Skip Navigation


Cardiovascular Research Advance Access originally published online on February 5, 2008
Cardiovascular Research 2008 78(3):476-484; doi:10.1093/cvr/cvn031
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/3/476    most recent
cvn031v2
cvn031v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Birinyi, P.
Right arrow Articles by Magyar, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birinyi, P.
Right arrow Articles by Magyar, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

Péter Birinyi1,{dagger}, András Tóth2,{dagger}, István Jóna1, Károly Acsai3, János Almássy1, Norbert Nagy2, János Prorok2, Iuliana Gherasim1, Zoltán Papp4, Zita Hertelendi4, Norbert Szentandrássy1, Tamás Bányász1, Ferenc Fülöp5, Julius Gy. Papp2,3, András Varró2,3, Péter P. Nánási1,* and János Magyar1

1 Department of Physiology, University of Debrecen, H-4012 Debrecen, PO Box 22, Hungary
2 Department of Pharmacology and Pharmacotherapy, University of Szeged, H-6701 Szeged, PO Box 427, Hungary
3 Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, H-6701 Szeged, PO Box 427, Hungary
4 Department of Cardiology, University of Debrecen, H-4012 Debrecen, PO Box 22, Hungary
5 Institute of Pharmaceutical Chemistry, University of Szeged, H-6701 Szeged, PO Box 121, Hungary

* Corresponding author. Tel: +36 52 416 634; fax: +36 52 432 289. E-mail address: nanasi{at}phys.dote.hu

Received 20 June 2007; revised 16 January 2008; accepted 25 January 2008

Time for primary review: 26 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: This study was designed to evaluate the effects of the Na+/Ca2+ exchange (NCX) inhibitor SEA0400 on Ca2+ handling in isolated canine ventricular myocytes.

Methods and results: Intracellular Ca2+ ([Ca2+]i) transients, induced by either field stimulation or caffeine flush, were monitored using Ca2+ indicator dyes. [Ca2+]i-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni2+-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca2+ release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca2+ sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca2+]i nor the amplitude of [Ca2+]i transients was significantly altered by SEA0400 up to the concentration of 1 µM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca2+]i, and it was more pronounced in reverse than in forward mode operation at every [Ca2+]i examined. The rate of decay of the caffeine-induced [Ca2+]i transients was decreased significantly by 1 µM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl2. Neither SR Ca2+ release and uptake nor cell shortening and Ca2+ sensitivity of the contractile proteins were influenced by SEA0400.

Conclusion: The lack of any major SEA0400-induced shift in Ca2+ transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca2+]i levels) and a concomitant reduction in Ca2+ influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca2+ current.

KEYWORDS Na+/Ca2+ exchanger; Calcium handling; NCX inhibitors; SEA0400; Dog ventricular myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
The Na+/Ca2+ exchanger (NCX) is known to be a crucial mechanism of Ca2+ extrusion from cardiac cells isolated from various mammalian species.1,2 Owing to its key role in Ca2+ homeostasis, agents specifically blocking NCX would be powerful tools for studies on cardiac Ca2+ handling. Furthermore, inhibitors of NCX were reported to prevent early and delayed after-depolarizations3,4 and reduce the ischaemia/reperfusion-induced cellular injuries and arrhythmias.5,6 The principal objection against therapeutic application of NCX blockers is based on the assumption that they elevate cytosolic Ca2+ concentration ([Ca2+]i)—as can be logically expected if NCX is the dominant mechanism of Ca2+ extrusion.7 Indeed, the selective NCX blocker SEA0400 was shown to increase [Ca2+]i and contractility in murine and rat myocardium.8,9 However, substantial differences are known to exist in Ca2+ handling between small rodents (mouse and rat) and larger mammalian species, including dog and human.

Recently we have shown that SEA0400 blocked effectively both inward and outward NCX currents in canine ventricular myocytes,10 and similar results were obtained in guinea pigs.11 Importantly, SEA0400 failed to block ion channels in the surface membrane, except for L-type Ca2+ current.10,11 There is no information, however, on its possible actions of SEA0400 on the chief determinants of intracellular Ca2+ handling and contractility, such as the RyR2 Ca2+ release channel and the Ca2+ pump in the sarcoplasmic reticulum (SR), or the Ca2+ sensitivity of the contractile machinery. This information would be an absolute requirement for its future application either as a research tool or a therapeutic agent. Therefore, in the present work, the effects of SEA0400 on intracellular Ca2+ handling were evaluated using isolated canine ventricular cells, a preparation most resembling human myocytes in electrophysiological properties.12,13 We found that partial blockade of NCX by SEA0400 fails to affect the [Ca2+]i transient and contractility in canine ventricular cells. This finding can well be explained by the limited inhibitory effect of SEA0400 on NCX (further attenuated by physiological [Ca2+]i levels) and the concomitant reduction in Ca2+ influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca2+ current.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Recording of [Ca2+]i transients and cell shortening in field-stimulated myocytes
Single-canine myocytes were obtained from hearts of adult mongrel dogs of either sex, weighing 10–20 kg, anesthetized with intravenous injections of 10 mg/kg ketamine hydrochloride (Calypsol) + 1 mg/kg xylazine hydrochloride (CP-Xilazin) using the segment perfusion technique, as described previously.12 Following digestion with 1 mg/mL collagenase (Worthington, type II) in Joklik solution (Sigma, St Louis, MO, USA) containing 50 µM Ca2+ for 30 min, the left ventricular free wall was cut into small pieces and the cells were released from the tissue by gentle agitation. The viable cells were sedimented in a plexiglass chamber, allowing for continuous superfusion with oxygenized Tyrode solution containing NaCl 144, KCl 4.0, CaCl2 1.5, MgCl2 0.53, NaH2PO4 0.33, HEPES 5, and glucose 5.5 mM at pH 7.4. Intracellular Ca2+ transient and cell shortening were recorded as described earlier.4 Briefly, the cells were incubated with 2 µM Fura-2-AM (Molecular Probes, Eugene, OR, USA) for 30 min. After this incubation period, the cells were superfused with normal Tyrode solution at 35°C. Myocytes were stimulated using an electronic stimulator (DS-R1, Fonixcomp Ltd, Hungary) at a constant frequency of 1 Hz through a pair of platinum electrodes. The chamber was attached to the stage of an inverted fluorescent microscope (IX71, Olympus, Japan). Cells were excited at 360 and 380 nm from a xenon arc lamp (Optosource, Cairn, UK). The excitatory wavelengths were selected using a galvanometric monochromator (Optoscan, Cairn, UK) at 100 kHz switch rate. The emitted light was band pass-filtered and detected by a photomultiplier tube. The demultiplexed optical signals were recorded and analysed using the Acquisition Engine software (Cairn, UK). Changes in [Ca2+]i were characterized by the ratio of the fluorescence intensities obtained at 360 and 380 nm excitation (F360/F380) following corrections for non-specific background and bleaching. Cell shortening was recorded using a video edge detector system (VED-105, Crescent Electronics, Sandy, UT, USA) and expressed as per cent of the diastolic cell length. Original traces of Ca2+ transient and cell shortening are presented as average of 5–10 consecutive recordings.

2.2 Recording of [Ca2+]i transients induced by caffeine
Canine ventricular myocytes were loaded with 8 µM Fluo-4 AM at room temperature for 30 min, were mounted in a low-volume imaging chamber (RC47FSLP, Warner Instruments, USA), and paced at 1 Hz. Optical measurements were performed using an Olympus IX 71 inverted fluorescence microscope. The dye was excited at 480 nm and fluorescence was measured at 535 nm by a photon counting module (H7828, Hamamatsu, Japan). Measurements were performed using the Isosys software (Experimetria, Hungary). [Ca2+]i transients were induced by applying 5 µL caffeine (10 mM) containing Tyrode solution for 6 s at a flow rate of 50 µL/min directly onto the cell surface from a micropipette. These micropipettes, having typical tip diameter of 100 µm, were positioned by a manipulator to ~100 µm distance from the cell. The control caffeine flush was repeated after 2 min, then 1 µM SEA0400 was superfused for 5 min, and the caffeine flushes were applied again. The 2 min period of time spent between the consecutive flushes was sufficient for the cells to recover from the caffeine transient, i.e. the two subsequent challenges yielded close to identical responses, otherwise data were discarded. The two control and two SEA0400 curves were corrected against dark current, non-specific background, and bleaching artefact, averaged, and normalized so as to have identical amplitudes. When studying the effect of Ni2+, the cells were exposed to 10 mM NiCl2 containing Tyrode for 15 s prior to the beginning of the caffeine pulse and throughout the caffeine exposure.

2.3 Measurement of Na+/Ca2+ exchange current in voltage-clamped myocytes
NCX current was recorded in voltage-clamped canine ventricular myocytes as described previously.10 Briefly, after establishing the whole-cell configuration in Tyrode solution, the cell was superfused with a special K+-free bath solution (containing NaCl 135, CsCl 10, CaCl2 1, MgCl2 1, BaCl2 0.2, NaH2PO4 0.33, TEACl 10, HEPES 10, and glucose 10 mM, pH 7.4) supplemented with 20 µM ouabain, 1 µM nisoldipine, and 50 µM lidocaine in order to block Na+, K+, Ca2+, and Na/K pump currents. The pipette solution contained CsOH 140, aspartic acid 75, TEACl 20, Mg-ATP 5, HEPES 10, NaCl 20, EGTA 20, and CaCl2 10 mM (pH 7.2). Under these conditions, Ca2+ concentration of the pipette solution was estimated to be 140 nM using the WinMaxC software.14 Varying the CaCl2 and EGTA (or in some experiments BAPTA) content of the pipette solution, free Ca2+ values were set to 55, 140, 500, and 1000 nM, respectively. NCX current was recorded using ramp pulses (having the speed of 100 mV/s) delivered at a rate of 0.05 Hz. The membrane was initially depolarized from the holding potential of –40 to +60 mV, then hyperpolarized to –100 mV, and finally, the membrane potential returned to the holding potential. Outward and inward NCX currents were determined during the descending limb of the ramp at 40 and –80 mV, respectively, as indicated by the arrows in Figure 3A and B. After taking the control record in K+-free solution, the cell was superfused with 1 µM SEA0400, and finally 10 mM NiCl2 was added in order to fully block the current. Thus, total NCX current was determined at both membrane potentials as a Ni2+-sensitive current by subtracting the third record from the first one. The fraction of block induced by SEA0400 was expressed as per cent of total NCX current. Outputs from the clamp amplifier (Axopatch-2B, Axon Instruments Inc., Foster City, USA) were digitized at 20 kHz using a Digidata 1200 A/D card (Axon Instruments) and stored for later analysis, which was performed under the control of pClamp 6.0 software (Axon Instruments).

2.4 Calcium flux measurements in sarcoplasmic reticulum membrane vesicles
Heavy SR (HSR) vesicles and the ryanodine receptor Ca2+ release channel (RyR2) were isolated from canine left ventricular myocardium as described earlier.15 The vesicles were actively loaded with Ca2+, and the Ca2+ efflux was determined by measuring the extravesicular Ca2+ concentration using a Fluoromax spectrofluorometer (Jobin-Yvon, NJ, USA) modified for absorption measurements by monitoring the transmittance at 710 and 790 nm and calculating the corrected absorbance change (A710–A790) as described earlier.16 In brief, vesicles were suspended in a medium containing 92.5 mM KCl, 1 mM MgCl2, 180 µM antipyrylazo III (ICN Biomedicals, Aurora, OH, USA), 54 µM CaCl2, and 18.5 mM K-MOPS, having a final protein concentration up to 260 µg/mL. Vesicles were actively loaded with Ca2+ in the cuvette by the addition of the appropriate amount of CaCl2, and the uptake was initiated by the addition of 1 mM ATP.

The effect of SEA0400 on SR Ca2+ release was assessed in two ways. After completion of the ATP-ADP conversion, a second Ca2+ injection was applied in order to activate the release channel by adjusting the extravesicular Ca2+ to 20 µM (‘no inhibition’ protocol). SEA0400 was applied following stabilization of the rate of Ca2+ efflux. The rate of Ca2+ release was measured and compared before and after the addition of SEA0400. In the case of the ‘no activation' protocol, the Ca2+ injection contained much less Ca2+ setting extravesicular Ca2+ concentration to 2 µM only, so the rate of baseline Ca2+ efflux was very low. Then SEA0400 was added to see whether there is an increase in Ca2+ release. Rate of Ca2+ efflux was calculated from the rate of change of the light intensity just prior to and after the application of SEA0400. At the end of the experiment, Ca2+ ionophore (2 µM A23187 [GenBank] ) was used to check Ca2+ load. Extravesicular Ca2+ concentration was calibrated using a very similar protocol, in the same medium without the addition of ATP, and in the presence of 2 µM A23187. [GenBank] Calibration curve was obtained by stepwise elevation of Ca2+ while the A710–A790 values were recorded. Free Ca2+ concentrations were calculated using the absolute stability constants and the computer program developed by Fabiato and Fabiato.17

Effect of SEA0400 on the initial rate of Ca2+ uptake was studied in light SR (LSR) vesicles using a similar protocol, but in this case, the vesicles were first incubated with a given concentration (0, 0.3, 1, or 3 µM) of SEA0400 for 15 min, then the Ca2+ uptake was initiated by the addition of ATP and subsequently Ca2+. The rate of Ca2+ uptake was determined by linear regression from the slope of light intensity changes using the time window between 30 and 150 s following the addition of Ca2+.

2.5 Planar lipid bilayer measurements
A CHAPS-solubilized single RyR2 molecule was incorporated into planar lipid bilayer.18 The bilayer was formed across a 250 µm aperture of a nolrene cup using a lipid mixture composed of phosphatidyl-ethanolamine, phosphatidyl-serine, and LS{alpha}S-phosphatidyl-choline in a ratio of 5:4:1. Lipids were dissolved in n-decane up to a final concentration of 20 mg/mL. Reconstitution was initiated in symmetric buffer solution (containing 250 mM KCl, 100 µM EGTA, 150 µM CaCl2, and 20 mM PIPES, pH 7.2) by adding a small aliquot of the solubilized RyR2 to one side of the bilayer chamber defined as the cytoplasmic (cis) side. Single-channel currents were recorded at room temperature (22–25°C) using an Axopatch 200 amplifier controlled by pClamp 6.03 software (Axon Instruments). The current was filtered at 1 kHz through an eight-pole low-pass Bessel filter and digitized at 3 kHz. Channels with conductance >400 pS were accepted as RyR2. Open probability (Po) values were calculated from minimum 2 min long segments of the current traces. After application of SEA0400 (always in the cis chamber), at least 5 min period of equilibration was allowed before the measurement.

2.6 Measurement of contractility in chemically skinned single-myocyte preparations
Small, frozen tissue blocks were first defrosted and mechanically disrupted in cell isolation solution containing MgCl2 1, KCl 145, EGTA 2, ATP 4, and imidazole 10 mM at pH 7.0. The suspension was incubated for 5 min in this solution supplemented with 0.3% Triton X-100 (Sigma), washed, and kept in cell isolation solution on ice for a maximum of 12 h. Subsequently, a demembranated single cardiomyocyte was mounted between two thin needles with silicone adhesive (Dow Corning, Midland, USA) and viewed under an inverted microscope (Axiovert 135, Zeiss, Germany) as described previously.19 One needle was attached to a force transducer element (SensoNor, Horten, Norway) and the other to an electromagnetic motor (Aurora Scientific Inc., Aurora, Canada). Measurements were performed at 15°C, and the average sarcomere length was adjusted to 2.2 µm.

Composition of relaxing and activating solutions used in force measurements was calculated as described previously.17,20 The pCa values (i.e. –log[Ca2+]) of the relaxing and activating solutions were 9.0 and 4.75, respectively. Solutions with intermediate Ca2+ levels were obtained by mixing activating and relaxing solutions. All solutions used for force measurements contained also MgCl2 1, MgATP 5, phosphocreatine 15, and N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid 100 mM. The ionic equivalent was adjusted to 150 with KCl at an ionic strength of 186. Isometric force was measured after the preparation had been transferred from the relaxing solution to a Ca2+-containing one. When a steady level of force was reached, the length of the myocyte was abruptly (within 2 ms) reduced by 20% and then quickly re-stretched. As a result, the force first dropped from the peak isometric level to zero and then started to re-develop. The passive component of force was determined in relaxing solution following the Ca2+ contractures. Ca2+-activated isometric force was calculated by subtracting the passive force from the peak isometric force, obtained at pCa = 4.75. After the first maximal activation at pCa = 4.75, the resting sarcomere length was re-adjusted to 2.2 µm, when it was necessary. This second maximal activation at pCa = 4.75 was used to calculate maximal isometric force (Pmax). Cells were subsequently exposed to a series of solutions with intermediate pCa values. The Ca2+-activated force measured at these intermediate levels of activation was normalized to Pmax, and data were fit to the Hill equation in order to estimate Ca2+ concentration, resulting in half-maximal force production (pCa50) as shown in Figure 6A. If at the end of this series re-exposure to pCa 4.75 yielded a Pmax <80% of the initial value, the measurement was discarded.

2.7 Statistics
Results are expressed as mean ± SEM values. Statistical significance of differences obtained between control and SEA0400 treated preparations was evaluated with Student’s t-test for paired or unpaired data, as relevant. Differences were considered significant when P was <0.05.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Effect of SEA0400 on [Ca2+]i transient and cell shortening in field-stimulated myocytes
Myocytes were stimulated at a constant frequency of 1 Hz through a pair of platinum electrodes. After establishing steady-state contractions and [Ca2+]i transients, the perfusion was switched to the solution containing 0.1, 0.3, or 1 µM SEA0400, and 5 min was allowed to develop full drug effects. Original recordings of [Ca2+]i transients and cell shortening before and after administration of SEA0400 are presented in Figure 1A and B. Average values obtained for the diastolic fluorescent ratio, F360/F380 amplitude, and cell shortening are summarized in Figure 1C–E. Data indicate that no significant change in these parameters was caused by SEA0400 in the 0.1–1 µM concentration range. Similarly, no significant change was observed in the decay time constant of the [Ca2+]i transient determined before and after the application of 1 µM SEA0400 (269 ± 41 and 266 ± 17 ms, respectively, n = 9).


Figure 1
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Effect of 0.1, 0.3, and 1 µM SEA0400 on intracellular Ca2+ transients (A) and cell shortening (B) in isolated canine ventricular cells stimulated at a constant frequency of 1 Hz. [Ca2+]i is expressed as a fluorescent ratio (F360/F380), and cell shortening is given as a fraction of diastolic cell length. Recordings were made from the same cells before (open circle) and 5 min after (filled circle) the application of SEA0400. Average values obtained in the presence of SEA0400 for the diastolic fluorescent ratio (C), F360/F380 amplitude (D), and cell shortening (E), all expressed as per cent of control. Numbers in parentheses denote the number of cells studied.

 
3.2 Effect of SEA0400 on [Ca2+]i transients induced by caffeine
Since the relaxation kinetics of a [Ca2+]i transient reflects the kinetic properties of Ca2+ elimination from the cytosol, NCX inhibition is accompanied with the elongation of the decay of [Ca2+]i transients induced by rapid application of caffeine.21 Therefore, the effect of 1 µM SEA0400 was studied on [Ca2+]i transients evoked by superfusion with 10 mM caffeine for 6 s. In order to compare the time course of decay, the transients were normalized to identical amplitudes and superimposed (Figure 2A). The decay time constant, determined using mono-exponential fit, was significantly increased by 1 µM SEA0400 from 1.03 ± 0.05 to 1.32 ± 0.06 s (P < 0.05, n = 10), whereas the respective time-matched control values (i.e. when SEA0400 was omitted from the solution) were 0.96 ± 0.05 and 0.95 ± 0.04 s (NS, n = 8). These results directly prove that under the given conditions, SEA0400 is capable to partially inhibit NCX. The NCX-blocking potency of SEA0400 can be qualitatively evaluated by comparing with that of 10 mM NiCl2, which is known to fully block the exchanger. As in the presence of NiCl2 the decaying limb could not be fitted exponentially, the time of half-relaxation was determined: it was increased by NiCl2 from 1.3 ± 0.1 to 10.1 ± 1.3 s (P < 0.05, n = 4). Although the effect of 1 µM SEA0400 on the rate of decay of the caffeine-induced [Ca2+]i transient was only a fraction of that observed with 10 mM NiCl2, it was clearly detectable. However, these results suggest that the NCX-blocking effect of SEA0400 may be relatively moderate when [Ca2+]i is elevated.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Representative superimposed records showing the effect of 1 µM SEA0400 (A) and 10 mM NiCl2 (B) on the time course of the [Ca2+]i transients elicited by superfusion of 10 mM caffeine for 6 s. Before the application of caffeine, the myocytes were continuously paced at a frequency of 1 Hz. Stimulation was suspended during the recording period.

 
3.3 Calcium-dependent effect of SEA0400 on Na+/Ca2+ exchange current
NCX current was recorded as a Ni2+-sensitive current using the descending limb of a ramp pulse changing slowly from 60 to –100 mV during 1.6 s. Outward and inward NCX current was determined at 40 and –80 mV, respectively (Figure 3A and B; for further details, see Methods section). One micro mole SEA0400 suppressed both inward and outward NCX current; however, suppression of the outward current was more pronounced than the inward current block. The effect of SEA0400 on NCX current was studied at various levels of [Ca2+]i (ranging from 55 nM to 1 µM) set by changing the CaCl2 and EGTA content of the pipette solution. The blocking effect of SEA0400 on NCX was reduced by 50% with increasing [Ca2+]i from 55 nM to 1 µM (Figure 3C). It must be noted, however, that 1 µM SEA0400 was able to block 28 ± 7% of the inward NCX current even in the presence of 1 µM [Ca2+]i.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Effect of SEA0400 on Na+/Ca2+ exchange current as a function of [Ca2+]i. Ramp protocols used to elicit Na+/Ca2+ exchange current (A) and superimposed current traces obtained in K+-free bath solution (control), in the presence of 1 µM SEA0400, and after addition of 10 mM NiCl2 in order to fully block Na+/Ca2+ exchange current (B). Outward (reverse mode) and inward (forward mode) Na+/Ca2+ exchange currents were determined during the descending limb of the ramp at 40 and –80 mV, respectively, as indicated by the arrows. Blocking effect of 1 µM SEA0400 on the inward (open symbols) and outward (filled symbols) Na+/Ca2+ exchange currents as a function of [Ca2+]i (C). Free Ca2+ was set to 55, 140, 500, and 1000 nM by varying the CaCl2 and EGTA content of the pipette solution. Symbols and bars represent mean ± SEM, numbers in parentheses denote the number of cells studied.

 
In theory, the missing effect of SEA0400 on myocyte shortening might be due to some direct effect of SEA0400 on SR Ca2+ handling, or by altering the Ca2+ responsiveness of the myofilaments. In the following, therefore, these possibilities are examined.

3.4 Effect of SEA0400 on sarcoplasmic reticulum calcium release
In HSR vesicle preparations, the effect of SEA0400 was studied by measuring Ca2+ release from the vesicles in the presence of 20 µM extravesicular Ca2+. This Ca2+ concentration makes the RyR2 partially open as seen from the increase of extravesicular Ca2+ concentration in Figure 4A. Addition of SEA0400 failed to alter the rate of release as revealed by the unchanged slope of the Ca2+ flux, indicating that the drug did not modify Ca2+ flux through the partially open channel (‘no inhibition’ protocol). The rate of Ca2+ release was 12.4 ± 0.9 nmol/min/mg protein before the application of SEA0400, whereas it was 11.8 ± 1.1, 13.1 ± 1.2, and 13.2 ± 1.2 nmol/min/mg protein in the presence of 0.3, 1, and 3 µM SEA0400, respectively (n = 7 for each).


Figure 4
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 Effect of SEA0400 on Ca2+ release [(A) and (B)] and Ca2+ uptake [(C) and (D)] in sarcoplasmic reticulum membrane vesicles. (A) Typical record of the ‘no inhibition’ protocol. After loading the vesicles with Ca2+ by addition of ATP in the presence of Ca2+, the extravesicular Ca2+ concentration was set to 20 µM by a second Ca2+ injection in order to activate RyR2 channels. The rate of release was calculated from the slope of the curve measured just before and after application of SEA0400. Light intensity, shown in the ordinate, is inversely related to extravesicular Ca2+ concentration. (B) The effect of SEA0400 on the resting (closed) RyR2 channels was studied using the ‘no activation’ protocol. In this case, after loading the vesicles, the extravesicular Ca2+ concentration was set to 2 µM. This caused only negligible activation of the channels, which was not modified by the subsequent application of SEA0400. Finally, the Ca2+content of the vesicles was fully released by the Ca2+ ionophore A23187 [GenBank] (2 µM). (C) Representative pair of superimposed recordings showing the initial rate of Ca2+ uptake as determined from the slope of the post-Ca2+ segment of the light intensity signal. Ca2+ uptake was initiated by addition of Ca2+ (arrows) in the presence or absence of 3 µM SEA0400 to increase the extravesicular Ca2+ concentration to 10 µM. (D) Average rate of Ca2+ uptake determined without SEA0400 pre-treatment or in the presence of 0.3, 1, and 3 µM SEA0400. Numbers in parentheses denote the number of independent determinations.

 
Similarly designed experiments were performed to study the effect of SEA0400 on closed RyR2 channels. In this case, lower, just subthreshold extravesicular Ca2+ concentrations were applied. The horizontal segment of the record indicates that the channels are practically closed. SEA0400 failed to induce any Ca2+ release, in contrast to the Ca2+ ionophore, A23187 [GenBank] (2 µM), which caused marked Ca2+ release, showing that the vesicles were indeed fully packed with Ca2+. A typical experiment performed using this ‘no activation’ protocol is depicted in Figure 4B. Under these experimental conditions, the rate of baseline Ca2+ release was 2.2 ± 0.31 nmol/min/mg protein before the application of SEA0400, whereas it was 2.2 ± 0.3, 2.3 ± 0.28, and 2.3 ± 0.3 nmol/min/mg protein in the presence of 0.3, 1, and 3 µM SEA0400, respectively (n = 6 for each).

3.5 Effect of SEA0400 on sarcoplasmic reticulum calcium uptake
Activity of the SR calcium pump was estimated by measuring the initial uptake rate of LSR vesicles by measuring time-dependent changes in extravesicular Ca2+ concentration under maximal pumping rate conditions. The uptake rate was determined in the absence and presence of 0.3, 1, and 3 µM of SEA0400 (Figure 4C and D). The initial uptake rates were identical with and without SEA0400, indicating that SEA0400 fails to modify the re-uptake of Ca2+ from the intracellular space to the lumen of the SR.

3.6 Effect of SEA0400 on the gating of single-RyR2 channels
Effect of SEA0400 on single-RyR2 channels was also studied using Müller-type bilayer, where the solubilized RyR2 was incorporated into the lipid membrane, and single-channel current was measured under voltage clamp conditions. According to the analogue records presented in Figure 5A, the channels were almost fully open in the presence of 50 µM Ca2+ on the cis side: their open probability (Po) was 0.978 ± 0.047 (n = 9). Reducing cis Ca2+ to 472 nM channels exhibited a Po of 0.043 ± 0.008 (n = 9). After addition of 3 µM SEA0400, the open probability failed to change significantly (Po = 0.032 ± 0.005, n = 6). It is also evident from Figure 5A that the conductance of RyR2 remained unaltered in the presence of SEA0400, since amplitudes of single-channel currents were the same. Determining the mean open time of the channel revealed that two components are necessary to fit the open-time histograms with a reasonable correlation coefficient. No significant differences were observed in the faster or slower time constants estimated before and after the application of 3 µM SEA0400, as shown in Figure 5B. In summary, SEA0400 failed to modify any of the characteristic parameters of RyR2 up to the concentration of 3 µM.


Figure 5
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 Effect of SEA0400 on solubilized RyR2 incorporated into a Müller-type lipid bilyer. (A) Single-channel currents measured at the holding potential of 57 mV, first in the presence of 50 µM Ca2+ at the cis side (upper trace); later, cis Ca2+ concentration was decreased to 472 nM by addition of K2H2EGTA (middle trace). This resulted in a marked reduction of the open probability of the channel. Finally, SEA0400 was added to a final concentration of 3 µM (lower trace). Markers C and O indicate current levels associated to the closed and open channel state, respectively. (B) Representative mean open time histograms constructed before the application of SEA0400 (from a 54 s long current trace) and in the presence of 3 µM SEA0400 (from a 20 s long recording period). Calculated mean open time values are given.

 
3.7 Effect of SEA0400 on Ca2+ sensitivity of contractile proteins in chemically skinned myocytes
As described in the Methods section, first the force–pCa relationship was determined by exposing the preparation to stepwise increases of external Ca2+ in order to determine the half-activation Ca2+ concentration, defined as pCa50. The value estimated for pCa50 in these preparations was 6.4 (Figure 6A). The maximal isometric Ca2+-activated force was 27.9 ± 9.8 kN/m2, and the passive force was 3.6 ± 1.7 kN/m2 in the four permeabilized cardiomycytes, each obtained from a different heart. Force development was measured in the absence and presence of 1 µM SEA0400 at the single Ca2+ concentration of pCa = 6.4. Therefore, any hypothetical SEA0400-dependent alteration in Ca2+-dependent force production was not limited by Ca2+ saturation of the contractile machinery. SEA0400 was dissolved in concentrated DMSO; therefore, control measurements in the presence of DMSO were also included. The Ca2+-activated force did not differ in the presence or absence of SEA0400 (0.55 ± 0.03 and 0.55 ± 0.04% of Pmax, respectively). Moreover, force was also about the same when only DMSO was administered (0.57 ± 0.03% of Pmax) at pCa = 6.4 (Figure 6B).


Figure 6
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 Effect of SEA400 on Ca2+ sensitivity of the contractile proteins. (A) Force–pCa relationship determined in four skinned canine myocyte preparations. Solid curve was obtained by fitting data to the Hill equation. (B) Contractile force (normalized to Pmax) measured at pCa = 6.4 in control, in the presence of DMSO, and 1 µM SEA0400. Numbers in parentheses denote the number of experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
In this study, we have demonstrated that—in contrast to murine and rat ventricular myocardium8,9—partial blockade of NCX by SEA0400 fails to affect the [Ca2+]i transient and contractility in canine ventricular cells. In the absence of any direct effect of SEA0400 on sarcoplasmic Ca2+ transport and the contractile proteins (as also shown in the present study), this is only possible if the net effect of SEA0400 on transmembrane Ca2+ transport is close to zero (i.e. the effects on the outward and inward Ca2+ fluxes are approximately equal). As it was previously shown, 1 µM SEA0400 blocked more than 50% of inward NCX current when [Ca2+]i was kept relatively low.10 Such significant inhibition of NCX current should lead to [Ca2+]i accumulation accompanied by increased [Ca2+]i transients and cell shortening, which changes clearly failed to occur. On the other hand, the NCX-blocking effect of SEA0400 in canine myocytes depended on the actual level of [Ca2+]i. Suppression of NCX current was reduced to half when [Ca2+]i was increased from 55 nM to 1 µM, resulting in a 28% of inhibition. Since [Ca2+]i is believed to reach much higher values in the subsarcolemmal fuzzy space, the blocking effect of SEA0400 on NCX may even be lower under physiological conditions. This moderate reduction of Ca2+ extrusion, however, still must be balanced by proportional reduction of Ca2+ influx in absence of inotropic response. Since 1 µM SEA0400 was shown to decrease ICa by 20% in canine ventricular cells,10 this balance could easily be achieved. At 0.3 µM concentration, where the SEA0400-induced ICa blockade was <5% while the inhibition of NCX was almost as strong as measured with 1 µM SEA0400,10 further mechanisms have to be involved in the maintenance of equilibrium. For instance, congruently with our present results, SEA0400 was shown to block the reverse mode action of NCX more effectively than its forward mode operation,22 which may result in the reduction of Ca2+ entry. The large reserve capacity of NCX, together with the activation of NCX-independent mechanisms of Ca2+ extrusion (via sarcolemmal-active Ca2+ pump) or SR-independent Ca2+ sequestration (via mitochondrial Ca2+ uptake), may also contribute to elimination of cytosolic calcium. The signal to turn on these compensatory mechanisms is apparently missing, since neither diastolic nor systolic [Ca2+]i was elevated by SEA0400. However, it must be borne in mind that moderate changes in subsarcolemmal [Ca2+]i are not likely detected by the conventional [Ca2+]i-monitoring techniques. Although none of the outlined mechanisms alone seems to be sufficient to account for the inability of 0.3 µM SEA0400 to modify [Ca2+]i transient and contractility in canine myocytes, their combined effects may likely explain our observations. The contribution of NCX-independent mechanisms to Ca2+ elimination is supported by the finding that relatively normal [Ca2+]i transients could be observed in embryonic heart tubes of NCX knockout mice.23

Our present results with SEA0400 apparently conflicts with the observations of Hobai et al. obtained with exchange inhibitory peptide in healthy canine heart cells.24 In that study 27% inhibition of NCX resulted in approximately 50% increase in systolic [Ca2+]i. This discrepancy can likely be attributed to the pharmacological properties of SEA0400 as discussed earlier, i.e. the predominantly reverse mode NCX inhibition combined with some suppression of ICa. However, the reaction of a failing heart to NCX inhibition may also be different from normal.

As it was mentioned earlier, markedly different results were obtained in small rodents, where 1 µM SEA0400 increased the magnitude of both [Ca2+]i transients and cell shortening.8,9 This may be due to the lack of a distinct plateau phase in their action potential—in contrast to dogs and humans. Consequently, NCX spends relatively longer time in the forward mode during the cardiac cycle in rodents than in larger animals, favouring increased Ca2+ extrusion, which in turn may lead to increased [Ca2+]i transient and force development when NCX is blocked. In our canine myocytes, however, SEA0400-induced changes in [Ca2+]i transient and contraction were absent, suggesting that results obtained in small rodents can be interpreted for dogs or humans only with a particular care.

Since the selectivity of SEA0400 has been questioned,23 it is crucial to know whether SEA0400 can directly modify sarcoplasmic Ca2+ fluxes. Our results indicate that SEA0400 fails to alter the conductance or gating kinetics of the RyR2 Ca2+ release channel, has no effect on the rate of sarcoplasmic Ca2+ release and re-uptake up to a concentration of 3 µM, or Ca2+ sensitivity of the myofilaments. These results, congruently with the unchanged [Ca2+]i transients, indicate that SEA0400 exerts no direct action on sarcoplasmic Ca2+ handling in canine ventricular cells. Therefore, 0.3 µM SEA0400, which had negligible effects on transmembrane ion currents,10 can be considered a selective inhibitor of NCX, and as such, is a suitable tool to study the effects of partial NCX inhibition in cardiac myocytes.

The [Ca2+]i-dependent action of SEA0400 on NCX, shown by us in patch-clamped canine myocytes, seems to be an important feature of the compound. Although it has already been observed in the case of outward NCX current (corresponding to the reverse mode operation of NCX) recorded from Xenopus oocytes,22,25 the present study is the first one to demonstrate this effect on the forward mode operation of NCX. Furthermore, our experimental conditions better approximated the physiological situation regarding the ionic composition of the extracellular and intracellular solutions applied. The [Ca2+]i-dependent inhibitory effect of SEA0400 on NCX may be related to the SEA0400-induced acceleration of Na+-dependent inactivation of the current, which is enhanced by [Na+]i and reduced by [Ca2+]i.26 This implicates that the effect of SEA0400 is augmented under conditions of high [Na+]i., as it was shown to occur in Xenopus oocytes expressing NCX 1.1 isoform.22 These results together may explain why the NCX blocker SEA0400 is able to prevent Ca2+ overload under conditions of ischaemia/reperfusion (when NCX, driven by the elevated [Na+]i, works predominantly in its reverse mode) without causing Ca2+ overload in healthy canine (and probably human) cardiac tissues, where NCX operates entirely in forward direction.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
Hungarian Research Found (OTKA-K68457, OTKA-K73160, OTKA-NI61902, OTKA-K61442); Hungarian Ministry of Health (ETT-060/2006, ETT-449/2006); National Research and Development Programmes (NKFP 1A/046/2004), Hungarian Ministry of Economy (GVOP-3.2.1-2004-04-0104/3.0); European Community (HPRN-CT-2002-00331, LSHM-CT-2005-018833, LSHM-CT-2005-018676). Z.P. holds a Bolyai Fellowship.


   Acknowledgements
 
The authors thank Mrs Vighné Katalin Horváth for the excellent technical assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res (2000) 87:275–281.[Free Full Text]
  2. Reuter H, Pott C, Goldhaber JI, Henderson SA, Philipson KD, Schwinger RH. Na+/Ca2+ exchange in the regulation of cardiac excitation–contraction coupling. Cardiovasc Res (2005) 67:198–207.[Abstract/Free Full Text]
  3. Watano T, Harada Y, Harada K, Nishimura N. Effect of Na+/Ca2+ exchanger inhibitor, KB-R7943 on ouabain-induced arrhythmias in guinea-pigs. Br J Pharmacol (1999) 127:1846–1850.[CrossRef][Web of Science][Medline]
  4. Nagy ZA, Virág L, Tóth A, Biliczki P, Acsai K, Bányász T, et al. Selective inhibition of sodium-calcium exchanger by SEA-0400 decreases early and delayed after depolarization in canine heart. Br J Pharmacol (2004) 143:827–831.[CrossRef][Web of Science][Medline]
  5. Yamamura K, Tani M, Hasegawa H, Gen W. Very low dose of the Na+/Ca2+ exchanger inhibitor, KB-R7943, protects ischemic reperfused aged Fisher 344 rat hearts: considerable strain difference in the sensitivity to KB-R7943. Cardiovasc Res (2001) 52:397–406.[Abstract/Free Full Text]
  6. Takahashi K, Takahashi T, Suzuki T, Onishi M, Tanaka Y, Hamano-Takahashi A, et al. Protective effects of SEA0400, a novel and selective inhibitor of the Na+/Ca2+ exchanger, on myocardial ischemia-reperfusion injuries. Eur J Pharmacol (2003) 458:155–162.[CrossRef][Web of Science][Medline]
  7. Sipido KR, Varro A, Eisner D. Sodium calcium exchange as a target for antiarrhythmic therapy. Handb Exp Pharmacol (2006) 171:159–199.[Medline]
  8. Tanaka H, Namekata I, Takeda K, Kazama A, Shimizu Y, Moriwaki R, et al. Unique excitation–contraction characteristics of mouse myocardium as revealed by SEA0400, a specific inhibitor of Na+–Ca2+ exchanger. Naunyn-Sch Arch Pharmacol (2005) 371:526–534.[CrossRef]
  9. Acsai K, Kun A, Farkas AS, Fülöp F, Nagy N, Balázs M, et al. Effect of partial blockade of the Na+/Ca2+ exchanger by SEA0400 on Ca2+ handling in isolated rat ventricular myocytes. Eur J Pharmacol (2007) 576:1–6.[CrossRef][Web of Science][Medline]
  10. Birinyi P, Acsai K, Bányász T, Tóth A, Horváth B, Virág L, et al. Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes. Naunyn Schmiedebergs. Arch Pharmacol (2005) 372:63–70.[CrossRef]
  11. Tanaka H, Nishimaru K, Aikawa T, Hirayama W, Tanaka Y, Shigenobu K. Effect of SEA0400, a novel inhibitor of sodium–calcium exchanger, on myocardial ionic currents. Br J Pharmacol (2002) 135:1096–1100.[CrossRef][Web of Science][Medline]
  12. Szentandrássy N, Bányász T, Bíró T, Szabó G, Tóth IB, Magyar J, et al. Apico-basal inhomogeneity in distribution of ion channels in canine and human ventricular myocardium. Cardiovasc Res (2005) 65:851–860.[Abstract/Free Full Text]
  13. Szabó G, Szentandrássy N, Bíró T, Tóth IB, Czifra G, Magyar J, et al. Asymmetrical distribution of ion channels in canine and human left ventricular wall: epicardium versus midmyocardium. Pflügers Arch (2005) 450:307–316.[CrossRef][Web of Science][Medline]
  14. Patton C, Thompson S, Epel D. Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium (2004) 35:427–431.[CrossRef][Web of Science][Medline]
  15. Esteve E, Smida-Rezgui S, Sarkozi S, Szegedi C, Regaya I, Chen L, et al. Critical amino acid residues determine the binding affinity and the Ca2+ release efficiacy of maurocalcine in skeletal muscle cells. J Biol Chem (2003) 278:37822–37831.[Abstract/Free Full Text]
  16. Sarkozi S, Szentesi P, Jona I, Csernoch L. Effects of cardiac glycosides on excitation-contraction coupling in frog skeletal muscle fibres. J Physiol (Lond) (1996) 495:611–626.[Abstract/Free Full Text]
  17. Fabiato A, Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) (1979) 75:463–505.[Medline]
  18. Mouton L, Ronjat M, Jona I, Villaz M, Feltz K, Maulet ZS. Skeletal and cardiac ryanodine receptors binds to the Ca2+-sensor region of the dihydropiridine receptor {alpha} 1C subunit. FEBS Lett (2001) 505:441–444.[CrossRef][Web of Science][Medline]
  19. van der Velden J, Klein LJ, Zaremba R, Boontje NM, Huybregts MA, Stooker W, et al. Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts. Circulation (2001) 104:1140–1146.[Abstract/Free Full Text]
  20. Papp Z, Szabo A, Barends JP, Stienen GJM. The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes. J Physiol (Lond) (2002) 543:177–189.[Abstract/Free Full Text]
  21. Bassani RA, Bassani JW, Bers DM. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol (Lond) (1992) 453:591–608.[Abstract/Free Full Text]
  22. Lee C, Visen NS, Dhalla NS, Le HD, Choptiany MIP, Gross G, et al. Inhibitory profile of SEA0400 [2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline] assessed on the cardiac Na+–Ca2+ exchanger, NCX1.1. J Pharmacol Exp Ther (2004) 311:748–757.[Abstract/Free Full Text]
  23. Reuter H, Henderson SA, Han T, Matsuda T, Baba A, Ross RS, et al. Knockout mice for pharmacological screening. Testing the specificity of Na+–Ca2+ exchange inhibitors. Circ Res (2002) 91:90–92.[Abstract/Free Full Text]
  24. Hobai IA, Maack C, O’Rourke B. Partial inhibition of sodium/calcium exchange restores cellular calcium handling in canine heart failure. Circ Res (2004) 95:292–299.[Abstract/Free Full Text]
  25. Bouchard R, Omelchenko A, Le HD, Choptiany P, Matsuda T, Baba A, et al. Effects of SEA0400 on mutant NCX1.1 Na+–Ca2+ exchangers with altered ionic regulation. Mol Pharmacol (2004) 65:802–810.[Abstract/Free Full Text]
  26. Iwamoto T, Watanabe Y, Kita S, Blaustein MP. Na+/Ca2+ exchange inhibitors: a new class of calcium regulators. J Cardiovasc Hematol Disord Drug Targets (2007) 7:188–198.[CrossRef]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Xu, C. S. Kappler, S. K. Mani, N. R. Shepherd, L. Renaud, P. Snider, S. J. Conway, and D. R. Menick
Chronic Administration of KB-R7943 Induces Up-regulation of Cardiac NCX1
J. Biol. Chem., October 2, 2009; 284(40): 27265 - 27272.
[Abstract] [Full Text] [PDF]

Home page
 EuropaceHome page
G. Antoons and K. R. Sipido
Targeting calcium handling in arrhythmias
Europace, December 1, 2008; 10(12): 1364 - 1369.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/3/476    most recent
cvn031v2
cvn031v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Birinyi, P.
Right arrow Articles by Magyar, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birinyi, P.
Right arrow Articles by Magyar, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?