1 1 Signaling responses after exposure to 5α-dihydrotestosterone or ...

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Dec 31, 2009 - estradiol (E2) or 5α-dihydrotestosterone (DHT) are understudied. ... and studies demonstrating testosterone and 5α-dihydrotestosterone (DHT).
Articles in PresS. J Appl Physiol (December 31, 2009). doi:10.1152/japplphysiol.00994.2009

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Signaling responses after exposure to 5α-dihydrotestosterone or 17β-estradiol in

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norepinephrine-induced hypertrophy of neonatal rat ventricular myocytes

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Yevgeniya E Koshman1,3, Mariann R. Piano2, Brenda Russell1 and Dorie W. Schwertz2

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Department of Physiology and Biophysics, University of Illinois at Chicago

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Department of Biobehavioral Health Science, University of Illinois at Chicago

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The Cardiovascular Institute. Loyola University Chicago, Stritch School of Medicine

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Running Title: Signaling differences with sex hormones in cardiac hypertrophy

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Corresponding author

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Brenda Russell, Ph.D.

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University of Illinois at Chicago. MC 901

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835 South Wolcott Avenue

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Chicago, IL 60612-7342

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Tel: (312) 413-0407

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Fax: (312) 996-6312

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email: [email protected]

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Copyright © 2009 by the American Physiological Society.

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ABSTRACT

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Androgens appear to enhance whereas estrogens mitigate cardiac hypertrophy. However,

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signaling pathways in cells for short (3 minute) and longer-term (48 hour) treatment with 17β-

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estradiol (E2) or 5α-dihydrotestosterone (DHT) are understudied. We compared the effect of

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adrenergic stimulation by norepinephrine (NE, 1 μM) alone or in combination with DHT (10nM)

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or E2 (10nM) treatment in neonatal rat ventricular myocytes (NRVMs) by cell area, protein

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synthesis, sarcomeric structure, gene expression, phosphorylation of extracellular signal-

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regulated (ERK) and focal adhesion (FAK) kinases, and phospho-FAK nuclear localization. NE

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alone elicited the expected hypertrophy, and strong sarcomeric organization, DHT alone gave a

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similar but more modest response whereas E2 did not alter cell size. Effects of NE dominated

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when used with either E2 or DHT with all combinations. Both sex hormones alone rapidly

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activated FAK but not ERK. Long-term or brief exposure to E2 attenuated NE-induced FAK

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phosphorylation whereas DHT had no effect. Neither hormone altered NE-elicited ERK

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activation. Longer-term exposure to E2 alone reduced FAK phosphorylation and reduced

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nuclear phospho-FAK whereas its elevation was seen in the presence of NE with both sex

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hormones. The mitigating effects of E2 on NE-elicited increase in cell size and hypertrophic

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effect of DHT in NRVMs are in accordance with results observed in whole animal models. This

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is the first report of rapid, non-genomic sex hormone signaling via FAK activation and altered

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FAK trafficking to the nucleus in heart cells.

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INTRODUCTION Sustained cardiac hypertrophy is associated with increased risk for arrhythmias, sudden

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death and heart failure (38, 62). Results from epidemiological studies indicate that prevalence

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of hypertrophy is lower in women (30, 40, 62) and given the same mechanical load on the

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heart, women are less likely to develop hypertrophy than men (16). Left ventricular wall

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thickness increases after menopause (1) with greater relative wall thickness found in

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postmenopausal women compared to age-, race- and weight-matched premenopausal women

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(33). Further, evidence suggests that hormone replacement therapy reduces left ventricular

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mass in postmenopausal women (44, 45). In addition, healthy men have larger hearts than

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age-matched women even when indexed to body size and the ventricular size-divergence

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develops following puberty (23, 53). These findings suggest that being female and/or female sex

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hormones diminish the cardiac hypertrophic response. Alternatively, male sex or androgenic

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hormones may contribute to and enhance hypertrophy; a possibility supported by the finding

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that testosterone elicits cardiac hypertrophy in sham-operated and post myocardial infarction

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(MI) male rats (46) and studies demonstrating testosterone and 5α-dihydrotestosterone (DHT)

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elicit hypertrophy in isolated neonatal rat ventricular myocytes (NRVMs) (3, 41).

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To our knowledge, a direct comparison of the influence of male and female sex

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hormones on extracellular signal-induced hypertrophy in cardiac myocytes has not been

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conducted; nor has there been a direct comparison of the effect of sex hormones on

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hypertrophic growth and signaling pathways. In this study, we examined the direct effect of 17

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β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on neonatal rat ventricular myocyte (NRVM)

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hypertrophy and the modulatory effect of sex hormones on norepinephrine (NE)-elicited

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hypertrophy. Myocardial hypertrophy develops in response to multiple initiating stimuli including

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mechanical stress (from hemodynamic overload) and extracellular signals (such as

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catecholamines and growth factors). Mechanical stress is communicated into the cell via 3

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integrins that link the extracellular matrix to a network of intracellular structural and signal-

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transducing proteins including a cytoplasmic tyrosine kinase called focal adhesion kinase (FAK).

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FAK phosphorylation and activation is well established to be associated with the hypertrophic

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response. Extracellular signals including endothelin-1, angiotensin II, and the well-studied

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alpha-adrenergic agonist, phenylephrine, and others activate multiple mitogen activated protein

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kinases (MAP kinases) including extracellular signal-related kinase (ERK). ERK has been

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established to have an important role in cardiac cell hypertrophy. Activation of hypertrophic

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pathways via different initiating signals is not independent but have complex downstream

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interactions, see reviews (6, 11, 13, 52).

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Given that estrogen is considered protective in the context of hypertrophy and

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androgens are thought to elicit and/or contribute to cardiac hypertrophy; the aims of this study

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were: 1) to compare the direct effects of 17 β-estradiol (E2) and 5α-dihydrotestosterone (DHT)

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on cardiac hypertrophy in isolated NRVMs, 2) to compare the effect of these two sex hormones

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on norepinephrine (NE)-induced hypertrophy, 3) to compare the effect of E2 and DHT on the

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activation of kinases previously shown to be involved in transduction of hypertrophic signals

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(ERK and FAK), and 4) to determine whether male and female sex hormone-elicited effects on

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ERK and FAK are the result of rapid steroid signaling.

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METHODS

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Cell culture. Hearts of 1-2 day-old Sprague-Dawley male and female rat pups (Harlan,

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Indianapolis, IN) were uses to isolate NRVM by step-wise collagenase digestions as reported

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previously (9). The cells were pre-plated for 1 hour to reduce non-myocyte contamination

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before incubation in phenol-free Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12

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HAM (Sigma, St. Louis, MO) without L-glutamine and with standard amino acid concentrations,

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palmitic acid (2.56 mg/L) and linoleic acid (0.84 mg/L), penicillin G (100 IU/ml), streptomycin

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(0.1 mg/ml), and amphotericin (0.25 µg/ml) with 5% Charcoal-dextran filtered steroid-free fetal

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bovine serum (FBS – Hyclone; Logan Utah). Cytosine-β-D-arabino-furanoside (ARA-C, 5

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μg/mL) was added to prevent fibroblast proliferation. After 24 hours in a 5% CO2, 37ºC

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incubator, unattached cells were removed by aspiration and the remaining cells were washed 3

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times with ITS Buffer (serum-free, phenol-free DMEM and with the addition of insulin (5 μg/ml),

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transferring (1 μg/ml) and selenium (5 ηg/ml) (ITS Premix; BD Biosciences, Bedford MA). The

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cells were maintained in ITS buffer with ARA-C and antibiotics throughout the experimental

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protocols. NRVM were plated at 30 to 60 percent confluency on 100mm Primaria culture dishes

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(BD Bioscience) unless otherwise stated. All experiments were performed in accordance with

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the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes

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of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional

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Animal Care and Use Committee.

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Experimental Protocols. In order to examine the effect of sex hormones on NE-elicited

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hypertrophy and to answer the question whether long-term or short-term hormone exposure is

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necessary for the modulation of the NE-induced hypertrophic response, three protocols were

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developed. Protocol #1 was designed to answer aims 1 and 2, i.e., to determine the effect of

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sex hormones on NRVM hypertrophy and to determine the effect of sex hormones on NE5

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induced NRVM hypertrophy. In Protocol #1 NRVMs were exposed NE [1µM] in the presence or

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absence of E2 [10nM] or DHT [10nM] for 48 hrs. Forty-eight hours of exposure to NE has been

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shown by others to reliably produce NRVM hypertrophy(54). Protocol #2 was designed to

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answer aim #3, i.e., to determine whether sex hormones alter NE-elicited activation of kinases

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shown to be involved in hypertrophic signaling. NE-elicited kinase activation is a rapid response

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whereas genomic steroid signaling (alteration in gene expression) may require hours to days.

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Therefore, after exposure to E2 or DHT (or no treatment) for 48 hrs, NRVM were stimulated with

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NE for 2 minutes. Protocol #3 was designed to answer aim #4, i.e., to determine whether short-

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term sex hormone exposure (rapid steroid signaling) altered NE-elicited kinase activation. Cells

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were incubated for 48 hours without treatment followed by exposure to E2 or DHT for one

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minute and then to NE (or no treatment) for another 2 minutes. Because early evidence

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suggests that the NE-elicited hypertrophic response is mediated through alpha1-adrenergic

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receptors(54), PE is frequently used experimentally as a hypertrophic signal. However, others

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find that both alpha- and beta-adrenergic receptors are involved in mediating the hypertrophic

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response(5). We chose to examine the effect of sex hormones on NE-elicited hypertrophy in

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NRVMs because it is a clinically relevant, endogenous mixed alpha- and beta-adrenergic

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hypertrophic stimulus and there is no known endogenous alpha-adrenergic ligand. However, we

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added phenylephrine (PE-10μM] as a positive control in all experiments. Thus all protocols

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included the following groups: no treatment (negative control), exposure to NE alone, E2 alone,

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E2 and NE, DHT alone, DHT and NE or PE alone.

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Drug treatment. Drugs were dissolved in phosphate buffered saline. E2 required

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dimethylsulfoxide (DMSO) at a final concentration of less than 0.001 percent to facilitate

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solution. E2, DHT and NE were obtained from Sigma Chemical Co. (St. Louis MO) and were

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added to 100mm culture dishes in 100μl aliquots. 6

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Cell surface area analysis. Myocyte surface area was determined in cells exposed to sex

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hormones and/or NE for 48 hours (Protocol #1). The concentration of DMSO was shown to

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have no effect on cell surface area. Cells were visualized by light microscopy (Nikon Microphot-

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FXA) with a 40X objective. Four randomly chosen fields of cells were selected from each

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100mm culture dish (2 dishes per treatment per experiment) and digitally photographed Spot RT

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CCD (Diagnostic Instruments). Cells within each field were outlined and 2-dimensional area

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was obtained using ImageJ software (National Institutes of Health; Bethesda, Maryland).

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Approximately 50 cells were measured for each treatment in each experiment and the final

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mean surface area for each treatment was determined from three separate experiments.

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Tritiated-leucine incorporation. NRVM were plated on 30 mm Primaria cell culture dishes.

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Sex hormones and NE and 0.5 μCi of 3H-leucine (L-[3,4,5-3H(N))] (PerkinElmer Life Sciences;

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Boston MA) were added together to the cells in 2 ml ITS buffer. Forty-eight hour later (Protocol

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#1) the cells were washed x 3 with PBS and then incubated with shaking at room temperature in

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PBS (0.5 ml) with sodium dodecylsulfate (SDS;10%) and trichloroacetic acid TCA; 5%). Cells

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were scraped off the dishes and a 0.25 ml sample was taken for liquid scintillation counting in

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Econo-Safe Liquid scintillation fluid (Research Products International, Mt. Prospect, IL).

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Counting error was less than 2%.

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Determination of mRNA. NRVM were treated with E2, DHT and NE (48 hrs- Protocol #1).

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RNA was isolated using TRIzol (Invitrogen) according to manufacturer instructions and

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concentration was determined by NanoDrop spectrophotometry (Thermo Fischer, Inc).

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Complementary DNA was synthesized from 1µg RNA using the SuperScript III First-Strand

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Synthesis System (Invitrogen). Real-time PCR was run in triplicate on 10 ng cDNA per well with 7

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ribosomal RNA (18S) as an endogenous control and detected by TaqMan® chemistry

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(fluorogenic 5' nuclease chemistry) using the AB7300 Real Time PCR System (Applied

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Biosystems). TAQMAN probe accession numbers (Applied Biosystems) for each RNA of

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interest were as follows: brain natriuretic peptide (Rn00580641_m1), atrial natriuretic peptide

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(Rn00561661_m1), skeletal actin (Rn00570060_g1), and beta MHC (Rn000568328_m1). The

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comparative CT method was used for relative quantification.

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Western blotting. After described NE and hormone treatments, cells were rinsed with cold

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PBS and then scraped in ice-cold lysis buffer with sodium vanadate (1mM). Protein

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concentrations were assessed using the Bradford assay (Pierce, Rockford, IL) and then equal

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amounts of protein were separated by 4-20% SDS-PAGE and transferred to nitrocellulose

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membranes (Hybond, Amersham, Arlington Heights, IL). Proteins were probed with appropriate

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antibodies including FAK mouse antibody (BD transduction, Lexington, KY), antibody to

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recognize phosphorylated FAK at tyrosine 397 (FAK pY397 rabbit; Biosource, Camarillo, CA),

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ERK 1,2 rabbit antibody (Promega, Madison, WI), and ERK 1,2 pTEpY rabbit antibody

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(Promega, Madison, WI). Corresponding horseradish peroxidase-conjugated goat anti-mouse or

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goat anti-rabbit secondary antibodies were visualized by enhanced chemiluminescence (ECL,

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Amersham, Arlington Heights, IL). The bands corresponding to specified proteins were

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quantified by laser densitometry. Samples were normalized to GAPDH antibody to insure equal

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loading.

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Immunochemistry and image analysis. NRVM were washed in Ca2+ and Mg2+ free

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phosphate buffered saline (PBS). Fixation was carried out by submersion in 4%

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paraformaldehyde/PBS (Fisher, Fairlawn, NJ) for 10 minutes at room temperature followed by a

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wash in 70% ethanol for 5 minutes. Cells were stored in 70% ethanol at -20o C until use. 8

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Immunostaining was performed as previously described (using anti-FAK antibody (BD

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Transduction, CA), Alexa-488 phalloidin (Molecular Probes, Carlsbad, CA), phospho-antibody

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anti-FAK pY397 (Biosource, Camarillo, CA), and appropriate fluorescence-labeled secondary

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antibodies. Membranes were then mounted on glass slides with 4’6’ –diamidino-2-phenylindole

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nuclear stain (DAPI; Vector Laboratories, Burlingame, CA) as an antifade agent. Fluorescently

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labeled cells were then viewed using a Zeiss Model LSM 510 laser scanning confocal

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microscope.

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Data analysis. All values are mean ± SEM, with n = 3-4 separate experiments. Data were

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analyzed using one-way ANOVA followed by a Bonferoni Multiple Comparison post hoc test

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using Graph Pad Prizm statistical software (San Diego, CA). Differences among means were

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considered significant at p

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