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Pregnancy |
Department of Biochemistry and Molecular Biology3
Department of Pediatrics,4 University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, oxytocin, pregnancy, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transient receptor potential (Trp) proteins constitute a large family of plasma membrane cation channels and are classified into three main subfamilies (TrpC, TrpV, and TrpM) on the basis of sequence homology [2, 3, 5, 6]. The TrpC proteins (TrpCs 1–7) implicated in capacitative Ca2+ entry are cation channels expressed in excitable and nonexcitable cells [1, 8, 9]. Some TrpC channels are also activated by diacylglycerol [7, 10, 11]. The TrpC channels have been implicated in cell growth, differentiation and modulation of membrane potential, and pacemaker activity [6, 12, 13]. The TrpC channels are postulated to have different properties in different cells, depending on whether they are comprised of homo- or heterotetramers of specific TrpC proteins, many of which can interact with other proteins [14].
Whereas capacitative Ca2+ entry and expression of mRNA for TrpCs 1, 3, 4, 6, and 7 have been reported in human myometrial cells [15–17], the consequences of this expression for myometrial Ca2+ dynamics have not been investigated in detail. In the present study, we examined properties of endogenous capacitative Ca2+ entry in myometrial cells and determined the effect of hTrpC3 overexpression on Ca2+ entry. We found that myometrial cells allowed entry of a number of divalent cations, at least one without the capacitative stimulus, and that they also respond to diacylglycerol. Moreover, overexpression of hTrpC3 enhanced the responses of the cells to all of these stimuli.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fura-2/acetoxymethylester (Fura-2-AM) and Pluronic F127 were obtained from Molecular Probes (Eugene, OR). SKF 96365 (1-{ß-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride), U73122, and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were obtained from Calbiochem (San Diego, CA), and thapsigargin, poly-lysine, oxytocin, and other compounds were obtained from Sigma (St. Louis, MO). Cell-culture medium and other reagents were obtained from Gibco BRL (Gaithersburg, MD). Matrigel was obtained from Collaborative Research, Inc. (Bedford, MA).
Cell Culture
PHM1 cells were derived from immortalized, late-term pregnant human myometrial cells [18]. Two sublines used in the present study were derived from the initial immortalization culture and behave similarly in all respects tested in the laboratory. PHM1-41 cells were used for all studies except those involving adenoviral infection, in which PHM1-31 cells were used. Cells were cultured in 100-mm dishes in Dulbecco minimal essential medium-high glucose (DMEM) with 10% (v/v) fetal calf serum (FCS), 50 U/ml of penicillin, 50 µg/ml of streptomycin, 0.1 mg/ml of G418 sulfate (Gibco BRL), and 2 mM L-glutamine (Sigma) and were used between passages 20 and 26. For Ca2+ experiments, cells were plated in 200-µl culture medium onto Matrigel-coated glass inserts in 35-mm MatTek dishes (MatTek, Ashland, MA). After 2 h, 2 ml of culture medium were added to the dishes. Alternatively, cells were plated on the dishes at 1 x 105 cells/ml in 1 ml of culture medium. Cells were used after 48–72 h.
Primary nonpregnant human smooth myocytes (Clonetics, East Rutherford, NJ) were cultured in SmGM-2 modified MCDB 131 medium as recommended by the supplier and were used at passages 5–6.
HEK293 cells infected with recombinant adenovirus expressing hTrpC3 were cultured in DMEM with 10% FCS, 50 U/ml of penicillin, 50 µg/ml of streptomycin, and 4 mM L-glutamine.
Recombinant Adenovirus Construction and Adenoviral Infection
Recombinant adenovirus expressing hTrpC3 was constructed using published methods [19]. Full-length hTrpC3 cDNA was obtained by reverse transcription-polymerase chain reaction from PHM1-41 mRNA, and identity to the published sequence was verified by direct sequencing. The hTrpC3 cDNA was digested with XbaI/KpnI and subcloned into the shuttle vector pAdTrack-CMV [19] containing a second promoter driving expression of an enhanced green fluorescent protein (eGFP) marker. The eGFP marker allowed identification of infected cells. The resultant plasmid was linearized with PmeI and subsequently transfected into Escherichi coli BJ5183 cells together with the adenoviral backbone plasmid pAdeasy-1 [19]. Recombinants were selected for kanamycin resistance, and recombination was confirmed by restriction endonuclease analysis. The recombinant plasmid was linearized with PacI and transfected into HEK293 cells to allow viral synthesis and packaging. Recombinant adenovirus was typically generated within 7–12 days after transfection. The HEK293 cells were subjected to freeze/thaw cycles and centrifuged at 500 x g, and the supernatant was used to prepare high-titer adenovirus stocks in 90% confluent HEK293 cells. Viruses were titered by limiting dilution; the titer of hTrpC3 recombinant adenovirus used in the present study was approximately 2 x 108 plaque-forming units (PFU)/ml. For Ca2+ experiments, a mixture of PHM1 cells (1 x 105) and hTrpC3 recombinant adenovirus (100 PFU/cell) were plated in 200 µl of culture medium onto Matrigel-coated glass inserts in 35-mm MatTek dishes. After 2 h, 2 ml of culture medium were added to the dishes. Cells were used within 48–72 h after infection.
Western Blot Analysis
PHM1 cells (2 x 106/10 ml) were plated in 100-mm dishes in culture medium and infected the following day at approximately 70% confluency with recombinant adenovirus at a multiplicity of infection (MOI) of 100 PFU/cell. Cells were harvested after 72 h, and crude cell membranes were prepared as described previously [17]. Western blot analysis was performed as described previously [17].
Measurement of Intracellular Calcium
Cells were loaded at room temperature for 30–35 min with Fura-2-AM (5 µM) in fluorescence buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5 mM MgCl2, 1 mM CaCl2, 10 mM Hepes, and 5 mM glucose; pH 7.4). After loading, the cells were washed twice in the same buffer and used after 30–45 min. Cells were perfused at 1 ml/min with fluorescence buffer as indicated. The Ca2+-free buffer included 100 µM EGTA, except in experiments using GdCl3, in which nominally Ca2+-free buffer was used. Primary myometrial cells were loaded similarly, except that 0.1% Pluronic F-127 was included in the buffer. Changes in intracellular free Ca2+ concentration ([Ca2+]i) in individual cells were measured at 340- and 380-nm excitation and 510-nm emission wavelengths in an InCyt2 imaging system (Intracellular Imaging, Inc., Cincinnati, OH). The same protocol was used for measurement of Sr2+ and Ba2+ entry; influx of these divalent cations is expressed as changes in the 340/380-nm fluorescence ratio, without estimation of their intracellular concentration [20]. Cells expressing eGFP were identified using an eGFP/fluorescein excitation filter (485 nm). We have previously determined that eGFP expression does not interfere with Fura-2-AM measurements (unpublished data).
When responses were similar between cells, the individual responses of 20–40 cells in one dish were averaged as indicated and the data expressed as the mean ± SEM of these average values for n dishes. Statistical analysis was performed by Student t-test, and significance was taken as a value of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Capacitative calcium entry exhibits different properties in different cell types. To characterize the endogenous activity in PHM1 cells, the cells were subjected to a perfusion protocol that allowed each cell to serve as its own control [17]. Basal intracellular calcium [Ca2+]i in PHM1 cells was 110 ± 7 nM (n = 21) in Ca2+-free medium and was not increased significantly by readdition of 1 mM extracellular Ca2+ (Fig. 1, A and B). Consistent with previous observations [17], thapsigargin (100 nM), an inhibitor of the endoplasmic reticulum Ca2+ ATPase (Fig. 1A), and oxytocin (100 nM), a stimulator of IP3 production (Fig. 1B), elicited [Ca2+]i, transients in the absence of extracellular Ca2+. Subsequent addition of 1 mM extracellular Ca2+ provoked a second rise in [Ca2+]i. The Ca2+ entry was seen on addition of extracellular Ca2+ following stimulation by lower concentrations of oxytocin (10–50 nM) as well but, on average, was smaller in magnitude (data not shown).
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SKF 96365 and gadolinium (Gd3+) were used to inhibit capacitative Ca2+ entry. SKF 96365, an imidazole derivative not without effects on L-type Ca2+ channels, has shown some selectivity in blocking receptor-mediated Ca2+ entry [21, 22]. The Gd3+ is a commonly used blocker of nonselective cation channels [23]. Relatively low concentrations of SKF 96365 (0.1–0.5 µM) and Gd3+ (10 µM) attenuated the second [Ca2+]i transient induced by thapsigargin and by oxytocin (Fig. 1, C and D). In addition, the phospholipase C-inhibitor U73122 (2 µM) completely blocked oxytocin stimulation of both extracellular Ca2+-independent and Ca2+-dependent increases in [Ca2+]i (Fig. 2).
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The permeability of PHM1 cells to other divalent cations under these conditions was also explored. Whereas the values for the dissociation constant (Kd) of Fura-2-AM for Ba2+ and Sr2+ (1.4 and 7.6 x 10-6 M, respectively) are higher than that for Ca2+ (0.14 x 10-6 M) [24], the entry of these cations can still be monitored as a change in fluorescence [25–27]. Addition of 1 mM Sr2+ in Ca2+-free medium did not change basal Fura-2-AM fluorescence (Fig. 3, A and B). Both thapsigargin and oxytocin elicited the expected fluorescence transients in the absence of extracellular Ca2+; addition of extracellular Sr2+ elicited a capacitative increase similar to that seen on addition of Ca2+ (Fig. 3, A and B). Consistent with this interpretation, SKF 96365 and Gd3+ were potent inhibitors of Sr2+ entry (Fig. 3, C and D).
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In contrast to the response to Sr2+, addition of Ba2+ provoked a marked increase in Fura-2 fluorescence in the absence of a capacitative Ca2+ stimulus (Fig. 4A). Consistent with the observation that Ba2+ is a poor substrate for cellular Ca2+ pumps [28–30] and is an inhibitor of K+ channels [31, 32], this increase was prolonged. This effect was inhibited by SKF 96365 (data not shown). After thapsigargin treatment, Ba2+ entry also increased (Fig. 4B), and this increase was inhibited in the presence of Gd3+ (Fig. 4C) and SKF 96365 (data not shown). SKF 96365 and Gd3+ also blocked Ba2+ entry in PHM1 cells following addition of oxytocin (Fig. 4D and data not shown).
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Primary human myometrial cells behaved similarly to PHM1 cells. They did not exhibit significant Ca2+ and Sr2+ entry in the absence of a capacitative stimulus but allowed entry of both ions in response to thapsigargin and oxytocin (Fig. 5, A and B). The cells also responded to a depolarizing concentration of KCl (Fig. 5A), suggesting the presence of voltage-sensitive Ca2+ channels in these cells. The thapsigargin-stimulated entry of Ca2+ was inhibited by Gd3+ (Fig. 5C) and was blocked by SKF 96365 (data not shown). Similar to PHM1 cells, primary myocytes allowed Ba2+ entry in the absence of the capacitative entry stimulus as well as after thapsigargin stimulation (Fig. 5D).
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OAG-Induced Capacitative Ca2+ Entry in PHM1 Cells
When overexpressed, hTrpCs 3, 6, and 7 are activated by diacylglycerol [7, 33]. Because PHM1 cells express mRNAs for all three Trps and express hTrpC3 and hTrpC6 protein [17], they might be expected to respond to diacylglycerol if these channels are functional. Consistent with this hypothesis, individual PHM1 cells responded to the cell-permeable diacylglycerol-analog OAG (100 µM) with an immediate increase in [Ca2+]i that was manifested as cell-specific, irregular patterns of Ca2+oscillations (Fig. 6A). SKF 96365 and Gd3+ blocked OAG-stimulated increases in [Ca2+] (Fig. 6B and data not shown, respectively). The effect was not seen in Ca2+-free buffer (data not shown) or after stimulation with oxytocin (Fig. 6C) at the cell densities used. These data are consistent with a stimulation by OAG of endogenous hTrpC channels in PHM1 cells. Similar effects also were seen in primary myometrial cells (data not shown).
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Capacitative Ca2+ Entry Is Enhanced by hTrpC3 Overexpression in PHM1 Cells
Although we have transiently transfected PHM1 cells by conventional means, the efficiency of expression is greatly enhanced using recombinant adenoviral constructs. For [Ca2+]i studies, PHM1 cells were infected with recombinant adenovirus encoding hTrpC3 plus eGFP protein expressed from a separate promoter or control vector encoding only the eGFP marker, and fluorescence was used to select the infected cells. We examined the result of hTrpC3 recombinant adenoviral infection in PHM1 cells at MOIs of 10, 100, and 1000. Transfection efficiencies ranged from approximately 30% (MOI = 10) to 80%–90% (MOI = 100 and 1000). At a MOI of 1000, however, the cells exhibited very bright fluorescence and aberrant behavior with respect to indicator loading and hydrolysis, so a MOI of 100 was chosen. Membranes prepared from cells overexpressing hTrpC3 showed markedly enhanced hTrpC3 expression compared to membranes from uninfected cells (Fig. 7A).
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Basal [Ca2+]i concentrations in eGFP-infected control and hTrpC3-infected PHM1 cells were not significantly different (121 ± 6 nM and 138 ± 9 nM, respectively; n = 8). In the absence of a capacitative entry stimulus, addition of 1 mM extracellular Ca2+ did not significantly change basal [Ca2+]i (133 ± 5 nM and 160 ± 14 nM, respectively; n = 4). Both cells expressing eGFP alone and those expressing eGFP and hTrpC3 responded to thapsigargin with a similar transient increase in [Ca2+]i (Fig. 7B). The influx of Ca2+ subsequent to the addition of 1 mM extracellular Ca2+ was significantly enhanced in cells overexpressing hTrpC3 compared to eGFP-expressing cells (maximal increases of 295 ± 16 nM and 493 ± 25 nM, respectively; n = 4; P < 0.05) (Fig. 7C), and SKF 96365 inhibited this increase (data not shown).
Cells expressing eGFP alone and cells expressing eGFP and hTrpC3 responded to 100 nM oxytocin with a similar initial, transient increase in [Ca2+]i in the absence of extracellular Ca2+ (maximal increases of 550 ± 60 nM and 578 ± 50 nM, respectively; n = 8), but the influx of Ca2+ subsequent to the addition of 1 mM extracellular Ca2+ was significantly enhanced in cells overexpressing hTrpC3 compared to eGFP-expressing cells (maximal increases of 201 ± 26 nM and 319 ± 42 nM, respectively; n = 8; P < 0.05) (Fig. 7C).
We also examined OAG-induced capacitative Ca2+ entry in PHM1 cells overexpressing hTrpC3. The eGFP control cells responded to 100 µM OAG with an immediate increase in [Ca2+]i oscillations similar to those observed in uninfected cells. Whereas the detailed responses varied between cells, measuring the integrated [Ca2+]i elevation over a period of 10 min allowed us to average data between cells and to assess the contribution of exogenous hTrpC3. Overexpression of hTrpC3 enhanced the OAG-stimulated [Ca2+]i response, expressed in relative units of fluorescence ratio/time, (9.0 ± 0.4 [n = 5] vs. 15.0 ± 1.0 [n = 6] relative units in eGFP controls vs. cells overexpressing hTrpC3, respectively; P < 0.05) (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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q-phospholipase C signal transduction pathways and by intracellular Ca2+ release [2, 3, 6]. The mechanisms are not completely understood, but they can involve direct stimulation of entry channels by the IP3-receptor or IP3 receptor-independent pathways [5, 34–37]. Capacitative Ca2+ entry has been implicated in a number of aspects of smooth muscle function, including proliferation, maintenance of tone, and pacemaker activity [2, 6, 12, 13, 38]. Overexpression of TrpC proteins increases capacitative Ca2+ entry, but the properties of this entry do not entirely replicate endogenous capacitative activity. The TrpC proteins were predicted to function as homo- or heterotetramers [39–41]. Individual TrpC proteins possess a number of interaction and binding motifs [6, 42–45]. These features may allow association of specific TrpC proteins with signaling scaffolds and account for the widely varying properties of capacitative Ca2+ entry in different cell types. Recent reports indicate that hTrpC1 can form channel complexes with hTrpCs 4 and 5 but not hTrpCs 3, 6, or 7 and that hTrpCs 3, 6, and 7 can form complexes with each other but not with hTrpCs 1, 4, or 5 [10, 46]. Human myometrium and immortalized pregnant human myometrial PHM1 cells express mRNA for hTrpCs 1, 3, 4, 6 and 7 and protein for hTrpCs 1, 3, 4, and 6 [15, 17]. Thus, channels comprised of TrpC1/4 and TrpC3/6/7 combinations might be present in myometrium, but the consequences of this expression on the properties on endogenous Ca2+ entry have not been explored.
Neither PHM1 nor primary myometrial cells exhibited Ca2+ or Sr2+ entry when the extracellular concentrations of these ions were raised from negligible to 1 mM. In contrast, both cell types allowed entry of Ca2+ and Sr2+ in response to oxytocin or thapsigargin that was inhibited by 0.1 µM SKF 96365 and 10 µM Gd3+. The median inhibitory concentration of SKF 96365 versus several agonists are in the range of 10–20 µM, showing that SKF 96365 acts as a "functional" antagonist and not on specific receptors [21]. The lanthanides Gd3+ and La3+ are frequently used as effective blockers, but they may affect Trp channels from the extracellular as well as from the cytosolic side of the plasma membrane [47]. Although SKF 96365 and Gd3+ are not entirely specific for capacitative Ca2+ channels, they have been widely used to inhibit receptor-operated, store-operated, and Trp-mediated Ca2+ entry [21, 22, 47–49]. The data are consistent with the expression in human myometrium of endogenous capacitative channels that accommodate both Ca2+ and Sr2+. At face value, these channels do not appear to exhibit significant spontaneous activation in the absence of a capacitative stimulus. Like PHM1 cells, human peripheral blood T lymphocytes, Jurkat cells, and PC12 adrenal cells allowed Sr2+ entry after capacitative stimuli in the absence of Trp overexpression, suggesting functional endogenous channels [50, 51]. In contrast, both HEK293 and DT40 B lymphocytes did not allow Sr2+ entry after a stimulus unless hTrpC3 was overexpressed [27, 37], indicating minimal endogenous channel activity.
In contrast to the entry of Ca2+ and Sr2+, Ba2+ readily entered both PHM1 and primary myometrial cells in the absence of a capacitative stimulus; this was inhibited by SKF 96365. The Ba2+ is a poor substrate for plasma membrane and endoplasmic reticulum Ca2+ pumps [29, 30], and it also inhibits K+ channels [31, 32]. Influx of Ba2+ in the absence of a capacitative stimulus could reflect endogenous Trp activity that is unmasked, in contrast to Ca2+ and Sr2+ entry as a result of either action. Myometrial Trp channels may be spontaneously active or activated as a consequence of basal phospholipase C activity or some undefined stimulus. Alternatively, the entry may represent a passive leak or entry through other types of channels. It is unlikely that L-type channels are involved. Although PHM1 cells lack significant L-type Ca2+ channel expression [52], the primary myometrial cells responded to KCl, which is suggestive of such expression, and yet showed capacitative cation-entry properties identical to those exhibited by PHM1 cells. The finding that oxytocin- and thapsigargin-stimulated Ba2+ entry was inhibited by SKF 96365 and Gd3+ favors the argument for some endogenous capacitative entry in myometrial cells. In contrast, HEK293 cells, DT40 cells, and COS cells allowed Ba2+ entry in response to a capacitative stimulus when Trps were overexpressed [27]. Jurkat and human peripheral blood T-lymphocytes, which express endogenous mRNA for TrpCs 1, 3, 4, and 6, allowed Ba2+ entry without Trp overexpression, but only in response to a stimulus [50]. These data, together with Ca2+ and Sr2+ entry data, illustrate the point that cation entry varies significantly between cell types and suggest that endogenous Trp channels may be relatively more active in myometrial cells than in some other cell types, despite endogenous Trp expression. It is interesting to note that whereas TrpCs 4, 6, and 7 are widely expressed in murine and canine smooth muscle, TrpC3 has a more limited distribution, notably in the canine renal artery [11]. Whether endogenous expression of hTrpC3 influences the properties of PHM1 cells is being investigated.
The TrpC-mediated Ca2+ entry can be activated directly by diacylglycerol independent of protein kinase C activation. Overexpressed TrpC3 and TrpC6 were activated by diacylglycerol in a protein kinase C-independent manner in CHO-K1 cells [7]. Also, TrpC3 was activated by OAG in DT40 B lymphocytes [36, 37], and mTrpC7 was activated by OAG in HEK293 cells [33]. On the other hand, TrpCs 1, 4, and 5 were unresponsive to diacylglycerol [7]. In none of these cases were responses to diacylglycerol seen in the absence of Trp overexpression or prolonged Ca2+ oscillations observed. In contrast, membrane-permeant OAG induced marked [Ca2+]i oscillations in both PHM1 cells and primary myometrial cells. The response to OAG is consistent with the expression of endogenous hTrpC3/6/7 channels in myometrial cells. Activation of endogenous cation entry by OAG has recently been reported in Jurkat cells and human peripheral blood T lymphocytes [50] and in adrenal PC12 cells expressing TrpCs 1–6 [51]. The Ba2+ entry observed in unstimulated conditions might reflect activation of endogenous channels by diacylglycerol produced as a result of basal phospholipase C activity. The fact that U73122 inhibits basal phosphoinositide turnover in PHM1 cells (unpublished results) is consistent with such an effect. The oscillations induced by OAG were not invoked by oxytocin under comparable conditions, although oxytocin can induce oscillations when the cells approach confluence [53; unpublished observations]. The [Ca2+]i oscillations may reflect a complex interplay of Ca2+ release and sequestration mechanisms that is particularly important in a phasic smooth muscle such as myometrium, and this is under investigation.
Overexpression of hTrpC3 in PHM1 cells did not increase Ca2+ entry in the absence of a capacitative stimulus, suggesting that this protein did not exhibit significant spontaneous activity under these conditions. In contrast, HEK293 cells stably expressing hTrpC3 exhibited significantly higher basal as well as agonist-stimulated influxes of Ca2+, Mn2+, Ba2+, and Sr2+ than control cells [27]. Nonetheless, PHM1 cells overexpressing hTrpC3 exhibited enhanced response to OAG and enhanced thapsigargin- and oxytocin-stimulated capacitative Ca2+ entry. The [Ca2+]i transients in myometrial cells are relatively short-lived, perhaps reflecting relatively active Ca2+ ATPase activities that attenuate responses relative to other cell types. With respect to qualitative differences, the actual configuration of the putative heterotetramers and/or their localization in the membrane relative to scaffolding complexes may differ between cell types and alter the relative sensitivity to GPCRs versus thapsigargin.
Depletion of Ca2+ stores with the endoplasmic reticulum Ca2+ pump-inhibitor thapsigargin activates Ca2+ influx independent of IP3-dependent Ca2+ release in PHM1 cells; these cells also respond to GPCR stimulation with Ca2+ entry. Because PHM1 cells do not express a significant amount of L-type, voltage-operated calcium channels, capacitative Ca2+ entry may be a significant contributor to this influx mechanism. Although primary myometrial cells may retain L-type channel expression, they show responses to capacitative stimuli similar to those of PHM1 cells. Therefore, although L-type channels are clearly important for sustained myometrial contractions, these data suggest that Trp channels may play a role in controlling myometrial intracellular free Ca2+ concentrations and may be important transducers of agonist-mediated signals that increase at the time of parturition and labor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Barbara M. Sanborn, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523. FAX: 970 491 7569; barbara.sanborn{at}colostate.edu
Received: 10 January 2003.
First decision: 3 February 2003.
Accepted: 3 April 2003.
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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