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J. Biol. Chem., Vol. 281, Issue 31, 21827-21836, August 4, 2006
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1
From the
Department of Cardiology, Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, SE1 7EH and ¶Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1, Aspenlea Rd., Hammersmith, London, W6 8LH United Kingdom
Received for publication, April 25, 2006 , and in revised form, June 2, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of 
-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Here we investigated the potential impact of this disulfide dimer formation on the function of PKA. PKA has two major forms (type I and type II), both of which exist as a tetramer comprising two catalytic and two regulatory subunits. There are two types of regulatory subunits (RI and RII), the presence of which in the PKA holokinase nominally defines the enzyme as type I or II, respectively. Recent studies have shown that the full dissociation of type I PKA in response to cAMP requires the presence of a substrate (7). This substrate-induced sensitization of type I PKA is not a feature of the type II enzyme (8). The regulatory subunits contain N-terminal sequences that are important for protein kinase A anchor protein (AKAP) binding. AKAPs are a diverse group of proteins that are found next to PKA substrate proteins and, thus, function to target PKA (9). Type I PKA is located in the cytosol, whereas type II is not as a result of being primarily bound (targeted) to AKAP proteins that are associated with various subcellular compartments, including the myofilaments in myocytes.
RI and RII have significant homology, but one notable difference is the presence of a pair of N-terminal cysteine residues in RI. These cysteines have been thought to form constitutively present interprotein disulfides between RI subunits (10), which align anti-parallel to each other and form disulfide bonds linking Cys-17 and -38 (in rat) of different RI molecules (11). However, here we report RI does not, in fact, exist constitutively as a disulfide dimer. The RI disulfide dimer only forms when prooxidizing conditions exist. In cardiac tissue this redox change induces a subcellular translocation and kinase activation, resulting in phosphorylation of multiple PKA substrates, which increases the amplitude of myocyte contraction. The disulfide formation and PKA activation are causatively linked, as a selective inhibitor of this kinase (H89) prevents these changes. This oxidant-induced modification and activation of PKA as well as phosphorylation of established PKA substrates occurs without elevations in cAMP, further supporting a functional consequence of the oxidative structural modification.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Fresh and Cultured Rat Myocyte Preparation—Calcium-tolerant adult rat ventricular myocytes (ARVM) were isolated from male Wistar rats (250–300 g) as before (12) and kept at room temperature in modified Tyrode buffer for 3 h or cultured overnight as described previously (13). Fresh ARVM were treated with H2O2 (1 µM–10 mM) (Sigma) for 5 min. Cells were centrifuged at 500 x g for 30s and then reconstituted in nonreducing SDS sample buffer containing maleimide (100 mM). In a separate experiments ARVM were treated with H2O2 (100 µM) or isoprenaline (100 nM) (Sigma) for 5 min with or without a 5-min pretreatment of H89 (10 µM) (Calbiochem) and again prepared in maleimide sample buffer. In cultured ARVM, after 18 h of incubation H2O2 (1 µM–1 mM) was added to the culture medium for 5 min. The culture medium was then aspirated, and the myocytes were scraped into 100 µl of maleimide sample buffer.
Crystalloid Perfusion of the Rat Heart—Hearts from male Wistar rats were prepared and buffer-perfused as before (3). After 25 min of aerobic perfusion, hearts were perfused at a constant flow with Krebs-Henseleit bicarbonate buffer containing H2O2 (1 µM–10 mM) or isoprenaline (100 nM) for 5 min or with H2O2 (100 µM) for 1–10 min. Ventricles were snap-frozen and homogenized (10 ml buffer/g tissue) on ice in 100 mM Tris-HCl, pH 7.2, plus maleimide (100 mM) and protease inhibitors (Complete C, Roche Applied Science) using a Polytron tissue grinder. An unfractionated aliquot was reconstituted in non-reducing maleimide SDS sample buffer. For subcellular fractions, the 10% ventricle homogenate was centrifuged at 21,000 x g for 5 min, and the supernatant was designated as cytosol. The pellet was resuspended in homogenization buffer containing 1% Triton X-100 and centrifuged as before, with the new supernatant enriched in membrane proteins and the pellet in myofilament and nuclear proteins.
Immunoblotting—Non-reducing SDS-polyacrylamide gel electrophoresis was carried out using the Bio-Rad mini protean III system. After electrophoresis samples were transferred to polyvinylidene difluoride using a Bio-Rad semidry blotter.
Antibodies Used in This Work—Primary antibodies used in these studies included PKA RI subunit (Calbiochem and Santa Cruz, Heidelberg, Germany), PKA-catalytic subunit (Transduction Laboratories, Oxford, UK), phospho-(Ser/Thr) PKA substrate (New England Biolabs, Hitchin, UK), phospho-phospholamban (Badrilla, Leeds, UK), phospho-troponin I (New England Biolabs, Hitchin, UK), and -MyHC (Santa Cruz). Horseradish peroxidase-linked secondary antibody and ECL reagent (both Amersham Biosciences) were used to visualize the proteins.
cAMP Assay—cAMP was measured in cells treated with H2O2 or isoprenaline using a Bridge-It cAMP designer fluorescence assay kit according to the manufacturer's guidelines (Mediomics). Myocyte aliquots were treated with H2O2 (1–1000 µM), isoprenaline (1–1000 nM), or control (modified Tyrode) for 5 min at room temperature. Cells were pelleted at 12,000 x g for 5 min, and the supernatant was discarded. 100 µl of "designer assay solution" was added to the pellet and vortexed. 90 µl of each sample was transferred to a 96-well black polypropylene microplate, covered, and incubated at room temperature for 30 min. The fluorescence intensity was read with a SpectraMax Gemini XPS fluorescence plate reader (Molecular Devices, Wokingham, UK) (excitation 
485 nm; emission 530 nm).
Gel Filtration—Ventricular tissue was homogenized as described above but without maleimide. 0.9 ml of the homogenate was supplemented with 0.1 ml of Triton X-100 (10% v/v), vortexed, and centrifuged at 21,000 x g for 5 min. A soluble fraction was separated on a 24 ml of Superose 12 column (Amersham Biosciences) using a Bio-Rad chromatograph collecting 0.75-ml fractions. Fractions were reconstituted in SDS sample buffer and analyzed for the distribution of the catalytic unit of PKA by immunoblotting.
cAMP-Agarose Affinity Capture and Protein Band Identifications—1 ml of Triton-soluble heart extract was prepared as above with maleimide buffer and diluted 10-fold with phosphate-buffered saline plus 1% Triton X-100 (PBS-T), supplemented with protease inhibitors, and rotated overnight at 4 °C with 100 µl of cAMP-agarose (A-7396, Sigma). The affinity matrix was spin-washed 5 times with 10 ml of PBS-T and reconstituted in nonreducing SDS sample buffer and resolved by SDS-PAGE. Unknown protein bands were identified as described previously (6, 14) by analysis of tryptic digests using a quadrupole-time of flight hybrid quadrupole/orthogonal acceleration time of flight spectrometer (Micromass, Manchester, UK) interfaced to a Micromass CapLC capillary chromatograph.
Immunoprecipitation—0.5 ml of Triton-soluble extracts were prepared as above and diluted 10-fold with PBS-T and additional protease inhibitors. This was rotated for 5 h at 4 °C with 5 µg of antibody to -myosin heavy chain (-MyHC) before rotation at 4 °C for 2 h with of 50 µl of protein G-Sepharose (Amersham Biosciences). After spin-washing (4x with PBS-T) the beads were reconstituted in SDS sample buffer and analyzed for the presence of -MyHC and PKA by immunoblotting.
PKA-Myofilament/Membrane Binding Assays—Ventricular tissue from aerobically perfused hearts was homogenized as above. The myofilaments and membranes from a 1-ml aliquot were pelleted by centrifugation (21,000 x g for 5 min) and spin-washed twice in PBS by resuspension and repelleting. The pellet was resuspended into 1 ml of PBS with protease inhibitors, and then 0.2-ml aliquots of this suspension was mixed with 0.8 ml of PBS containing 1 mg of type I PKA holoenzyme (Biaffin, Kassel, Germany) with or without 100 mM dithiothreitol. The RI subunit of the kinase was supplied in the oxidized interprotein disulfide state, and 100 mM treatment efficiently reduced it to the free thiol state. After rotation at 4 °C for 2 h, the myofilaments and membrane fraction were repelleted, and the soluble fraction was reconstituted in SDS sample buffer. The pellet was then resuspended in PBS-T and centrifuged at 21,000 x g for 5 min to pellet the myofilaments but leave the Triton-soluble membranes in the supernatant. Both of these fractions were mixed with SDS sample buffer, and the relative distribution of the added recombinant PKA was determined using an RI subunit antibody (Santa Cruz).
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Immunofluorescence and Confocal Microscopic Imaging—ARVM were cultured and prepared for immunofluorescence studies essentially as described previously (15). Briefly, ARVM were cultured overnight on sterile laminin-coated glass coverslips and exposed to H2O2 (100 µM) or modified Tyrode for 5 min. The myocytes were washed with PBS and fixed with 4% paraformaldehyde, PBS for 10 min. After washing cells were permeabilized with 0.1% Triton X-100, PBS for 20 min. ARVM were again washed and blocked with 1% bovine serum albumin, PBS for 60 min. After removal of blocking buffer, ARVM were then incubated with 1% bovine serum albumin, PBS containing PKA RI antibody (1:100) for 60 min. ARVM were then washed twice and incubated in the dark with the secondary antibody Alexa Fluor 488 (goat anti-rabbit, 1:100) to detect cellular PKA RI. ARVM were then washed twice, and the coverslips were mounted on slides with fluorescent mounting medium (Dako Cytomation, Denmark). The slides were viewed on an inverted laser scanning microscope (LSM510, Carl Zeiss Inc) equipped with a 40x/1.3 NA Plan-Neofluar oil immersion objective lens (Carl Zeiss, Inc.), and images were acquired and processed using LSM510 software (Version 2.01).
Statistical Analysis—Differences between groups were assessed using analysis of variance with further analysis using Dunnett's test or the Student-Newman-Keuls test (for multiple comparisons). Paired t tests were used to analyze amplitude data from myocyte contractility studies. p < 0.05 was considered significant. Data points shown are the means ± S.E.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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cAMP Concentration Does Not Change after H2O2 Treatment—Treatment of ARVM with isoprenaline caused a 2.3-fold change in fluorescence due to increased cAMP concentration (see Fig. 3). In comparison, H2O2 treatment did not cause any alterations in fluorescence/cAMP in these cells.
PKA RI Translocation—Fig. 4a shows PKA RI subcellular distribution in the isolated heart after treatment with 100 µM H2O2 for 1–10 min. PKA RI is a cytosolic monomer that undergoes time-dependent dimerization during H2O2 treatment. The RI dimer translocates to the myofilament compartment, with some evidence on long Western blot exposures of a small increase in the membrane fraction also. This RI translocation was also seen in ARVM after H2O2 treatment without any translocation of PKA RII subunit (not shown). Isoprenaline treatment did not induce disulfide formation or subcellular translocation. This translocation was confirmed in immunofluorescence confocal imaging studies shown in Fig. 4b. There is a clear translocation of PKA RI to the nucleus of myocytes treated with H2O2. This increased nuclear localization was also accompanied by a less striking increase in myofibrillar localization as evidenced by an enhancement in the striated pattern of the H2O2-treated cells. However, although the nuclear translocation is very pronounced and robust, the enhanced filament staining is a more subjective interpretation and is a common issue encountered when interpreting immunofluorescence micrographs of adult ventricular myocytes. We examined this increased affinity of the RI disulfide dimer for the myofilament/nuclear fraction in in vitro reconstitution studies. Fig. 4c shows PKA RI in the oxidized disulfide, but not the reduced thiol, state has affinity with the myofilament/nuclear fraction. This demonstrates the importance of disulfide formation for subcellular relocalization during H2O2 treatment.
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Immunoblot analysis of the regulatory subunits in these samples was also undertaken. However, because these subunits remain as dimeric complexes regardless of oxidation state, the molecular mass only changes from 200 to 100 kDa during kinase activation and dissociation. The Superose 12 column is unable to resolve adequately over this range, and so, as expected, there is little variation in the distribution of the regulatory subunit between treatment groups (not shown).
cAMP-agarose Affinity Capture and Co-immunoprecipitation Studies—We investigated the possibility that formation of the PKA RI disulfide dimer leads to increased affinity with a substrate or associated binding protein, acting as a targeting event that co-localizes kinase and substrate. Such an event would also account for the H2O2-mediated translocation. Consequently, we used cAMP-agarose to affinity-purify the regulatory subunit of PKA along with any proteins that are differentially associated after H2O2 treatment. Fig. 6a shows a Western blot of the affinity-purified preparations probed with a PKA RI antibody, demonstrating binding of both the reduced form and oxidized disulfide form of RI. Fig. 6b shows a Coomassie-stained gel of cAMP-agarose affinity preparations, demonstrating increasing amounts of a high molecular weight protein as the H2O2 dose increased. This protein was identified as -MyHC in four separate experiments and, as discussed below, highlights a novel role for this protein as an AKAP. In reciprocal experiments we immunoprecipitated -MyHC to further test its interaction with RI as it dimerizes (Fig. 6c). Again, we observed an increased association of -MyHC with RI in ARVM in a H2O2 dose-dependent manner. Similar results were also obtained with immunoprecipitations from isolated heart preparation (not shown).
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-adrenergic stimulation, which in the heart includes increased force of contraction. This occurs via phosphorylation of the same proteins we have shown here, which were phosphorylated by PKA in response to H2O2. We, therefore, hypothesized that H2O2 would increase contractility of cardiac myocytes. Fig. 7 shows the results for the amplitude of contraction in the ARVM treated with H2O2 with or without a pretreatment with H89. H2O2 (100 µM) significantly increased the amplitude of contraction (0.204 µm) compared with control myocytes (0.111 µm). This doubling of contraction amplitude was sensitive to H89 (1 µM), indicating the H2O2-induced changes in contraction are also PKA-dependent. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We previously observed that PKA RI exists in the non-disulfide state in control tissue, only forming the disulfide dimer in response to pro-oxidants (6). We could make these observations as our samples were prepared with maleimide, which alkylates the thiol group and prevents subsequent oxidative disulfide formation. The N terminus of the RI molecule where the dimer interaction occurs contains the cysteine residues (Cys-17 and -38 in rat) that form disulfides. This N-terminal dimerization event is at the opposite end from the cAMP binding sites, which are involved in holoenzyme dissociation and activation. Our studies with cAMP-agarose (Fig. 6) show that both the reduced and oxidized disulfide forms of the RI subunit bind cAMP.
The oxidative disulfide formation in RI is associated with subcellular translocation of type I PKA from the cytosol to the nuclear and myofilament compartment and to a lesser extent, a membrane fraction as demonstrated by immunoblotting of subcellular fractions of heart tissue and immunofluorescence confocal imaging of intact myocytes (Fig. 4). In this connection it is of note that Boeshans et al. (16) demonstrated that a primary difference between bovine cardiac cytosolic RI and that associated with the membrane was that the latter "had a higher extent of disulfide bond formation in the N-terminal dimerization domain," although they concluded, "This was not likely the cause for membrane localization." However, our observations suggest that the presence of the disulfide is important for subcellular localization. This redistribution is accompanied by phosphorylation of established PKA substrate proteins in these fractions, including troponin I, myosin-binding protein C, and phospholamban. These phosphorylation events are sensitive to the inhibitor H89, demonstrating phosphorylation is likely mediated by PKA. Other kinases will likely be activated by peroxide, but these would have to also phosphorylate established PKA substrates and at the same time be sensitive to H89. It is also notable that the profile of phosphorylation detected using the pan-specific PKA-substrate antibody is very similar regardless of H2O2 or isoprenaline treatment, although the latter intervention gave stronger signals, perhaps explained by simultaneous activation of type II PKA. It is possible that future mutagenesis studies involving the replacement of either or both of the redox-active cysteines in RI would help to unequivocally link disulfide formation to kinase activation. However, how such mutants will behave is difficult to anticipate with certainty. It remains possible that amino acid substitutions could alter the RI dimer interaction, possibly constitutively activating the kinase if the RI interaction was fully disrupted. Also, because this is the AKAP binding domain, cysteine replacement mutants may have altered interactions with these targeting proteins, adding further complexity to the study of oxidant-induced activation involving disulfide formation.
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-stimulation. However, there was no elevation in cAMP after H2O2 treatment (Fig. 3), consistent with studies demonstrating inhibition of adenylate cyclase by oxidants (17, 18). So disulfide formation activates type I PKA without an elevation in cAMP, and this is associated with subcellular relocalization to compartments containing PKA substrates. To obtain another index of the activity status of PKA after H2O2 intervention, we assessed the molecular complex size of the enzyme by gel filtration chromatography. Our interpretation of these results is that under basal conditions the catalytic subunit is part of an intact tetrameric complex (containing two catalytic and two regulatory subunits) and, therefore, elutes in early fractions. After H2O2 treatment the catalytic subunit is released from the complex and is free to elute in its monomeric state in later fractions. The decrease in PKA complex size, highlighting the dissociation of the catalytic subunit from the holoenzyme complex, provided additional evidence of kinase activation after H2O2 treatment. We can explain these observations regarding oxidant-induced activation of this kinase in light of recent studies demonstrating that type I PKA, in contrast to type II, is activated by substrate-induced sensitization to cAMP (7, 8). Also, type I is generally accepted to activate at lower cAMP concentration than type II. Thus, the translocation of type I PKA during oxidative stress is crucially important for its activation, because it brings the kinase close to its physiological substrates. We reasoned disulfide formation in type I PKA must increase the affinity of the kinase with its substrates or with an associated protein that enables localization with substrate. AKAPs are a diverse array of proteins that allow molecular targeting of PKA to its multiple and variously distributed substrates (9). Type I PKA is generally cytosolic and not constitutively associated with AKAP proteins, unlike the type II isoform that is pretargeted to AKAP-substrate complexes. To investigate the possibility that the redox status of type I PKA regulates its affinity with substrate complexes, we compared the ability of type I PKA in the reduced or disulfide state to bind cardiac myofilament/membrane preparations. These studies verified that the oxidized, but not the reduced, form of type I PKA binds to the myofilament compartment (Fig. 4c). This demonstrates that disulfide formation in the RI subunit operates as a redox switch controlling distribution in both this in vitro binding assay and in cells. To further understand this targeting event, we undertook affinity purifications of the regulatory subunit of PKA from control- or H2O2-treated preparations using cAMP-agarose, which we knew bound reduced and oxidized disulfide forms of RI. We looked for proteins unique to preparations from tissue treated with H2O2, as any such proteins might represent an AKAP that binds the disulfide form of RI to effect subcellular translocation. This was considered a possibility because the regulatory subunits of PKA contain AKAP binding sequences, which are in proximity to the redox active cysteines.
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-MyHC as a protein that behaved in this way. Although this protein is not an established AKAP, our identification of it interacting with PKA would highlight this function. -MyHC has a well defined role in cardiac muscle contractility, but its function as an AKAP is rational as it would localize PKA close to its myofilament substrates troponin I and myosin-binding protein C. Indeed, these proteins were phosphorylated after H2O2 treatment (Fig. 2) at the same time as the RI disulfide dimer forms and translocates to the myofilaments by, probably, -MyHC. Recent studies have demonstrated that several myosins function as AKAPs (19, 20). The rod domain of MyHC contains an amphipathic -helix stabilized in a coiled-coil configuration. This helix could function like amphipathic helices found in AKAPS and serve to localize PKA, although in this case it would be dependent on RI being in an oxidized disulfide state. Further evidence for -MyHC functioning as an AKAP that localizes oxidized PKA comes from immunoprecipitation studies showing that RI co-purified with -MyHC progressively more as the dose of H2O2 was increased in cells (Fig. 6). It is also notable that -MyHC has been identified as a protein kinase G-anchoring protein (21), providing further support for this interaction as a targeting mechanism.
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-adrenergic stimulation is mediated by cAMP-dependent phosphorylation of substrates by PKA, including those proteins we showed were phosphorylated after H2O2 treatment. Consequently, one of the predicted consequences of H2O2 treatment would, therefore, be increased myocyte contractility, which was what we observed. This contrasts other work demonstrating negative inotropic responses with H2O2 treatment (22, 23), although in some studies initial increases in contractility were observed (24). However, many of these studies showing H2O2-induced loss of contractility were carried with higher concentrations or for extended treatment times. Furthermore, these studies had the underlying hypothesis that oxidants adversely affect cardiac function, perhaps leading to the use of H2O2 at concentrations that produce the anticipated detrimental effects. However, in one study, using lower concentrations similar to those used here, H2O2 also substantially enhanced contractility (25). Although we were not able to measure phosphorylation or activation of the L-type calcium channel in these studies, previous work demonstrated its activation in cardiac myocytes after H2O2 treatment (26). This would lead to increased intracellular calcium that would further contribute to the inotropic effect of H2O2 treatment. The catalytic subunit of PKA is also susceptible to cysteine oxidation and is inhibited by S-thiolation, which is enhanced when the kinase is activated (27, 28). We have not assessed catalytic subunit S-thiolation here, but we did not detect any shift in the migration of the catalytic subunit in any of our H2O2-treated samples. This is in contrast to the observations of Humphries et al. (27), who found oxidative stress was associated with both interprotein disulfide bond formation (large gel shift to a higher mass) as well as intramolecular disulfide formation, which caused a faster migrating species and a small shift down the gel. We have also previously screened for proteins susceptible to cysteine-targeted oxidation including S-thiolation (3–6, 14) and found no evidence of catalytic subunit modification, although we could have missed it due to low abundance. However, it is clear from the studies here that in cardiac cells and tissues exposure to H2O2 activates type I PKA.
Our novel observations may help shed some light on the teleological purpose of two isoforms of PKA. It is possible that type I PKA activity is primarily coupled to the cellular redox state and is activated by H2O2, whereas the role of type II is the classical activation in response to cAMP via -adrenergic stimulation. This work also highlights how type I PKA, which is predominantly cytosolic under basal conditions, is able to phosphorylate its substrates located elsewhere in the cell. Activation of PKA independently of increases in cAMP has important implications for our view of -receptor signaling. Clearly, many cellular events can lead to increases in cellular oxidants such as H2O2 (1, 2). The oxidant load of cells not only increases during times of increased metabolic activity but via many neurohormonal pathways that couple to phosphorylation-dependent oxidase activation (29, 30). As highlighted in Fig. 8, this oxidant-induced activation of PKA via RI interprotein disulfide formation provides a mechanism whereby the redox status of cells can integrate into control mechanisms involving phosphoregulation. Many disease states are characterized by oxidative stress, and one consequence of our observations is that such tissues might have abnormal or chronic stimulation of type I PKA, a possibility that warrants further attention.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Cardiology, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK. Tel.: 44-20-7188-0969; Fax: 44-20-7188-0970; E-mail: philip.eaton{at}kcl.ac.uk.
2 The abbreviations used are: PKA, protein kinase A; AKAP, protein kinase A anchor protein; ARVM, adult rat ventricular myocytes; PBS-T, phosphate-buffered saline plus 1% Triton X-100; -MyHC, -myosin heavy chain.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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