Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma
Francisco R. González-Pacheco,
Carlos Caramelo,
Maria Ángeles Castilla,
Juan J. P. Deudero,
Javier Arias1,
Susana Yagüe,
Sonsoles Jiménez,
Rafael Bragado and
Maria Victoria Álvarez-Arroyo
Servicio de Nefrología and Servicio de Inmunología, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, and
1 Hospital Clínico Universitario, Universidad Complutense, Instituto Reina Sofía de Investigación Nefrológica (IRSIN), Madrid, Spain
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Abstract
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Background. Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLC
) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2.
Methods. The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLC
.
Results. The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLC
with a concentration- and time-profile similar to that of [Ca2+]i mobilization.
Conclusions. The present study reveals that H2O2 activates PLC
on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca2+]i mobilization, shape-changing actions and damage by H2O2 in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H2O2 and may help to design pharmacologically protective strategies.
Keywords: calcium signal; hydrogen peroxide; phospholipase C gamma; vascular smooth muscle cells
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Introduction
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The effects of reactive oxygen species (ROS) on cells of the vascular wall are critical in the response to ischaemia/reperfusion and, of utmost importance, in the behaviour of the vascular smooth muscle cells (VSMC) forming the neointimal layer in atherosclerotic vessels [1]. The sources of ROS in these conditions appear to be the vascular cells themselves, as well as leukocytes adhering to and infiltrating the vessels, with H2O2 being a paradigmatic molecule of oxidative aggression to the cells. These factors are of critical importance in the pathogenesis of a number of renal diseases, with special reference to vascular pathologies, diabetes, vasculitis and acute renal failure.
In spite of extensive efforts in the last decade to characterize the mechanisms of ROS-induced injury, many aspects of the response of VSMC to ROS are still unknown. In this regard, even though a Ca2+-release response by H2O2 has been described in VSMC [2,3], data about the actual mechanisms by which ROS might both activate and injure VSMC are still incomplete. The available studies support the theory that H2O2 stimulates a rather complex signalling network, which involves protein kinase C (PKC), mitogen activated protein kinase (MAP-K) and phosphatidyl inositol 3 kinase (PI3K) [3]. Additional data are available on the putative effector mechanisms of ROS on glomerular mesangial cells (GMC), a cell type resembling VSMC both structurally and biologically. The exposure of mesangial cells to ROS induces several activation sequences. In these cells, both a dantrolene-inhibitable and an extracellular Ca2+-related increase in [Ca2+]i were observed upon exposure to H2O2 [46]. Other experiments disclosed that GMC planar surface area reduction and autocrine proliferation mechanisms are triggered by ROS [6].
Based on the aforementioned data, the area in which information is particularly lacking is that of coupling VSMC exposure to ROS to the main VSMC activation mechanism, i.e. Ca2+-related signalling. Insufficient evidence is available on the actual triggering mechanism leading to the [Ca2+]i-related signalling sequence of ROS on VSMC; as mentioned before, this sequence may differ considerably from that of the better known receptor mediated pathways [3,79]. However, none of the cascades of protein phosphorylation initiated by ROS and described in recent years has been directly related to triggering the [Ca2+]i response in VSMC [9]. Nevertheless, some of the pathways that become activated by the action of ROS as well as by other mediators, namely angiotensin II (Ang II), may induce the activation of PLC
[10,11]. PLC
is capable of triggering Ca2+ release in several cellular systems by catalysing the production of inositol trisphosphate from membrane phospholipids [16,17]. This type of PLC is a putative candidate to be activated by agents that, like H2O2, differ from receptor-ligand-related, G protein-driven signal transduction. The possibility that PLC
is involved in H2O2-induced [Ca2+]i release in VSMC has been suggested [12] but, to our knowledge, it has not yet been specifically demonstrated. The present study was, therefore, designed to examine the mechanisms of activation of VSMC by H2O2, with focus on the [Ca2+]i mobilization response through tyrosine phosphorylation-mediated signal transduction pathways, with particular reference to PLC
.
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Materials and methods
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VSMC culture
Human VSMC were isolated and cultured from venous samples of multiorgan donors by a modification of methods described elsewhere [8]. The culture in these conditions yields a homogeneous cell population devoid of endothelial cells and fibroblasts [8]. For the [Ca2+]i experiments, cells were grown on round 13 mm glass coverslips. All experiments were performed between the second and sixth trypsinization. In previous setting-up studies, no differences in terms of phenotypes, Ca2+ response and cytochemical characterization were detected between VSMC obtained and cultured from donor veins from several territories and those from arterial vessels (González Pacheco F.R., unpublished observations).
Measurement of cytosolic free Ca2+ concentration ([Ca2+]i)
The measurements of [Ca2+]i transients were performed as described previously by using the fluorescent indicator Fura-2 (Sigma, Madrid, Spain) [8]. Fura2 fluorescence was measured at 37°C using a fluorescence spectrophotometer (LS-50B Perkin-Elmer, Foster City, CA, USA) at an emission wavelength of 500 nm and excitation wavelengths of 342 and 380 nm.
Experimental manoeuvres
H2O2 was used as a paradigm of extracellular ROS-induced transient challenge. In terms of the possible pathophysiological projection of the experimental manoeuvres, Brouwer et al. demonstrated that H2O2 can replace for ischaemia/reperfusion in the setting of in vivo organ injury [13]. For analysing the different pathways involved in H2O2-dependent Ca2+ mobilization, experiments were performed using cells pre-incubated (minimum 30 min, maximum 60 min) or not with the different agents or media as follows: the tyrosine-kinase inhibitor genistein (10100 µM; Sigma), the PLC pathway inhibitor, U73122, (1 µM; Calbiochem Corp., La Jolla, CA, USA) [14,15] and the intracellular Ca2+-release blocker dantrolene (50 µM; Procter and Gamble Pharmaceuticals, Weiterstadt, Germany) [8]. Furthermore, experiments were done using media with no Ca2+ plus 100 µM Mn2+, and no Ca2+ plus EGTA (2 mM); in the latter two cases, no pre-incubation was done with these media [8]. Similar experiments using Ang II (0.1 µM) as the Ca2+ mobilization agent were done as a positive control. Fura2 measurements were performed between 14 and 21 days of culture, when cells had formed a confluent monolayer with a density of 1.52x106 cells/dish. In all the experiments, samples containing the vehicle of the different agents were included as controls.
Assessment of H2O2 -induced VSMC morphological changes
As described previously, morphological changes were assessed as a model of the functional effects of H2O2 on VSMC [7,8]. Briefly, non-confluent VSMC were exposed to minimum essential medium (MEM+1% foetal calf serum, room temperature) and photographed (Phase Contrast Diaphot, Nikon, Japan) before and after 10 min of adding H2O2 (250 and 500 µM, final concentration) or vehicle (MEM, pH 7.4). Changes in cell shape were analysed by planimetry, as described previously [7,8]. The number of cells analysed for each condition ranged from 212 to 278, in at least five different experiments. In the studies using genistein (100 µM) or U73122 (1 µM), the cells were pre-treated with the drugs from 30 min before exposure to H2O2. In all the cases, the researchers were blinded for the treatment received by the cells. Time-control experiments were performed using either no treatment or treatment with genistein or U73122 alone. As a further assessment of the effects of U73122, additional images of the cells were obtained at 18 h of the H2O2 challenge. On these images, cell count was performed on a minimum of five photographs, corresponding to different microscopic fields.
PLC
immunoprecipitation and immunoblotting
These experiments were performed according to modifications of methods described previously [16]. Confluent human VSMC were incubated for 48 h with MEM plus 1% FCS. Cells were then exposed to 250 µM H2O2 for 30 s and 5 min in serum-free medium at 37°C. The 30 s time was chosen with the purpose of demonstrating the presence or absence of PLC
-tyrosine phosphorylation according to the time-profile of the response observed in the experiments on [Ca2+]i (see Results). To stop the activation, the monolayers were washed (2x) with ice-cold phosphate-buffered saline containing 400 µM Na3VO4, 5 mM EDTA and 10 mM NaF. Cells were then lysed in lysis buffer (25 mM HEPES pH 7.5, 1.5 mM Cl2Mg, 300 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.1% Triton X-100, 50 mM NaF, 1 mM Na3VO4, 100 µg/ml PMSF, 10 µg/ml leupeptin, 30 min, 4°C). Similar amounts of protein (100 µg, BCA method; Pierce Chemical Co, Rockford, IL, USA) were subjected to 8% SDSPAGE and transferred to a nitrocellulose membrane (Bio-Rad, Richmond, CA, USA) by standard procedures. The membranes were blocked in a solution of 10% non-fat dry milk in phosphate-buffered saline phis Tween 20 (PBT) (80 mM HPO4Na2, 20 mM H2PO4Na, 100 mM NaCl, 0.05% Tween 20 pH 7.5) for 1 h and rinsed three times with PBT. The membrane was then incubated with a specific anti-PLC
mAb (1/250 in PBT, Transduction Laboratories, Lexington, KY, USA) and subsequently incubated with a peroxidase-labelled goat anti-mouse Ig (1/6000, Bio-Rad, Richmond, CA, USA) in PBT for 1 h at room temperature. The membranes were processed by enhanced chemiluminescence, according to the manufacturer's specifications (Amersham Pharmacia Biotech, Barcelona, Spain). To analyse tyrosine phosphorylation of total proteins, membranes were stripped with 62.5 mM TrisHCl, pH 6.7, 2% SDS and 100 mM ß-mercaptoethanol for 15 min at 55°C, and reprobed with the anti-phosphotyrosine mAb PY20 (1/500 in PBT, Transduction Laboratories) as above. Quantifying of ECL images was done by densitometry (Molecular Dynamics, Sunnyvale, CA, USA) and values of tyrosine phosphorylated PLC
were normalized for those obtained from the corresponding total PLC
. VSMC stimulated for 2 min with Ang II (0.1 µM) [15] were used as positive controls for induced tyrosine phosphorylation of PLC
. A Jurkat cells extract provided by the manufacturer was used to ascertain further the location of the PLC
band. In the experiments with genistein, VSMC were pre-incubated with this drug (100 µM) for 60 min at 37°C before being stimulated with H2O2. To ascertain further the specificity of the findings, a similar blotting procedure was performed on samples that had been previously immunoprecipitated. For immunoprecipitation, 250 µg of total lysate were precleared with 50 µl of 20% protein GSepharose beads (Amersham Pharmacia Biotech) and afterwards with normal mouse serum and Protein G-Sepharose. Immunoprecipitation was carried out with 3 µg of specific anti-PLC
mAb pre-bound to protein GSepharose beads, for 1 h at 4°C. The immunoprecipitates were recovered by a 10 s microcentrifuge pulse and washed (x6) with lysis buffer. Sepharose beads were equilibrated in SDSPAGE sample buffer including 5% ß-mercaptoethanol and then heated for 5 min at 95°C before loading the gel. Transfer of proteins and detection of PLC
was performed as described above.
Statistics
Unless specified otherwise, results correspond to a minimum of five experiments of each type. Changes in variables for the different incubations and concentrationresponse curves were analysed by one-way and two-way analysis of variance for repeated measures and subsequent Scheffe's test. Comparisons between two groups of data were done by Students t-test for unpaired observations. A P value of <0.05 was considered significant.
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Results
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Characteristics of the H2O2-induced changes in [Ca2+]i
H2O2 induced a concentration-dependent increase in [Ca2+]i, with a [Ca2+]i peak of 354±33 nM, 545±96 nM and 789±177 nM with 50, 250 and 500 µM H2O2, respectively (P<0.01 for all three concentrations with respect to baseline). The highest level of [Ca2+]i was reached at 32±7 s from the challenge with H2O2. A decrease in [Ca2+]i levels occurred thereafter, reaching a sustained phase with a small increase in [Ca2+] with respect to the baseline within 6090 s (Figure 1
). Of major relevance for clarifying the sequence of mechanisms of H2O2-induced activation of VSMC, the [Ca2+]i mobilization peak was blocked after VSMC pre-treatment with the tyrosine kinase inhibitor, genistein (100 µM) and the specific PLC inhibitor, U73122 (Table 1
, Figure 1
). Genistein pre-incubation had a smaller effect on the Ang II-induced [Ca2+]i peak (29±3% blockade, n=4, P<0.01 with respect to the percentage inhibition of the H2O2-induced peak). A marked inhibition of the H2O2 (250 µM)-induced [Ca2+]i peak was also obtained with lower concentrations of genistein (10 µM, 94±3% blockade, n=4, P=NS with respect to the effect of 100 µM genistein). Additional features of the Ca2+ response are shown in Figure 1
and Table 1
, as follows. (i) The pre-treatment with dantrolene (50 µM) markedly inhibited the [Ca2+]i response. (ii) When VSMC were challenged with H2O2 in the absence of extracellular Ca2+ (no Ca2+, 2 mM EDTA medium), a decrease in the magnitude of the [Ca2+]i peak occurred, but the sustained component of the [Ca2+]i increase was abolished. (iii) The existence of a significant H2O2-induced activity of a divalent cation channel was suggested by the blockade of the [Ca2+]i sustained response by Mn2+ (Table 1
).

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Fig. 1. Representative traces of VSMC [Ca2+]i transients obtained in different conditions. (A) H2O2 (250 µM). (B) H2O2 (250 µM) on VSMC incubated in the presence of U73122 (1 µM, 30 min pre-incubation). (C) Angiotensin II (0.1 µM). Arrows denote the moment of exposure to H2O2.
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VSMC shape change
Additional experiments were performed to characterize the functional implications of the observed changes in [Ca2+]i. These experiments showed that the percentage of cells undergoing significant shape changes under H2O2 exposure was markedly diminished after pre-incubation with genistein (Figure 2A
). A similar inhibitory effect of genistein was observed when H2O2 was used at 500 µM concentration (72±3% inhibition of the cell shape change induced by H2O2, n=4, P<0.01). In comparison, a significantly smaller inhibition of the Ang II-induced cell shape change was obtained after genistein pre-incubation (23±5% inhibition of the 250 µM H2O2-induced shape change; n=5, P<0.01). In addition, and in accordance with the effects observed in [Ca2+]i mobilization, a marked inhibition of VSMC shape change was also obtained with pre-incubation with U73122 (1 µM) (Figure 2A
). Similarly, as with genistein, U73122 (1 µM) significantly inhibited (80±5%, P<0.001) the VSMC shape change induced by 500 µM H2O2. Furthermore, a major inhibition of 250 µM H2O2-induced VSMC shape change was observed in the presence of dantrolene (50 µM) (Figure 2A
).

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Fig. 2. (A) Per cent of VSMC shape change, as induced by H2O2 (250 µM). H2O2 (250 µM) on cells pre-incubated (30 min) with genistein, U73122 (1 µM) or dantrolene (50 µM). (B) Image of VSMC 24 h after treatment with H2O2 (250 µM), plus (lower panel) or minus (upper panel) pre-treatment with the phospholipase C pathway antagonist, U73122 (1 µM). *P<0.01 with respect to VSMC treated with H2O2 alone.
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An additional result of potential practical importance was obtained by analysing the VSMC image at 18 h after H2O2 challenge. As shown in Figure 2B
, H2O2 (250 µM) provoked an intense detachment of VSMC; this deleterious effect was significantly inhibited in VSMC pre-incubated in the presence of U73122 (1 µM) before exposure to H2O2 (250 µM) (Figure 2B
). The cell count (mean±SD) at 18 h is shown in Table 2
. No study of this type was performed using genistein or dantrolene, due to the cytotoxic properties of these compounds in prolonged incubations (González Pacheco et al., unpublished observations). No effect of U73122 alone (1 µM) was detected in similar experiments (P=NS for cell count with no treatment; Table 2
).
Role of PLC
tyrosine phosphorylation in signal transduction
With the premise of the inhibitory effect of genistein and U73122 on [Ca2+]i peak and VSMC cell shape change, additional studies were conducted to examine the putative mechanisms linking tyrosine phosphorylation and [Ca2+]i mobilization in H2O2-stimulated VSMC. These experiments showed that the exposure of VSMC to H2O2 (100, 250 and 500 µM, 30 s) stimulated the tyrosine phosphorylation of a number of bands (data not shown). To analyse the possible link between these phosphorylations and Ca2+ mobilization, we tested whether PLC
was one of the proteins tyrosine phosphorylated under H2O2 challenge (250 µM). In agreement with this hypothesis, after immunoprecipitation, one of the tyrosine-phosphorylated bands was identified by immunoblotting with the specific mAb (Figure 3A
) as PLC
. The densitometric evaluation of the level of PLC
tyrosine phosphorylation in the H2O2-challenged VSMC indicated a marked stimulation (Figure 3B
). Furthermore, the phosphorylation of the band identified as PLC
was inhibited by genistein (100 µM) (Figure 3A
and 3B
). In the measurements taken at 5 min, in cells incubated with no inhibition of naturally occurring phosphatases, the tyrosine phosphorylation of PLC
was no longer detected (data not shown).
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Discussion
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The present study presents data on the action of H2O2 on the vascular wall. Even though the results were obtained under experimental conditions designed to resemble those in short-duration ischaemia/reperfusion, conclusions can be extended to other diseases involving vascular pathology of the kidney. As a main finding, our results provide evidence relating the H2O2-induced [Ca2+]i transient with the activation of PLC
through a tyrosine kinase-mediated pathway. Basically, a key role of tyrosine phosphorylation on the [Ca2+]i-mobilizing effect of H2O2 on VSMC was revealed, which suggests that the involved pathway includes PLC activation and, more specifically, the stimulation of the tyrosine-phosphorylation-activated isoform of PLC, i.e. PLC
.
To our knowledge, this is the first demonstration that PLC
is directly involved in the effects of exogenous H2O2 on VSMC and, moreover, that inhibition of PLC-dependent pathways leads to important functional consequences. PLC
is the only isoform of PLC that becomes activated by tyrosine phosphorylation, leading to inositol trisphosphate production and [Ca2+]i release from intracellular stores [11]. Using a specific mAb, our studies demonstrate the induction of PLC
tyrosine phosphorylation by H2O2. Of practical importance in terms of VSMC contraction and potential pharmacological applicability, our findings demonstrated that the H2O2-induced [Ca2+]i peak is abolished by genistein and U73122. However, since our study was focused on PLC
, the putative activation of other isoforms, although less probable, cannot be completely ruled out.
González-Rubio et al. have found in GMC [7] and Taher et al. in VSMC [9] that ROS induce tyrosine phosphorylation of several proteins in VSMC and GMC. Among others, these proteins include pp60-src and MAP-K. pp60-src has protein kinase activity on PLC
[10,11]. The same or other mechanisms can be involved in more prolonged effects, such as growth factor production. In this regard, recent data from our laboratory have shown that ROS induce both the production of vascular endothelial growth factor (VEGF) and the expression of VEGF receptors on VSMC [17,18].
The fact that both the [Ca2+]i peak and VSMC shape change were inhibited by either genistein or U73122 strongly supports the possibility that the two effects are driven through a tyrosine phosphorylation-activated PLC. The results on tyrosine phosphorylation further support that PLC
is the particular isoform involved in the H2O2 effect. U73122 inhibits PLC-mediated pathways distal to receptor binding and is therefore useful as a blocker of all PLC-mediated mechanisms [14,15]. Of further interest, the fact that U73122 blocked the cell-injuring effect of H2O2 provides a relevant clue for the investigation into the H2O2-mediated damage of vascular cells. Taken together, the effects of U73122 suggest that this compound may constitute a potential tool to interfere with in vivo vascular actions of ROS.
The effects on [Ca2+]i found with H2O2 are coincident with those reported by other authors, who described that the challenge of VSMC with H2O2 induces a rapid increase in [Ca2+]i, followed by a decrease to a new, higher than the baseline level [3]. The effects of dantrolene, Ca2+-free and Mn2+ media suggest that a main component of the [Ca2+]i transient involves the release from intracellular stores to the cytosol; this component is followed by a long lasting increase, which is dependent on Ca2+ entry from the extracellular fluid; the use by this mechanism of a divalent cation channel transporting both Ca2+ and Mn2+ is suggested in the present study by the quenching effect obtained in the presence of Mn2+. The results obtained with genistein and U73122 (see above) and with no Ca2+ media suggest that an outside-in Ca2+ transport is activated by tyrosine phosphorylation, and using PLC-mediated mechanisms. In the case of Ang II, the role of tyrosine phosphorylation-related mechanisms appears to be of less influence than for Ang II, as determined by the magnitude of the inhibition of [Ca2+]i transients and shape change obtained with genistein. These differences were rather predictable, based on the higher importance in the case of Ang-II of G-protein-driven pathways, which transduce their signals through tyrosine-kinase-independent isoforms of PLC, i.e. PLCß [19].
Our data have potentially relevant consequences for the understanding of the cellular mechanisms of action of ROS in organ injury and for the design of new treatment strategies for situations in which oxidative aggression is involved in VSMC contraction and injury, e.g. acute and chronic vascular damage in ischaemia/reperfusion or nephrosclerosis and organ transplantation.
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Acknowledgments
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The present study was supported by grant numbers 97/0341 and 97/0597 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS), by PM 95/0047 and PM 98/0063 from the Comisión Interministerial de Ciencia y Tecnología, by Comunidad Autónoma de Madrid (CAM 07/016/96 and 07/055/96), the Instituto Reina Sofia de Investigación Nefrológica (IRSIN) and Caramelo S.A. (La Coruña, Spain). M.V.A.A. is a researcher of FISS, F.R.G.P. and J.J.P.D. are fellows from the Fundación Conchita Rábago. S.Y. is a fellow of the Spanish Ministry of Health and M.A.C. is a fellow of IRSIN. The authors wish to thank Dr Carmen Gómez Guerrero for help in setting up the PLC
techniques and to Drs Diego Rodriguez Puyol and Jesús Egido for their critical reading of the manuscript.
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Notes
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Correspondence and offprint requests to: Maria Victoria Álvarez-Arroyo, Instituto de Investigaciones Médicas, Fundación Jiménez Díaz, Universidad Autónoma, Avenida Reyes Católicos 2, E-28040 Madrid, Spain. Email: mvarroyo{at}fjd.es 
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Received for publication: 28. 6.01
Revision received 25.10.01.