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J. Biol. Chem., Vol. 277, Issue 19, 17108-17111, May 10, 2002

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Tyrosine 537 within the Na⁺,K⁺-ATPase -Subunit Is Essential for AP-2 Binding and Clathrin-dependent Endocytosis^*

Stefania Cotta Doné, Ingo B. Leibiger, Riad Efendiev^§, Adrian I. Katz^¶, Barbara Leibiger, Per-Olof Berggren, Carlos H. Pedemonte^§, and Alejandro M. Bertorello

From the  Department of Molecular Medicine, Karolinska Institutet, The Rolf Luft Centrum for Diabetes Research, Karolinska Hospital, 171 76 Stockholm, Sweden, the ^§ College of Pharmacy, University of Houston, Houston, Texas 77204, and the ^¶ Department of Medicine, University of Chicago, Chicago, Illinois 60637

Received for publication, February 8, 2002, and in revised form, February 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In renal epithelial cells endocytosis of Na⁺,K⁺-ATPase molecules is initiated by phosphorylation of its ₁-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na⁺,K⁺-ATPase ₁-subunit. We identified a conserved sequence (Y⁵³⁷LEL) within the ₁-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na⁺,K⁺-ATPase ₁-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent -subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased ₁-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na⁺,K⁺-ATPase activity. Because phorbol esters increase Na⁺,K⁺-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na⁺,K⁺-ATPase endocytosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In renal epithelial cells the Na⁺,K⁺-ATPase is located within the basolateral domain (1) and shuttles between the plasma membrane and intracellular organelles during its regulation by G protein-coupled receptor signals (2-6). In renal epithelial cells, inhibition of Na⁺,K⁺-ATPase activity by DA¹ is mediated by the removal of active molecules from the plasma membrane. This endocytic traffic of Na⁺,K⁺-ATPase molecules to endosomal compartments occurs via a clathrin vesicle-dependent mechanism (2) and requires PKC. PKC -dependent phosphorylation of a serine (Ser¹⁸) residue within the catalytic ₁-subunit (7, 8) does not result in inactivation of the enzyme while in the plasma membrane but is critical for initiating its endocytosis (9). The resulting conformational changes within the N-terminal segment of the ₁-subunit (as a consequence of Ser¹⁸ phosphorylation) favor the activation of class I_A phosphoinositide 3-kinase (PI 3-kinase), possibly by promoting its interaction with a proline-rich motif located upstream of the PKC phosphorylation site (10, 11).

Activation of PI 3-kinase is critical for Na⁺,K⁺-ATPase endocytosis as it favors the binding of AP-2 to the ₁-subunit and thereby promotes clathrin recruitment (11). While phosphorylation of the ₁-subunit is important for activation of PI 3-kinase, AP-2/clathrin recruitment, and endocytosis, the reversal of this effect by other agonists does not appear to be controlled by dephosphorylation of the -subunit (12). In contrast, G protein-coupled receptor signals that oppose Na⁺,K⁺-ATPase endocytosis, such as -adrenergic agonists, do so by increasing the levels of inositol hexakisphosphate, thereby preventing the interaction of AP-2 with the Na⁺,K⁺-ATPase -subunit (12). Thus, regulation of the ₁-subunit-AP-2 interactions appears to be an important on/off mechanism for regulating Na⁺,K⁺-ATPase activity and its availability at the plasma membrane in response to G protein-coupled receptor signals. The present study was therefore performed to identify the Na⁺,K⁺-ATPase ₁-subunit recognition domain(s) that interacts with AP-2.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Antibodies against GFP were purchased from CLONTECH (Palo Alto, CA). Clathrin antibody was from Oxford Biotechnology (Kidlington, UK). AP-2 antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Dopamine was purchased from Solvay Pharmaceuticals GmbH (Hannover, Germany). All other reagents were of highest available grade.

Plasmid Construction-- To obtain plasmid pCMV.GFP-Na⁺,K⁺-ATPase we first introduced a NruI site into the 5'-untranslated region of the -subunit of the rat Na⁺,K⁺-ATPase in pCMVouabain (PharMingen) by site-directed mutagenesis. The GFP0 cDNA, which lacks the stop codon, was obtained from pB.CMV.GFP0 (13) and was inserted in-frame in pCMVouabain-NruI following digestion with NruI and ClaI. Mutants of the un-tagged and the N-terminally GFP-tagged Na⁺,K⁺-ATPase were created by site-directed mutagenesis by exchanging nucleotides as follows: Y50F (TAC versus TTC), Y255F (TAC versus TTC), Y469F (TAC versus TTC), Y537F (TAC versus TTC), Y537A (TAC versus GCC), Y679F (TAC versus TTC), L499A (CTG versus GCT), and L554A (CTT versus GCT). All mutations were introduced by employing the QuikChange Mutagenesis Kit from Stratagene, and oligonucleotides bearing the respective nucleotide exchanges were synthesized at Genset. All constructs were verified by DNA sequence analysis.

Cell Culture and Transfection-- OK cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum, penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively) and 2 mM glutamine in a 5% CO₂ incubator at 37 °C. Cells were transfected with various expression constructs using the LipofectAMINE technique (LipofectAMINE, Invitrogen) (14, 15). Two days after transfection the cells were transferred to a medium containing 5 µM ouabain (Sigma). Because the native Na⁺,K⁺-ATPase is inhibited by this concentration of ouabain, only OK cells expressing the transfected rodent ₁-subunit survive. Therefore, the resistant clones (expressing the transfected rat ₁-subunit mutants) were expanded and grown in DMEM supplemented with 5 µM ouabain. Expression of the native Na⁺,K⁺-ATPase ₁-subunit was negligible (7).

Determination of Na⁺,K⁺-ATPase Activity-- Na⁺,K⁺-ATPase activity was determined from the ouabain-inhibitable ⁸⁶Rb⁺ transport (nmol of Rb/mg of protein/min). To assess the effect of DA, cells were preincubated with 5 µM monensin (Sigma) for 30 min as described by Seri et al. (16) and then with 1 µM DA (5 min) before assay. Measurements of Na⁺,K⁺-ATPase-mediated ⁸⁶Rb⁺ transport were performed as described previously (5, 15).

Preparation of Clathrin Vesicles-- Vesicles were isolated as described previously (2, 17). Briefly, after incubation with 1 µM DA or vehicle (Hanks' medium), OK cells were homogenized using a motor pestle homogenizer (Kimble-Kontes, Vineland, NJ) in 1 mM EGTA, 0.5 mM MgCl₂, 0.1 M Mes, and 0.2 mg/ml NaN₃, titrated to pH 6.5 with NaOH. The homogenate was centrifuged at 85,000 × g for 1 h, and the pellet was resuspended in the same buffer and applied to a discontinuous sucrose gradient (w/v): 60%, 50%, 40%, 10%, and 5%. Samples were then centrifuged at 80,000 × g for 75 min and collected from the 10-40% interface; they were then washed in homogenization buffer and centrifuged at 85,000 × g for 1 h. Wheat germ agglutinin was added to a concentration of 1 mg:10 mg of protein and incubated overnight at 4 °C. The agglutinated material was sedimented at 20,000 × g for 15 min.

Immunoprecipitation-- OK cells were incubated in the presence or absence of 1 µM DA for 2 min at 25 °C. Thereafter, the medium was replaced by immunoprecipitation buffer (in mM: 100 NaCl, 50 Tris-HCl, 2 EGTA, 1 phenylmethylsulfonyl fluoride, 5 mg/ml of protease inhibitors (aprotinin, leupeptin, antipain), 1% Triton X-100 (pH 7.5)), and the samples were transferred to ice. The cells were disrupted by homogenization with a motor pestle homogenizer. Immunoprecipitation of AP-2 was performed as described previously (12). In brief, aliquots (500 µg protein) were incubated overnight at 4 °C with 6 µg of a polyclonal antibody raised against the C subunit of AP-2 and the simultaneous addition of excess protein A-Sepharose beads (Amersham Biosciences, Uppsala, Sweden). Protein content was determined according to Bradford (18). Samples were analyzed by SDS-PAGE using the Laemmli buffer system (19). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA), and Western blots were performed using an antibody against the Na⁺,K⁺-ATPase ₁-subunit (20) and developed with an ECL Plus (Amersham Biosciences, Amersham, UK) detection kit.

Confocal Microscopy-- Monitoring of GFP fluorescence was performed as described previously (21). Briefly, OK cells expressing the GFP-tagged Na⁺,K⁺-ATPase -subunit were grown on 24-mm glass coverslips in supplemented DMEM. For fluorescence imaging, the coverslip was placed in a perfusion chamber and mounted on an inverted fluorescence microscope Leica DMIRB (Leica Lasertechnik GmbH, Heidelberg, Germany). The cells were maintained throughout the experiment in Hanks' medium at 37 °C. The fluorescence of GFP-tagged Na⁺,K⁺-ATPase -subunit was monitored by confocal laser scanning fluorescence microscopy using an argon/krypton laser at 488 nm (Leica TCS NT, Leica Lasertechnik GmbH, Heidelberg, Germany). The following filter settings were used: dichroic mirror TK 500, emission filter BP525/50, and 63× lens (Leica PL APO 63×/1.32-0.6 oil). Images were processed using Adobe Photoshop software.

Statistical Analysis-- Comparison between two experimental groups was made with the nonpaired Student's t test. p < 0.05 was considered significant.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Using site-directed mutagenesis in intact cells we identified a sequence within the Na⁺,K⁺-ATPase -subunit that interacts with AP-2 and demonstrated that this interaction is essential for DA-dependent regulation of Na⁺,K⁺-ATPase activity and endocytosis.

Clathrin-dependent endocytosis of Na⁺,K⁺-ATPase molecules in response to G protein-coupled receptor signals requires the interaction of AP-2 with the Na⁺,K⁺-ATPase ₁-subunit (13, 14). The AP-2 molecules recognize and bind to short consensus motifs present in membrane proteins to be internalized (22) and thereby participate in the recruitment of clathrin to the site of endocytosis. Many AP-2-target interactions involve the AP-2 µ-chain binding to a consensus NPXY or YppØ motif (23), where Y is tyrosine, X is any amino acid, p are preferentially positively charged residues, and Ø is a residue with a bulky hydrophobic chain. Analysis of the Na⁺,K⁺-ATPase ₁-subunit sequence revealed several intracellular sites for possible interaction with AP-2 (Fig. 1, left panel), whereas the -subunit has one consensus motif that is located within the putative transmembrane domain.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Potential endocytic sequences within the Na+,K+-ATPase 1-subunit. Several tyrosine motifs were identified within the N terminus and the cytoplasmic loops between M1-M2 and M4-M5 (left panel). Na+,K+-ATPase activity was determined in OK cells stably expressing the 1-subunit in which either tyrosine residue was replaced by a phenylalanine (right panel). Cells were incubated with 1 µM DA or vehicle (Hanks' medium) for 5 min at 23 °C. Each bar represents the mean + S.E. of six determinations performed in triplicate. *, p < 0.05.

The tyrosine residues identified in all those potential AP-2 binding motifs were initially mutated to phenylalanine. This mutation eliminates only the hydroxyl groups but keeps the aromatic characteristic of the amino acid side chain. OK cell lines stably expressing the Na⁺,K⁺-ATPase bearing either of these mutations were generated, and determination of Na⁺,K⁺-ATPase activity (a reflection of endocytosis, Ref. 9) in response to DA was used as read-out. Whereas nonstimulated Na⁺,K⁺-ATPase activity was similar in all groups (mutants and wild type), only OK cells expressing the Na⁺,K⁺-ATPase-Y537F mutant demonstrated a significant, although not complete, reduction in the inhibitory response to DA (Fig. 1, right panel). We studied this motif further by introducing a different mutation, Tyr⁵³⁷  Ala (Y537A); since alanine is an aliphatic amino acid the mutation, Tyr  Ala should totally eliminate any possible interaction between AP-2 and the amino acid side chain. The experiments were performed in OK cells stably expressing the rat Na⁺,K⁺-ATPase ₁-isoform carrying a GFP tag at the N terminus (-WT). The presence of this tag did not affect the basal nonstimulated Na⁺,K⁺-ATPase activity (Fig. 2A). Furthermore, DA decreased enzyme activity (Fig. 2A), enhanced the interaction of the ₁-subunit with AP-2 (Fig. 2B), and promoted the endocytosis of active molecules in clathrin-coated vesicles (CCV) (Fig. 2C). Introduction of the Y537A mutation (-Y537A) did not affect the level of ₁-subunit expression, as evident from images of the intrinsic GFP fluorescence using confocal microscopy (Fig. 3A) and Western blot analysis using a GFP- or -subunit antibody (Fig. 3B).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of DA on OK cells expressing stably the Na+,K+-ATPase 1-subunit tagged with GFP. A, OK cells (stably expressing the 1-subunit with (open bars) or without (closed bars) GFP) were incubated with 1 µM DA or vehicle (Hanks' medium) for 5 min at 23 °C, and Na+,K+-ATPase activity was determined. Each bar represents the mean + S.E. of four independent determinations performed in triplicate. **, p < 0.01. B, OK cells transfected with the Na+,K+-ATPase bearing GFP in it's -subunit were incubated with DA as indicated in A. Western blot analysis with an antibody against GFP (1:500) was performed in the immunoprecipitated material obtained with an AP-2 antibody. C, Na+,K+-ATPase 1-subunit abundance was determined by Western blot analysis in CCV from OK cells expressing the Na+,K+-ATPase 1-subunit bearing GFP. Before CCV preparation, OK cells were treated with DA as described in the legend to A. The data are representative of four experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effect of DA on OK cells expressing the Na+,K+-ATPase bearing the Y537A mutation in the -subunit. A, confocal images of OK cells transfected with the Na+,K+-ATPase 1-subunit bearing the Y537A mutation (-Y537A) or the wild type (-WT). B, Western blot analysis of cell homogenates (30 µg) performed with a GFP antibody (1:500). C, determination of Na+,K+-ATPase activity (nmol of Rb/mg of protein/min) in OK cells treated with 1 µM DA for 5 min at 23 °C (closed bars) or vehicle (Hanks' medium, open bars). Bars represent the mean + S.E. of four independent experiments performed in triplicate. *, p < 0.05. D, OK cells transfected with the Na+,K+-ATPase -subunit wild type (-WT) or Y537A mutant (-Y537A) were incubated with DA as indicated in C. Western blot analysis was performed with an antibody against the Na+,K+-ATPase 1-subunit in the immunoprecipitated material obtained with an AP-2 antibody (left panel). Western blot was performed with an AP-2 antibody in the immunoprecipitated material with a Na+,K+-ATPase antibody (right panel). E, Na+,K+-ATPase 1-subunit abundance in clathrin vesicles prepared from cells that have been treated with 1 µM DA (5 min at 23 °C) or vehicle (Hanks' medium). Western blots were developed with an antibody against GFP (1:500). Data are representative of five independent experiments.

Cells expressing either the wild type or mutated GFP-tagged Na⁺,K⁺-ATPase isoforms both had comparable catalytic activity, determined as the rate of ouabain-sensitive Rb⁺ transport in intact OK cells (Fig. 3C). However, while DA inhibited Na⁺,K⁺-ATPase in OK cells expressing the wild type ₁-subunit, it failed to induce a significant change in enzyme activity in OK cells expressing the -Y537A mutant (Fig. 3C). This mutation, contrary to -Y537F, completely blocked the inhibitory effect of DA on Na⁺,K⁺-ATPase activity.

Co-immunoprecipitation assays were performed to further establish whether the Y537A mutation has indeed rendered the Na⁺,K⁺-ATPase catalytic ₁-subunit unable to recognize AP-2 molecules, thus leading to deficient endocytosis in response to DA (Fig. 3D). Incubation with DA increased the amount of Na⁺,K⁺-ATPase ₁-subunit immunoprecipitated with an AP-2 antibody in -WT, and, as predicted, this interaction was absent in -Y537A cells (Fig. 3D, left panel). In another set of experiments, using the same strategy, we demonstrated an increase in AP-2 from the immunoprecipitated material with a Na⁺,K⁺-ATPase antibody in DA-treated -WT, but not -Y537A, cells (Fig. 3D, right panel). The sequence identified (Y⁵³⁷LEL) is highly conserved among several species and Na⁺,K⁺-ATPase isoforms. Because the two amino acids adjacent to Tyr are not positively charged residues, the motif YLEL cannot be considered a typical "Tyr-based" consensus sequence. Thus, it is possible that its interaction with AP-2 in intact cells and in response to a physiological agonist (dopamine) may be facilitated by, or involve the presence of, other accessory proteins present in the endocytic machinery. At present it is difficult to establish the AP-2/Na⁺,K⁺-ATPase structural relationship, although some predictions can be made. Whereas the crystal structure of the Na⁺,K⁺-ATPase is still unknown, the structure of skeletal muscle sarcoplasmic reticulum calcium ATPase (SERCA) ₁-subunit (which appears to share many common structural features with the Na⁺,K⁺-ATPase) has recently been determined (24). The amino acids of SERCA corresponding to the Na⁺,K⁺-ATPase sequence Y⁵³⁷LEL are on the exterior of the molecule N-domain, which contains the nucleotide binding site. If the site within the Na⁺,K⁺-ATPase is as exposed as in the SERCA molecule, it may suggests that it is the activation of AP-2 and not the exposure of the "endocytic sequence" that is regulated by the action of dopamine.

Because the decrease in Na⁺,K⁺-ATPase activity elicited by DA is exclusively mediated by internalization of active Na⁺,K⁺-ATPase molecules (9), we further examined whether the absence of Tyr⁵³⁷ within the ₁-subunit resulted in its deficient clathrin-dependent endocytosis. Na⁺,K⁺-ATPase ₁-subunit abundance in clathrin vesicles was determined in vesicles prepared from -WT or -Y537A cells that were previously treated with DA. Characterization of CCV preparations was performed, and the results were similar to the ones previously described in our laboratory (2). The incubation time chosen (2.5 min) reflects the maximal incorporation of Na⁺,K⁺-ATPase molecules in CCV obtained from renal proximal tubule cells incubated with DA (2). DA treatment significantly increased the Na⁺,K⁺-ATPase abundance in -WT, whereas it failed to do so in CCV derived from -Y537A cells (Fig. 3E). Na⁺,K⁺-ATPase immunoreactivity was present in CCV prepared from the -Y537A mutants despite lacking the ability to bind AP-2. This could represent a population of Na⁺,K⁺-ATPase molecules present in CCV that originate from the recruitment pathway (recycling endosomes) in their way to the plasma membrane (25). Because the latter is a process that is mediated by AP-1, its interaction site within the Na⁺,K⁺-ATPase appears not to be shared with the AP-2 binding motif.

In contrast to DA, phorbol esters stimulate Na⁺,K⁺-ATPase activity in OK cells (5, 15), and this effect is mediated by increasing the number of Na⁺,K⁺-ATPase molecules within the plasma membrane (5). Interestingly, a phorbol ester (PMA) also stimulated enzyme activity (nmol of Rb/mg of protein/min) in OK cells bearing the Y537A mutation (-WT, vehicle: 9.9 ± 0.6 versus 1 µM PMA, 16.3 ± 1.2 and -Y537A; vehicle: 9.7 ± 0.7 versus 1 µM PMA, 14.0 ± 0.8, n = 3 in all groups), indicating that the stimulatory mechanisms remained intact and that the Y537A mutation appears to affect specifically the inhibitory response to DA. Additionally, these results suggest that the interaction of AP-1 with the Na⁺,K⁺-ATPase molecule that is needed for its recruitment to the plasma membrane in response to phorbol esters (5) requires a different recognition sequence within the ₁-subunit.

Whereas the AP-2 µ-chain interacts with a NPXY or YppØ motif, the -chain of AP-2 recognizes instead dileucine motifs ()(2-4)xLL, where () is usually a negatively charged residue and x a polar residue (22). Further analysis of the Na⁺,K⁺-ATPase ₁-subunit sequence revealed the presence of two possible interacting dileucine motifs (EPKHLL⁴⁹⁹ and L⁵⁵⁴LLPDE). Na⁺,K⁺-ATPase activity was determined in -WT cells and in cells stably expressing the ₁-subunit carrying a mutation Leu⁴⁹⁹  Ala (-L499A) or Leu⁵⁵⁴  Ala (-L554A). The presence of these mutations within the Na⁺,K⁺-ATPase ₁-subunit neither affected the inhibitory action of dopamine nor the stimulatory effect of phorbol esters (Table I).

View this table: [in this window] [in a new window]   Table I Na,K-ATPase activity (nmol Rb/mg of protein/min) in response to dopamine or PMA in OK cells transfected with the Na,K-ATPase -subunit bearing a mutation within two different dileucine motifs

In summary, the present study demonstrates the Y⁵³⁷LEL motif as the recognition site within the Na⁺,K⁺-ATPase molecule (catalytic -subunit) for its interaction with AP-2, a mandatory link in the signaling cascade that translates G protein-coupled receptor activation into clathrin-dependent endocytosis of Na⁺,K⁺-ATPase molecules. Moreover, our data indicate that this motif is not involved in the stimulation/recruitment of Na⁺,K⁺-ATPase molecules (AP-1-dependent) to the plasma membrane induced by phorbol esters and supports the concept that endocytosis and recruitment are two processes involving separate target motifs within the Na⁺,K⁺-ATPase.

	ACKNOWLEDGEMENTS

We thank M. J. Caplan for the gift of Na⁺,K⁺-ATPase antibody and T. Moede for useful discussions.

	FOOTNOTES

^* This work was supported in part by funds from the Swedish Research Council, the Swedish Heart and Lung Foundation, Åke Wibergs Stiftelse, Karolinska Institutet (to A. M. B.), Novo Nordisk Fond, National Institutes of Health Grant DK 53460, and by American Heart Association (Texas Affiliate) Grant 0050801Y (to C. H. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Medicine, L3, Karolinska Hospital, 171 76 Stockholm, Sweden. Tel.: 46-8-5177-9453; Fax: 46-8-5177-9450; E-mail: Alejandro.Bertorello@molmed.ki.se.

Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M201326200

	ABBREVIATIONS

The abbreviations used are: DA, dopamine; AP-2, adaptor protein-2; PKC, protein kinase C; PI 3-kinase, phosphoinositide 3-kinase; OK, opossum kidney; GFP, green fluorescent protein, CCV, clathrin-coated vesicles; SERCA, skeletal muscle sarcoplasmic reticulum calcium ATPase; Mes, 2-(N-morpholino)ethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; WT, wild type; PMA, phorbol 12-myristate 13-acetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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