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Molecular & Cellular Proteomics 7:1007-1018, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Comparative Proteomics Analysis of Vascular Smooth Muscle Cells Incubated with S- and R-Enantiomers of Atenolol Using iTRAQ-coupled Two-dimensional LC-MS/MS^*,S

Jianjun Sui^,, Jianhua Zhang^,¶, Tuan Lin Tan, Chi Bun Ching^|| and Wei Ning Chen^||

From the School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Atenolol is a β₁-selective drug, which exerts greater blocking activity on β₁-adrenoreceptors than on β₂-adrenoreceptors, with the S-enantiomer being more active than R-enantiomer. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using an iTRAQ-coupled two-dimensional LC-MS/MS approach. Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b₅ reductase, and -N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with the S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b₅ reductase in the intracellular anabolic activity was validated by NAD⁺/NADH assay with a higher ratio of NAD⁺/NADH correlating with a higher proportion of NAD⁺. The down-regulation of the calcium-binding proteins was possibly involved in the lower intracellular Ca²⁺ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca²⁺ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modeling. Our preliminary results thus provide molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.

There are three known types of β-adrenoreceptors, designated as β₁, β₂, and β₃. Particularly β₁- and β₂-adrenoreceptors are well known pharmacologically. β₁-Adrenoreceptors are located mainly in the heart and in the kidneys, whereas β₂-adrenoreceptors are located mainly in the lungs, gastrointestinal tract, vasculature, and uterus; however, both receptors appear to be present in these organs (10,11). β-Blockers are a class of drugs that compete with endogenous and exogenous β-adrenergic agonists. They inhibit the normal epinephrine-mediated sympathetic actions and reduce the effect of excitement/physical exertion on heart rate and force of contraction, dilation of blood vessels, and opening of bronchi. Their specific effects depend on their selectivity for β₁-adrenoreceptors or β₂-adrenoreceptors. Atenolol is a so-called β₁-selective drug, which means that it exerts greater blocking activity on β₁-adrenoreceptors than on β₂-adrenoreceptors. It has been shown that in humans a plasma level of 1 µg/ml atenolol is associated with a 30% reduction of exercise-accelerated heart rate and that a clear linear relationship could be obtained between log plasma concentration and percentage of reduction in heart rate (12). Despite the well documented blocking action of catecholamines on β-adrenoreceptors, concerns regarding their potential adverse effects such as β-blockers at usual doses carrying an unacceptable risk of provoking type 2 diabetes (13) and particularly the underlying molecular mechanism remain to be addressed. To date most β-blockers are administered as the racemic mixture, although only the S-enantiomer is active at the receptor. The mechanism of action of β-blockers has been studied at the gene expression level (14). However, little is known about their action at the protein level. It will be of great interest to detect changes in quantitative protein profiles and to infer biological function from the observed patterns. Proteomics analysis has been widely used to establish cellular signaling pathways in response to various external stimuli, including comparing normal and diseased conditions (15–18). Established methods for relative quantitation of proteins involve isotope-coded affinity tag (19) and chemical and enzymatic modifications (20). Recently an MS/MS-based quantitation method (iTRAQ) has been developed (21). The system enables up to four samples to be analyzed within one experiment. They are differentially isotopically labeled such that all derivatized peptides will have an identical mass and LC retention time after tagging. Following CID MS/MS analysis of the precursor ion, the four reporter groups appear as distinct ions (m/z 114–117). The relative concentration of the peptides is derived from the relative intensities of the reporter ions. In this study, we used a fourplex multiplex strategy to simultaneously detect and quantify differences in expression levels of proteins in untreated vascular smooth muscle cells and those incubated with the S- and R-enantiomers of atenolol, respectively, that reflect the pharmacologic action of enantiomers. To identify proteins from a complex mixture, a two-dimensional (2D) application was used. In this approach, a strong cation exchange (SCX) column was used for the first dimension, a reversed-phase column was used for the second, and two identical enrichment columns were used for trapping the peptides.

Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas some metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b₅ reductase, and -N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with S-enantiomer than those incubated with the R-enantiomer. The findings of our investigation of the detected proteins with differential expression levels provide molecular evidence on the anabolic activity and blocking effect induced by atenolol incubation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Cultures—
Vascular smooth muscle cells, A7r5 cells obtained from the American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin). Cells were maintained at 37 °C in an atmosphere of 5% CO₂. All culture media and media supplements were purchased from Invitrogen. After reaching 80% confluence the cells were incubated with the S-enantiomer and the R-enantiomer of atenolol at a concentration of 20 µM, respectively, for 24 h in the absence of serum.

MTT Assay—
Cytotoxic concentrations were determined by the MTT reduction test. After chemical exposure, the medium was removed, and cells were incubated for 3 h with 5 mg/ml MTT dissolved in PBS. MTT was cleared out, and the formazan salts were solubilized in 100 µl of DMSO. Plates were read at 570 nm against a 660-nm reference wavelength on a microplate reader (Benchmark Plus). The cell viability was expressed as a percentage of the corresponding control value.

Cell Lysis, Protein Digestion, and Labeling with iTRAQ Reagents—
Cells were harvested and lysed in 150 µl of 8 M urea, 4% (w/v) CHAPS, and 0.05% SDS (w/v) on ice for 20 min with regular vortexing. Protein was centrifuged at 15,000 x g for 60 min at 4 °C, supernatant was removed, and protein was quantified using the 2-D Quant kit (GE Healthcare). A standard curve was made using BSA as a control. A total of 100 µg of each sample was precipitated by the addition of 4 times the sample volume of cold (–20 °C) acetone to the tube for 2 h and carefully decanting the supernatant. The protein pellets were then dissolved in the solution buffer and denatured, and cysteines were blocked as described in the iTRAQ protocol (Applied Biosystems). Each sample was then digested with 20 µl of 0.25 µg/µl sequencing grade modified trypsin (Promega) solution at 37 °C overnight and labeled with the iTRAQ tags as follows: control A7r5, iTRAQ 114; A7r5 incubated with the S-enantiomer of atenolol, iTRAQ 115; and A7r5 incubated with the R-enantiomer of atenolol, iTRAQ 117. The labeled samples were then pooled before analysis.

On-line 2D Nano-LC-MS/MS Analysis—
The analysis was performed on an Agilent 1200 nanoflow LC system (Agilent Technologies) interfaced with a QSTAR XL mass spectrometer (Applied Biosystems/MDS Sciex). In the first dimension 3 µl of the combined peptide mixture was loaded onto the PolySulfoethyl A SCX column (0.32 x 50 mm, 5 µm) and was eluted stepwise by injecting salt plugs of 10 different molar concentrations of 10, 20, 30, 40, 50, 60, 80, 100, 300, and 500 mM KCl solution. In the second dimension, while the 10-port valve was in position 1 (Fig. 1a), the sequentially eluted peptides from the SCX column were trapped onto the Zorbax 300SB C₁₈ enrichment column 1 (0.3 x 5 mm, 5 µm) and washed isocratically with buffer A (5% acetonitrile and 0.1% formic acid) at 0.005 ml/min for 100 min to remove any excess reagent. Meanwhile the peptides bound on Zorbax 300SB C₁₈ enrichment column 2 during the previous run were eluted with buffer B (0.1% formic acid) and buffer C (a nanoflow gradient of 5–80% acetonitrile + 0.1% formic acid) at a flow rate of 500 nl/min. Further separation was achieved onto the analytical Zorbax 300SB C₁₈ reversed-phase column (75 µm x 50 mm, 3.5 µm). In the next run, while the 10-port valve was switched to position 2 (Fig. 1b), column 1 was switched into the solvent path of the nanopump, and column 2 was used to trap the newly eluted peptides from SCX. Altogether 11 runs were performed to finish one experiment. For MS/MS analysis, survey scans were acquired from m/z 300 to 1500 with up to two precursors selected for MS/MS from m/z 100 to 2000 using dynamic exclusion, and the rolling collision energy was used to promote fragmentation. To gain statistical evidence for differential expression of proteins another two separate experiments were performed as described above.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Flow diagram for on-line nano-2D LC. a, the 10-port valve was in position 1: bypass of SCX column, washing of enrichment column 1, and analysis of peptides from enrichment column 2. b, the 10-port valve was in position 2: bypass of SCX column, washing of enrichment column 2, and analysis of peptides from enrichment column 1.

NAD⁺/NADH Assay—
Each of the three types of A7r5 cells (control cells, the cells incubated with S-enantiomer of atenolol at a concentration of 20 µM, and the cells incubated with R-enantiomer of atenolol at a concentration of 20 µM) were cultured for 24 h to reach nearly 80% confluence in the absence of serum for the NAD⁺/NADH quantification assay (BioVision). Three independent experiments were performed for each type of cells. A total of 4 x 10⁵ cells were used in each assay, and intracellular NAD⁺/NADH was extracted with 400 µl of NAD⁺/NADH extraction buffer by subjecting the cells to two cycles of freeze/thaw (20 min on dry ice followed by 10 min at room temperature). To detect total NAD⁺ and NADH (NADt), 50 µl of each extracted sample was transferred into a 96-well plate in duplicates. The plate was incubated at room temperature for 5 min in the presence of 100 µl of NAD Cycling Mix to allow the conversion of NAD⁺ to NADH. To detect NADH, 200 µl of extracted solution was taken from each sample and heated at 60 °C for 30 min in a heating block. Under these conditions, all NAD⁺ was decomposed, whereas NADH remained intact. Then 50 µl of each NADH sample was taken into a 96-well plate in duplicates. Subsequently 10 µl of NADH developing solution was added into each well. After 40 min in the dark, the plates were read at 450-nm wavelength on a microplate reader (Benchmark Plus). The ratio of NAD⁺/NADH was calculated as follows: (NADt – NADH)/NADH.

Real Time PCR—
Each of the three types of A7r5 cells were cultured for RNA isolation using an RNeasy minikit (Qiagen). Three independent experiments were performed for each type of cells. DNase was used to treat RNA to remove genomic DNA contamination. The iSCRIPT One-step RT-PCR kit (Bio-Rad) was used for the precise real time quantification of RNA targets. SYBR Green was used in real time PCR as a dye to emit the fluorescence signal. Primers that were specific to the following cDNAs that displayed changes in the respective protein level (Table I), calmodulin, protein S100-A11, protein S100-A4, annexin A6, and nestin, are shown in Table I. These primers were designed to yield amplification products within 300 bp in size to reduce nonspecific binding of SYBR Green. Quantification was performed by calculating the fluorescence density during the amplification cycle. The real time PCR was carried out in an IQ5 multicolor real time PCR detection system (Bio-Rad). The cycling program was as follows: the initial cycle with 10 min at 50 °C and 5 min at 95 °C followed by 40 cycles with 10 s at 95 °C and 30 s at 60 °C. The disassociation analysis was routinely carried out by acquiring a fluorescence reading for each 1 °C increase from 55 to 95 °C. Microsoft Excel-formatted data including amplification analysis, experimental report, melting curve analysis, and threshold cycle number were provided automatically by DNA IQ5 optical system software version 2.0 (Bio-Rad). The -fold changes were calculated as shown in the following formula: Sample Ct = Ct_sample – Ct_β-actin; Ct = Sample Ct – control Ct; -Fold of sample versus control = 2^Ct.

Intracellular Ca²⁺ Concentration Measurements—
A Fluo-4 NW Calcium Assay kit (Invitrogen) was used to measure intracellular Ca²⁺ concentration on a fluorometer (Tecan) following the manufacturer's protocols. Briefly each of the three types of A7r5 cells was cultured separately in a 96-well plate. The growth medium was replaced with 100 µl/well Fluo-4 dye solution containing probenecid to prevent extrusion of the dye out of cells. The plate was incubated at 37 °C for 30 min and then at room temperature for an additional 30 min. The assay was done at 494 nm for excitation and at 516 nm for emission.

Immunostaining and Fluorescence Microscopy—
Each of the three types of A7r5 cells were seeded separately on glass coverslips for 24 h, washed with prewarmed PBS, and fixed with 1 ml of 3% paraformaldehyde, PBS followed by another wash with PBS. Cells were then permeabilized with 0.2% Triton X-100, PBS and blocked with 10% FBS, 0.1% Triton-X100, PBS followed by a wash with PBS. To probe the structure of cytoskeleton, the fixed and permeabilized A7r5 cells were incubated with FITC-phalloidin for 1 h. The coverslip was mounted with mounting solution, and the fluorescence imaging of cells stained with actin was performed with a Pascal 5 confocal microscope (Pascal 5, Carl Zeiss). The sample was excited by an argon-ion laser with a wavelength of 488 nm, and the emitted light was detected with a band pass filter of 520 nm.

Data Analysis and Statistics—
False discovery rate and q-value methodology have recently been applied in proteomics studies to estimate an acceptable level of true or false positives within the calls of significance (22,23). In this study Student's t tests were performed if the quantitative information met the criteria for further statistical analysis as described above and was in a minimum of three independent experiments. In addition, the statistical difference for the real time PCR analysis, the quantification of the protein level by Western blot analysis, and the measurement of intracellular Ca²⁺ concentration was also evaluated by Student's t test for paired or unpaired data as appropriate, and p < 0.05 denoted a statistical difference.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
MTT Assay and Concentration of Atenolol—
Drugs have been shown to have an impact on cell growth in vitro as cell death has been observed depending on the concentration of drugs and the time of incubation (24). Disturbances in metabolism and cell signaling as a result of cell death, however, may be different in nature to normal cellular responses to exposure to drugs. In the case of drug treatment, such cell death-related signaling would not be relevant for drugs that were designed and synthesized for non-anticancer applications. As the purpose of this study was to establish the cellular protein profile in response to the non-anticancer drug, it was critical to minimize the cell death associated with the incubation of drugs. To determine the appropriate concentration of atenolol used in our study, A7r5 cells were incubated with an increasing concentration of the respective enantiomer of atenolol, and the viability was examined by the MTT assay. Results shown in Fig. 2 indicated that either of the two enantiomers of atenolol was toxic to the cells with a drastic decrease in cell viability for any concentration higher than 50 µM. In addition, the overall comparison of the MTT values obtained showed that the R-enantiomer of atenolol was somewhat less toxic than the S-enantiomer of atenolol. Our results also indicated that no significant effect on the cell viability was observed for those concentrations below 25 µM. For this study the concentration of 20 µM was chosen in all experiments.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Cell viability of A7r5 cells after 24 h of exposure to R- and S-enantiomer of atenolol, respectively.

View larger version (23K): [in this window] [in a new window]   FIG. 3. A representative MS/MS spectrum showing a peptide, ADQLTEEQIAEFK, from calmodulin. The ion assignments are as follows: control A7r5, iTRAQthis 114; A7r5 incubated with the S-enantiomer of atenolol, iTRAQ 115; and A7r5 incubated with the R-enantiomerof atenolol, iTRAQ 117.

View larger version (27K): [in this window] [in a new window]   FIG. 4. A representative MS/MS spectrum showing a peptide, DLESDIIGDTSGHFQK, from annexin A6. The ion assignments are as follows: control A7r5, iTRAQ 114; A7r5 incubated with the S-enantiomer of atenolol, iTRAQ 115; and A7r5 incubated with the R-enantiomer of atenolol, iTRAQ 117.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Distribution of all proteins identified after iTRAQ labeling and tandem mass spectrometry into different functional categories.

Up-regulation of Metabolic Enzymes—
Anabolism is the set of metabolic pathways that construct molecules from smaller units, which is the opposite of catabolism. One common feature of anabolic metabolic pathways is the oxidation of cofactors such as NADH to NAD⁺. Anabolic enzymes with such a feature in our study included aspartate aminotransferase (mitochondrial), which facilitates the conversion of aspartate and -ketoglutaric acid to oxaloacetate and glutamate (25); glutathione S-transferase P, which catalyzes the conjugation of reduced glutathione via the sulfhydryl group to electrophilic centers on a wide variety of substrates (26) and is considered to contribute to the phase II biotransformation of xenobiotics (27); and NADH-cytochrome b₅ reductase, which functions in the desaturation and elongation of fatty acids (28,29), cholesterol biosynthesis (30), and drug metabolism (31). NADH-cytochrome b₅ reductase is involved in the oxidation of NADH to NAD⁺ in the following catalytic reaction: NADH + 2 ferricytochrome b₅ = NAD⁺ + H⁺ + 2 ferrocytochrome b₅.

To strengthen the involvement of NADH-cytochrome b₅ reductase in cellular response to the individual enantiomers of atenolol, the level of intracellular NAD⁺ and NADH was measured. Results shown in Table III indicated that the ratio of NAD⁺/NADH was significantly higher in cells incubated with the S-enantiomer of atenolol (with an average of 1.248) compared with the level in either control cells (with an average of 0.773) or cells incubated with the R-enantiomer of atenolol (with an average of 0.863). The higher ratio of NAD⁺/NADH would correlate with a higher proportion of NAD⁺, indicative of higher anabolic activity.

To substantiate the down-regulation as quantitated by iTRAQ-coupled 2D LC-MS/MS, the level of two proteins (calmodulin and annexin A6) was examined by Western blot analysis. Similarly to the iTRAQ analysis, results shown in Fig. 6 indicated that the level of the calmodulin and annexin A6 was lower in cells incubated with the S-enantiomer of atenolol compared with the level in the control cells. In addition, the level of annexin A6 showed no statistically significant changes in R-enantiomer of atenolol-incubated cells compared with control cells (p > 0.05), whereas the level of calmodulin in cells incubated with the R-enantiomer of atenolol was found to be significantly different compared with the control cells (p < 0.05). Quantification of the protein level based on Western blot analysis indicated that the reduction in these two proteins was more dramatic than that in iTRAQ analysis (Fig. 6b). One possible explanation for the observed discrepancy between iTRAQ and Western blot analyses could be that it is due to the normalization of total protein content leading to less dramatic values for lower abundance proteins in iTRAQ quantitation.

View larger version (21K): [in this window] [in a new window]   FIG. 6. a, Western blot analysis of protein levels of calmodulin and annexin A6 in S-enantiomer- and R-enantiomer-incubated cells. Lane Control, cells in the absence of atenolol; Lane S, cells incubated with the S-enantiomer of atenolol; Lane R, cells incubated with the R-enantiomer of atenolol. b, quantification of the protein level based on Western blot analysis. The ratio between S-enantiomer-incubated cells and control cells (S:C) as well as between R-enantiomer-incubated cells and control cells (R:C) is shown. The p values indicated statistical significance of the observed differences with p < 0.05 considered as statistically significant and p > 0.05 considered as not statistically significant.

View larger version (49K): [in this window] [in a new window]   FIG. 7. Real time PCR analysis of mRNA levels of genes coding for calcium-binding proteins in S-enantiomer and R-enantiomer of atenolol-incubated cells. The ratio between the S-enantiomer-incubated and control cells (S:C) as well as between the R-enantiomer-incubated and control cells (R:C) is shown. The p values indicated statistical significance of the observed differences with p < 0.05 considered as statistically significant and p > 0.05 considered as not statistically significant.

View larger version (53K): [in this window] [in a new window]   FIG. 8. The immunofluorescence images of control A7r5 cell (A), S-enantiomer of atenolol-incubated A7r5 cells (B), and R-enantiomer of atenolol-incubated A7r5 cells (C). The scale bar represents 20 µm.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One up-regulated metabolic enzyme in Table II is glutathione S-transferase P. The rat glutathione S-transferase P has been found to be dramatically up-regulated in its expression in preneoplastic and neoplastic cells (33,34) and is widely used as a specific marker in the basic analysis of chemical carcinogenesis (35). Another metabolic enzyme associated with lipid and drug metabolism is NADH-cytochrome b₅ reductase. It has been well documented that the membrane-bound cytochrome b₅ is located in the endoplasmic reticulum where it can accept an electron from NADH-cytochrome b₅ reductase. Reduced cytochrome b₅ then provides reducing equivalents for the biosynthesis of selected lipids and drugs (36). We performed the NAD⁺/NADH assay comparing the cellular anabolism in S-enantiomer-incubated cells with that in R-enantiomer-incubated cells and control cells. Interestingly the NAD⁺/NADH ratio in cells incubated with the S-enantiomer of atenolol was about 61% higher than that for control cells, and the ratio in R-enantiomer-treated cells was 12% higher than that for control cells; these results were in line with the protein level for NADH-cytochrome b₅ reductase in individual type of cells (about 65 and 16% higher in S-enantiomer-treated cells and R-enantiomer-treated cells, respectively). The increase in the anabolic activity preceded by higher levels of metabolic enzymes such as NADH-cytochrome b₅ reductase therefore provided molecular evidence on the metabolic effect associated with atenolol treatment.

The Ca²⁺ ion is a highly versatile intracellular signal regulating many different cellular functions, including fertilization, cell cycle, apoptosis, muscle contraction, vision, and memory. In eukaryotic cells, cytoplasmic Ca²⁺ entry and outflow are governed by two sources: intracellular stores such as the endoplasmic reticulum and extracellular Ca²⁺ that enters the cell through various transporters on the plasma membrane (37). The Ca²⁺ flux machinery, consisting of ion channels, pumps, and exchangers, gives rise to highly localized and transient Ca²⁺ signals that are, in turn, transduced by calcium-binding proteins acting on various enzymes and downstream effector proteins. The ubiquitous calcium-binding protein calmodulin plays a crucial role in various cellular signaling cascades through regulation of numerous target proteins in a Ca²⁺-dependent manner. It can activate the Ca²⁺ pump of plasma membranes by interacting with a domain next to its carboxyl terminus (38) and can interact with GRK5, a member of the GRK family that is associated with homologous desensitization of G protein-coupled receptor, to reduce GRK5 binding to the membrane (39). Annexins are a family of proteins that bind in a calcium-dependent manner to phospholipid membranes. It was reported that annexin A6 increased the activity of the sarcoplasmic reticulum Ca²⁺-ATPase (40). S100-A11 is a member of the S100 family of calcium-binding proteins that is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100-A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins such as annexin A6. It was hypothesized that an increase in cytosolic free Ca²⁺ leads to formation of a complex of S100-A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma (41). S100-A4 is another member of the S100 family, which is associated with active stress fibers (probably regulating contraction) and the sarcoplasmic reticulum (regulating Ca²⁺ homeostasis) (42). The down-regulation of the calcium-binding proteins was therefore closely correlated with the decrease in the intracellular Ca²⁺ concentration. The lower intracellular Ca²⁺ concentration in A7r5 cells incubated with S-enantiomer of atenolol would induce less control of actin-myosin-based contraction in A7r5 cells that was mediated by the myosin light chain kinase-calmodulin complex (43). The cells incubated with S-enantiomer of atenolol spread much more (Fig. 8) compared with the other two types of cells, indicative of less contraction and more relaxation.

Nestin is an intermediate filament protein expressed in dividing cells during the early stages of development in the central nervous system and myogenic and other tissues and can play a complex role in regulation of the assembly and disassembly of intermediate filaments together with vimentin or -internexin and in remodeling of the cell (44). Tropomyosin in muscle and non-muscle occurs in tight association with actin filaments, and in skeletal and cardiac muscle tropomyosin plays a central role in regulation of contraction through mediation of the calcium response of the troponin complex to actin filaments (45). However, the physiological function of tropomyosin in smooth muscle cells and non-muscle cells is not fully understood due in part to the absence of troponin. It has been suggested that tropomyosin is involved in stabilization of actin filaments, cytoskeletal modeling, and cell motility (46), and β-tropomyosin plays a significant role in the process of phenotypic modulation of smooth muscle cells (47). Unlike nestin and β-tropomyosin, which were down-regulated in A7r5 cells incubated with atenolol, tubulin -2 chain was up-regulated. Cooper and co-workers (48–50) reported that the isolated myocytes showing contractile dysfunction were accompanied by increased cytoskeletal stiffness characterized by an augmentation of the amount of total tubulin and an elevated degree of polymerization. However, the role of tubulin in the development of hypertrophy and heart failure has been questioned by Collins et al. (51) and Bailey et al. (52).

Transforming protein RhoA is a small GTPase protein that directly stimulates actin polymerization through activation of diaphanous-related formins (DRF proteins). DRF proteins stimulate addition of actin monomers to the fast growing end of actin filaments. DRFs act together with Rho kinases (ROCKs) to mediate Rho-induced stress fiber formation (53). In addition, ROCKs induce actomyosin-based contractility and phosphorylate several proteins involved in regulating myosins and other actin-binding proteins (54). It was reported that inhibition of the RhoA/Rho kinase pathway is effective in reducing pulmonary hypertension (55). Bi et al. (56) reported that the expression level of RhoA plays a crucial role in regulating the contractility of cultured vascular smooth muscle cells. 14-3-3 protein / was up-regulated in S-enantiomer-incubated cells and was found to be significantly up-regulated in breast tumor cells (57).

Collectively, calmodulin plays a crucial role in activating the Ca²⁺ pump of plasma membranes, and the lower calmodulin expression level in A7r5 cells incubated with S-enantiomer of atenolol possibly led to the lower intracellular calcium concentration in those cells compared with the other two types of cells. It can be inferred from the above discussion that the lower calmodulin expression level can increase GRK5 binding to the membrane, resulting in overdesensitization of G protein-coupled receptor. In turn, a relative lower intracellular Ca²⁺ concentration in S-enantiomer-incubated cells would induce less interaction of S100-A11 with annexin A6, resulting in less physical connection between the plasma membrane and the cytoskeleton. The Ca²⁺ signals were transduced by calcium-binding proteins acting on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in regulation of the cytoskeletal modeling and cell contraction in conjunction with S100-A4. In addition a lower RhoA expression level in S-enantiomer-incubated cells might result in less actin polymerization and thus induce less contractility with DRF proteins and ROCKs compared with control cells and R-enantiomer-incubated cells.

In summary, our results demonstrate that the application of quantitative proteomics based on iTRAQ is an effective approach to evaluate cellular changes with atenolol. We were able to identify and quantify some proteins with differential protein expression levels from A7r5 cells incubated with individual enantiomers of atenolol. Our preliminary results indicate significant alterations of proteins that were involved in the intracellular anabolism, Ca²⁺ signal transduction pathway. This provides molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol treatment.

	FOOTNOTES

 
Received, October 9, 2007, and in revised form, January 10, 2008.

Published, MCP Papers in Press, February 11, 2008, DOI 10.1074/mcp.M700485-MCP200

¹ The abbreviations used are: GPR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; 2D, two-dimensional; iTRAQ, isobaric tags for relative and absolute quantification; SCX, strong cation exchange; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DRF, diaphanous-related formin; ROCK, Rho kinase; NADt, total NAD⁺ and NADH.

^* This work was supported in part by Academic Research Funds, Ministry of Education, Singapore (Grant RG44/06 to W. N. C.).

^S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

Both authors contributed equally to this work.

Recipient of a graduate research scholarship from Nanyang Technological University.

^¶ A research assistant in the School of Chemical and Biomedical Engineering, Nanyang Technological University.

^|| To whom correspondence should be addressed. Tel.: 65-63162870; E-mail: WNChen{at}ntu.edu.sg; CBChing{at}ntu.edu.sg

	REFERENCES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Johnson, M. (2006 ) Molecular mechanisms of β2-adrenergic receptor function, response, and regulation. J. Allergy Clin. Immunol. 117, 18 –24[CrossRef][Medline]

2. Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. (1989 ) Identification of two serine residues involved in agonist activation of the β-adrenergic receptor. J. Biol. Chem. 264, 13572 –13578[Abstract/Free Full Text]

3. Hamm, H. E. (1998 ) The many faces of G protein signaling. J. Biol. Chem. 273, 669 –672[Free Full Text]

4. Nishikawa, M., Lanerolle, P., Lincoln, T. M., and Adelstein, R. S. (1984 ) Phosphorylation o f mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase. J. Biol. Chem. 259, 8429 –8436[Abstract/Free Full Text]

5. Somlyo, A. P., and Somlyo, A. V. (1994 ) Signal transduction and regulation in smooth muscle. Nature 372, 231 –236[CrossRef][Medline]

6. Brown, A.M. (1990 ) Regulation of heartbeat by G protein-coupled ion channels. Am. J. Physiol. 259, H1621 –H1628[Medline]

7. Palczewski, K. (1997 ) GTP-binding-protein-coupled receptor kinases. Eur. J. Biochem. 248, 261 –269[Medline]

8. Krupnick, J. G., and Benovic, J. L. (1998 ) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu. Rev. Pharmacol. Toxicol. 38, 289 –319[CrossRef][Medline]

9. De Vries, L., and Farquhar, M. G. (1999 ) RGS proteins: more than just GAPs for heterotrimeric G proteins. Trends Cell Biol. 9, 138 –144[CrossRef][Medline]

10. Minneman, K. P., Hedberg, A., and Molinoff, P. B. (1979 ) Comparison of β-adrenergic receptor subtypes in mammalian tissues. J. Pharmacol. Exp. Ther. 211, 502 –508[Free Full Text]

11. Stiles, G. L., Taylor, S., and Letkowitz, R. J. (1983 ) Human cardiac β-adrenergic receptor subtype heterogeneity delineated by direct radioligand binding. Life Sci. 33, 476 –483

12. Brown, H. C., Carruthers, M. D., Johnston, G. D., Kelly, J. G., McAinsh, J., McDevitt, D. G., and Shanks, R. G. (1976 ) Clinical pharmacologic observations on atenolol, a β-adrenoceptor blocker. Clin. Pharmacol. Ther. 20, 524 –534[Medline]

13. Taylor, E. N., Hu, F. B., and Curhan, G. C. (2006 ) Antihypertensive medications and the risk of incident type 2 diabetes. Diabetes Care 29, 1065 –1070[Abstract/Free Full Text]

14. Herndon, D. N., Dasu, M. R. K., Wolfe, R. R., and Barrow, R. E. (2003 ) Gene expression profiles and protein balance in skeletal muscle of burned children after β-adrenergic blockade. Am. J. Physiol. 285, E783 –E789

15. Shiio, Y., Donohoe, S., Yi, E. C., Goodlett, D. R., Aebersold, R., and Eisenman, R. N. (2002 ) Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21, 5088 –5096[CrossRef][Medline]

16. Han, D. K., Eng, J., Zhou, H., and Aebersold, R. (2001 ) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946 –951[CrossRef][Medline]

17. Griffin, T. J., Gygi, S. P., Ideker, T., Rist, B., Eng, J., Hood, L., and Aebersold, R. (2002 ) Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae. Mol. Cell. Proteomics 1, 323 –333[Abstract/Free Full Text]

18. Tan, T. L., and Chen, W. (2005 ) A proteomics analysis of cellular proteins associated with HBV genotype-specific HBX: potential in identification of early diagnostic markers for HCC. J. Clin. Virol. 33, 293 –298[CrossRef][Medline]

19. Hansen, K. C., Schmitt-Ulms, G., Chalkley, R. J., Hirsch, J., Baldwin, M. A., and Burlingame, A. L. (2003 ) Mass spectrometric analysis of protein mixtures at low levels using cleavable ¹³C-isotope-coded affinity tag and multidimensional chromatography. Mol. Cell. Proteomics 2, 299 –314[Abstract/Free Full Text]

20. Zappacosta, F., and Annan, R. (2004 ) N-terminal isotope tagging strategy for quantitative proteomics: results-driven analysis of protein abundance changes. Anal. Chem. 76, 6618 –6627[Medline]

21. Ross, P., Huang, Y., Marchese, J., Williamson, B., Parker, K., Hattan, S., Khainovski, Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., and Pappin, D. (2004 ) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154 –1169[Abstract/Free Full Text]

22. Karp, N. A., McCormick, P. S., Russell, M. R., and Lilley, K. S. (2007 ) Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in-gel electrophoresis. Mol. Cell. Proteomics 6, 1354 –1364[Abstract/Free Full Text]

23. Benjamini, Y., Drai, D., Elmer, G., Kafkafi, N., and Golani, I. (2001 ) Controlling the false discovery rate in behavior genetics research. Behav. Brain Res. 125, 279 –284[CrossRef][Medline]

24. Witczak, Z. J., Kaplon, P., and Dey, M. (2003 ) Thio sugars. VII. Effect of 3-deoxy-4-S-(β-D-gluco- and β-D-galactopyranosyl)-4-thiodisaccharides and their sulfoxides and sulfones on the viability and growth of selected murine and human tumor cell lines. Carbohydr. Res. 338, 11 –18[CrossRef][Medline]

25. Dennis, S. C., and Clark, J. B. (1977 ) The pathway of glutamate metabolism in rat brain mitochondria. Biochem. J. 168, 521 –527[Medline]

26. Douglas, K. T. (1987 ) Mechanism of action of glutathione-dependent enzymes. Adv. Enzymol. Relat. Areas Mol. Biol. 59, 103 –167[Medline]

27. Oshino, N., Imai, Y., and Sato, R. (1971 ) A function of cytochrome b5 in fatty acid desaturation by rat liver. microsomes. J. Biochem. 69, 155 –167[Abstract/Free Full Text]

28. Keyes, S. R., and Cinti, D. L. (1980 ) Biochemical properties of cytochrome b5-dependent microsomal fatty acid elongation and identification of products. J. Biol. Chem. 255, 1357 –1364

29. Hales, B. F., and Huang, C. (1994 ) Regulation of the Yp subunit of glutathione S-transferase P in rat embryos and yolk sacs during organogenesis. Biochem. Pharmacol. 47, 2029 –2037[CrossRef][Medline]

30. Reddy, V. V. R., Kupfer, D., and Capsi, E. (1977 ) Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes: evidence for the participation of microsomal cytochrome b5. J. Biol. Chem. 252, 2797 –2801[Abstract/Free Full Text]

31. Hildebrandt, A., and Estabrook, R. W. (1971 ) Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Arch. Biochem. Biophys. 143, 66 –79[CrossRef][Medline]

32. Rasanen, J., and Jouppila, P. (1995 ) Uterine and fetal hemodynamics and fetal cardiac function after atenolol and pindolol infusion. Eur. J. Obstet. Gynecol. Reprod. Biol. 62, 195 –201[CrossRef][Medline]

33. Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I., and Sato, K. (1984 ) Purification, induction, and distribution of placental glutathione S-transferase: a new marker enzyme for preneoplastic cells in rat chemical hepatocarcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 85, 3964 –3968

34. Sato, K. (1990 ) Glutathione S-transferases as markers of preneoplasia and neoplasia. Adv. Cancer Res. 52, 205 –255

35. Ito, N., Hasegawa, R., Imaida, K., Hirose, M., and Shirai, T. (1996 ) Medium-term liver and multi-organ carcinogenesis and chemopreventive agents. Exp. Toxicol. Pathol. 48, 113 –119[Medline]

36. Canova, D. E., and Waskell, L. (1984 ) The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5. J. Biol. Chem. 259, 2541 –2546[Abstract/Free Full Text]

37. Bootman, M. D., and Berridge, M. J. (1995 ) The elemental principles of calcium signaling. Cell 83, 675 –678[CrossRef][Medline]

38. Shull, G. E, and Greeb, J. (1988 ) Molecular cloning of two isoforms of the plasma membrane Ca²⁺-transporting ATPase from rat brain. J. Biol. Chem. 263, 8646 –8657[Abstract/Free Full Text]

39. Sallese, M., and Iacovelli, L. (2000 ) Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins. Biochim. Biophys. Acta 1498, 112 –121[Medline]

40. Krasavchenko, K. S., Melgunov, V. I., Akimova, E. I., and Krasheninnikov, I. A. (2006 ) Annexin A6: interaction with sarcoplasmic reticulum proteins. Biol. Membr. 23, 339 –345

41. Chang, N., Sutherland, C., Hesse, E., Winkfein, R., Wiehler, W. B., Pho, M., Veillette, C., Li, S., Wilson, D. P., Kiss, E., and Walsh, M. P. (2007 ) Identification of a novel interaction between the Ca²⁺-binding protein S100A11 and the Ca²⁺- and phospholipid-binding protein annexin A6. Am. J. Physiol. 292, C1417 –C1430[CrossRef]

42. Heizmann, C. W., and Cox, J. A. (1998 ) New perspectives on S100 proteins: a multi-functional Ca²⁺-, Zn²⁺- and Cu²⁺-binding protein family. Biometals 11, 383 –397[CrossRef][Medline]

43. Holzapfel, G., Wehland, J., and Weber, K. (1983 ) Calcium control of actin-myosin based contraction in Triton models of mouse 3T3 fibroblasts is mediated by the myosin light chain kinase (MLCK)-calmodulin complex. Exp. Cell Res. 148, 117 –126[CrossRef][Medline]

44. Michalczyk, K., and Ziman, M. (2005 ) Nestin structure and predicted function in cellular cytoskeletal organization. Histol. Histopathol. 20, 665 –671[Medline]

45. Lees, M., and Helfman, D. (1991 ) The molecular basis for tropomyosin isoform diversity. BioEssays 13, 429 –437[CrossRef][Medline]

46. Lin, J. J., Warren, K. S., Wamboldt, D. D., Wang, T., and Lin, J. L. (1997 ) Tropomyosin isoforms in non-muscle cells. Int. Rev. Cytol. 170, 1 –38[Medline]

47. Girjes, A. A., Keriakous, D., and Cockerill, G. W. (2002 ) Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells. Int. J. Biochem. Cell Biol. 34, 505 –515[CrossRef][Medline]

48. Tsutsui, H., Ishihara, K., and Cooper, G. (1993 ) Cytoskeletal role for contractile dysfunction of hypertrophied myocardium. Science 260, 682 –687[Abstract/Free Full Text]

49. Tsutsui, H., Tagawa, H., and Kent, R. L. (1994 ) Role of microtubules in contractile dysfunction of hypertrophied cardiocytes. Circulation 90, 533 –555[Abstract/Free Full Text]

50. Tagawa, H., Wang, N., Narishige, T., Ingber, D. E., Zile, M. R., and Cooper, G. (1997 ) Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ. Res. 80, 281 –289[Abstract/Free Full Text]

51. Collins, J. F., Pawloski-Dahm, C., and Davie, M. G. (1996 ) The role of the cytoskeleton in left ventricular pressure overload hypertrophy and failure. J. Mol. Cell. Cardiol. 28, 1435 –1443[CrossRef][Medline]

52. Bailey, B. A., Dipla, K., Li, S., and Houser, S. R. (1997 ) Cellular basis of contractile derangements of hypertrophied ventricular myocytes. J. Mol. Cell. Cardiol. 29, 1823 –1835[CrossRef][Medline]

53. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003 ) Cell migration: integrating signals from front to back. Science 302, 1704 –1709[Abstract/Free Full Text]

54. Riento, K., and Ridley, A. J. (2003 ) ROCKs: multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4, 446 –456[CrossRef][Medline]

55. Guilluy, C., Sauzeau, V., Rolli-Derkinderen, M., Guerin, P., Sagan, C., Pacaud, P., and Loirand, G. (2005 ) Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension. Br. J. Pharmacol. 146, 1010 –1018[CrossRef][Medline]

56. Bi, D., Nishimura, J., Niiro, N., Hirano, K., and Kanaide, H. (2005 ) Contractile properties of the cultured vascular smooth muscle cells—the crucial role played by RhoA in the regulation of contractility. Circ. Res. 96, 890 –897[Abstract/Free Full Text]

57. Zang, L., Toy, D. P., Hancock, W. S., Sgroi, D. C., and Karger, B. L. (2004 ) Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and ¹⁶O/¹⁸O isotopic labeling. J. Proteome Res. 3, 604 –612[CrossRef][Medline]

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