provide insights into novel anti-metastatic therapeutic targets. Fascin-1 is an actin-bundling protein fundamental to all physiological or pathological processes that require cell migration. It is responsible for cross-linking actin microfilaments during the formation of actin-rich cellular structures at the leading edge of migrating cells such as filopodia, lamellipodia and invadopodia. While most epithelial tissues express low levels of Fascin-1, it is dramatically elevated in the majority of cancers and its expression has been associated with more aggressive disease and decreased overall survival. Hence, it has been proposed as a potential anti-cancer target. In the present review, we studied recent literature with regard to Fascin-1 expression in different cancers, its role in altering the mechanical properties of cancer cells, promoting cancer cell migration, invasion and metastasis and the effect of its inhibition, via various pharmacological inhibitors, in eliminating metastasis in vitro and/or in vivo. Recent studies corroborate the notion that Fascin-1 is critically involved in metastasis and prove that it is a valuable anti-metastatic target that is worth investigating further.

Computer-Aided Drug Design of ß-Secretase, γ-Secretase and Anti-Tau Inhibitors for the Discovery of Novel Alzheimer's Therapeutics.

Aging-associated neurodegenerative diseases, which are characterized by progressive neuronal death and synapses loss in human brain, are rapidly growing affecting millions of people globally. Alzheimer's is the most common neurodegenerative disease and it can be caused by genetic and environmental risk factors. This review describes the amyloid-ß and Tau hypotheses leading to amyloid plaques and neurofibrillary tangles, respectively which are the predominant pathways for the development of anti-Alzheimer's small molecule inhibitors. The function and structure of the druggable targets of these two pathways including ß-secretase, γ-secretase, and Tau are discussed in this review article. Computer-Aided Drug Design including computational structure-based design and ligand-based design have been employed successfully to develop inhibitors for biomolecular targets involved in Alzheimer's. The application of computational molecular modeling for the discovery of small molecule inhibitors and modulators for ß-secretase and γ-secretase is summarized. Examples of computational approaches employed for the development of anti-amyloid aggregation and anti-Tau phosphorylation, proteolysis and aggregation inhibitors are also reported.

Ginkgo biloba L. flavonoids inhibit CYP 2A5; potential dietary supplement for nicotine replacement therapy enhancement.

Smoking is a public health concern, and even though smoking cessation methods exist, nicotine replacement therapy (NRT) is often ineffective. Smoking behavior is related to the nicotine metabolizing enzyme (NME) P450 2A6 (mouse 2A5) polymorphisms. Accordingly, fast metabolizers are nicotine dependent, and have low quitting rates compared to slow metabolizers. In this study we examined the ability of Ginkgo biloba L (GB) and its constituents to inhibit the NME, using mouse liver microsomes containing the 2A5 enzyme. Our results indicate that GB can inhibit 2A5 (25% inhibition at 5%v/v), with the flavonoids quercetin, isorhamnetin, and kaempferol being responsible for this inhibition (23.5%, 10.7%, 25.2% inhibition at 60 ng/µL, respectively). Importantly, the flavonoids inhibited 2A5 via mechanism based inhibition (for quercetin 30 ng/µl inhibition increased from 20.8% to 26.9% within 15 minutes). Our results suggest that GB if consumed on a regular basis can help NRT enhancement particularly in fast nicotine metabolizers.

Effects of 2-methoxyestradiol on hydrogen peroxide induced neuronal cell death and tau hyperphosphorylation.

AIMS: Alzheimer's Disease (AD) is characterized by progressive cognitive impairment, and memory loss. It has been shown that depletion of estrogens renders women vulnerable to AD with menopause women presenting higher risk for AD development than men. However, women under hormone replacement therapy (HRT) with 17ß-estradiol (E2) show lower risk for AD, implying that E2 may be protective. It has been shown that E2 exerts its effects through the estrogen receptor (ER) but also via its biologically active metabolites, 2-hydroxyestradiol (2OH), and 2-methoxyestradiol (2ME). We hypothesized that the neuroprotective effects of E2 are partly attributed to its metabolites. MATERIALS AND METHODS: SH-SY5Y neuronal cells were subjected oxidative stress (OS) cell death by hydrogen peroxide (H2O2), in the presence or absence of E2, 2ME and 2OH. Viability was assessed by trypan blue and thiazolyl blue tetrazolium bromide assays, intracellular OS with the Dichlorodihydrofluorescein Diacetate (DCFDA) assay, and Bax, p53 and PUMA quantified by RT-PCR. Tau hyperphosphorylation was studied by western blot. KEY FINDINGS: E2 and its metabolites 2OH and 2ME protect from cell death as assessed by the viability assays. Their effect was partly attributed to their antioxidant properties evidenced by the reduction of intracellular OS. Treatment with 2ME resulted in a reduction of Bax, but not p53 or PUMA in cells challenged with OS. Finally, 2ME was able to inhibit tau hyperphosphorylation as well. SIGNIFICANCE: E2 protects neuron cells partly through its metabolites. Further studies are needed to fully delineate the mechanism for this protection.

Cheminformatics and virtual screening studies of COMT inhibitors as potential Parkinson's disease therapeutics.

Introduction: Parkinson's Disease (PD) is a neurodegenerative central nervous system (CNS) disorder characterized by dopaminergic neuron degeneration with consequent reduction in striatal dopamine (DA) levels that leads to motor symptoms. Catechol-O-methyltransferase (COMT, E.C 2.1.1.6) inactivates dopamine and other substrates bearing catechol through the methylation of a hydroxyl group. COMT inhibition can block metabolism of catecholamines including DA. Since the increase in DA bioavailability is dependent on the inhibition of DA metabolism at the periphery, the development of COMT inhibitors as adjuvants to levodopa/aromatic amino acid decarboxylase (AADC) inhibitor treatment improves the clinical benefits of PD symptomatic treatment significantly.Areas covered: This review focuses on the contribution of computational studies to develop novel COMT inhibitors as therapeutics of Parkinson's disease with substantially improved efficacy.Expert opinion: The increasing use of in silico methods and the development of new chemoinformatic tools in combination with the knowledge gained from the development of different inhibitors studied both in silico, in vitro and in vivo, could help solve a number of issues related to the shortcomings of currently marketed treatments. They can also aid to open new avenues for centrally acting COMT inhibitors, and perhaps irreversible inhibitors, to be tested for PD and other neurological diseases.

Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts.

In many solid tumours a desmoplastic reaction takes place, which results in tumour tissue stiffening due to the extensive production of extracellular matrix (ECM) proteins, such as collagen, by stromal cells, mainly fibroblasts (FBs) and cancer-associated fibroblasts (CAFs). In this study, we investigated the effect of collagen stiffness on pancreatic FBs and CAFs, particularly on specific cytoskeleton properties and gene expression involved in tumour invasion. We found that cells become stiffer when they are cultured on stiff substrates and express higher levels of alpha-smooth muscle actin (α-SMA). Also, it was confirmed that on stiff substrates, CAFs are softer than FBs, while on soft substrates they have comparable Young's moduli. Furthermore, the number of spread FBs and CAFs was higher in stiffer substrates, which was also confirmed by Ras-related C3 botulinum toxin substrate 1 ( RAC1) mRNA expression, which mediates cell spreading. Although stress fibres in FBs become more oriented on stiff substrates, CAFs have oriented stress fibres regardless of substrate stiffness. Subsequently, we demonstrated that cells' invasion has a differential response to stiffness, which was associated with regulation of Ras homologue family member ( RhoA) and Rho-associated, coiled-coil containing protein kinase 1 ( ROCK-1) mRNA expression. Overall, our results demonstrate that collagen stiffness modulates FBs and CAFs cytoskeleton remodelling and alters their invasion properties.

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform.

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

A1 adenosine receptor upregulation accompanies decreasing myocardial adenosine levels in mice with left ventricular dysfunction.

BACKGROUND: It is well known that adenosine levels are increased during ischemia and protect the heart during ischemia/reperfusion. However, less is known about the role of adenosine-adenosine receptor (AR) pathways in hearts with left ventricular dilation and dysfunction. Therefore, we assessed adenosine levels and selective AR expression in transgenic mice with left ventricular systolic dysfunction secondary to overexpression of tumor necrosis factor-alpha (TNF 1.6). METHODS AND RESULTS: Cardiac adenosine levels were reduced by 70% at 3 and 6 weeks of age in TNF 1.6 mice. This change was accompanied by a 4-fold increase in the levels of A1-AR and a 50% reduction in the levels of A2A-AR. That the increase in A1-AR density was of physiological significance was shown by the fact that chronotropic responsiveness to the A1-AR selective agonist 2-chloro-N6-cyclopentanyladenosine was enhanced in the TNF 1.6 mice. Similar changes in adenosine levels were found in 2 other models of heart failure, mice overexpressing calsequestrin and mice after chronic pressure overload, suggesting that the changes in adenosine-AR signaling were secondary to myocardial dysfunction rather than to TNF overexpression. CONCLUSIONS: Cardiac dysfunction secondary to the overexpression of TNF is associated with marked alterations in myocardial levels of adenosine and ARs. Modulation of the myocardial adenosine system and its signaling pathways may be a novel therapeutic target in patients with heart failure.

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.

Permitted Daily Exposure of the Androgen Receptor Antagonist Flutamide.

This report aims to determine the permitted daily exposure (PDE) of flutamide, an androgen receptor blocker, as directed by guideline EMA/CHMP/CVPM/SWP/169430/2012 that came into effect on June 2015. A literature review was conducted to identify toxicity studies of flutamide. Hazards and sensitive endpoints were determined. Based on the no adverse effect levels (NOAELs) and lowest observed adverse effect levels (LOAELs) reported from both reproductive, developmental, and 28-day toxicity studies the PDE was calculated. Most of the toxicity studies converge toward a NOAEL of 1 mg/kg/d that translates to a PDE of 0.1 mg/d. However, taking into consideration the worst case scenarios for additional safety a PDE of 0.025 mg/d (25 µg/d) was calculated based on a reported NOAEL of 0.25 mg/kg/d. A PDE of 0.05 mg/d (50 µg/d) was also calculated from reproductive/developmental toxicity studies, which is in close agreement with the PDE from the 28-day toxicity studies. Considering the lowest PDE of 0.025 mg/d, residual flutamide at this dose is unlikely to pose any risk to humans. Nonmonotonic dose response (NMDR) effects of flutamide were not supported by literature. Oral route of administration was considered.

Targeting Inflammation to Improve Tumor Drug Delivery.

Inefficient delivery of drugs is a main cause of chemotherapy failure in hypoperfused tumors. To enhance perfusion and drug delivery in these tumors, two strategies have been developed: vascular normalization, aiming at normalizing tumor vasculature and blood vessel leakiness, and stress alleviation, aiming at decompressing tumor vessels. Vascular normalization is based on anti-angiogenic drugs, whereas stress alleviation is based on stroma-depleting agents. We present here an alternative approach to normalize tumor vasculature, taking into account that malignant tumors tend to develop at sites of chronic inflammation. Similarly to tumor vessel leakiness, inflammation is also characterized by vascular hyperpermeability. Therefore, testing the ability of anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs (NSAIDs) or inflammation resolution mediators, as an alternative means to increase tumor drug delivery might prove promising.

Ras Suppressor-1 (RSU-1) in Cancer Cell Metastasis: Friend or Foe?

Metastasis to distant organs and not the primary tumor itself is usually the cause of death for cancer patients. Hence, studying the key molecules and molecular pathways involved in metastasis are essential. Metastasis is a complex process in which cancer cells detach from the original tumor, migrate, and invade through surrounding tissues and metastasize to other sites of the body through the circulation. The cell-extracellular matrix (ECM) adhesion proteins play a fundamental role in this process as cancer cells need to weaken their adhesions to dissociate from the ECM as well as the neighboring cells within the tumor and finally form new adhesions and invade surrounding tissues. Ras suppressor-1 (RSU-1) was originally identified as a suppressor of Ras-dependent oncogenic transformation and found to be localized to cell-ECM adhesions where it binds to PINCH-1, a focal adhesion involved in cell survival. Although RSU-1 was connected to cancer early on, little is known about its expression in various cancer types or its role in metastasis. In this article, we review the recent literature regarding the expression of RSU-1 in various cancer types and its potential role in metastasis, discussing interesting findings and issues that still need to be addressed.

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.

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.

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.

Ras suppressor-1 promotes apoptosis in breast cancer cells by inhibiting PINCH-1 and activating p53-upregulated-modulator of apoptosis (PUMA); verification from metastatic breast cancer human samples.

Metastasis, responsible for most deaths from breast cancer (BC), is a multistep process leading to cancer cell spread. Extracellular matrix (ECM)-related adhesion and apoptosis resistance play pivotal role in metastasis. Ras suppressor-1 (RSU-1) localizes to cell-ECM adhesions and binds to pro-survival adhesion protein PINCH-1. Little is known about the role of RSU-1 in BC. In the present study, we investigated the role of RSU-1 in BC metastasis using two BC cell lines that differ in terms of their metastatic potential and a set of 32 human BC samples from patients with or without lymph node metastasis. We show that RSU-1 is upregulated in the aggressive MDA-MB-231 cells compared to MCF-7 and that its silencing by siRNA leads to upregulation of PINCH-1, induction of proliferation and reduction of apoptosis through downregulation of the pro-apoptotic gene p53-upregulated-modulator-of-apoptosis (PUMA). Our findings in the cell lines were further validated in the human BC tissues where normal adjacent tissues were used as controls. We demonstrate for the first time, that RSU-1 expression is upregulated in metastatic BC samples and downregulated in non-metastatic while it is negatively correlated with PINCH-1 and positively correlated with PUMA expression, suggesting that a pro-apoptotic mechanism is in place in metastatic BC samples and identifying RSU-1 as a potentially interesting molecule that needs to be evaluated further as a novel BC metastasis biomarker.

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.