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Originally published In Press as doi:10.1074/jbc.M407470200 on July 26, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40634-40639, September 24, 2004
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Ca2+ Controls Functional Expression of the Cardiac K+ Transient Outward Current via the Calcineurin Pathway*

Emeline Perrier, Romain Perrier, Sylvain Richard, and Jean-Pierre Bénitah{ddagger}

From the INSERM U637, CHU A. de Villeneuve, 34295 Montpellier, France

Received for publication, July 6, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transient outward K+ current (Ito) modulates transmembrane Ca2+ influx into cardiomyocytes, which, in turn, might act on Ito. Here, we investigated whether Ca2+ modifies functional expression of Ito. Whole-cell Ito were recorded using the patch clamp technique in single right ventricular myocytes isolated from adult rats and incubated for 24 h at 37 °C in a serum-free medium containing various Ca2+ concentrations ([Ca2+]o). Increasing the [Ca2+]o from 0.5 to 1.0 and 2.5 mM produced a gradual decrease in Ito density without change in current kinetics. Quantitativereverse transcriptase-PCR showed that a decrease of the Kv4.2 mRNA could account for this decrease. In the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM)-loaded myocytes (a permeant Ca2+ chelator), Ito density increased significantly when cells were exposed for 24 h to either 1 or 2.5 mM [Ca2+]o. Moreover, 24-h exposure to the Ca2+ channel agonist, Bay K8644, in 1 mM [Ca2+]o induced a decrease in Ito density, whereas the Ca2+ channel antagonist, nifedipine, blunted Ito decrease in 2.5 mM [Ca2+]o. The decrease of Ito in 2.5 mM [Ca2+]o was also prevented by co-incubation with either the calmodulin inhibitor W7 or the calcineurin inhibitors FK506 or cyclosporin A. Furthermore, in myocytes incubated for 24 h with 2.5 mM [Ca2+]o, calcineurin activity was significantly increased compared with 1 mM [Ca2+]o. Our data suggest that modulation of [Ca2+]i via L-type Ca2+ channels, which appears to involve the Ca2+/calmodulin-regulated protein phosphatase calcineurin, down-regulates the functional expression of Ito. This effect might be involved in many physiological and pathological modulations of Ito channel expression in cardiac cells, as well other cell types.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
K+ channels play critical roles in a wide variety of physiological processes including regulation of heart rate and contraction. Among other cardiac K+ currents, the transient outward K+ current (Ito) is crucial because it controls the amplitude of the plateau phase and the duration of the action potential. As a consequence, Ito strongly modulates transmembrane Ca2+ entry and, thereby, the excitation-contraction coupling (1). Thus, any change in Ito has profound pathophysiological consequences, often leading to the generation of life-threatening arrhythmias. For example, changes in the density and/or the properties of Ito occur in conjunction with myocardial damage or disease, including acute or chronic diabetes mellitus, hypertrophy induced by pressure or volume overload, and cardiac failure (2). Unidentified common pathway(s) may underlie a down-regulation of the expression of Ito channels in all these pathological conditions. A possible candidate for triggering such channel remodeling could be the altered level of intracellular Ca2+ ([Ca2+]i).

Even if defined as Ca2+-independent, by contrast to the 4-aminopyridine-resistant Ca2+-activated transient outward chloride current, Ito might be regulated by Ca2+. A modulation of Ito by extracellular Ca2+ ([Ca2+ ]o) induced by depolarizing shifts in the gating parameters has been documented (3). Moreover, Ito inactivation in human atrial myocytes might be controlled by [Ca2+]i-dependent processes, involving Ca2+/calmodulin-dependent protein kinase II (CaMKII)1 (4). At the molecular level, shal-type voltage-gated K+ channels (in rat predominantly Kv4.2 (5)) are the pore-forming subunits of the Ito channel that are regulated by auxiliary subunits (5, 6). Interestingly, the regulatory subunits KCHiPs and the neuronal calcium sensor (NCS) that modulate Ito expression and kinetics are Ca2+-binding proteins. Thus, Ca2+ controls the gating of Kv4-KChiP (7) or Kv4-NCS-1 complexes (8). In addition to these acute effects, Ca2+ also affects cardiac gene expression via excitation-transcription coupling. It is now well recognized that Ca2+ entry into neuronal cells through voltage or ligand-gated channels triggers neuronal activity-dependent gene expression critical for neurobiological adaptive changes (9). In cardiac cells, Ca2+-sensitive transcription factors may play a key role in maintaining the contractile phenotype and contribute to adaptive or pathological changes in the structural or functional properties of the heart (10). Notably, long term changes in intracellular Ca2+ level have been linked to altered functional expression of the ion channel. In cultured neonatal cardiomyocytes, increases in [Ca2+]i cause a fall in density of the Na+ current (11). Exposure of adult rat ventricular myocytes in culture to high Ca2+ increases Ca2+ channel mRNA and protein abundance, producing a corresponding change in the L-type Ca2+ current (ICa) (12).

In the present study, we hypothesized that Ca2+ regulates the functional expression of Ito. Experimental manipulations of Ca2+ entry via ICa, in adult rat ventricular myocytes incubated for 24 h, showed that increased cytosolic Ca2+ decreases Ito and down-regulates Kv4.2 mRNA expression, which involves Ca2+/calmodulin-regulated protein phosphatase calcineurin cascade.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Isolation and Incubation—Cardiac ventricular myocytes were isolated from adult male Wistar rats (250–280 g) using an enzymatic perfusion method described previously (13). Taking into account the electrical heterogeneity, only the right ventricle was selected (14). Myocytes were incubated for 24 h at 37 °C in Tyrode's solution supplemented with 100 IU/ml penicillin and 0.1 µg/ml streptomycin as described previously (13). Before electrophysiological recordings, myocytes were washed out for 10 min to minimize any acute effects of the drugs used during the incubation.

Drugs—The acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), Bay K8644, and tacrolimus (FK506) were purchased from Calbiochem; Nifedipine was from Bayer; 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62) was from Sigma; n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) was from Research Biochemicals Inc.; and cyclosporin A (CsA) was generously provided by Novartis.

Electrophysiology—Whole-cell currents were monitored at 0.1 Hz with an Axopatch 1D amplifier and recorded with a pCLAMP-7 (Axon Instruments) at 21–23 °C. Series resistance was electronically compensated (40–60%). The Ito, defined as the 3 mM 4-aminopyridine-sensitive current, was recorded as described previously (13). Because cell size variation might account for differences, current amplitudes were normalized to cell capacitance (Cm) and expressed as densities (picoampere/picofarad). No difference in the Cm of myocytes in the different incubation conditions was observed (Table I).


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  		TABLE I
Cell capacitances and Ito kinetic parameters after 24-h incubation in different conditions

The voltage-dependent availability were fit to a Boltzmann distribution with , the potential of half-inactivation, and kinac, the slope factor. The time to peak values, , were determined as the time from the onset of depolarization at +40 mV to the time of maximal current amplitude and reflect activation rates. The inactivation kinetics were determined by fitting the decay phase of the current traces to a monoexponential function with , time constant at +40 mV. pF, picofarads.

 
Reverse Transcription and Real-time PCR—Total RNA was extracted using the TRIzol method (InVitrogen) from whole dish of cardiomyocytes (samples), and its integrity was analyzed by electrophoresis with a chip-based RNA analysis system (Agilent Technologies). To obtain cDNA, 200 ng of RNA was reverse-transcripted using the Taqman Gold RT-PCR kit (Applied Biosystems). Real-time PCR analysis was done with an iCycler iQ detection system (Bio-Rad) using a specific mix containing specific primers and probe and AmpliTaq Gold DNA polymerase, as described elsewhere (15, 16). Each real-time quantitative PCR assay was performed twice using n samples.

Calcineurin Activity—Cellular calcineurin phosphatase activity was measured on cell extract using the Calbiochem Calcineurin Cellular Activity Assay Kit according to the manufacturer's instructions. The fraction of total phosphatase activity due to calcineurin was determined by detection of free phosphate released in absence or presence of EGTA buffer. Colorimetric measure (assay with Malachite Green) was done at 620 nm on a plate reader (Dynatech MR 5000).

[Ca2+]i Measurement—After 24-h incubation, cells were loaded with 2.5 µM Fura-2 AM (Molecular Probes) for 30 min at 37 °C. Cells were then rinsed in respective storage solution (1 or 2.5 mM [Ca2+]o), and fluorescence measurements were made using the MetaFluor imaging system (Universal Imaging Corp.). [Ca2+]i was calculated as reported previously (11); the used dissociation constant of Fura-2 for Ca2+ was 0.141 µM.

Statistical Analysis—Data were presented as mean ± S.E. Unpaired Student's t test was used for comparisons. A value of p < 0.05 was accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
High Extracellular Ca2+ Decreases Ito and Kv4.2 mRNA Expression—Fig. 1A, left, shows typical examples of the 4-aminopyridine-sensitive Ito recorded in myocytes incubated for 24 h at three different [Ca2+]o. Ito density showed a gradual decrease correlating with [Ca2+]o increase from 0.5 to 1.0 and 2.5 mM. On average, the current density-voltage relationships show statistically significant variations for all voltages above +10 mV depending on the [Ca2+]o (Fig. 1A, right). Detailed analysis revealed that these variations were not related to changes in voltage- or time-dependent properties of Ito during the 24-h incubation (Table I).



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  		FIG. 1.
24-h incubation with increasing [Ca2+]o produces a gradual decrease in Ito density and Kv4.2 mRNA level. A, left panel, typical pattern of Ito generated by myocytes isolated from the right ventricle of the heart and incubated 24 h at 37 °C with 0.5 (up), 1 (medium), or 2.5 mM (down) [Ca2+]o. Right panel, plots of averaged Ito densities (maximum current amplitude normalized to Cm) versus membrane potential obtained from cells incubated 24 h at 37 °C with 0.5 (gray shaded circles, n = 19), 1 (solid black circles, n = 16), or 2.5 mM [Ca2+]o (white circles, n = 22). Lines were fitted by eye to the data. B, quantitative real-time RT-PCR analysis of Kv4.2 after 24-h incubation with increased [Ca2+]o. Amplitude histograms show the relative expression Kv4.2 gene mRNA in isolated myocytes incubated 24 h with 0.5, 1, or 2.5 mM [Ca2+]o. Data were normalized to the amount of mRNA coding for cyclophilin present in the same cell extracts and then expressed as percentage of ratio observed in cells incubated with 1 mM [Ca2+]o. Columns represent mean values of n experiments. *, p < 0.05; **, p < 0.005; and ***, p < 0.0005 versus 1 mM [Ca2+]o.

 
We next investigated the expression level of Kv4.2, quantified by real-time RT-PCR after 24-h incubation of the cells at the three different [Ca2+]o. When normalized to the amount of cyclophylin mRNA present in the same cell extracts, we observed a gradual decrease of Kv4.2 mRNA expression (Fig. 1B). This did not exclude changes in other message levels, such as Kv4.3 or KChIP. Overall, these experiments suggested that the high [Ca2+]o-induced decrease in Ito density correlates with a decrease in Kv4.2 mRNA expression and, therefore, seems to involve gene regulation.

Intracellular Ca2+ Buffering Prevents Down-regulation of Ito—Increased [Ca2+]o is expected to cause an increase in cytosolic Ca2+ ([Ca2+]i) (11, 12). To check it, we evaluated the mean diastolic [Ca2+]i using Fura-2 AM in myocytes incubated 24 h. [Ca2+]i was significantly (p < 0.005) higher in cells incubated with 2.5 mM [Ca2+]o (228.8 ± 3.1 nM (n = 27)) than with 1 mM [Ca2+]o (100.7 ± 1.1 nM (n = 24)). Then we repeated our electrophysiological experiments on cells incubated 24 h in either 1 or 2.5 mM [Ca2+]o in the presence of the membrane-permeant calcium chelator, BAPTA-AM. As shown in Fig. 2, 10 µM BAPTA-AM not only increased Ito in cells exposed to 1 mM [Ca2+]o but also blunted the decrease expected at 2.5 mM [Ca2+]o (Fig. 1). Thus, decreased [Ca2+]i resulted in a significant increase of Ito slope conductance.



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  		FIG. 2.
Decrease in Ito density is mediated by increase [Ca2+]i through ICa. Pattern of Ito and bar graphs of pooled Ito slope conductance () in myocytes incubated 24 h with 1 mM [Ca2+]o (left) in the absence of drugs or co-incubated with either a membrane-permeant Ca2+ chelator (BAPTA-AM) or an ICa agonist (Bay K8644) and with 2.5 mM [Ca2+]o (right) in the absence of drugs or co-incubated with either BAPTA-AM or an ICa antagonist (Nifedipine). The Ito-voltage relationships (from +10 to +60 mV) under each condition were fitted with a linear function to estimate . n, number of experiments. (*, p < 0.05 versus 1 mM [Ca2+]o; #, p < 0.05 versus 2.5 mM [Ca2+]o).

 
Ca2+ Channels Mediate Ca2+-dependent Down-regulation of Ito—The mechanisms by which [Ca2+]o promotes an increase in [Ca2+]i may involve greater leak across the sarcolemma through L-type Ca2+ channels or stimulation of the Na+/Ca2+ exchanger working in the "reverse mode" (17). Even if small, the open probability of the L-type Ca2+ channel at rest is not null as assessed by single channel recording (18). Moreover, increasing the driving force across the membrane in presence of higher [Ca2+]o will enhance channel conductance (19). We measured Ito in myocytes incubated for 24 h with either a Ca2+ channel agonist (Bay K8644) or a Ca2+ channel antagonist (Fig. 2, +Nifedipine). Incubation with 1 µM Bay K8644 in the presence of 1 mM [Ca2+]o decreased Ito, thereby mimicking the effect of incubation at high [Ca2+]o (2.5 mM). In contrast, 24-h incubation with nifedipine (0.1 µM) blunted the decrease of Ito observed at high [Ca2+]o (Fig. 2). It is worth noting that none of those treatments altered Ito kinetics and voltage dependence (Table I).

These experiments suggested, therefore, that down-regulation of the functional expression of Ito occurs mostly by modulation of [Ca2+]i through L-type calcium current.

Ca2+-induced Down-regulation of Ito Involves the Calcineurin Pathway—Many of the actions of Ca2+ are mediated through its interaction with CaM. CaM serves as an intracellular sensor for Ca2+ and selectively activates specific down-stream signaling pathways in response to local changes in [Ca2+]i (20). To focus on the Ca2+-triggered pathway involved in the down-regulation of Ito, one group of cells was treated with the Ca2+/CaM inhibitor W7 (21). The effect of 1 µM W7 in the presence of 2.5 mM [Ca2+]o is shown in Fig. 3. W7 prevented the down-regulation of Ito induced by high Ca2+, with no change in the kinetics and in the voltage dependence of Ito (Table I). This finding suggested that Ca2+ modulation of Ito involves CaM.



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  		FIG. 3.
The calmodulin/calcineurin-signaling pathway is involved in Ca2+-dependent decrease of Ito. A, Representative examples of Ito families and an amplitude histogram of pooled in myocytes incubated 24 h with 2.5 mM [Ca2+]o in the absence of drugs or co-incubated with either a Ca2+-calmodulin complex inhibitor (W7), a calmodulin kinase II inhibitor (KN-62), or calcineurin inhibitors (FK506 or CsA). Values are expressed in percentage of of cells incubated with 1 mM [Ca2+]o B, 24-h incubation with increased [Ca2+]o produced calcineurin activity increase. Bar graph of calcineurin activity, represented by the amount of phosphate released, in myocytes incubated 24 h at 37 °C with either 1 or 2.5 mM [Ca2+]o is shown. **, p < 0.005 and ***, p < 0.0005 versus 2.5 mM [Ca2+]o.

 
Two Ca2+/CaM-dependent enzymes that have major effects on cardiac muscle function are the CaMKII (22) and the phosphatase calcineurin (23). These enzymes have distinct Ca2+ sensitivities, partner proteins, and subcellular localizations that enable them to discriminate between different types of Ca2+ signals and regulate different functions (9).

To characterize further the Ca2+ pathway for long term regulation of Ito, we used the CaMKII inhibitor KN-62 that has been reported to specifically inhibit Ca2+/CaM protein kinase isoforms (24). Exposure of cells to 1 µM KN-62 in 2.5 mM [Ca2+]o for 24 h did not prevent the down-regulation of Ito (Fig 3A). In contrast, incubation with tacrolimus (FK506, 1 µM) or CsA (25 µM), known to block calcineurin (25), prevented Ito down-regulation (Fig. 3A). Neither the timenor the voltage-dependent properties of Ito were changed after these treatments (Table I).

Exposure of cardiomyocytes to higher doses of W7 (26), FK506, or CsA (27) might exert direct acute effects on ICa and/or Ito. To confirm calcineurin involvement, we checked its activity in myocytes incubated for 24 h in either 1 or 2.5 mM [Ca2+]o, using a colorimetric assay. Calcineurin activity determined by the amount of phosphate release was increased significantly in 2.5 mM [Ca2+]o-treated cells (Fig. 3B), suggesting that enhancement of calcineurin activity after Ca2+ treatment mediates Ito down-regulation. We concluded, therefore, that the Ca2+/CaM-regulated protein phosphatase, calcineurin, is involved in the long term Ca2+-dependent down-regulation of Ito.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that increased Ca2+ influx through L-type Ca2+ channels causes an increase in [Ca2+]i, which up-regulates the Ca2+/calcineurin pathway and leads to a down-regulation of the expression of Ito (Kv4.2 gene) in isolated adult rat ventricular myocytes. Fig. 4 summarizes our data.



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  		FIG. 4.
Schematic illustration of the possible signaling pathway for Ca2+-dependent control of Ito functional expression. Increased [Ca2+]o involves Ca2+ entry through L-type Ca2+ channels. Indeed, Bay K8644 induces Ito decrease, whereas nifedipine blunts it. The Ca2+ entry leads to an [Ca2+]i increase, since BAPTA prevents Ito decrease. The rise in [Ca2+]i activates CaM. Hence, W7 blunts Ito decrease in high [Ca2+]o. The CaMKII pathway is not involved, since KN-62 has no effect. The Ca2+-calmodulin complex might activate calcineurin (CaN). Indeed, FK506 or cyclosporin A prevents Ito down-regulation by Ca2+.

 
Long term changes in cardiac cellular excitability can be generated by regulating the expression of Ito channel genes (1, 5, 6, 28). This was well demonstrated by use of transgenic and targeted deletion strategies in mice (29). On the other hand, electrical activity might affect K+ channel gene expression. It has been reported that membrane depolarization or Bay K8644 (30) enhances expression of the rapid inactivating Shaker K+ channel Kv1.4 mRNA related to Ito in newborn rat cardiomyocytes (31). Chronic membrane depolarization of cultured neonatal myocytes reduces Ito density without affecting either current kinetics or voltage dependence (32). Our data suggest that Ca2+ influx through voltage-dependent Ca2+ channels is a route for modulation of functional expression of Ito. Indeed, the Ca2+ channel agonist Bay K8644 mimics the effect of high [Ca2+]o on Ito (decrease), whereas the Ca2+ channel antagonist nifedipine has the opposite effect (increase) analogous to the effect of reducing [Ca2+]o.

We demonstrate here that the calmodulin inhibitor, W7, blunts the decrease in Ito at high [Ca2+]o. An emerging body of work indicates that the Ca2+-calmodulin complex acts as an important second messenger for various signals, including angiotensin II, endothelin-1, {alpha}-adrenergic agents, and a mechanical stretch that triggers hypertrophic growth of the myocardium (33, 34). Interestingly, these modulators regulate both Kv4 channel current densities and mRNA levels. For example, angiotensin II has been implicated in the modulation of Ito (35). An autocrine/paracrine release of ET-1 modulates Kv4.2 and Kv1.2 (35). Thyroid status also regulated Ito density and Kv4.2 mRNA levels (36). Thus, some of the effects on Ito in response to a variety of endogenous modulators and hormones known, or postulated, to modulate cardiac function might involve changes in Ca2+ signaling, in particular, related to ICa regulation. In support of this view, we recently demonstrated that aldosterone-induced down-regulation of Ito functional expression occurs secondary to modulation of Ca2+ signaling (increased ICa) (13).

Members of the diverse superfamily of voltage-activated K+ channels are modulated by phosphorylation. Such modulations are catalyzed by a variety of protein kinases including CaMKII. In atrial myocytes, phosphorylation of the K+ channel by CaMKII affects the fast inactivation kinetics (4). In the present study we did not observe either kinetic modification of Ito with the different treatments or effects of CamKII inhibitor on the Ca2+ down-regulation of Ito. However, our experiments were conducted in the presence of millimolar levels of EGTA in the pipette that will attenuate acute intracellular Ca2+ variations. More recently, CaMKII phosphorylation of neuronal Kv4.2 at the C terminus has been shown to affect cell-surface expression of A-type K+ channels (37). Our results did not rule out this effect in cardiomyocytes but show that increased diastolic [Ca2+]i effect on Ito seems CamKII-independent.

Calcineurin inhibition by CsA has been shown to prevent the decreases in mRNA levels of Kv4.2 and Kv4.3 and Ito density after myocardial infarction (38). Moreover, in contrast to a previous report (39), overexpression of calcineurin decreases the density and function of the depolarization-activated K+ currents, and the density of Ito is restored by CsA treatment in transgenic mice (40). Consistent with these observations, we report here that calcineurin inhibition with either FK506 or CsA at low concentrations prevents Ca2+ down-regulation of Ito at high [Ca2+]o.

Changes in the cytoplasmic free Ca2+ concentration constitute one of the main routes by which information is transferred from extracellular signals received by animal cells to intracellular sites. In heart, these changes have been implicated in regulating diverse physiological and pathological processes. Numerous studies indicate that alterations in intracellular Ca2+ signaling are a primary stimulus for the hypertrophic response (41, 42). As reported, the Ca2+/calmodulin-activated phosphatase calcineurin and its downstream transcriptional effector calcineurin-nuclear factor of activated T-cells (NFAT) have been implicated as transducers of the pathological hypertrophic response (43). Importantly, decreased Ito and down-regulated expression of ventricular Kv4 mRNAs are consistent findings in the hypertrophic myocardium, which can lead to increases in Ca2+ influx through L-type Ca2+ channels during longer action potential (44). On the other hand, we suggest that, in this pathological condition, increased diastolic [Ca2+]i might induce down-regulation of Ito. Thus, Ca2+ influx is under control Ito through action potential duration influence, resulting in elevated intracellular Ca2+ levels, which in turn regulates Ito and then reshapes action potential waveforms favoring Ca2+ influx. We summarize this mechanism as Ca2+-induced Ca2+ entry. More generally, our finding, if extended to other cell types, might help to understand how cells coordinate the expression of K+ channel for regulation of excitability.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: INSERM U637, CHU A. de Villeneuve, 34295 Montpellier, France. Tel.: 33-4-67-41-52-38; Fax: 33-4-67-41-52-42; E-mail: benitah{at}montp.inserm.fr.

1 The abbreviations used are: CaMKII, Ca2+/calmodulin-dependent protein kinase II; BAPTA-AM, acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CsA, cyclosporin A; RT, reverse transcriptase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Michel F. Rossier for the help with real-time RT-PCR and Dr. Ana Maria Gomez for careful reading and helpful discussion.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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