Skip Navigation

Cardiovascular Research 1999 44(3):477-487; doi:10.1016/S0008-6363(99)00236-9
© 1999 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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 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 arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hobai, I. A
Right arrow Articles by Levi, A. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hobai, I. A
Right arrow Articles by Levi, A. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1999, European Society of Cardiology

Coming full circle

Membrane potential, sarcolemmal calcium influx and excitation–contraction coupling in heart muscle

Ion A Hobai1,* and Allan J Levi

Cardiovascular Research Laboratories, Bristol Heart Institute, Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK

* Corresponding author. Tel.: +1-410-614-4825; fax: +1-410-955-7953 ionhobai{at}welchlink.welch.jhu.edu

Received 10 December 1998; accepted 25 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Experiments in multicellular...
 3 Experiments in isolated...
 4 Summary and conclusions
 References
 
In heart muscle, strong evidence shows that excitation–contraction coupling involves Ca-induced Ca-release. However, under some conditions, single heart cells show Ca release and contraction which is not correlated with Ca entry via the Ca channel, suggesting a second Ca-independent release mechanism. Similar observations were made in early, pioneering studies using voltage-clamped multi-cellular preparations. We review the influence that experimental preparations and conditions have had on excitation–contraction coupling theory over the last 20 years.

KEYWORDS Calcium channel; Membrane potential; Contractile function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Experiments in multicellular...
 3 Experiments in isolated...
 4 Summary and conclusions
 References
 

"It is a capital mistake to theorise before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts."

Sir Arthur Conan Doyle, 1891

In 1999, if one conducted an opinion poll amongst researchers who work on cardiac excitation–contraction coupling (ECC), the most accepted mechanism would be that calcium (Ca) ion entry through the L-type Ca channels is able to induce a large Ca release from the sarcoplasmic reticulum (SR). It is believed widely that the rise in cytoplasmic [Ca] (Cai) which activates the contractile proteins is due (at least mostly) to Ca release from the SR [1]. Moreover, most researchers would use the working hypothesis that the SR release during a normal cardiac action potential (AP) is triggered by Ca entry through L-type Ca channels (Ca-induced Ca-release, CICR; [2–4]). There might be some voices suggesting the involvement of other mechanisms, but they would probably be regarded as an exception to the general rule.

However, science is not "democratic" in this way (as politics can be). A theory does not take "power" and become "true" because a majority of cardiac researchers might vote for this theory as the most likely at one time-point. Each theory must be supported by experimental evidence, and tests of the hypothesis must continue to be consistent. The rationale for most types of ECC experiments has been the following: If Ca entry during the L-type Ca current (ICa,L) is the main trigger for SR release, then it follows that under conditions of a similar SR Ca load, the release of Ca should follow ICa,L closely. There should be no SR release without ICa,L or ICa,L without release. Moreover, under conditions when ICa,L increases or decreases, SR release might be expected to change in parallel with ICa,L (if SR load remains constant during the experiment, and as long as the release mechanism is not saturated).

Three of the many ways of varying the amplitude of ICa,L are: (i) changing the pulse potential; (ii) changing the prepulse membrane potential; or (iii) applying ICa,L agonists or antagonists. In this review, we will discuss first the evidence provided by experiments in multicellular cardiac preparations and then experiments in isolated cardiac cells.

Most previous experiments using these three procedures have been considered generally as demonstrating a strict correlation between ICa,L amplitude and SR release, and thus being consistent with ICa,L-induced CICR. However, when we looked closely at the literature we found (surprisingly) that this does not appear to be the case for a number of studies. The aim of this review was, therefore, to present these different and sometimes contradictory results, obtained in different preparations and under different experimental conditions, in an open-minded way and without bias.

We wish to emphasise an important point about our aim in this review — we intend to focus more on presenting the experimental data, rather than on their interpretation. This may be a little different from the approach usually taken in reviews, but we considered that:

(i) It may be useful to survey a wide range of previous data, before attempting to develop a theory which is able to explain all the observations. [For example, a threshold for SR release more negative than for ICa,L in multicellular preparations might be accounted for by a trigger mechanism other than ICa,L, or else by a voltage escape (see Section 2.1). However, the same observation made in isolated cells, where the possibility of a voltage escape is minimised, would suggest that this might due to a physiological mechanism rather than an artefact (Section 3.1).]

(ii) There is a lack of consensus at present among heart researchers about whether one ECC theory may be less or more probable, or influential, than another. Some of these discordant views are based on real differences in data obtained by different groups, but there may also be an element of personal bias involved. Strong views are natural and are, perhaps, even beneficial for the whole cardiac muscle field. However, any disagreement should not extend to the actual published experimental data, which are independent of personal beliefs. In the present review, our aim was to present the various diverse and controversial experimental results that have been obtained. After this, it becomes reasonable to consider the possible theories.


   2 Experiments in multicellular cardiac preparations
Top
 Abstract
 1 Introduction
 2 Experiments in multicellular...
3 Experiments in isolated...
 4 Summary and conclusions
 References
 
Before the "era" of isolated heart cells, voltage-clamp experiments were performed in Purkinje fibres [5–7] or in strips of ventricular muscle [8–12] (see Table 1). Before discussing the results, a mention is needed of the technical difficulties involved. The voltage-clamp of multicellular preparations depended on the following factors:


View this table:
[in this window]
[in a new window]

  		Table 1 Experimental conditions used in ECC studies performed in multicellular preparationsa

 
(i) That the intracellular resistivity of the tissue (i.e. the cytoplasmic resistivity plus that of intercalated disks) is low as compared to membrane resistivity, so that the interior of all the cells is maintained at the same potential;

(ii) That the resistivity of the extracellular fluid (present in the narrow clefts between the cells) is reduced as compared to that of cell membrane, so that all the cells have the same exterior potential;

(iii) That the amplifier is able to provide sufficient electrical current rapidly during the flow of large rapid currents;

(iv) That the ion concentration in the extracellular spaces within the clefts remained constant during an experiment, especially on a short time scale [13,14].

These points were discussed in an extensive review of voltage-clamp studies on multicellular preparations by Johnson and Lieberman [15] in 1971 and were tested experimentally in a number of studies [16–19]. It is beyond the scope of this review to discuss these issues again. Nevertheless, without trying to minimise them, we ought to mention the following points:

(i) Many research groups [5,7,10,20,21] performed control experiments to check the spatial voltage control of their preparation — they usually found a less than 5% difference of membrane voltage at a point remote from the voltage measuring electrode.

(ii) The main results of these early ECC studies were reproduced in a number of laboratories working on different preparations — which is (at least tentatively) inconsistent with the presence of a substantial artefact. In addition, results similar to these early ones have now been observed in more recent studies using voltage-clamped isolated cells, in which spatial clamp and solution exchange problems are minimised.

2.1 The voltage-dependence of contraction
In experiments performed in multicellular preparations, the voltage-dependence of tension was assessed using three different types of voltage protocols, admirably synthesised and compared by Gibbons and Fozzard [7]. These experiments measured either:

(i) The steady-state contraction elicited during a train of pulses (e.g. [7,8,10,22,23]);

(ii) The first contraction after a long (1–5 min) rest (e.g. [5–7,22]);

(iii) The contraction elicited by a test pulse after a train of standard conditioning pulses (e.g. [7,9]).

Steady-state tension had a threshold of activation around –30 mV, close to the threshold of ICa,L [7,8,10,22,23] (note also that in Ref. [8] a "foot" of 5% maximal contraction was elicited between –60 and –40 mV). The behaviour at positive potentials was less reproducible — tension either increased [7,24], remained at a plateau [8,10] or decreased [22]. The complicated nature of these types of experiment was clearly appreciated at that time. Beeler and Reuter [8] showed that an important factor determining the amplitude of steady-state contraction during trains of pulses at different potentials was the Ca loading of the cell, and not the trigger mechanism. Thus, the voltage-dependence of steady-state contraction did not represent the voltage-dependence of the trigger mechanism, but rather the voltage-dependence of the mechanisms governing SR Ca load [7,8,10,22].

In contrast, the protocols which investigated the voltage-dependence of contraction after a long rest or after a train of conditioning pulses provided information about the voltage-dependence of trigger mechanisms, since SR Ca load was the same for each test pulse. For illustration of these data, see Fig. 1A,B. The voltage-dependence of the post rest contraction was similar in the studies from various laboratories [5–7,22]; contraction began to develop around –60 mV and reached a maximum around 0 mV. A similar threshold of activation of contraction (~–60 mV) was reported for contractions elicited after a train of conditioning pulses [7,9]. In addition, large contractions could (usually) be elicited at positive potentials, such as +50 or +60 mV [7,9] — see Fig. 1A,B). Since the threshold of activation of contraction was more negative than the threshold of ICa,L, this led to the general view at the time as stated by Beeler and Reuter [8]: "calcium ions activating contraction can be released [...from the SR] by an unknown mechanism which is dependent on depolarisation but apparently independent of the flow of ICa". However, this conclusion was a little vulnerable to the possibility of artefacts, such as poor voltage control. With hindsight, alternative pathways for Ca entry might also have been involved, such as T-type Ca channels [25], or reverse sodium (Na)/Ca exchange [26,27]. However, similar observations have now been made in isolated cells (Section 3.1), where these alternative hypotheses could be tested.


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Voltage-dependence of activation of contraction in multicellular preparations. A. Voltage-dependence of activation of contraction elicited by the first pulse after a long rest. Data were normalised for the plateau or maximal value and were fitted with a Boltzmann activation function: Tension (normalised)=1/(1+exp[(V0.5–Vm)/k]) where: V0.5 is half-maximal activation potential and k is the slope of the activation curve. Predicted V0.5 and k are given below. Data from: {square}: [5]; V0.5=–42.0±1.0; k=5.7±0.8; {blacksquare}: [6]; not fitted; {circ}: [7]; V0.5=–30.1±2.7; k=14.9±2.6. B. Voltage-dependence of activation of contraction elicited after a train of conditioning pulses. Data from: bullet: [9]; V0.5=–17.4±2.3; k=16.3±2.5; {diamond}: [7]; V0.5=–30.4±1.7; k=13.6±1.6.

 
2.2 Dependence of contraction on the prepulse potential
Cardiac muscle contraction was dependent not only on the pulse potential but also on the membrane potential immediately before the pulse (the prepulse potential). Beeler and Reuter [8] reported that contraction was maximal from a prepulse potential of –80 mV and decreased with less negative potentials — from a prepulse potential of –40 mV almost no tension was elicited. For an illustration of these data, see Fig. 2A. Similar results were obtained by Gibbons and Fozzard, [6] and New and Trautwein, [10] (Fig. 2B). As ICa,L only began to inactivate at –40 mV, New and Trautwein [10] concluded that "there is a potential-dependent tension-determining process which is unrelated to the slow inward current [i.e. ICa,L]".


Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Voltage-dependence of inactivation of contraction in multicellular preparations. A. Reproduced from Ref. [8] with permission. Tension (upper traces), membrane currents (middle traces) during voltage-clamp pulses (lower traces) to +10 mV. Prepulse voltages are shown for each panel, holding potential was –79 mV. Clamp steps applied at 0.33/s. B. Voltage-dependence of inactivation. Data were normalised for the maximum and fitted with a Boltzmann inactivation function, Tension (normalised)=1–1/(1+exp[(V0.5–Vm)/k]); where: V0.5 is half-maximal inactivation potential and k is the slope. Predicted V0.5 and k are given below. Data from: {square}: [8]; V0.5=–51.8±0.7; k=7.3±0.6; {blacksquare}: [6]; V0.5=40.4±0.7; k=6.9±0.7; {circ}: [7]; V0.5=–53.0±1.5; k=8.4±1.5; bullet: [22]; V0.5=–32.1±1.8; k=10.4±2.1.

 
The nature of this inactivation process remained obscure. A major role was thought to be played by inactivation of the trigger mechanism, although a part could be played by the intracellular cycle of release and reuptake of Ca [6]. The conclusion of Beeler and Reuter [8] remained valid for many years: "Attempts in the present study to correlate restoration [from inactivation] of contraction with the restoration of either the sodium system or the calcium system, which are both activated and inactivated during the depolarisation, failed completely. The factor responsible for release of calcium from intracellular binding sites during depolarisation also seems to undergo an activation–inactivation cycle but its nature remains to be solved". It may also be worth noting that this important observation that contraction was inactivated by a prepulse potential of –40 mV may be less easy to explain in terms of an artefact such as poor spatial clamp, or ion accumulation/depletion in the intercellular clefts.

2.3 Effect of blocking ICa,L
Bass made one of the first studies [12] investigating the effect of ICa,L blockers on ventricular muscle contraction. A train of test stimuli was applied first in control and then after applying 1 mM nickel (Ni) or 1 mM cobalt (Co). Ni and Co had no effect on the strength of the first test beat but decreased the steady-state contraction (to ~50% of control). From recent experiments, 1 mM Ni is known to inhibit the L-type Ca channel by 80% [28] and the effect on the steady-state contraction (and probably SR Ca load) was consistent with an inhibitory effect of Ni and Co on ICa,L. The effect of Ni or Co on the AP shape (the AP was shortened and with a less marked plateau) was identical for the first and subsequent test beats, suggesting that the Ni and Co effect was not use-dependent. Bass [12] concluded that "inward Ca current through the surface membrane must play a major part in loading the stores rather than in directly mediating contraction". In the context of ideas current at the time, Bass saw his results as inconsistent with a "direct" role for Ca entry in activating the myofilaments, but they appear just as difficult to explain if ICa,L-induced SR release was activating contraction.

These experiments were repeated by McDonald et al. [11] showing that 2 mM Co inhibited 33–46% of tension. The authors felt at the time that this was in sharp contrast with the results of Bass [12] but still did not explain how ~60% of the contraction might be left when ICa,L was blocked by 2 mM Co.

In conclusion, at the beginning of the 1980s, the experiments performed in multicellular preparations had suggested that ICa,L may be responsible for loading the SR with Ca, but were not always consistent with the involvement of ICa,L in triggering SR release. Tritthart et al. [22] stated that: "We can exclude a Ca-induced Ca-release as being the primary cause for activation, since (...) activation already occurs at the threshold potential of INa, where Ca is assumed not to carry much of the inward current". Other studies came to the same conclusion when investigating the voltage-dependence of inactivation of contraction [6,8,10] and the effect of ICa,L blockers [12].

Nevertheless, some studies did find a correlation between ICa,L and contraction. New and Trautwein [10] reported that the voltage-dependence of steady-state contraction had a similar threshold as ICa,L. However, they also found that contraction remained large at high positive potentials, which was not consistent with ICa,L-induced SR release. They suggested that "it is not the entering calcium which elicits the accompanying contraction, but rather the conductance change of the slow channel which could release calcium from intracellular binding sites" [10]. Furthermore, Trautwein et al. [29] reported that contraction (elicited with 30 ms depolarisations) had a voltage-dependence of activation and inactivation close to that of ICa,L. In multicellular preparations, Gibbons and Fozzard [23] and, more recently Arlock and Wolhfart [21] have observed a similar voltage-dependence of ICa,L and contraction, consistent with a role for ICa,L-induced CICR.


   3 Experiments in isolated cardiac cells
Top
 Abstract
 1 Introduction
 2 Experiments in multicellular...
 3 Experiments in isolated...
4 Summary and conclusions
 References
 
The early-to-mid 1980s saw four major and largely simultaneous developments:

(i) Increasing use of the technique for isolating single heart cells [30–33];

(ii) The patch-clamp technique, allowing low resistance access to the inside of the cells. This provided improved voltage control and also the possibility of dialysing substances into and out of cells [34];

(iii) Fluorescent Ca indicators (as Fura-2 and Indo-1 [35,36]);

(iv) The elegant demonstration of CICR by Fabiato and Fabiato in skinned cardiac cells [2,4,37–39].

The use of isolated myocytes minimised any spatial inhomogeneity of membrane potential, allowed faster exchange of external solution, and minimised ion accumulation/depletion artefacts. Balanced against these advantages was the uncertainty that single heart cells subjected to enzymatic and mechanical dispersion during the isolation procedure behave similarly to cells in vivo. Moreover, in the case of whole cell patch-clamp, there was also the possibility of dialysing important intracellular constituents out of the cell. The experiments performed initially in single cells provided a clear demonstration of the presence of an ICa,L-activated CICR mechanism (see below).

3.1 The voltage-dependence of contraction/SR release
The first paper investigating the voltage-dependence of contraction in single heart cells was by London and Krueger in 1986 [40] and reported the similarity between the voltage-dependence of contraction and the voltage-dependence of ICa,L. When Ca indicators began to be used to assess directly Ca release from the SR, the voltage-dependence of SR release was, under some experimental conditions, shown to be similar to the voltage-dependence of ICa,L (see Table 2 for Refs.). Typically, in isolated heart cells (depending on experimental conditions — see below) the threshold of activation of SR release was between –40 and –30 mV, similar to the threshold of activation of ICa,L. Cai transients generally reached a peak value near +20 mV and, in parallel with ICa,L, decreased at more positive potentials. Depolarisations to high positive potentials (between +60 and +100 mV) did not elicit a Cai transient. This "bell-shaped" voltage-dependence of contraction/SR release was (at least) consistent with the hypothesis that ICa,L might be the only trigger for CICR.


View this table:
[in this window]
[in a new window]

  		Table 2 Published full papers investigating the voltage-dependence of contraction/SR release in isolated cardiac cells under various experimental conditionsa

 
However, recently it began to become clear that the voltage-dependence of SR release was "bell-shaped" only if (at least) one of the following conditions was used (see Table 2 for Refs.):

(i) Internal K was replaced by caesium (Cs) (to block K currents and thus allow more selective recordings of ICa,L). Nevertheless, it has been shown that in cells dialysed with a Cs-based solution, the voltage-dependence of SR release was closer to a bell shape as compared to when a K-based solution was used (all other conditions being the same; [41,42]). One possible interpretation may be that K (as well as other ions [43]) may provide the counter-ion movement into the SR to compensate for the charge transferred out of the SR during Ca release [44,45]. Indirect evidence has also been shown that internal Cs may decrease the sensitivity of SR release channels for Ca and the "gain" of CICR [46].

(ii) The pipette solution used was Na-free (see Table 2). This minimised reverse Na/Ca exchange and thus decreased this possible Ca entry pathway. At the same time, reduction of Ca entry through Na/Ca exchange decreased the Ca load of the SR, but evidence suggested that this may be secondary in importance to the reduction of trigger Ca entry at positive potentials [27].

(iii) Experiments were performed at room temperature. Unlike skeletal muscle, the mammalian heart is always at 37°C. Both the Ca channels and the Na/Ca exchange are temperature-dependent (a Q10 — i.e. the change in activity produced by a 10°C change in temperature — of ~2–3 has been reported for ICa,L [47,48] and of ~4 for Na/Ca exchange [49]). Thus, in the many studies performed at room temperature, the Na/Ca exchange (especially) will be greatly reduced and this might be involved in the decrease in the SR release triggering at positive potentials.

When all these three conditions were kept "physiological" (i.e. experiments using a K-based, 5–15 mM Na containing pipette solution and a physiological temperature of 35–37°C), the voltage-dependence of SR release in heart cells had a "sigmoid" shape, with high levels at positive potentials (see Table 2 for Refs.).

The magnitude of Ca entry via L-type Ca channels between +60 and +100 mV may be difficult to measure (selectively measured ICa,L reversed between +60 and +80 mV [50], but the calculated Ca equilibrium potential (ECa) was +122 mV, if Cai=100 nM and external [Ca]=1 mM). However, there is little doubt that ICa,L is greatly reduced at these positive potentials as compared to peak ICa,L (at ~+20 mV). With ECa of ~+120 mV, and using a simple linear extrapolation for driving force, ICa,L recorded at +60, +80 and +100 mV would be 60, 40 and 20% of maximal ICa,L, respectively (this is, however, likely to be a large overestimation — see Ref. [51]). If we also take into account that the "gain" of CICR has been shown to be greatly reduced at positive potentials [52], then the SR release triggered by ICa,L between +60 and +100 mV should be only a small fraction of the SR release triggered at +20 mV. Therefore, the studies which reported a sigmoid voltage-dependence of SR release were able to suggest that, besides ICa,L, there may be other Ca entry mechanisms capable of triggering CICR (such as reverse Na/Ca exchange — see Table 2 for Refs.).

Alternative Ca entry pathways have been proposed to function at negative potentials as well. Leblanc and Hume suggested that Na ions entering via the Na channel may accumulate in the subsarcolemmal space and activate reverse Na/Ca exchange [26] (if one could exclude a "voltage escape" during the flow of INa [53,54]. Ca may enter via T-type Ca channels [55], tetrodotoxin (TTX)-sensitive Ca channels [56,57] or even (as proposed very recently) phosphorylated Na channels (the so-called "slip-mode conductance" [58]).

An interesting new development began with the report by Ferrier and Howlett [59] that in undialysed guinea-pig myocytes (voltage-clamped with sharp microelectrodes) the threshold of contraction was at –60 mV, thus more negative than the ICa,L threshold [59,60] (see Fig. 3). These authors suggested that, besides Ca entry induced CICR, heart cells might possess another SR release mechanism — which was proposed to be independent of transmembrane Ca entry, and perhaps directly controlled by membrane voltage. Similar results were obtained using patch-clamped cells dialysed with a pipette solution which contained cAMP [61–63]. The SR release had a threshold at –60 mV and a half-maximal voltage close to –40 mV, whereas the threshold of activation for ICa,L was –30 mV. Large Cai transients were obtained at +100 mV. Consistent with the hypothesis that this release may not be triggered by Ca entry, the threshold of SR release was not changed in the presence of 120 µM cadmium (Cd)+100 µM Ni (which blocked both ICa,L and T-type Ca current, ICa,T; [55,63]). In these experiments, 90 µM TTX was also present, which excluded other possible sources of Ca entry, as INa-induced reverse Na/Ca exchange [26], the TTX-sensitive Ca channels [56,57] or the phosphorylated Na channels [58].


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Contraction of isolated, undialysed myocytes. Data from Ref. [60]. Experiments performed in isolated guinea pig myocytes, voltage-clamped (discontinuous single-electrode voltage-clamp) using high-resistance microelectrodes (18–25 M{Omega}). Shown are: {circ}; voltage-dependence of unloaded cell shortening. Holding potential was –70 mV, in the presence of Lidocaine. Note the threshold of contraction was –60 mV and the voltage-dependence curve was sigmoid. Data were fitted with a Boltzmann function, with V0.5=–41.6±1.1; k=6.9±1.0. bullet; Voltage-dependent inactivation of contraction. After various prepulse potentials, cell contraction was elicited with a depolarisation to –40 mV. Data were fitted with a Boltzmann function, with V0.5=–47.0±0.1; k=4.1±0.1.

 
These new studies provide a link with the older ECC studies carried out in multicellular preparations, since the voltage-dependence of contraction/SR release in isolated heart cells (either undialysed or dialysed with pipette solution containing cAMP) appears similar to that reported in multicellular preparations (see Section 2.1). Importantly, the voltage threshold of SR release/contraction in isolated cells was more negative that the threshold of ICa,L. In the multicellular preparations one possible artefact might have been poor voltage control. However this was less likely to be problematic in isolated cell studies using the discontinuous single electrode voltage-clamp [59,60] or patch-clamp with or without blockers of INa and ICa,L [62]. Consequently, these newer studies have raised the possibility that there may be another ECC mechanism in cardiac muscle, in addition to CICR. Possibly, this second mechanism might be similar to the voltage-sensor mechanism thought to function in skeletal muscle [64]. The proposal is that the second ECC mechanism may function alongside CICR, and that depending on experimental conditions such as temperature and second messenger levels, one mechanism may appear to be more or less dominant [59,60,62,65]. (It is not the aim of this review to discuss the arguments for and against the existence of a "voltage-sensor-type" mechanism in heart muscle. For more details, the reader is invited to consult the original papers, and also a review [66].)

3.2 The dependence of SR release/contraction on the prepulse membrane potential
Unlike in multicellular preparations, SR release/contraction of isolated cells classically showed no [67,68] or little [42,69] change when the prepulse was varied between –80 and –40 mV. This was consistent with the idea that SR release is triggered by CICR following Ca entry via L-type Ca channels (or L-type Ca channels with Na/Ca exchange).

However, in undialysed cells [61] or in cells patch-clamped with a cAMP containing pipette solution [70] contraction/SR release was greatly reduced from a prepulse potential of –40 mV as compared to –80 mV. SR release elicited in the presence of Ni (which blocked CICR induced by either ICa,L or the Na/Ca exchange) was maximal from –80 mV, and more than 50% reduced from –40 mV, whereas little ICa,L inactivation occurred over this potential range. As a control experiment, we confirmed that there was no change in the SR Ca load from different prepulse potentials [70] or that Ni block of ICa,L was not dependent on the prepulse potential [28]. (Moreover, the other possible pathway for Ca entry, the Na/Ca exchanger, has no known voltage dependent inactivation and could not explain the reduction in SR release.)

Therefore, the contraction/SR release of isolated cells can (under some conditions) show a voltage-dependence of inactivation, similar to that observed in multicellular preparations (see Section 2.2). The voltage range was more negative (by about 20–30 mV) than that for ICa,L inactivation. This would appear to be inconsistent with the hypothesis that the trigger for SR release under these conditions might be ICa,L with or without the participation of the Na/Ca exchange.

3.3 The effect of blocking Ca entry pathways
Experiments in isolated cells, performed at room temperature, showed that SR release/contraction was blocked when ICa,L was blocked with 100–300 µM Cd [68,71,72]. These Cd concentrations were thought to be selective blockers of ICa,L, (but note they can also block 20–50% of Na/Ca exchange — see Ref. [73]). This result was considered consistent with the hypothesis that (at room temperature) ICa,L-induced CICR is the only trigger mechanism for SR Ca release. Similar results were obtained when ICa,L was blocked with organic Ca channel blockers [71,74–76].

However, at a physiological temperature of 37°C, a number of studies showed that blocking ICa,L with Cd or organic blockers did not abolish SR release/contraction [42,72,77–81]. In some studies (which used a cAMP-free pipette solution), the Cd insensitive component of SR release could be inhibited by 3–5 mM Ni (which also blocked the Na/Ca exchange [49]). Thus, these studies at 37°C supported a role for Ca entry via reverse Na/Ca exchange as a trigger for SR release [42,72,77].

Experiments performed in our laboratory in cells dialysed with cAMP-containing pipette solution have shown that a large component of SR release can still be elicited by membrane depolarisation, even when Ca entry into the cells was inhibited with 5–8 mM Ni. In control experiments, (with ICa,L measured selectively) we have confirmed that rapid application of Ni to an isolated myocyte is able to block ICa,L and the effect is largely not use-dependent [28]. Fig. 4 illustrates a typical experiment in which a mixture of 5 mM Ni and 100 µM Cd (each of them able to block ICa,L completely; in addition, 5 mM Ni blocked Na/Ca exchange, [49]) was rapidly applied to the cell. It can be seen that membrane depolarisation elicited a large SR Ca release in the absence of detectable ICa,L. A large SR release in the absence of any detectable ICa,L could be elicited in cells from rat, rabbit or guinea pig hearts [65] and their relative contribution to the total SR release was dependent on internal cAMP, saturating at 50–100 µM (concentration in the pipette solution) [65]. Interestingly, the Ni-insensitive component of SR release was reduced if cells were dialysed with a Cs-based pipette solution (rather than the more physiological K-based; I.A. Hobai and A.J. Levi, unpublished results). If this is put together with the fact that this second ECC mechanism may be largely inactivated at –40 mV, then it is possible to see how studies using a Cs-based pipette solution, at room temperature, and a holding potential of –40 mV may have observed ICa,L-induced CICR as the only ECC mechanism present even under β-adrenergic stimulation [46,53,69].


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 SR release in the presence of Ca entry blockers. An experiment performed in isolated guinea pig myocytes, patch-clamped using a K-based pipette solution which contained 100 µM cAMP, at 37°C. Shown are Cai transients and membrane currents elicited by a depolarisation from –60 mV to +20 mV, in the presence of 350 µM Lidocaine. Bottom traces show the same current traces superimposed and on a faster time base. In the presence of 5 mM Ni and 100 µM Cd, ICa,L was blocked but membrane depolarisation still elicited a large Cai transient (I.A. Hobai and A.J. Levi, unpublished data).

 
One possible interpretation of these results was that the SR release elicited in the presence of Ca entry blockers might be triggered by a second release mechanism which was activated by depolarisation and independent of Ca entry. An alternative possibility is that cAMP may increase the gain of CICR so that a small (undetectable) residual ICa,L might be able to trigger the large SR release observed [46] — although some evidence suggested this might not be the case [61,82].

These results were, again, similar to those obtained in multicellular preparations [12]. Multicellular preparations have also been used again recently. Thornton and Paterson [83] have shown that isolated guinea pig papillary muscle preparations continued to contract for many minutes in the presence of external 5 mM Ni.


   4 Summary and conclusions
Top
 Abstract
 1 Introduction
 2 Experiments in multicellular...
 3 Experiments in isolated...
 4 Summary and conclusions
References
 
One stimulus for this review article was the emergence of recent data [59,60,65] challenging the current view that heart ECC involves only Ca entry-induced CICR. This classical view has been supported by experiments showing that contraction and SR Ca release in isolated cells can (under some conditions) be closely related to ICa,L amplitude. These experiments suggested strongly that ICa,L-induced CICR can be a trigger of SR release. Nevertheless, they did not prove that ICa,L-induced CICR was the only trigger mechanism for SR release.

Apparently inconsistent with the possibility that ICa,L and Ca entry-induced CICR is the only trigger for SR release, it now seems clear that contraction/SR release in cells either undialysed, or dialysed with a cAMP-containing pipette solution, could be elicited by membrane depolarisation when little or no Ca entry occurs. Large SR releases could be elicited at potentials negative to the ICa,L threshold, and also at high positive potentials (close to ECa [59,60]) in the presence of ICa,L blockers (such as Cd or Ni [65]). Moreover, contraction/SR release was inactivated at potentials negative to the threshold for ICa,L inactivation [60,61,70]

Interestingly, these results appear somewhat similar to data obtained in the initial studies on heart ECC, performed in multicellular preparations in the early 1970s. The importance of these older and pioneering results (which themselves were not entirely consistent with ICa,L-induced CICR being the only ECC mechanism) has been minimised in the last 10 to 15 years, because of possible technical artefacts. Although similar observations now made in isolated cells might tend to suggest there may not have been a major artefactual influence, this nevertheless remains a possibility. Other possibilities exist which might explain these early multicellular results, from alternative pathways for Ca entry to a "skeletal muscle type" ECC mechanism. The detailed discussion of these other possibilities is a complex task for the future. However, if this review has provided an impetus to future experimentation and discussion, we would consider our main aim to have been achieved.

Time for primary review 21 days.


   Acknowledgements
 
Original experiments presented here and performed in our laboratory in Bristol were supported by grants from the Wellcome Trust, British Heart Foundation and The United Bristol Healthcare NHS Trust. The authors would like to thank Drs. Corne Kros and Jules Hancox (University of Bristol) and Clive Orchard (University of Leeds) for helpful discussions on the manuscript and encouragement. We are indebted to J.Q. Zhu, S.E. Howlett and G.R. Ferrier (Halifax, Canada), who made the first observations on the modulatory effect of internal cAMP on SR release and kindly shared their knowledge with us in Summer 1996. Our fruitful collaboration is acknowledged.


   Notes
 
1 Present address: Department of Medicine, Johns Hopkins University, Baltimore, USA. Back


   References
Top
 Abstract
 1 Introduction
 2 Experiments in multicellular...
 3 Experiments in isolated...
 4 Summary and conclusions
 References
 

  1. Bers D.M. Excitation–contraction coupling and cardiac contractile force. (1991) Dordrecht: Kluwer.
  2. Fabiato A. Simulated calcium current can both cause calcium loading in, and trigger calcium release from, the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol (1985) 85:291–320.[Abstract/Free Full Text]
  3. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol (1983) 14:C1–C14.
  4. Fabiato A., Fabiato F. Ca-induced release of Ca from the sarcoplasmic reticulum of skinned cells from adult human, dog, rabbit, rat and frog hearts and from foetal and new-born rat ventricles. Ann New York Acad Sci (1978) 307:491–522.[Medline]
  5. Fozzard H.A., Hellam D.C. Relationship between membrane voltage and tension in voltage-clamped cardiac Purkinje fibres. Nature (1968) 218:588.[CrossRef][Medline]
  6. Gibbons W.R., Fozzard H.A. Voltage dependence and time dependence of contraction in sheep cardiac Purkinje fibres. Circ Res (1971) 28:446–460.[Abstract/Free Full Text]
  7. Gibbons W.R., Fozzard H.A. Relationships between voltage and tension in sheep cardiac Purkinje fibres. J Gen Physiol (1975) 65:345–365.[Abstract/Free Full Text]
  8. Beeler G.W., Reuter H. The relation between membrane potential, membrane currents and activation of contraction in ventricular myocardial fibres. J Physiol (1970) 207:211–229.[Abstract/Free Full Text]
  9. Ochi R., Trautwein W. The dependence of cardiac contraction on depolarisation and slow inward current. Pflug Arch (1971) 323:187–203.[CrossRef][Web of Science][Medline]
  10. New W., Trautwein W. The ionic nature of slow inward current and its relation to contraction. Pflug Arch (1972) 334:24–38.[CrossRef][Web of Science][Medline]
  11. McDonald T.F., Pelzer D., Trautwein W. Does the calcium current modulate the contraction of the accompanying beat? Circ Res (1981) 49:576–583.[Abstract/Free Full Text]
  12. Bass O. The decay of potentiated state in sheep and calf ventricular fibres — influence of agents acting on transmembrane Ca flux. Circ Res (1976) 39:396–399.[Abstract/Free Full Text]
  13. Cohen I.S., Kline R. K+ fluctuations in the extracellular spaces of cardiac muscle. Circ Res (1982) 50:1–16.[Free Full Text]
  14. Kunze D.L. Rate-dependent changes in extracellular potassium in the rabbit atrium. Circ Res (1976) 41:122–127.[Web of Science]
  15. Johnson E.A., Lieberman M. Heart: excitation and contraction. Ann Rev Physiol (1971) 33:532.
  16. Beeler G.W., Reuter H. Voltage clamp experiments on ventricular myocardial fibres. J Physiol (1970) 207:165–190.[Abstract/Free Full Text]
  17. Rougier O., Vassort G., Stampfli S. Voltage clamp experiments on frog atrial heart muscle fibres with the sucrose gap technique. Pflug Arch (1968) 301:91–108.[CrossRef][Web of Science]
  18. Fozzard H.A., Hiraoka M. The positive dynamic current and its inactivation in cardiac Purkinje fibres. J Physiol (Lond) (1973) 234:569–586.[Abstract/Free Full Text]
  19. Fozzard H.A., Schoemberg M. Strength–duration curves in cardiac Purkinje fibres effects of liminal length and charge distribution. J Physiol (1972) 226:593–618.[Abstract/Free Full Text]
  20. Morad M., Trautwein W. The effect of the duration of the action potential on contraction of the mammalian heart muscle. Pflug Arch (1968) 299:66–82.[CrossRef][Web of Science]
  21. Arlock P., Wohlfart B. Force production following transient potential changes in voltage-clamped myocardium. Acta Physiol Scand (1990) 140:63–72.[Web of Science][Medline]
  22. Tritthart H., Kaufmann R., Volkmer H., Bayer A.K., Krause K.H. Calcium movement controlling myocardial contractility. Pflug Arch (1999) 338:207–231.[CrossRef]
  23. Gibbons W.R., Fozzard H.A. Slow inward current and contraction of sheep cardiac Purkinje fibres. J Gen Physiol (1975) 65:367–384.[Abstract/Free Full Text]
  24. Simurda J., Simurdova M., Bravny P., Sumbera J. Slow inward current and action potentials of papillary muscle under nonsteady state conditions. Pflug Arch (1976) 362:209–218.[CrossRef][Web of Science][Medline]
  25. Sipido K.R., Carmeliet E., Van de Werf F. T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes (Abstract). J Physiol (1998) 508:439–451.[Abstract/Free Full Text]
  26. Leblanc N., Hume J.R. Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. Science (1990) 248:372–376.[Abstract/Free Full Text]
  27. Levi A.J., Li J., Litwin S.E., Spitzer K.W. Effect of internal sodium and cellular calcium load on voltage-dependence of the Indo-1 transient in guinea-pig ventricular myocytes. Cardiovasc Res (1996) 32:534–550.[Abstract/Free Full Text]
  28. Hobai I.A., Hancox J.C., Levi A.J. Inhibition of the L-type Ca channel by external Ni2+ in guinea-pig ventricular myocytes dialysed with cAMP-free and cAMP-containing solutions (Abstract). J Physiol (1998) 511P:80P.
  29. Trautwein W., McDonald T.F., Tripathi O. Calcium conductance and tension in mammalian ventricular muscle. Pflug Arch (1975) 354:55–74.[CrossRef][Web of Science][Medline]
  30. Powell T., Terrar D.A., Twist V.W. Electrical properties of individual cells isolated from adult rat ventricular myocardium. J Physiol (1980) 302:131–153.[Abstract/Free Full Text]
  31. Dow J.C., Harding N.G.L., Powell T. Isolated cardiac myocytes: 1. Preparation of adult myocytes and their homology with intact tissue. Cardiovasc Res (1981) 15:483–514.[Free Full Text]
  32. Powell T., Twist V.W. A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium. Biochem Biophys Res Commun (1976) 72:327–333.[Web of Science][Medline]
  33. Gould R.P., Powell T. Intact isolated muscle cells from the adult rat heart. J Physiol (1972) 225:16P–19P.[Medline]
  34. Hamill O.P., Marty A., Neher E., Sakmann B., Sigworth F.J. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflug Arch (1981) 391:85–100.[CrossRef][Web of Science][Medline]
  35. Tsien R.Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures. Biochemistry (1980) 19:2396–2404.[CrossRef][Web of Science][Medline]
  36. Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of calcium indicators with greatly improved fluorescent properties. J Biol Chem (1985) 260:3440–3450.[Abstract/Free Full Text]
  37. Fabiato A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol (1985) 85:247–289.[Abstract/Free Full Text]
  38. Fabiato A., Fabiato F. Activation of skinned cardiac cells: subcellular effects of cardioactive drugs. Eur J Cardiol (1973) 1/2:143–155.
  39. Fabiato A., Fabiato F. Calcium release from the sarcoplasmic reticulum. Circ Res (1977) 40:119–129.[Free Full Text]
  40. London B., Krueger J.W. Contraction in voltage-clamped, internally perfused single heart cells. J Gen Physiol (1986) 88:475–505.[Abstract/Free Full Text]
  41. Levi A.J., Mitcheson J.S., Hancox J.C. The effect of internal sodium and caesium on phasic contraction of patch-clamped rabbit ventricular myocytes. J Physiol (1996) 492:1–19.[Abstract/Free Full Text]
  42. Wasserstrom J.A., Vites A.M. The role of Na–Ca exchange in activation of excitation–contraction coupling in rat ventricular myocytes. J Physiol (1996) 493:529–542.[Abstract/Free Full Text]
  43. Kourie J.I., Foster P.S., Dulhunty A.F. Inositol polyphosphates modify the kinetics of a small chloride channel in skeletal muscle sarcoplasmic reticulum (Abstract). J Membr Biol (1997) 157:147–158.[CrossRef][Web of Science][Medline]
  44. Cukierman S., Yellen G., Miller C. The K+ channel of sarcoplasmic reticulum. Biophys J (1985) 48:477–484.[Web of Science][Medline]
  45. Fink M., Veigel C. Calcium uptake and release modulated by counter-ion conductances in the sarcoplasmic reticulum of skeletal muscle. Acta Physiol Scand (1996) 156:397.[CrossRef][Web of Science][Medline]
  46. Hussain M., Orchard C.H. Sarcoplasmic reticulum Ca2+ content, L-type Ca2+ current and the Ca2+ transient in rat myocytes during β-adrenergic stimulation. J Physiol (1997) 505:385–402.[Abstract/Free Full Text]
  47. Mitchell M.R., Powell T., Terrar D.A., Twist V.W. Characteristics of the second inward current in cells isolated from rat ventricular muscle. Proc R. Soc. (1983) B219:447–469.[Web of Science]
  48. Cavalie A, McDonald F, Pelzer D, Trautwein W. Temperature-induced transitory and steady-state changes in calcium current of guinea-pig ventricular myocytes. Pflug Arch 1999;405.
  49. Kimura J., Miyamae S., Noma A. Identification of sodium–calcium exchange current in single ventricular cells of guinea-pig. J Physiol (1987) 384:199–222.[Abstract/Free Full Text]
  50. Lee K.S., Tsien R.W. Reversal of current through calcium channels in dialysed single heart cells. Nature (1982) 297:501.[CrossRef][Medline]
  51. Stern M.D. Theory of excitation–contraction coupling in cardiac muscle. Biophys J (1992) 63:497–517.[Web of Science][Medline]
  52. Wier W.G., Egan T.M., López-López J.R., Balke C.W. Local control of excitation–contraction coupling in rat heart cells. J Physiol (1994) 474:463–471.[Abstract/Free Full Text]
  53. Sham J.S.K., Cleemann L., Morad M. Gating of the cardiac Ca2+ release channel: The role of Na+ current and Na+–Ca+ exchange. Science (1992) 255:850–853.[Abstract/Free Full Text]
  54. Hancox J.C., Levi A.J. Calcium transients which accompany the activation of sodium current in rat ventricular myocytes at 37°C: a trigger role for reverse Na/Ca exchange activated by membrane potential. Pflug Arch (1995) 430:887–893.[CrossRef][Web of Science][Medline]
  55. Tytgat J., Vereecke J., Carmeliet E. A combined study of Na current and T-type Ca current in isolated cardiac cells. Pflug Arch (1990) 417:142–148.[CrossRef][Web of Science][Medline]
  56. Lemaire S., Piot C., Seguin J., Nargeot J., Richard S. Tetrodotoxin-sensitive Ca2+ and Ba2+ currents in human atrial cells. Recept Channels (1995) 3:71–81.[Web of Science][Medline]
  57. Aggarwal R., Shorofsky S.R., Goldman L., Balke C.W. Tetrodotoxin-blockable calcium currents in rat ventricular myocytes; a third type of cardiac cell "sodium" current. J Physiol (1998) 505:353–369.[CrossRef][Web of Science]
  58. Santana L.F., Gomez A.M., Lederer W.J. Ca2+ flux through promiscuous cardiac Na+ channels: Slip mode conductance. Science (1998) 279:1027–1033.[Abstract/Free Full Text]
  59. Ferrier G.R., Howlett S.E. Contractions in guinea-pig ventricular myocytes triggered by a calcium-release mechanism separate from Na and L-currents. J Physiol (1995) 484:107–122.[Abstract/Free Full Text]
  60. Howlett S.E., Zhu J.Q., Ferrier G.R. Contribution of a voltage-sensitive calcium release mechanism to contraction in cardiac ventricular myocytes. Am J Physiol (1997) 274:H1–H16.[Web of Science]
  61. Ferrier G.R., Zhu J.Q., Redondo I., Howlett S.E. Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes. J Physiol (1998) 513:185–201.[Abstract/Free Full Text]
  62. Levi AJ, Ferrier GR. Ca release activated by membrane depolarisation in the absence of Ca entry in mammalian heart (Abstract). Biophys J 1997;72(2):A161-Tu-Pos130.
  63. Hobai I.A., Hu X.W., Levi A.J. Voltage dependence of Ca release in guinea pig ventricular myocytes dialysed with cAMP (Abstract). Biophys J (1999) 76:A463.
  64. Rios E., Pizarro G. Voltage sensor of excitation–contraction coupling in skeletal muscle. Physiol Rev (1991) 71:849–908.[Free Full Text]
  65. Hobai I.A., Howarth F.C., Pabbathi V.K., et al. "Voltage-activated Ca release" in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP. Pflug Arch (1997) 435:164–173.[CrossRef][Web of Science][Medline]
  66. Howlett S.E., Ferrier G.R. The voltage-sensitive release mechanism: a new trigger for cardiac contraction (Abstract). Can J Physiol Pharmacol (1997) 75:1044–1057.[CrossRef][Web of Science][Medline]
  67. Beuckelmann D.J., Wier W.G. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J Physiol (1988) 405:233–255.[Abstract/Free Full Text]
  68. Bouchard R.A., Clark R.B., Giles W.R. Regulation of unloaded cell shortening by sarcolemmal sodium–calcium exchange in isolated rat ventricular myocytes. J Physiol (1993) 469:583–599.[Abstract/Free Full Text]
  69. Cleemann L., Morad M. Role of Ca2+ channel in cardiac excitation–contraction coupling in the rat: evidence from Ca2+ transients and contraction. J Physiol (1991) 432:283–312.[Abstract/Free Full Text]
  70. Hobai I.A., Patel K.C.R., Perchenet L., Levi A.J. Availability of "voltage-activated Ca2+ release" depends on pre-pulse membrane potential in guinea-pig ventricular myocytes at 37°C (Abstract). J Physiol (1998) 509P:144P.
  71. Bouchard R.A., Clark R.B., Giles W.R. Role of sodium–calcium exchange in activation of contraction in rat ventricle. J Physiol (1993) 472:391–413.[Abstract/Free Full Text]
  72. Vornanen M., Shepherd N., Isenberg G. Tension–voltage relations of single myocytes reflect Ca release triggered by Na/Ca exchange at 35°C but not at 23°C. Am J Physiol (1994) 267:C623–C632.[Web of Science][Medline]
  73. Hobai I.A., Bates J.A., Howarth F.C., Levi A.J. Inhibition by external Cd2+ of Na/Ca exchange and L-type Ca channel in rabbit ventricular myocytes. Am J Physiol (1997) 272:H2164–H2172.[Web of Science][Medline]
  74. Barcenas-Ruiz L., Wier W.G. Voltage-dependence of intracellular [Ca2+]i transients in guinea pig ventricular myocytes. Circ Res (1987) 61:148–154.[Abstract/Free Full Text]
  75. Sipido K.R., Callewaert G., Carmeliet E. [Ca2+] transients and [Ca2+]i dependent chloride current in single Purkinje cells from rabbit heart. J Physiol (1993) 468:641–667.[Abstract/Free Full Text]
  76. Wetzel G.T., Chen F., Klitzner T.S. Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes. Am J Physiol (1995) 268:H1723–1733.[Web of Science][Medline]
  77. Levi A.J., Spitzer K.W., Kohmoto O., Bridge J.H.B. Depolarisation-induced Ca entry via Na–Ca exchange triggers SR release in guinea-pig cardiac myocytes. Am J Physiol (1994) 266:H1422–H1433.[Web of Science][Medline]
  78. Levi A.J., Brooksby P., Hancox J.C. A role for depolarisation-induced calcium entry on the Na–Ca exchange in triggering intracellular calcium release and contraction in rat ventricular myocytes. Cardiovasc Res (1993) 27:1677–1690.[Abstract/Free Full Text]
  79. Kohmoto O., Levi A.J., Bridge J.H.B. Relation between reverse Na–Ca exchange and sarcoplasmic reticulum calcium release in guinea-pig ventricular cells. Circ Res (1994) 74:550–554.[Abstract/Free Full Text]
  80. Levi A.J., Li J., Spitzer K.W., Bridge J.H.B. Effect on the Indo-1 transient of applying Ca2+ channel blocker for a single beat in voltage-clamped guinea-pig cardiac myocytes. J Physiol (1996) 494:653–673.[Abstract/Free Full Text]
  81. Levi A.J., Issberner J. Effect on the Fura-2 transient of rapidly blocking the Ca channel in externally stimulated rabbit heart cells. J Physiol (1996) 493:19–37.[Abstract/Free Full Text]
  82. Hobai I.A., Levi A.J. The relation between L-type Ca current and intracellular Ca release in guinea pig cardiac myocytes and the effect of internal cAMP (Abstract). Biophys J (1999) 76:A463.
  83. Thornton J.M., Paterson D.J. Effect of nickel on contraction in the isolated guinea-pig papillary muscle (Abstract). J Physiol (1997) 504:90P.
  84. Callewaert G., Cleemann L., Morad M. Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes. Proc Natl Acad Sci USA (1988) 85:2009–2013.[Abstract/Free Full Text]
  85. Nuss H.B., Houser S.R. Sodium calcium exchange mediated contractions in feline ventricular myocytes. Am J Physiol (1992) 263:H1161–H1169.[Web of Science][Medline]
  86. Cannell M.B., Berlin J.R., Lederer W.J. Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science (1987) 238:1419–1423.[Abstract/Free Full Text]
  87. DuBell W.H., Lederer W.J., Rogers T.B. Dynamic modulation of excitation–contraction coupling by protein phosphates in rat ventricular myocytes. J Physiol (1996) 493:793–800.[Abstract/Free Full Text]
  88. DuBell W.H., Houser S.R. Voltage and beat dependence of Ca2+ transient in feline ventricular myocytes. Am J Physiol (1989) 257:H746–H759.[Web of Science][Medline]
  89. Wier W.G., Yue D.T. Intracellular calcium transients underlying the short term force–interval relationship in ferret ventricular myocardium. J Physiol (1986) 376:507–530.[Abstract/Free Full Text]
  90. Hancox J.C., Levi A.J. Na–Ca exchange tail current indicates voltage dependence of the Cai transient in rabbit ventricular myocytes. J Cardiovasc Electrophys (1995) 6:455–470.[Web of Science][Medline]

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. Neurophysiol.Home page
R. J. Haedo and J. Golowasch
Ionic Mechanism Underlying Recovery of Rhythmic Activity in Adult Isolated Neurons
J Neurophysiol, October 1, 2006; 96(4): 1860 - 1876.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. L. De Jongh, V. Ramanathan, B. K. Hoffmeister, and R. A. Malkin
Left ventricular geometry immediately following defibrillation: shock-induced relaxation
Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H815 - H819.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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 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 arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hobai, I. A
Right arrow Articles by Levi, A. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hobai, I. A
Right arrow Articles by Levi, A. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?