AJP - Regu AJP: Heart and Circulatory Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 279: R1856-R1864, 2000;
0363-6119/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hove-Madsen, L.
Right arrow Articles by Tort, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hove-Madsen, L.
Right arrow Articles by Tort, L.
Vol. 279, Issue 5, R1856-R1864, November 2000

Na+/Ca2+-exchange activity regulates contraction and SR Ca2+ content in rainbow trout atrial myocytes

Leif Hove-Madsen, Anna Llach, and Lluis Tort

Department of Physiology, Cell Biology and Immunology, Faculty of Science, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have used the whole cell configuration of the patch-clamp technique to measure sarcolemmal Ca2+ transport by the Na+/Ca2+ exchanger (NCX) and its contribution to the activation and relaxation of contraction in trout atrial myocytes. In contrast to mammals, cell shortening continued, increasing at membrane potentials above 0 mV in trout atrial myocytes. Furthermore, 5 µM nifedipine abolished L-type Ca2+ current (ICa) but only reduced cell shortening and the Ca2+ carried by the tail current to 66 ± 5 and 67 ± 6% of the control value. Lowering of the pipette Na+ concentration from 16 to 10 or 0 mM reduced Ca2+ extrusion from the cell from 2.5 ± 0.2 to 1.0 ± 0.2 and 0.5 ± 0.06 amol/pF. With 20 µM exchanger inhibitory peptide (XIP) in the patch pipette Ca2+ extrusion 20 min after patch break was 39 ± 8% of its initial value. With 16, 10, and 0 mM Na+ in the pipette, the sarcoplasmic reticulum (SR) Ca2+ content was 47 ± 4, 29 ± 6, and 10 ± 3 amol/pF, respectively. Removal of Na+ from or inclusion of 20 µM XIP in the pipette gradually eliminated the SR Ca2+ content. Whereas ICa was the same at -10 or +10 mV, Ca2+ extrusion from the cell and the SR Ca2+ content at -10 mV were 65 ± 7 and 80 ± 4% of that at +10 mV. The relative amount of Ca2+ extruded by the NCX (about 55%) and taken up by the SR (about 45%) was, however, similar with depolarizations to -10 and +10 mV. We conclude that modulation of the NCX activity critically determines Ca2+ entry and cell shortening in trout atrial myocytes. This is due to both an alteration of the transsarcolemmal Ca2+ transport and a modulation of the SR Ca2+ content.

caffeine; L-type calcium current; cardiac; excitation-contraction coupling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN MAMMALIAN CARDIAC MYOCYTES activation of contraction occurs through Ca2+-induced Ca2+ release with the sarcoplasmic reticulum (SR) contributing with the major part of the total Ca2+, whereas the transsarcolemmal Ca2+ flux primarily serves as a trigger for the Ca2+-induced Ca2+ release (12). Furthermore, Ca2+ current is thought to be the main Ca2+ trigger source under physiological conditions (5, 7, 24, 28). Na+/Ca2+ exchanger (NCX) also has been reported to contribute to the activation of contraction under certain experimental conditions (10, 15, 23, 37). The relative contribution of transsarcolemmal Ca2+ flux and the SR to the activation of contraction varies among species (13, 19, 30). Thus in the rabbit and guinea pig heart transsarcolemmal Ca2+ flux has been estimated to account for 30-35% of the total Ca2+ (3, 32), whereas the SR contributes with more than 90% in the rat heart (3, 7). It recently has been reported that overexpression of the NCX in transgenic mice results in a larger contribution of the NCX to the activation of contraction and an increased SR Ca2+ load (31). It does, however, seem clear that the contribution of the NCX to the activation of contraction is limited under physiological conditions in mammalian myocytes (23, 28, 29, 31), although quantitative studies of Ca2+ transport by the NCX in intact cell, under physiological conditions, are difficult because of interference from the SR and the L-type Ca2+ current (ICa). In contrast to this, transsarcolemmal Ca2+ flux has been reported to play a more important role in the activation of contraction in the neonate (21, 38) and the lower vertebrate heart (11, 33, 36). The evidence for the importance of transsarcolemmal Ca2+ flux in the lower vertebrate heart has to a large extent been based on ultrastructural studies (27) and quantitative information from the amphibian heart (14, 26), whereas the information available for other lower vertebrates has been largely qualitative (11, 33). Recent studies in a few teleost species have, however, quantified transsarcolemmal Ca2+ transport in isolated myocytes (20, 35, 36). Furthermore, characterization of Ca2+ transport by the trout NCX has been done in vitro (34), and recently the NCX from trout heart has been cloned and expressed in oocytes (39). Together, these results suggest that sarcolemmal Ca2+ flux may indeed also be important in the carp heart (35, 36), although ICa alone is not sufficient to activate contraction in trout cardiomyocytes (20). In agreement with this, we also have found that the SR could contribute significantly to the activation of contraction in trout cardiac myocytes (16, 19). Furthermore, these results suggested that the NCX could account for about 50% of the transsarcolemmal Ca2+ influx in trout. The present study was designed to quantify Ca2+ transport by the NCX and to determine its role in the activation of contraction and SR Ca2+ loading in an animal species where the Ca2+ influx through the NCX is expected to be quantitatively important.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Trout (Oncorhynchus mykiss) atrial myocytes were obtained by enzymatic digestion of the heart as described previously (18). The experimental protocol for cell isolation has been approved by the Ethical Commission on Animal and Human Experimentation at Univestitat Autònoma de Barcelona (DARP 1017). Briefly, the fish were killed by a blow to the head and decapitation. A cannula was then inserted into the ventricle and the heart was rinsed for 5-10 min and then perfused for 40 min at 20°C with a nominally Ca2+-free Tyrode containing 50 µM EGTA, 37.5 µM Ca2+, 0.1 mg/ml collagenase (Yakult, Japan), 0.1 mg/ml trypsin (Sigma), and 0.5 mg/ml BSA. At the end of the digestion, the atrium was cut off the ventricle, agitated gently, and filtered through a nylon mesh. Ca2+ was gradually increased to 750 µM, and cells were stored at 6°C.

Electrophysiological measurements. Ionic currents were measured using a software driven patch-clamp amplifier (EPC-9, Heka, Germany). After seal formation, the cell was lifted up from the bottom of a petri dish and placed in front of one of eight capillaries containing the desired extracellular solution. Standard internal and external solutions were used to eliminate Na+ and K+ and Na+-K+ pump currents. The internal medium contained (in mM) 100 CsCl, 3.1 Na2ATP, 4 MgCl2, 5 Na2 phosphocreatine, 0.42 Na2GTP, 0.025 EGTA, 10 HEPES, and 20 tetraethylammonium. pH was adjusted to 7.2 with CsOH. The external medium contained (in mM) 107 NaCl, 20 CsCl, 1.8 MgCl2, 4 NaHCO3, 0.8 NaH2PO4, 1.8 CaCl2, 10 HEPES, 5 glucose, and 5 pyruvate. pH was adjusted to 7.4 with NaOH. Na+ current in trout atrial cells could be eliminated with 1 µM TTX. Exchanger inhibitory peptide (XIP) was a generous gift from Dr. K. D. Phillipson.

Experimental protocols. The cell was stimulated continuously, using a 200-ms depolarization from a holding potential of -80 to 0 mV given every 8 s. This stimulation protocol allowed examination of ICa and the tail current (Itail) elicited on repolarization to -80 mV. Furthermore, the dependency of the membrane currents on membrane potential was examined by consecutive 200-ms depolarizations to different test potentials from a holding potential of -80 mV. Inhibition of the NCX was done by inclusion of 20 µM exchanger inhibitory peptide in the pipette solution or by changing the pipette solution to a nominally Na+-free solution during the experiment. Unless otherwise stated, the Ca2+ carried by Itail was obtained from the time integral of the difference between the Itail and the steady-state holding current at -80 mV.

Measurement of cell contraction. Cell contraction was recorded on videotape, and the maximal cell shortening was determined manually from the video monitor off line.

Statistics. Statistical significance of the results was tested using Student's t-test for paired or unpaired samples.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Quantification of Ca2+ carried by the NCX in trout atrial myocytes. To characterize the current that can be attributed to the NCX (INCX), we first used a standard 200-ms depolarization to 0 mV from a holding potential of -80 mV given every 8 s. After stabilization of the ionic currents, the ICa amplitude was determined as the difference between the peak inward current and the current measured at the end of the pulse. Furthermore, the current measured at the end of the depolarizing pulse was used as a measure of the INCX amplitude. Figure 1A shows a typical current trace elicited by a 200-ms depolarization from -80 to 0 mV under control conditions (a), in the presence of 5 µM nifedipine (Nif, b), and with Nif + 10 mM NiCl2 (c). Figure 1B shows the effects of Nif and NiCl2 on ICa and the current at the end of the depolarization. Notice that Nif abolished ICa, whereas Itail was reduced and the current at the end of the pulse was unaffected. Addition of NiCl2 together with Nif strongly reduced Itail and abolished the current at the end of the depolarization. Figure 1C compares the Ca2+ carried by Itail and cell shortening in the absence and presence of Nif. On average Nif reduced the Ca2+ carried by Itail and the cell shortening to 67 ± 6 (n = 15) and 66 ± 5% (n = 10) of their respective control values. Addition of both Nif and NiCl2 abolished contraction (not shown).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1.   Effects of nifedipine (Nif) and NiCl2 on ionic current and contraction. A: representative current traces obtained under control conditions (a), with 5 µM Nif (b), and in the presence of Nif + 10 mM NiCl2 (c). The time scale corresponds to 500 ms. B: time course of current inhibition by Nif and NiCl2. , peak inward current; open circle , current measured at the end of the depolarization. The points a, b, and c correspond to the current traces shown in A. C: average cell shortening and Ca2+ extrusion during tail current (Itail). Open bars, cell shortening normalized to the resting cell length (n = 10); closed bars, amount of Ca2+ extruded during Itail. Con, control.

Figure 2A shows superimposed current traces recorded in the absence and the presence of 5 µM Nif. To obtain the net current resulting from Ca2+ entry through ICa, the Nif trace was subtracted from the corresponding control trace (Fig. 2B). The time integral of this net trace during depolarization and repolarization (Fig. 2C) gives the Nif-sensitive Ca2+ entry through ICa and the corresponding Nif-sensitive Ca2+ extrusion during repolarization. Figure 3 compares the sarcolemmal Ca2+ entry and extrusion arising from ICa and the NCX. As seen in Fig. 3A, the amount of Ca2+ entering through ICa was not significantly different from the Nif-sensitive Ca2+ extrusion, suggesting that the cell was near a steady-state condition. The same approach was therefore used to determine the amount of Ca2+ carried by the NCX, by subtraction of the current measured with Nif + NiCl2 from that measured in the presence of Nif alone. Figure 3B shows that also the Nif-insensitive transsarcolemmal Ca2+ entry and extrusion were similar. A comparison of the amount of Ca2+ carried by ICa and INCX (Fig. 3C) shows that the contribution by the two mechanisms is similar when the cell is depolarized from -80 to 0 mV. Furthermore, the sum of the amount of Ca2+ entering through ICa and INCX (labeled In) is not significantly different from the total amount of Ca2+ extruded by the NCX during Itail (labeled Out).


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2.   Measurement of Ca2+ influx and efflux. A: representative current traces obtained in the absence () and presence (open circle ) of 5 µM Nif. B: net current trace obtained by subtraction of the Nif trace from the control trace in A. C: the Nif-sensitive Ca2+ influx and efflux was obtained from the time integral of the net trace in B. The time scale corresponds to 500 ms.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Comparison of sarcolemmal Ca2+ entry and extrusion. A: average Nif-sensitive Ca2+ entry and efflux. B: average Nif-insensitive but NiCl2-sensitive Ca2+ entry and efflux. C: comparison of the sarcolemmal Ca2+ influx through L-type Ca2+ channels and Na+/Ca2+ exchanger (NCX) with the total sarcolemmal Ca2+ efflux. L-type Ca2+ current (ICa) denotes Ca2+ influx due to ICa and current attributed to NCX (INCX), Ca2+ influx due to reverse mode NCX activity. In, the sum of these 2 parameters; Out, obtained from the difference between Itail in control and with Nif + NiCl2. All values were normalized to the cell capacitance (n = 7).

Figure 4 shows the dependency of ICa and INCX on the test potential used to stimulate the cell. Figure 4A shows superimposed current traces in the absence (open circle ) and presence () of 5 µM Nif at increasing test potentials. Nif inhibited ICa at all potentials, whereas the current at the end of the depolarization was not affected. Furthermore, Itail increased steadily with membrane potential both in the absence and the presence of Nif. Figure 4, B and C, summarizes the voltage dependency of the amount of Ca2+ carried by ICa and INCX, respectively (n = 7). The amount of Ca2+ carried by the two currents was determined as described in Fig. 2. The voltage dependency of the amount of Ca2+ carried by Itail under control conditions is shown in Fig. 4D. If the cell is at steady state, the transsarcolemmal Ca2+ influx should be balanced by the Ca2+ extrusion from the cell and Ca2+ influx through the NCX can therefore be calculated as the difference between the total Ca2+ extruded from the cell (i.e., Itail) and the Ca2+ entering through ICa. The voltage dependency of this difference current is shown in Fig. 4E. Notice that the voltage dependency of the amount of Ca2+ carried by INCX was different depending on the approach used (Fig. 4, C and E). Possibly, the difference is due to the fact that the pharmacological approach in Fig. 4C excludes an interaction between ICa and INCX, whereas it is present when using the approach in Fig. 4E. When normalizing the Ca2+ carried by ICa and INCX (from Fig. 4E) to the Ca2+ carried by Itail, the relative amount of Ca2+ carried by ICa reaches a maximum at -10 mV, corresponding to 54 ± 10% of the total Ca2+ extruded during Itail.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4.   Comparison of the voltage dependency of the Ca2+ transport by ICa, INCX, and Itail. A: representative current traces at -20, 0, +20, and +50 mV in the absence () and presence (open circle ) of 5 µM Nif. Time scale bar corresponds to 500 ms. B-D: relationship between the amount of Ca2+ carried by the ionic currents shown and voltage (n = 7). ICa, INCX, and Itail were obtained as described in Figs. 2 and 3. Voltage scale ranges from -80 to +50 mV. E: relationship between the Ca2+ carried by NCX was also obtained as the difference between Itail and ICa.

Because the amount of Ca2+ carried by the NCX depends critically on membrane potential, contraction is expected to show a similar voltage dependency if the NCX participates in the activation of contraction. We therefore examined the relationship between the measured ionic currents and the cell shortening elicited by a depolarization. Figure 5A compares the voltage dependency of cell shortening in the absence and presence of 5 µM Nif. Notice that both the charge carried by Itail (Fig. 4D) and the contraction increased with more positive membrane potentials (Fig. 5A). As can be seen from Fig. 5B there was a linear relationship between the Ca2+ carried by Itail and the cell shortening when ICa was inhibited with Nif. A linear regression through the points obtained in the absence of Nif when depolarizing the cell to potentials between -60 to -10 mV gave a slope of -22%amol/pF, whereas the regression line in the presence of Nif had a slope of -11%amol/pF. This suggests that a given amount of Ca2+ delivered by ICa is more efficient at activating contraction as the same amount of Ca2+ delivered by the NCX.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5.   Dependency of cell shortening on membrane potential and Ca2+ entry. A: dependency of cell shortening on membrane potential. Cell shortening was measured in the absence (open circle ) and the presence of 5 µM Nif (down-triangle). B: relationship between Ca2+ carried by Itail and cell shortening in the absence (open circle ) and the presence of 5 µM Nif (down-triangle). Values were normalized to the resting cell length (n = 7). Solid lines represent regression lines for control (-60 to -10 mV) and Nif (-40 to +50 mV).

Because simultaneous inhibition of SR Ca2+ uptake and the NCX prevents relaxation of trout cardiac myocytes, we examined the influence of manipulation of the NCX activity on the transsarcolemmal Ca2+ flux and on the SR Ca2+ load. First, we used an intracellular perfusion system that allows changes in the pipette Na+ concentration ([Na+]) during an experiment. Figure 6A shows ionic currents elicited by a standard 200-ms depolarization before (a) and after (b and c) changing the pipette solution from 16 to 0 mM. Notice that Itail diminished significantly when Na+ was eliminated from the pipette solution. Figure 6B shows how the amount of Ca2+ carried by Itail () diminished after changing the pipette solution to a nominally Na+-free solution. For comparison, the amount of Ca2+ carried by ICa in the same experiment (open circle ) did not change much, suggesting that the decrease was not due to a general rundown of ionic currents. On average, the Ca2+ carried by Itail (after a 200-ms depolarization to 0 mV) was reduced to 44 ± 5% of the value with 16 mM Na+ (n = 6) 20 min after switching to a nominally Na+-free solution. In contrast, the Ca2+ carried by ICa was 108 ± 11% of the value with 16 mM Na+. Figure 6C summarizes the voltage dependency of the amount of Ca2+ carried by Itail with three different [Na+] in the patch pipette. Inclusion of 20 µM XIP in the pipette solution also caused a gradual decline in the amount of Ca2+ carried by Itail. By 20 min after establishment of the whole cell configuration, the Ca2+ carried by Itail had declined to 39 ± 8% of the value at patch break, whereas Ca2+ entry through ICa was 113 ± 3% of the start value (n = 3).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of intracellular perfusion with 0 Na+ on Ca2+ influx and efflux. A: representative current traces before (a) and after (b and c) changing the pipette solution from 16 to 0 mM Na+. The time scale corresponds to 400 ms. B: time course of the changes in the amount of Ca2+ carried by ICa (open circle ) and Itail () when the pipette Na+ concentration ([Na+]) is changed from 16 mM to a nominally Na+-free solution. a, b, and c, data points from the current traces shown in A. C: relationship between membrane potential and the amount of Ca2+ carried by Itail at 3 different pipette [Na+]. Number of experiments is given in parentheses.

To calculate the effect of the pipette [Na+] on the SR Ca2+ content, we used intermittent brief exposures of the cells to 10 mM caffeine (Caf). Figure 7A shows Caf-induced NCX currents before and 5 and 15 min after changing the pipette [Na+] from 16 to 0 mM. As can be seen, the current amplitude diminished after switching to 0 mM Na+, and Fig. 7B shows the reduction in the amount of Ca2+ carried by the Caf-induced NCX current. On average, the SR Ca2+ content 20 min after changing the pipette solution to 0 Na+ was reduced to 57 ± 9% of the initial value with 16 mM Na+ in the pipette (P < 0.001, n = 6). Figure 7C compares the dependency on the pipette [Na+] of the amount of Ca2+ carried by Itail after a 200-ms depolarization to +50 mV and the amount of Ca2+ released from the SR with 10 mM Caf. Both the amount of Ca2+ extruded during Itail and that released from the SR decreased significantly with lower [Na+] in the patch pipette.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of intracellular Na+ on Itail and sarcoplasmic reticulum (SR) Ca2+ content. A: NCX currents elicited by exposing the myocyte to 10 mM caffeine (Caf) before and 5 and 15 min after changing the pipette [Na+] from 16 to 0 mM. B: amount of Ca2+ released from the SR by Caf was estimated from the time integrals of the currents shown in A. The time scale in A and B corresponds to 3 s. C: amount of Ca2+ carried by Itail after a 200-ms depolarization to +50 mV (open circle ) and the corresponding amount of Ca2+ released by Caf () is shown as a function of the pipette [Na+]. The number of experiments is given above symbols. For statistical significance, values with 10 mM Na+ were compared with values with 16 mM: * P < 0.05, *** P < 0.0001 and 0 mM with 10 mM Na+, ++ P < 0.05 for both values.

Finally, to examine the Ca2+ extrusion across the sarcolemma and Ca2+ uptake in the SR under conditions where SR Ca2+ content and uptake could be determined, we first cleared the SR Ca2+ content with a brief exposure of the cell to 10 mM Caf. The cell was then stimulated with 20 depolarizations to -10 mV at a rate of 0.5 Hz. After this, the cell was again exposed to Caf to determine the Ca2+ taken up by the SR. The cell was then stimulated with another 20 depolarizations to +10 mV followed by a final exposure to 10 mM Caf. Figure 8A shows schematically the stimulation protocol, and Fig. 8B shows the corresponding Ca2+ movements. Ca2+ entry through L-type Ca2+ channels during each depolarization was calculated from ICa and Ca2+ extrusion at the resting potential was calculated from Itail as described in Fig. 2. The bar diagram in Fig. 8B corresponds to the cumulative sum of Ca2+ entry through ICa and Ca2+ extrusion during Itail. Thus a bar above the 0 line in Fig. 8B corresponds to a net Ca2+ gain in the cell, whereas a bar below the 0 line corresponds to a net Ca2+ loss from the cell. Notice that although there was an apparent cumulative net Ca2+ loss after 20 pulses, Caf was still able to liberate a large amount of Ca2+ from the SR. This then suggested that an additional amount of Ca2+ had entered the cell through the NCX during each depolarization. To calculate the Ca2+ entering through the NCX, the total amount of Ca2+ entering through ICa (labeled ICa), extruded by the NCX (labeled Itail) and liberated from the SR (labeled Caf) after the 20-stimulation pulses was calculated (Fig. 8C). Because the sarcolemmal Ca2+ pump plays a minor role in trout heart cells (18) and the SR was emptied before the first depolarization, the total amount of Ca2+ entering the cell through the NCX (labeled INCX in Fig. 8C) should equal the total amount extruded during Itail plus the amount liberated from the SR minus the total amount entering through ICa. The values obtained with 20 depolarizations to -10 and +10 mV are shown as the open and the solid bars for each of the four parameters. Notice that the amount of Ca2+ entering through the NCX with a depolarization to -10 mV was 48 ± 11% of the value obtained when depolarizing +10 mV (P < 0.01, n = 6). In the same way the amount of Ca2+ extruded by Itail and released from the SR at -10 mV was 65 ± 7 and 80 ± 4% of the corresponding values at +10 mV (P < 0.01, n = 6 for both values). On the contrary, no significant difference was found for the amount of Ca2+ entering through ICa at the two potentials. When comparing the amount of Ca2+ accumulated by the SR with the total amount of Ca2+ entering the cell (which should correspond to Itail + Caf), we found that the SR accumulates about 45% of the total Ca2+, whereas the remaining 55% are extruded from the cell by the NCX. This value was not significantly different at the two potentials examined (43 ± 4% at -10 mV and 49 ± 5% at +10 mV, n = 6).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 8.   Dependency of sarcolemmal Ca2+ movements and SR Ca2+ content on membrane potential. A: stimulation protocol. , brief exposure to 10 mM Caf. B: corresponding net Ca2+ movements. A bar above 0 represents a net Ca2+ gain and below 0 a net Ca2+ loss from the cell. The bar below the Caf exposure represents the Ca2+ released from the SR. C: cumulative Ca2+ movements due to Ca2+ entry through ICa, Ca2+ extrusion by the NCX during Itail, and Ca2+ released from the SR by Caf. Ca2+ entry due to reverse mode NCX (INCX) was calculated as Itail + Caf - ICa. Open bars, values obtained at -10 mV; solid bars, at +10 mV (n = 6). * Significant difference between values obtained at -10 and +10 mV (P < 0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+ influx through the NCX. All the experimental protocols used in this study support a significant contribution of the NCX to the activation of contraction in trout atrial myocytes. Thus the relationship between voltage and cell shortening shows that contraction increases steadily with more positive membrane potentials both in the absence and presence of Nif. This is in contrast to mammalian cardiac myocytes where the relationship is bell shaped and both cell shortening and Ca2+ transient follow the relationship between ICa and voltage (5, 7, 24). Furthermore, with a 200-ms depolarization pulse to 0 mV, both cell shortening and the amount of Ca2+ carried by Itail are only reduced by about 35% when ICa is inhibited with 5 µM Nif. This agrees with results from the neonate mammalian heart (38) that appears to depend more strongly on transsarcolemmal Ca2+ entry (21, 38). Finally, inhibition of the NCX with XIP or by perfusion the cell with an Na+-free solution reduced both the sustained outward current at the end of the depolarization and Itail during repolarization to -80 mV.

Quantification of Ca2+ influx through the NCX. To determine quantitatively the contribution of the NCX to the activation of contraction we used two different approaches. The first approach was based on a pharmacological dissection of ICa and the NCX using Nif to inhibit ICa and Nif + NiCl2 to eliminate both ICa and INCX. Theoretically, the Ca2+ entering through the NCX when ICa is inhibited should then match the Ca2+ extruded by the NCX during Itail on return to -80 mV when the cell is at a steady state. In agreement with this, we find that there is no significant difference between transsarcolemmal Ca2+ entry and extrusion due to either ICa or INCX (see Figs. 2 and 3). Furthermore, the sum of the Ca2+ entering through the ICa and the NCX is not significantly different from the total Ca2+ extruded during Itail (see Fig. 3). With this pharmacological dissection ICa and INCX could account for 41 and 59% of the sarcolemmal Ca2+ influx, respectively (at 0 mV and with 16 mM Na+ in the patch pipette).

The use of a pharmacological dissection of the ionic currents could, however, bias the quantification of the sarcolemmal Ca2+ entry. Thus inhibition of ICa with Nif may lead to an overestimation of the Ca2+ influx through the NCX because it eliminates Ca2+ entry through ICa and possibly also Ca2+ release from the SR. To avoid this, we used another approach where the Ca2+ entry through ICa was subtracted from the time integral of Itail (described in Fig. 4). With this approach Ca2+ entry through the NCX, at 0 mV, was 51% of the Ca2+ carried by Itail. The small difference between the Ca2+ entry through the NCX with the two approaches then suggests that ICa has a relatively small effect on Ca2+ entry through the NCX at 0 mV. At potentials negative to -10 mV ICa does, however, appear to produce a more significant dampening of Ca2+ entry through the NCX (see Fig. 4E), which coincides with the steeper relationship between total sarcolemmal Ca2+ entry and contraction at these potentials (Fig. 5).

It is important to point out that while the Ca2+ entry through the NCX may account for about 50% of the transsarcolemmal Ca2+ flux (Figs. 1 and 6), this only amounts to 20-25% of the total Ca2+ required to activate a normal contraction in trout atrial myocytes (Ref. 19 and Fig. 8). In contrast to this, inhibition of ICa with 5 µM Nif only reduced contraction by 35%, suggesting that NCX can account for 65% of a normal contraction. In this respect it should, however, be borne in mind that the contraction measured in the presence of Nif can result from both Ca2+ entry through the NCX and Ca2+ release from the SR.

Dependency of Ca2+ influx through the NCX on [Na+] and membrane potential. As expected from an electrogenic NCX (2, 9, 22), the relative contribution of the NCX to the activation of contraction depended on both the membrane potential and the [Na+] in the patch pipette. Thus a reduction in the pipette Na+ from 16 to 10 mM decreased Ca2+ entry through NCX by 58%, resulting in an increase in the relative contribution of ICa to the activation of contraction. In agreement with this, we previously have found that changes in the extracellular [Na+] alter the steady-state contraction in multicellular trout ventricular preparations (17). In the same way an increase in the membrane potential, used to activate cell shortening, from -10 to +10 mV increased the Ca2+ entering through the NCX, whereas Ca2+ influx through ICa was unaltered (see Fig. 8). The net result of increasing the membrane potential was therefore a 40% increase in the total transsarcolemmal Ca2+ influx, whereas the relative contribution of ICa was diminished by 30%. Indeed, the relationship between voltage and transsarcolemmal Ca2+ flux shows that the relative amount of Ca2+ carried by ICa in trout reaches a maximum at -10 mV (see Fig. 4) where it accounts for 54% of the total Ca2+ influx. Between +10 and +20 mV, which is the plateau range for the action potential in trout at 20°C (25), ICa only accounts for 27-39% of the total Ca2+ entry across the sarcolemma.

This then suggests that relatively small alterations in the action potential shape or duration may have a significant impact on the Ca2+ entry through the NCX and indirectly on the SR Ca2+ load. In this respect, the relative importance of the NCX would be expected to increase further at lower temperatures, where the action potential is significantly longer (25), whereas the activity of the NCX is expected to decrease little due to its low-temperature sensitivity (34).

The present results agree with results from carp ventricle (36) with respect to the relative contribution of L-type Ca2+ channels and NCX to the total sarcolemmal Ca2+ influx. The results are, however, contrary to results from carp in the sense that the SR is able to accumulate about 45% of the Ca2+ entering the cell in trout atrial myocytes, whereas Ca2+ accumulation is considered insignificant in carp (36). The results are also contrary to data from mammalian species where ICa determines the amplitude of the intracellular Ca2+ transient and the cell shortening by triggering Ca2+ release from the SR (4, 6, 8, 24). A significant Ca2+ entry through the NCX during rest and the latter part of the Ca2+ transient has, however, been found in transgenic mice overexpressing the NCX (31).

Despite the steady increase in transsarcolemmal Ca2+ transport with increasing membrane potential, the relationship between transsarcolemmal Ca2+ transport and contraction shown in Fig. 5 indicates that this relationship is steeper at potentials where Ca2+ mainly enters through L-type Ca2+ channels than potentials where influx through NCX dominates. Indeed, when ICa is inhibited with Nif, the slope of the relationship is uniform for all potentials and only one-half of the slope obtained with ICa contributes to Ca2+ influx. Together, these results then suggest that in trout either the Ca2+ entering through ICa is more accessible to the myofilaments or that sarcolemmal Ca2+ influx triggers Ca2+ release from the SR and that ICa is more efficient at doing this than INCX. The latter case is well established in mammalian species (1, 5, 28, 29).

Ca2+ influx through the NCX and SR Ca2+ loading. We have shown previously that long depolarizations of the trout cardiomyocyte can load the SR with Ca2+ (18). This loading increases with the length of the depolarization and the membrane potential at which loading occurs, suggesting that the loading occurs by Ca2+ influx through the NCX. It is therefore possible that the NCX is important in the regulation of the SR Ca2+ load. The stimulation pulses used in that study were unphysiologically long, but the present study shows that the NCX does also regulate the SR Ca2+ load with physiological stimulation pulses. Thus the steady-state SR Ca2+ content depended critically on the pipette [Na+]. Furthermore, the SR Ca2+ load reached after 20 stimulation pulses was 20% smaller with a depolarization to -10 mV than that with a depolarization to +10 mV, although the Ca2+ entry through ICa was similar (see Fig. 8). Finally, inhibition of the NCX with XIP or a nominally Na+-free pipette solution led to a gradual depletion of the SR Ca2+ content.

In conclusion, our results show that the NCX plays a central role in the regulation of the cytosolic Ca2+ and contraction in trout atrial myocytes. This regulation occurs both through a direct regulation of the Ca2+ entering through the NCX during depolarization and indirectly through a regulation of the SR Ca2+ content. Thus the sarcolemmal Ca2+ entry depends critically on the intracellular [Na+] and the membrane potential, with the entry at -10 mV being one-half of that at +10 mV. In the same way, the SR Ca2+ content increases when the NCX is stimulated while a gradual but complete loss of the SR Ca2+ content occurs when it is inhibited.

Perspectives

The understanding of the regulation of cytosolic Ca2+ in the lower vertebrate heart and in the fish heart in particular has improved strongly during the last few years due to a burst in the quantitative information about Ca2+ transport across the sarcolemma and Ca2+ release from the SR. Until now, the focus has largely been on L-type Ca2+ channels and Ca2+ release from the SR. The present characterization of the Na+/Ca2+-exchange current and its role in the regulation of contraction and SR Ca2+ content should therefore contribute with another important piece of information. Together with the recent cloning of the trout heart NCX (39) this also should provide the basis for a further exploration of its importance in the regulation of the cytosolic Ca2+ and contraction in the lower vertebrate heart.


    ACKNOWLEDGEMENTS

We thank Dr. K. D. Phillipson for generously providing XIP and Marc Puigcerver for excellent animal care.


    FOOTNOTES

This work was supported by grants from the Spanish Ministry of Education and Culture (Incorporación de doctores) to L. Hove-Madsen, MAR97 0408-C02-02 to L. Tort, and Generalitat de Catalunya to A. Llach (1998FI 00176).

Address for reprint requests and other correspondence: L. Hove-Madsen, Unitat de Fisiologia Animal, Dept. Biologia Cel.lular i Fisiologia, Facultad de Ciencies, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain (E-mail: Leif{at}cc.uab.es).

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

Received 3 February 2000; accepted in final form 28 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adachi-Akahane, S, Lu L, Li Z, Frank JS, Phillipson KD, and Morad M. Calcium signalling in transgenic mice overexpressing cardiac Na+-Ca2+ exchanger. J Gen Physiol 109: 717-729, 1997[Abstract/Free Full Text].

2.   Barcenas-Ruiz, L, Beuckelmann DJ, and Wier WG. Sodium-calcium exchange in heart: membrane currents and changes in [Ca2+]i. Science 238: 1720-1722, 1987[Abstract/Free Full Text].

3.   Bassani, JWM, Bassani RA, and Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol (Lond) 476: 279-293, 1994[Abstract/Free Full Text].

4.   Bassani, JWM, Yuan W, and Bers DM. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol Cell Physiol 269: C1313-C1329, 1995.

5.   Beuckelman, D, and Wier WG. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J Physiol (Lond) 405: 233-256, 1988[Abstract/Free Full Text].

6.   Bouchard, RA, Clark RB, and Giles WR. Role of sodium-calcium exchange in activation of contraction in rat ventricle. J Physiol (Lond) 472: 391-413, 1993[Abstract/Free Full Text].

7.   Bouchard, RA, Clark RB, and Giles WR. Effects of action potential duration on excitation-coupling in rat ventricular myocytes. Circ Res 76: 790-801, 1995[Abstract/Free Full Text].

8.   Cheng, H, Lederer WJ, and Channell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740-744, 1993[Abstract/Free Full Text].

9.   Crespo, LM, Grantham J, and Channell MB. Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes. Nature 345: 618-621, 1990[Medline].

10.   Dipla, K, Mattiello JA, Margulies KB, Jeevanandam V, and Houser SR. The sarcoplasmic reticulum and the Na+/Ca2+ exchanger both contribute to the Ca2+ transient of failing human ventricular myocytes. Circ Res 84: 435-444, 1999[Abstract/Free Full Text].

11.   Driedzic, WR, and Gesser H. Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis and low temperature. Physiol Rev 74: 221-257, 1994[Free Full Text].

12.   Fabiato, A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol Cell Physiol 245: C1-C14, 1983[Abstract/Free Full Text].

13.   Fabiato, A, and Fabiato F. Calcium induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat and frog hearts and from fetal and newborn rat ventricles. Ann NY Acad Sci 307: 491-522, 1978[Medline].

14.   Fan, J, Shuba YM, and Morad M. Regulation of cardiac sodium-calcium exchanger by beta -adrenergic agonists. Proc Natl Acad Sci USA 93: 5527-5532, 1996[Abstract/Free Full Text].

15.   Harrison, SM, and Boyett MR. The role of the Na+-Ca2+ exchanger in the rate-dependent increase in contraction in guinea-pig ventricular myocytes. J Physiol (Lond) 482: 555-566, 1995[ISI][Medline].

16.   Hove-Madsen, L. The influence of temperature on ryanodine sensitivity and the force-frequency relationship in the myocardium of rainbow trout. J Exp Biol 167: 47-60, 1992[Abstract/Free Full Text].

17.   Hove-Madsen, L, and Gesser H. Force frequency relation in the myocardium of rainbow trout. J Comp Physiol [B] 159: 61-69, 1989[Medline].

18.   Hove-Madsen, L, Llach A, and Tort L. Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes. Am J Physiol Regulatory Integrative Comp Physiol 275: R2070-R2080, 1998[Abstract/Free Full Text].

19.   Hove-Madsen, L, Llach A, and Tort L. Quantification of calcium release from the sarcoplasmic reticulum in trout ventricular myocytes. Pflügers Arch 438: R545-552, 1999.

20.   Hove-Madsen, L, and Tort L. L-type Ca2+ current and E-C coupling in single atrial myocytes from rainbow trout. Am J Physiol Regulatory Integrative Comp Physiol 275: R2061-R2069, 1998[Abstract/Free Full Text].

21.   Huynh, TVF, Chen GT, Wetzel GT, Friedman WF, and Klitzner TS. Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal and adult heart cells. Circ Res 70: 508-515, 1992[Abstract/Free Full Text].

22.   Kimura, J, Noma A, and Irisawa H. Na-Ca exchange current in mammalian heart cells. Nature 319: 596-597, 1986[Medline].

23.   Litwin, SE, Li J, and Bridge JHB Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J 75: 359-371, 1998[Abstract/Free Full Text].

24.   López-López, JR, Shalklock PS, Balke CW, and Wier WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268: 1042-1045, 1995[Abstract/Free Full Text].

25.   Møller-Nielsen, T, and Gesser H. Sarcoplasmic reticulum and excitation coupling at 10 and 20°C in rainbow trout myocardium. J Comp Physiol [B] 162: 526-534, 1992.

26.   Morad, M, Goldman YE, and Trentham DR. Rapid photochemical inactivation of Ca2+-antagonists shows that Ca2+ entry directly activates contraction in frog heart. Nature 304: 635-638, 1981.

27.   Santer, RM. Morphology and innervation of the fish heart. Adv Anat Embryol Cell Biol 89: 1-102, 1985[Medline].

28.   Sham, JSK, Cleemann L, and Morad M. Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na+-Ca2+ exchange. Science 255: 850-853, 1992[Abstract/Free Full Text].

29.   Sham, JSK, Cleemann L, and Morad M. Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci USA 92: 121-125, 1995[Abstract/Free Full Text].

30.   Sham, JSK, Hatem SN, and Morad M. Species differences in the activity of the Na+-Ca2+ exchanger in mammalian cardiac myocytes. J Physiol (Lond) 488: 623-631, 1995[ISI][Medline].

31.   Terracciano, CMN, de Souza AI, Phillipson KD, and MacLeod KT. Na+-Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+-Ca2+ exchanger. J Physiol (Lond) 512: 651-667, 1998[Abstract/Free Full Text].

32.   Terraciano, CMN, and MacLeod KT. Measurements of Ca2+ entry and sarcoplasmic reticulum Ca2+ content during the cardiac cycle in guinea pig and rat ventricular myocytes. Biophys J 72: 1319-1326, 1997[Abstract/Free Full Text].

33.   Tibbits, GF, Moyes CD, and Hove-Madsen L. Excitation-contraction coupling in the teleost heart. Fish Physiol XIIA: 267-304, 1992.

34.   Tibbits, GF, Phillipson KD, and Kashihara H. Characterization of myocardial Na+-Ca2+ exchange in rainbow trout. Am J Physiol Cell Physiol 262: C411-C417, 1992[Abstract/Free Full Text].

35.   Vornanen, M. Sarcolemmal Ca influx through L-type Ca channels in ventricular myocytes of a teleost fish. Am J Physiol Regulatory Integrative Comp Physiol 272: R1432-R1440, 1997[Abstract/Free Full Text].

36.   Vornanen, M. Na+/Ca2+ exchange current in ventricular myocytes of fish heart: contribution to sarcolemmal Ca2+ influx. J Exp Biol 202: 1763-1775, 1999[Abstract].

37.   Vornanen, M, Shepard N, and Isenberg G. Tension-voltage relations of single myocytes reflect Ca release triggered by exchange at 35°C but not at 23°C. Am J Physiol Cell Physiol 267: C623-C632, 1994[Abstract/Free Full Text].

38.   Wetzel, GT, Chen F, and Klitzner TS. Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes. Am J Physiol Heart Circ Physiol 268: H1723-H1733, 1995[Abstract/Free Full Text].

39.   Xue, X-H, Hryshko LV, Nicoll DB, Phillipson KD, and Tibbits GF. Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger. Am J Physiol Cell Physiol 277: C693-C700, 1999[Abstract/Free Full Text].

Am J Physiol Regul Integr Comp Physiol 279(5):R1856-R1864
0363-6119/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. E. Molina, H. Gesser, A. Llach, L. Tort, and L. Hove-Madsen
Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R388 - R395.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
U. Schotten, S. de Haan, S. Verheule, E. G.A. Harks, D. Frechen, E. Bodewig, M. Greiser, R. Ram, J. Maessen, M. Kelm, et al.
Blockade of atrial-specific K+-currents increases atrial but not ventricular contractility by enhancing reverse mode Na+/Ca2+-exchange
Cardiovasc Res, January 1, 2007; 73(1): 37 - 47.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
G. L. J. Galli, E. W. Taylor, and H. A. Shiels
Calcium flux in turtle ventricular myocytes
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2006; 291(6): R1781 - R1789.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
A. Llach, J. Huang, F. Sederat, L. Tort, G. Tibbits, and L. Hove-Madsen
Effect of {beta}-adrenergic stimulation on the relationship between membrane potential, intracellular [Ca2+] and sarcoplasmic reticulum Ca2+ uptake in rainbow trout atrial myocytes
J. Exp. Biol., March 15, 2004; 207(8): 1369 - 1377.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
L. Hove-Madsen, A. Llach, and L. Tort
The function of the sarcoplasmic reticulum is not inhibited by low temperatures in trout atrial myocytes
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R1902 - R1906.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. L. Elias, X.-H. Xue, C. R. Marshall, A. Omelchenko, L. V. Hryshko, and G. F. Tibbits
Temperature dependence of cloned mammalian and salmonid cardiac Na+/Ca2+ exchanger isoforms
Am J Physiol Cell Physiol, September 1, 2001; 281(3): C993 - C1000.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hove-Madsen, L.
Right arrow Articles by Tort, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hove-Madsen, L.
Right arrow Articles by Tort, L.

HOME HELP