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Cardiovascular Research 2001 52(1):76-83; doi:10.1016/S0008-6363(01)00363-7
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Copyright © 2001, European Society of Cardiology

On the source of Ca2+ activating the tonic component of contraction of myocytes of guinea pig heart

Krzysztof Emanuel, Urszula Mackiewicz and Bohdan Lewartowski*

Department of Clinical Physiology, Medical Centre of Postgraduate Education, Marymoncka 99, 01-813 Warsaw, Poland

blew{at}cmkp.edu

* Corresponding author. Tel.: +48-22-834-0367; fax: +48-22-864-0834

Received 7 March 2001; accepted 21 May 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Contractions of isolated, single myocytes of guinea pig heart stimulated at 37°C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca2+ activating the tonic component. Methods: Experiments were performed at 37°C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of –40 to +5 mV. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Results: Superfusion of 5 mmol/l Ni2+ during 30 s pause did not inhibit subsequent biphasic Ca2+ transients and contractions despite inhibition of Ca2+ current and Na+/Ca2+ exchange. KB-R7943 (5 µmol/l) or intracellular dialysis with 0 Na+ solution, both of which inhibit reversed Na+/Ca2+ exchange, decreased amplitude of Ca2+ transients and contractions by ~40%. The ratio of amplitudes of tonic to phasic component was increased by Ni2+ and was not changed by KB-R7943 or 0 Na+i. Ryanodine (200 µmol/l) inhibited both components of contractions in cells superfused with Ni2+. The phasic component but not the tonic component was inhibited by 20 µmol/l nifedipine in cells superfused with Ni2+. Conclusions: Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca2+ current or by the reverse mode Na+/Ca2+ exchange as currently proposed in literature. Rather, it is activated by Ca2+ released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca2+ fraction activating the phasic component of contraction.

KEYWORDS Myocytes; Contractile function; e–c Coupling; Calcium (cellular)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiomyocytes of guinea pig heart stimulated at 37°C by cellular action potentials (APs) or voltage clamps longer than 100 ms show biphasic Ca2+ transients and contractions: the phasic component relaxing before the end of the stimulating pulse is followed by a tonic component relaxing upon repolarization (Fig. 1). At room temperature these components merge forming an apparently monophasic Ca2+ transients and contractions.


Figure 1
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  		Fig. 1 Biphasic contractions of single myocytes of guinea pig heart. (A) Cellular action potentials (top records) and contractions (shortening downwards). Stimulation rate 30–210/min. (B) Membrane currents (top) and contractions activated by voltage protocol shown above the record. Stimulation rate 60/min. Duration of the longer pulse 700 ms. (C) Membrane currents and Indo 1-AM fluorescence. (D) Indo1-AM fluorescence of a field-stimulated cell.

 
Biphasic nature of myocardial contraction noticed for the first time by Kavaler [1] has been investigated by numerous authors (for an early review see Ref. [2]) under various experimental conditions in multicellular preparations and in single isolated cardiac myocytes. There is a general consensus that initial, phasic component is initiated by Ca2+ released from the SR by membrane potential [3] or by ICa [4–6]. This is based on the findings that interventions, which eliminate SR from the process of excitation–contraction coupling inhibit the phasic component of contraction. The source of Ca2+ activating the second, slow or tonic component is, however, controversial. Two main hypotheses are proposed: (1) the tonic component is activated directly by ICa [7–11] or due to release of Ca2+ from the SR by ICa [3,5,6]; and (2) the tonic component is activated by Ca2+ influx by the reverse mode Na+/Ca2+ exchange [12–14].

In this paper, we used the experimental protocol described in the previous paper [4] enabling to activate nearly normal, biphasic Ca2+ transients and contractions without apparent activation of ICaL. This eliminated the first hypothesis. Since the tonic component was not inhibited by the blockers of the reverse mode Na+/Ca2+ exchange or decrease in intracellular Na+ concentration, also the second hypothesis seems unlikely. Since both components of contraction were readily inhibited by 200 µmol/l ryanodine (Ry) it seems that they both are activated by Ca2+ released from the SR. The kinetics and mechanism of release of these two Ca2+ fractions are, however, different.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell isolation
Guinea pigs of both sexes weighing 250–300 g were injected i.p. with 2500 U heparin followed 30 min later by an overdose of pentobarbital sodium. After the heart was rapidly excised and washed in cold Tyrode solution, the aorta was cannulated and retrogradely perfused for 3 min with nominally Ca2+ free solution containing 100 µmol/l (EGTA) (for composition of solutions see below). Initial washout period was followed by 10–15 min of perfusion with Ca2+ free Tyrode solution containing 15 mg collagenase B (Boehringer) and 3 mg protease (Sigma) per 50 ml. Thereafter the ventricles were cut from the atria and placed in a 50-ml beaker containing the same solution, disrupted with pincettes into small strands and agitated. The cell suspension was filtered through the nylon mesh, and allowed to sediment. The supernatant was discarded and cells were washed twice with Tyrode solution, the Ca2+ concentration being increased gradually to 1 mmol/l.

2.2 Cells superfusion and recording of contractions
Cells were placed in the 0.5-ml superfusion chamber mounted on the stage of an inverted microscope (Nikon Diaphot) and allowed to attach to its glass bottom. The chamber was perfused at a rate of ~2 ml/min. Three lines of perfusion solution heated up to the inlet enabled to change its composition within ~30 s. Temperature within the chamber was kept at ~37°C. The TV camera was mounted in the side port of the microscope and the cell length monitored by video edge-tracking system designed and built by John Parker (Cardiovascular Laboratories, School of Medicine, UCLA).

2.3 Recording of Indo 1-AM fluorescence
A Nikon mercury lamp was used as a source of illumination for epifluorescence. A concentric diaphragm enabled illumination of a small fragment of a cell. The fluorescent light was passed to the 405-nm DE35 and 495-nm DE20 photomultipliers mounted in the holders attached to the side port of the microscope. The ratio of 410- to 495-nm fluorescence was obtained from the output of Dual Channel Ratio Fluorometer (Biomedical Instrumentation Group, University of Pennsylvania). Cells were loaded with Indo 1-AM form as described by Spurgeon et al. [15]: 12.5 µl of a solution containing 50 µl of 1 mmol/l Indo 1-AM dissolved in dimethyl sulfoxide, 2.5 µl of 25% Pluronic, and 75 µl of bovine calf serum was added to 500 µl of cell suspension. Cells were incubated 5–15 min at room temperature, washed in Tyrode solution and placed in superfusion chamber. No attempt was made to convert the fluorescence ratio to Ca2+ concentration.

We were not able to record Ca2+ transients and contractions simultaneously in one cell.

2.4 Recording of the ionic currents
The currents were recorded using whole cell clamp method. Pipettes of 1.8–2.2 M{Omega} resistance were pulled by the programmed Flaming/Brown Puller Model — 97 from borosilicate glass capillaries. Pulses from a holding potential of –40 to +5 mV (duration 300 ms) were applied at 0.5–1 Hz. Currents were recorded using an Axopatch 1-D amplifier controlled by the V-clamp software and V-clamp computer interface designed by D.R. Matteson (Department. of Biophysics, University of Maryland).

Cellular APs were recorded with the same amplifier set to current clamp mode. Stimulating, slightly suprathreshold pulses of 5 ms duration were delivered to the cells through the recording electrode.

2.5 Solutions
For cells isolation and throughout the experiments we used Tyrode solution of the following composition (in mmol/l): 144 NaCl, 5 KCl, 1 MgCl2, 0.43 NaH2PO4, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 11 glucose and 5 sodium pyruvate. The pH of the solution was adjusted with NaOH to 7.3 for cells isolation and to 7.4 for experiments. In the experiments CaCl2 was added to concentration of 3 mmol/l. The patch pipettes were filled with a solution containing (in mmol/l): 135 KCl, 5 NaCl, 1 MgCl2, 10 HEPES, 4 Mg ATP, 0.05 8-Br-cAMP. In one series of experiments LiCl was used instead of NaCl. Experimental protocols will be for clarity described in detail in the Results section.

2.6 Statistical evaluation
The quantitative results are presented as means±S.D. Student’s t test for paired samples was used in order to check statistical significance of differences between the means.

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


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Normal Ca2+ transients and contractions
Fig. 1A shows the cellular APs and contractions of ventricular myocytes of guinea pig heart stimulated at the rate of 30–210/min. Contractions consisted of two components: a phasic one and a tonic one. The tonic component was visible also at the stimulation rates close to the physiological rate of in situ guinea pig heart (140–180/min), which suggests that it was not an artifact resulting from the low stimulation rates used in most of experiments. Fig. 1B and C show the Ca2+ currents activated by the pulses from the holding potential of –40 to +5 mV at the rate of 60/min and the respective contractions and Ca2+ transient. Both signals consisted of two components. The phasic component of contraction began after ~10 ms delay with respect to the onset of Ca2+ current and relaxed well before the end of depolarization. The smaller tonic component was superposed on the relaxing phasic component. It could be flat or have a form of additional contraction separated from the first one by a dip (Fig. 1B). Relaxation of the tonic component was initiated by repolarization and was prolonged by its delay (Fig. 1B). In most of cells the amplitude of the tonic component increased slowly during depolarization with long pulses (Fig. 1B), in others the record was flat (Fig. 3B). Myocytes showed biphasic Ca2+ transients also when field-stimulated by short (5 ms) electrical suprathreshold pulses (Fig. 1D).


Figure 3
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  		Fig. 3 The effect of 5 mmol/l Ni2+ on membrane currents (top), Ca2+ transients (A) and contractions (B) of single myocytes of guinea pig heart stimulated at the rate of 60/min by the voltage pulses shown above the records. Ni2+ superfused from the beginning of the preceding 30 s break in stimulation. Duration of the longer pulse 700 ms.

 
3.2 The tonic component was not inhibited by the blockers of ICa or of the reversed Na+/Ca2+ exchange
This has been shown by several experimental protocols, which are detailed below.

3.2.1 The effect of Ni2+ on Ca2+ transients and contractions
We investigated the effects on the tonic component of 5–10 mmol/l Ni2+ used as blocker of ICa and reversed Na+/Ca2+ exchange [16,17]. The blocking effect of Ni2+ on ICa has been shown in a number of papers [4,16], however, its effect on the reverse mode Na+/Ca2+ exchange has not been, to our knowledge, systematically investigated. Therefore, we tested first the effects of 5 mmol/l Ni2+ on reverse mode Na+/Ca2+ exchange. Cells loaded with Indo 1-AM were incubated for 30 min with 10–6 mol/l thapsigargin (TG) and 10 µmol/l Ry as used by Satoh et al. [18]. Then they were placed in superfusion chamber, and voltage clamped at holding potential of –40 mV. The pulses to +100 mV activated slow increase in fluorescence relaxing upon repolarization. It was completely or nearly completely inhibited by 5 mmol/l Ni2+ (Fig. 2A).


Figure 2
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  		Fig. 2 The effect of 5 mmol/l Ni2+ and 5 µmol/l KB-R7943 on Na+/Ca2+ exchange reversed by positive voltage. (A, B) Indo 1-AM fluorescence of two separate, voltage clamped myocytes of guinea pig heart pretreated with 10–6 mol/l thapsigargin and 10 µmol/l ryanodine. Increases in fluorescence induced by the voltage pulses shown above the records delivered at the rate of 60/min. (A) right panel: Ni2+ superfused for 1 min. (B) right panel: KB-R7943 superfused for 1 min.

 
Increase in [Ca2+]i by the pulses to +100 mV in cells with blocked SR, and Na+ channel inactivated by holding potential of –40 mV could result only from Ca2+ influx by reversed Na+/Ca2+ exchange. So we infer from these experiments that if any component of Ca2+ transients or contractions stimulated by depolarizing pulses is due to the reverse mode Na+/Ca2+ exchange it should be strongly or completely inhibited by 5 mmol/l Ni2+.

In order to investigate the effect of Ni2+ on the tonic component of contractions we repeated experiments described in detail in the preceding paper [4] showing that it is possible to activate nearly normal contractions without apparent activation of Ca2+ current. Cells were stimulated for 60 s by the pulses from the holding potential of –40 to +5 mV (duration 300 or 700 ms) and Ca2+ transients or contractions recorded. Stimulation was stopped for 30 s and cells immediately superfused with 5 mmol/l Ni2+. This maneuver trapped intracellular Ca2+ within the myocyte. Despite of inhibition of the Ca2+ current, stimulation resumed after 30 s activated nearly normal, biphasic Ca2+ transients and contractions (Fig. 3A, B). The amplitude of the phasic component decreased to 82% of pre-Ni2+ control, whereas that of the tonic component was not significantly affected. Hence, the ratio of the amplitudes of the tonic to phasic component significantly increased (Table 1). The contractions were stable over several minutes. Ni2+ consistently affected the tonic component stimulated by the longer pulses. In cells in which amplitude of the tonic component was increasing during control long depolarization, the increase became steeper. In cells in which the control amplitude was not increasing, it increased during Ni2+ superfusion (Fig. 3B). In order to make sure that Na+/Ca2+ exchange was completely blocked, after obtaining contractions in five cells superfused with 5 mmol/l Ni2+ we increased Ni2+ concentration to 10 mmol/l. This resulted in slowing of the kinetics of the phasic component, which partially merged with the tonic component. Nevertheless the tonic component was clearly seen in most cells superfused with 10 mmol/l Ni2+ (not shown).


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  		Table 1 The effect of Ni2+, KB-R7943 and intracellular dialysis with 0 Na+ solution on amplitude of components of contractions of single ventricular myocytes of guinea pig hearta

 
3.2.2 The effect of KB-R7943 on contractions and Ca2+ transients
Recently it has been reported that the compound known under the symbol KB-R7943 selectively blocks reversed mode Na+/Ca2+ exchange [18–20]. First we checked this effect using the protocol described for the experiments with Ni2+ (Fig. 2B). We found that 5 µmol/l KM-R7943 completely inhibited increase in fluorescence stimulated by voltage pulses to +100 mV.

In order to test the effect of KB-R7943 on contractions and Ca2+ transients, cells were superfused with Tyrode solution and stimulated by the pulses to +5 mV of the duration of 300 or 700 ms. Superfusion of 5 µmol/l KB-R7943 resulted in rapid acceleration of inactivation of Ca2+ current which stabilized within ~15 s and in a decrease in both components of contraction by ~40%. The latter effect stabilized within a few minutes. The ratio of the amplitude of the tonic component measured at the moment of repolarization after 300 ms to the peak amplitude of the phasic component did not change significantly although the latter became more flat (Fig. 4A and Table 1). In cells in which amplitude of tonic component was increasing during 700 ms depolarization it became flat. Similar results were obtained in cells loaded with Indo 1-AM (Fig. 4B). In cells in which 700-ms pulses resulted in a gradual increase in [Ca2+]i the signal became flat. In others it even slightly declined. The effects of KB-R7943 were partly reversible upon washout (Fig. 4A).


Figure 4
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  		Fig. 4 The effect of selective inhibition of reversed Na+/Ca2+ exchange by 5 µmol/l KB-R7943 (A, B) or intracellular dialysis with 0 Na+ solution (C) on membrane currents (top records) and contractions (A, C) or Ca2+ transients (B) of single myocytes of guinea pig heart stimulated by the voltage protocol pertaining to all records shown in panel A. Stimulation rate 60/min. KB-R7943 superfused for 4 min. Zero Na+i dialysis for 4 min since the rupture of sarcolemma by the micropipette.

 
3.2.3 The effect of low [Na]i on contractions
The reverse mode Na+/Ca2+ exchange may be also inhibited by decrease of intracellular Na+ concentration. Therefore, Na+-free internal solution was used instead of that containing 5 mmol/l Na+. The effect of 0 Na+ on contractions was very similar to that of KB-R7943. In experiments in five cells, we recorded the monotonous and proportional decrease of amplitude of both components as the internal dialysis proceeded after rupture of the membrane. The ratio of the amplitudes of the tonic and phasic components did not change, although the latter became more flat (Fig. 4C and Table 1).

3.3 The tonic component can be separated from the phasic component

1. As shown also in the preceding paper [4], in cells superfused with Ni2+, 20 µmol/l nifedipine blocked the phasic component of contraction leaving the tonic component apparently intact. This experiment was now repeated in seven cells (Fig. 5A).
2. In some cells superfused with Ni2+, the phasic component was absent and only the tonic component appeared (Fig. 5B).


Figure 5
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  		Fig. 5 Isolation of the tonic component of contraction (A) or Ca2+ transients (B) from the phasic component. (A) the effect of 20 µmol/l nifedipine superfused over the cell pretreated with 5 mmol/l Ni2+. (B) in this cell, unlike in the cell shown in Fig. 3A, superfusion with Ni2+ resulted in inhibition of the phasic component of Ca2+ transient and only the tonic component was left.

 
3.4 Both components of contraction are inhibited by 200 µmol/l ryanodine
As shown in Fig. 6A, 3 min superfusion of the normal cell with 200 µmol/l Ry which blocks the ryanodine receptors (RyRs) [21,22] resulted in changing of rapid, biphasic contraction into a slow, monophasic contraction. However, the total amplitude of contraction was not much affected consistent with the results of Janiak et al. [23]. Apparently, contractions in cells superfused with 200 µmol/l Ry were activated by Ca2+ influx. In order to eliminate the effect of Ca2+ influx, we tested the effect of Ry on components of contraction in seven cells superfused with Ni2+ according to protocol described above. After obtaining biphasic contractions, 200 µmol/l Ry was added to the bath. This resulted in a progressive decrease of both components of contraction, which disappeared within 1–3 min. In most cells the decrease of the phasic component was more rapid than that of the tonic component (Fig. 6B).


Figure 6
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  		Fig. 6 The effect of 200 µmol/l ryanodine on membrane currents (top records) and contractions in normal cell (A) and in the cell pretreated with 5 mmol/l Ni2+ (B). The voltage protocol pertains to all records. Stimulation rate 60/min.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results of this work do not support the two main hypotheses concerning the source of Ca2+ activating the slow, tonic component of Ca2+ transient and contraction of single, isolated myocytes of guinea pig heart. This component was left intact in cells superfused with 5–10 mmol/l Ni2+, which blocks the Ca2+ currents. It might be argued that block of the Ca2+ current was not complete and that there was some residual Ca2+ influx not visualized in our records. However, it is highly improbable that such a negligible Ca2+ current left in cells superfused with Ni2+ was able to activate directly the contractile proteins as strongly as the full-size, intact current or to induce Ca2+ release from the SR. Thus, the direct or indirect activation of the tonic component by ICa seems unlikely.

As shown in Fig. 2A, 5 mmol/l Ni2+ blocked almost completely Na+/Ca2+ exchange reversed by the pulses to +100 mV. Thus, if the tonic component of contraction produced by the pulses from –40 to +5 mV were activated by the reverse mode Na+/Ca2+ exchange, 5–10 mmol/l Ni2+ should have blocked it, which was not the case. Our results are at variance with those of O’Neill et al. [13], who were able to block with 5 mmol/l Ni2+ the tonic component of contraction produced by depolarization in isolated rat myocytes. The differences in species, temperature (room temperature vs. 37°C) and experimental protocol may be the reason of this discrepancy.

The amplitude of the tonic component of contraction stimulated by 300-ms pulses was not selectively affected by KB-R7943 shown to block the reverse mode Na+/Ca2+ exchange ([18–20] and our experiments) or by intracellular dialysis with 0 Na+ solution. These results are at variance with those of Gaughan et al. [12]. They claimed that the tonic component of contraction of the myocytes isolated from the explanted human hearts was produced by the reverse mode Na+/Ca2+ exchange: it was voltage dependent, was inhibited by internal cell dialysis with 0 Na+ solution and by KB-R7943. However, these experiments were performed in the myocytes isolated from the failing human hearts, which may differ very much from the myocytes of the hearts of young, healthy guinea pigs.

The effects of 5 mmol/l Ni2+ differed from those of KB-R7943 or internal cell dialysis with 0 Na+ solution. Contractions in cells superfused with Ni2+ were stable, whereas both components of contractions of the cells superfused with KB-R7943 or dialyzed with 0 Na+ declined. The tonic component became flat during 300-ms pulses or even declined during 700-ms pulses in cells superfused with KB-R7943, whereas Ni2+ had opposite effect in this respect. These differences most likely depend on differences in the effect of Ni2+ and two other interventions on Na+/Ca2+ exchange. Ni2+ blocks non-selectively bidirectional Na+/Ca2+ exchange and Ca2+ currents trapping intracellular Ca2+ within the myocyte. KB-R7943 and 0 Na+ internal dialysis block selectively the reverse mode Na+/Ca2+ exchange whereas the Ca2+ out mode is little affected (KB-R7943) or stimulated (0 Na+i). This, and acceleration of inactivation of ICa by KB-R7943 might lead to progressive depletion of cellular Ca2+.

Both components of contraction in cells superfused with Ni2+ (i.e. with the Ca2+ currents inhibited) were readily inhibited by 200 µmol/l Ry, which blocks the RyRs [21,22]. This might look as a contradiction of the results of our previous experiments [4], in which TG inhibited strongly but in some cells not completely the tonic component of contractions in cells superfused with Ni2+. However, it takes a long time for TG to block completely the SR Ca2+ uptake in cells superfused at 37°C. Since TG does not affect the RyRs, there might be some residual Ca2+ flux through the SR. Ry (200 µM) blocks readily RyRs thereby interrupting the flux. The experiments with Ry suggest that Ca2+, activating both components of contraction, is released from the SR. However, the mechanism and kinetics of release of these two Ca2+ fractions are different. Recently, Ferrier et al. [24] found that Ca2+ activating the sustained component of contraction initiated by a ‘voltage-sensitive release mechanism’ (VSRM) is released from the SR. VSRM was inactivated in our experiments by the holding potential of –40 mV [25]. Thus, we investigated a different event. Nevertheless, despite the important differences in approach and experimental protocols, our results concerning the source of Ca2+ activating the tonic component of contractions activated by depolarizations from a holding potential of –40 mV are similar to those of Ferrier et al. [24] concerning sustained component of contractions activated by VSRM.

The mechanism of activation of the phasic component in cells superfused with Ni2+ has been investigated and discussed in detail in our preceding paper [4]. In normal cells it is activated by Ca2+ released through the RyRs of the SR, CIRC with some contribution of DHPRs acting as voltage sensors being the dominant mechanism of their activation.

Upstroke of the phasic component of Ca2+ transient and contraction in normal cells and in cells superfused with Ni2+ was rapid and relaxation was independent on the membrane voltage (it was completed well before the repolarization). The tonic component developed slowly and its relaxation was repolarization-dependent. The different pattern of the two components of contraction suggests that Ca2+ fractions activating them are released from morphologically different sources and/or through the channels of different properties. Similar conclusion concerning the source of two components of Ca2+ transients of isolated myocytes of human atria was reached by Hatem et al. [26]. They proposed that the rapidly rising component results from the release of Ca2+ from the junctional SR whereas the slow component results from release of Ca2+ from the corbular SR. This might be the case also in ventricular myocytes of guinea pig heart. Also, the theoretical considerations have led Peskoff and Langer [27] to the notion that release of calcium from corbular SR should affect the profile of cellular calcium concentration. Hatem et al. [26] proposed that the Ca2+ channels of junctional SR are activated by ICa whereas the channels of corbular SR would be activated by Ca2+ released from junctional SR diffusing from the subsarcolemmal space deeper into the cytosol. However, in our experiments the phasic and tonic components were activated quite independently since they could be completely separated (Fig. 5). Thus, the tonic component could not be activated by the same Ca2+ pool, which activated the phasic component. In contrast to the phasic component, RyRs releasing Ca2+ producing the tonic component were not activated by DHPRs acting as Ca2+ channels or voltage sensors since the tonic component was not inhibited by nifedipine (Fig. 5). This is consistent with the corbular terminal cisternae being remote from sarcolemma [28]. Also Jorgensen et al. [29] proposed that Ca2+ release from the junctional and the corbular sarcoplasmic reticulum is regulated by different signals. What might be, then, the mechanism of activation of Ca2+ channels activating the tonic component of contraction?

Tanna et al. [30] found that binding of a reversible ryanoid with single sheep RyRs incorporated into planar phospholipid bilayer is affected by the holding potential. According to these authors this effect results from voltage-dependent conformational changes in the Ca2+ release channels. It is tempting to speculate that RyR of the corbular SR might be directly activated by the voltage pulses. Although Tanna et al. [30] did not see any effect of the respective steady state holding potentials on the open probability (Po) of the channels, they might be more responsive to the rapid changes in voltage, as they are more sensitive to rapid changes in Ca2+ concentration than to its steady state concentration [31].

In conclusion, we propose that the tonic component of contractions of single ventricular myocytes of guinea pig heart is activated mostly by Ca2+ released from SR, however, the kinetics and mechanism of its release are different from those of Ca2+ pool activating the phasic component of contraction.

Time for primary review 22 days.


   Acknowledgements
 
The authors are greatly indebted to Prof. Glenn A. Langer for reading the manuscript and helpful remarks. An expert and devoted technical contribution of Ms. Jadwiga Dermanowska is gratefully acknowledged. This work has been supported by the Grant No 4 PO5A 01818 of the State Committee for Scientific Research.


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

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