Br. J. Pharmacol. (1990),101, 399-405
,'-.
Macmillan Press Ltd, 1990
The effects of ryanodine and caffeine on Ca-activated current in guinea-pig ventricular myocytes 'Edward White & 2Derek A. Terrar University Department of Pharmacology, South Parks Road, Oxford, OX1 3QT 1 Action potentials from guinea-pig single ventricular myocytes were interrupted by application of a 300ms voltage clamp to -40mV in order to evoke the Ca-activated tail current which is thought to be carried by Na: Ca exchange. Stimulation frequency was 1 Hz and temperature 36°C. 2 The actions of ryanodine (1 ,UM and 10 UM) and caffeine (1 mm and 10 mM) on Ca-activated tail currents were investigated. 3 Exposure to 10 mm caffeine and ryanodine reduced tail currents associated with very abbreviated (12 ms duration) action potentials and greatly reduced the difference between first and steady-state tail currents at this action potential duration. These observations were interpreted in terms of suppression of Ca release from the sarcoplasmic reticulum (SR) stores. 4 Tail current decay during the voltage clamp is thought to reflect the fall in [Ca]i which accompanies muscle relaxation. Current decay is dependent on Ca extrusion via Na: Ca exchange and on Ca accumulation by the SR stores. Time constants of tail current decay were seen to decrease with increasing action potential duration. This relationship was not affected by 1 mm caffeine or 1 pM ryanodine. Ryanodine at 10puM and 10nmm caffeine abolished this relationship and increased the time constants of current decay. An increase in the time constant of tail current decay was thought to reflect a reduction in the rate of Ca accumulation by the sarcoplasmic reticulum. 5 The actions of caffeine and ryanodine on the Ca-activated tail currents are consistent with a dosedependent leakage of Ca from the SR Ca stores. The Ca-activated tail current appears to be a useful tool in the study of Ca homeostasis.
Introduction A Ca-activated 'tail' current has been demonstrated by several groups in single myocytes from amphibian hearts (Hume & Uehara, 1986; Cambell et al., 1988) and in mammalian hearts following either voltage clamp depolarizations (Fedida et al., 1987; Mitchell et al., 1987; Giles & Shimoni, 1989) or interruption of action potentials (Egan et al., 1989; Terrar & White, 1989). There is general agreement that the major component of the tail current is carried by electrogenic Na: Ca exchange operating in the Ca extrusion mode. However, the presence of a Ca-activated non-specific cation conductance (Ehara et al., 1988) in some situations cannot be discounted. Properties of Na: Ca exchange in single guinea-pig ventricular cells have been described by Kimura et al. (1987), BarcenasRuiz et al. (1987) and Beuckelmann & Wier (1989). In the heart Na:Ca exchange is thought to be a major mechanism in Ca homoeostasis (Mullins, 1979; Chapman, 1983; Noble, 1986). The time course of the decay of the Ca-activated current is thought to reflect the fall in cytosolic Ca which accompanies relaxation of cardiac muscle. The fall in cytosolic Ca is believed to result from the actions of Na:Ca exchange (which extrudes Ca from the cell) and the sarcoplasmic reticulum (SR) adenosine-5'-triphosphatase (ATP-ase, which loads Ca into the SR for re-release); see for e.g. Hilgemann & Noble (1987). It is predicted that the tail current will be sensitive to actions which influence these mechanisms. In support of this hypothesis, it has been shown that the non-specific Na:Ca exchange inhibitor dichlorobenzamil can slow the decay of tail current (Lipp & Pott, 1988). Agents active upon SR function also influence tail current decay, e.g. adrenaline speeds the decay of the tail current (Fedida et al., 1987). Caffeine slows the tail current decay (Giles & Shimoni, 1989; Terrar & White, 1989) 1 Present address: Lab. d'Electrophysiologie et de Pharmacologie Cellulaires, Universit6 de Tours, Parc de Grandmont, F-37200, Tours, France. 2 Author for correspondence.
but the effects of ryanodine are less clear (compare Fedida et al., 1987; Giles & Shimoni, 1989; White & Terrar, 1990). In contrast the cardiac glycoside strophanthidin (which is not thought to exert a direct influence on SR function) can increase the magnitude of the tail current without influencing the decay (White & Terrar, 1990). Clearly the Ca-activated tail current should be useful in studying processes which modulate the systolic intracellular Ca transient of cardiac tissue. Ryanodine and caffeine are important pharmacological tools in the study of Ca regulation in the heart (for ryanodine see Sutko & Willerson, 1980; Sutko et al., 1985; Meissner, 1986; Rousseau et al., 1987: for caffeine, Blinks et al., 1972; Chapman & Loety, 1976; Hess & Weir, 1984; Rousseau & Meissner, 1989). To date there is no quantitative comparative study of the effects of ryanodine and caffeine on the Caactivated tail current in single myocytes. The major aim of this work was to make such a study. A second aim was to explore the possibility that the study of Ca-activated current in the presence of these drugs might shed further light on the roles of Na: Ca exchange and SR function in Ca homeostasis.
Methods Experiments were performed on single cardiac ventricular myocytes isolated from guinea-pig. Guinea-pigs were stunned then killed by cervical dislocation. Hearts were excised and single cells isolated as previously described by Powell et al. (1980) and Mitchell et al. (1984a). After isolation cells were superfused with a oxygenated solution at 36°C containing (mM): NaCl 118.5, NaHCO3 14.5, KCl 4.2, K2H2PO4 1.2, MgSO4 1.2, glucose 11.1 and CaC12 2.0. Ca-activated tail currents were evoked by use of the technique of interrupted action potentials. This procedure was used to produce an envelope of tail currents. Currents were analysed with the VCAN software package provided by Dr. J. Dempster. Both these techniques have been described in detail previously (Terrar & White, 1989; White & Terrar, 1990).
400
E. WHITE & D.A. TERRAR
Briefly, action potentials were evoked at a frequency of 1 Hz and were interrupted at various times after the upstroke by application of a 300 ms voltage clamp to -40 mV. The pattern of stimulation was always eight full action potentials followed by eight interrupted action potentials. The onset of the voltage clamp evoked a Ca-activated tail current which decayed during the voltage clamp pulse. By fitting a single exponential to the decay of the current (using the VCAN package) the magnitude of the current at the point of interruption of the action potential together with the time constant of the decaying current could be estimated. This technique allowed the separation of Ca-activated tail current from the rapid current transient (thought to consist of capacity currents and current carried by rapidly de-activating Ca channels) which is also evoked by onset of the voltage clamp. In some control cells the first tail current associated with an interrupted action potential duration of 12ms was seen to rise for a few ms before falling. In these cases a single exponential described the falling section of the current. Current amplitude was extrapolated by eye (White & Terrar, 1990). The rise in tail current following repolarization is thought to reflect continued release of Ca from the SR and has also been described by Egan et al. (1989) and Giles & Shimoni (1989). Throughout this work a tail current 'transient' refers to the relationship between current amplitude and action potential duration (e.g. Figures 1 and 2). 'Decay' of a tail current refers to the fall in current during a single voltage clamp pulse at -40mV (e.g. Figures 5 and 6). Cells were exposed to caffeine (1 mM or 10 mM) or ryanodine (1 fiM or 10uM). These drug concentrations were chosen as they represent those most commonly used to achieve an effect on SR function (see references in the final paragraph of Introduction). Exposure was achieved by inclusion of the agent in the superfusing solution and was therefore continuous unless otherwise indicated. It was assumed that under these conditions the effects of both caffeine and ryanodine had reached a steady state before the records presented in the results were collected. It should be stressed in particular that the responses to caffeine were distinct from the 'caffeine contractures' evoked upon initial exposure to this agent (e.g. Chapman & Tunstall, 1983). It was a constant concern that the experiments should be performed at as close to physiological conditions as the experimental design would allow. For this reason the action potential was used to generate the calcium transient in favour of more controllable voltage clamp steps. As the processes under study are know to be temperature sensitive (see, for example, Egan et al., 1989; Giles & Shimoni, 1989), it was felt important that these studies be carried out at 360C. Statistical tests were made by either paired or unpaired t tests as appro-
priate. Results
Tail current transients Action potentials were stimulated at a frequency of 1 Hz. After eight normal action potentials, eight action potentials were interrupted by a 300ms voltage clamp pulse to -40mV. This procedure evoked the Ca-activated tail current which is thought to be carried by Na:Ca exchange. Varying the time of action potential interruption allowed the generation of an envelope of tail currents. Such an envelope is shown in Figure 1. It can be seen that in this cell, current amplitude (following the initial rapid current transient) decreased with increasing action potential duration. The tail current decayed to a steady state level within the 300 ms period for which the voltage
clamp was applied. Amplitude of the first evoked tail current for each action potential duration gives a tail current transient which is thought to reflect qualitatively the cytosolic Ca transient (Egan et al., 1989; Terrar & White, 1989). Figure 2 (c and d,
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9
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Figure 1 Membrane potential (upper traces) and membrane current (lower traces) in guinea-pig ventricular myocytes. Envelope of Caactivated tail current, constructed by superimposing records for the first tail currents evoked by interrupting action potentials after 12, 52, 102 and 152ms with a 300ms voltage clamp to -40mV. In all figures zero current is indicated by the level of current before and after the voltage clamp. Stimulation frequency 1 Hz, 360C.
dotted lines) describes mean tail current transients from 18 cells. Several characteristic features about the time course of this transient should be noted: (1) even very short (12 ms) action potentials can evoke a large tail current; (2) the transient peaks approximately 50ms into the action potential; (3) there is a rapid fall in the transient to a low level before the end of action potential repolarization. An explanation of our hypothesis of events which determine the shape of this 'control' transient is crucial to the interpretation of the results. It is predicted that the ability of short (12 ms) action potentials to trigger tail currents of almost equal amplitude to those found at the peak of the tail current transient is due to a substantial release of Ca from intracellular SR stores. The fall in the transient after the peak is due to a combination of Ca removal from the cytoplasm by extrusion from the cell by Na:Ca exchange and Ca re-uptake into SR stores. If these assumptions are correct then the tail current transients and the time course of tail currents during the voltage clamp should be affected by ryanodine and caffeine as both are thought to influence SR function. Figure 2c gives transients for cells exposed to 1 or 10mM caffeine (6 cells in each case). The transient in cells exposed to 1 mm caffeine was similar to control except that the fall in the transient appeared to be slowed (cf. points after 152 ms). In the presence of 10mM caffeine the transient was altered in a manner described by Terrar & White (1989) under slightly different conditions (2.5 mm Ca, 0.3 Hz). Currents associated with 12 ms action potentials were reduced (in both absolute amplitude and relative to peak current) and the fall in the transient was greatly delayed. It should also be noted that while exposure to 10 mm caffeine resulted in action potentials with a characteristic delayed phase of repolarization (see Terrar & White, 1989) this was not observed in cells exposed to 1 mm caffeine. If it is assumed that exposure to 10 mm caffeine results in an absence of normal SR function, then the ability of cells to generate Ca-activated tail currents of comparable magnitude to control cells may be associated with the large increase in Ca influx via Ca channels associated with exposure to 10mM caffeine (Terrar & White, 1989). Figure 2d shows the transients from cells exposed to 1 CM (10 cells) or 1pOaM ryanodine (6 cells). The transients reveal a major depression of current associated with the shortest action potentials and a reduction of peak current, although current appeared to remain close to peak levels for longer than control before falling. The early depression of current is consistent with the removal of a stores component of Ca release which normally allows rapid development of the tail current transient (presumably reflecting a rapid rise of cytosolic Ca). Tail currents in the presence of 1 ,UM ryanodine were
Ca-ACTIVATED CURRENT IN GUINEA-PIG VENTRICLE
401
b
a
I
0
0 mnV~
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Action potential duration (ins) Action potential duration (ins) Figure 2 Representative action potentials in guinea-pig ventricular myocytes. (a) Control (0), in the presence of 1 mm caffeine (0) and 1O mm caffeine (0). (b) Control (El), in the presence of 1 FM ryanodine (0) and 10pUM ryanodine (0). Mean Ca-activated tail current transients for control cells are represented by (El --- El) in (c) and (d) (n = 18 cells) and for cells exposed to: (c) 1 mM caffeine (0. n = 6 cells); 10mM caffeine (0, n = 6 cells); (d) 1UM ryanodine (0, n = 1O cells); 10pIM ryanodine (0, n = 6 cells). Note depression of tail currents at 12ms in cells exposed to ryanodine and 1OmM caffeine. Mean tail currents at 202 ms were calculated from those cells which had not repolarized beyond -40 mV after this time (control, n = 7; 1 mm caffeine, n = 3; 10mM caffeine, n = 6; 1fM ryanodine, n = 2 only and hence not included; 10pgM ryanodine, n = 4). In the presence of 1 mm caffeine tail currents were significantly larger than control at 102ms (P < 0.05), 152ms (P < 0.01) and 202 ms (P < 0.05). Tail currents in 10mM caffeine were smaller than control at 12 ms (P < 0.001), larger at 102 ms (P < 0.05), 152 ms and 202 ms (P < 0.001). In the presence of 1jUM ryanodine tail currents were smaller than control at 12ms (P < 0.001) and 52ms (P < 0.01). In 10pM ryanodine, currents were smaller at 12ms and 52ms (P < 0.001) and larger at 152ms (P < 0.05) and 202ms (P < 0.01), P calculated by unpaired t tests. Exposure to 10mM caffeine and 10pM ryanodine prolonged the transient. In (c) and (d) vertical lines indicate s.e.
not significantly different from those in the presence of 1Opam ryanodine up to 152 ms.
First/steady state currents It was consistently observed that in control cells the level of tail current associated with the first action potential of 12ms duration could not be sustained by the following similarly abbreviated action potentials (P < 0.001) (Figure 3a). It may be that the first shortened action potential can trigger Ca release from stores (filled by the preceding full action potentials). As a consequence of reduced Ca entry through Ca channels and increased Ca-extrusion via Na:Ca exchange (which both result from the early repolarization), this high level of stores filling may not be maintained during the subsequent shortened action potentials and hence a reduction in current occurs. Figure 3b shows the differences between first and steady state current remains despite exposure to 1 mm caffeine (P < 0.001). This supports the hypothesis that in these experiments controlled release of Ca persists in cells treated with 1 mm caffeine. In contrast the response is abolished or reduced in cells exposed to either 10mm caffeine (Figure 3c), 1aum ryanodine (Figure 3d) or 10puM ryanodine (Figure 3e) (P > 0.05 in all 3 treatments). Figure 4 gives mean first and steady state currents. Responses in the control and 1 mm caffeine cells are clearly different from those during other treatments.
Although the magnitude of currents in the presence of ryanodine is much smaller than control there was a reduction of current at the steady state in some cells (5 out of 10 cells exposed to 1 atm ryanodine and 5 out of 6 of those exposed to 10pM). One explanation is that this observation reflects a small ryanodine insensitive component of stores release. In some cells no difference in first and steady state current was observed but in others a difference was seen despite prolonged (over 1 h) exposure to ryanodine. It was also observed that despite prolonged exposure a difference in first and steady state current was often seen when cells were first stimulated.
This difference disappeared or reduced with further stimulation and was thought to be related to the use-dependent nature of ryanodine action (Mitchell et al., 1984b; Malecot & Katzung, 1987). For this reason cells were stimulated for at least 1 min before collection of tail current records. From these results it seems that the modulation of a large first steady-state response in very abbreviated action potentials may be a useful tool in the study of Ca release by the SR.
Decay of tail currents After the onset of the voltage clamp pulse the Ca-activated current decayed at a steady state. It has been shown that the decay of the current speeds up as the length of the associated action potential (Terrar & White, 1989) or voltage clamp depolarization (Giles & Shimoni, 1989) increases. This effect is demonstrated by the records in Figure 1. Time constants of current decay decreased with increasing action potential duration as follows: 36, 27, 18, 6ms. This observation has been explained in terms of increasing activity of the Ca uptake procedure by Giles & Shimoni (1989). Figure 5 shows tail currents recorded after action potentials of 52 ms for control and cells exposed to caffeine and ryanodine. The greatest divergence from control is seen in the cell exposed to 10 mm caffeine (Figure Sc). Mean relationships between current decay and action potential duration in the presence of caffeine are shown in Figure 6a. The decrease in time constant with increasing action potential duration was maintained in cells exposed to 1 mm caffeine but not in the presence of the higher dose of the drug. Exposure to 10mm caffeine greatly increased time constants of currents associated with action potentials of over 12 ms duration. Figure 6b gives values for cells exposed to ryanodine. Time constants appeared little affected by 1 aUM ryanodine. In the presence of 10Mm ryanodine time constants were qualitatively similar to those in the presence of lOmm caffeine though faster. The very large standard error of current decay associated with 12 ms action potentials in the presence of 10,uM ryanodine is probably due to the difficulty in fitting
402
E. WHITE & D.A. TERRAR Control
a
Control
a T = 27 ms
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Caffeine 1 mM
Caffeine 1 mm
T=
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c
Caffeine 10 mM T =
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Ryanodine 1 JLM d
Ryanodine 1 ALM
T= 24 ms
e
F,0wpr.,-- -TW-rI
Ryanodine 10 FM *e
Ryanodine 10 FkM
T= 50 ms
Figure 3 First (arrows where distinguishable) and steady state tail currents associated with action potentials interrupted after 12ms for:
(a) control; (b) 1mM caffeine; (c) 10mM caffeine; (d) 1pM ryanodine; (e) 10pM ryanodine. Note responses in (a) and (b) are clearly different from others. Stimulation frequency 1 Hz, 36°C.
c -
NS
-
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a)
C._ 40 0
riNS
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Control
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mM
Caff
10 mM Caff Treatment
1
|
~NS
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10 FM
Ryan
Ryan
Figure 4 Mean first (open columns) and steady state (solid columns) Ca-activated current associated with action potentials interrupted after 12 ms for control (n = 18 cells); 1 mM caffeine (Caff, n = 6 cells); 10mM caffeine (n = 6 cells); 1pM ryanodine (Ryan, n = 10 cells) and 10pM ryanodine (n = 6 cells). *** P < 0.001, NS = not significant, paired t tests. Bars show s.e.mean.
Figure 5 Ca-activated tail currents associated with action potentials interrupted after 52 ms. Time constants of current decay (r) are given. (a) Control; (b) 1 mm caffeine; (c) 10mM caffeine; (d) 1pM ryanodine and (e) 10pM ryanodine. Stimulation frequency 1 Hz, 36°C.
an exponential to such small currents, given the signal to noise ratio of these records. One further point should also be noted: it was often observed that in control cells the time constant of the first tail current associated with action potentials of 12 ms was greater than that of the steady state (P < 0.05); for example, time constants of currents in Figure 3a were 36 and 30 ms for first and steady state respectively. The first abbreviated action potential is thought to be associated with a large release of SR Ca but reduced Ca entry. It would be predicted that this situation leads to a net extrusion of Ca from the cell by Na: Ca exchange until (during subsequent abbreviated action potentials) a steady state is established (at which point Ca entry must equal Ca extrusion, Hilgemann & Noble, 1987). It is possible that the increased amount of Ca extruded during the first abbreviated action potential results in a slower time constant of current decay as compared with the steady state. In order to test the above observations more fully we also recorded tail currents in some cells which were subsequently exposed to either 1 pM ryanodine or 10 mm caffeine (3 cells for each treatment). In all cases the observed changes in action potential shape, tail current transients, first steady-state and time constant of current decay were consistent with the above observations. This was also true in the case of 3 cells which
Ca-ACTIVATED CURRENT IN GUINEA-PIG VENTRICLE a 100
403
a
-
E 80-
m
0) 60 en
40 0)
o 020-
100 mV 1 nA
100 Ms 200 Action potential duration (ms) 100
0
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b
b 100
-1
at
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>' 804) a)
_0
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+-.
40 60-
c
0) E
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Action potential duration (ms)
Figure 6 Mean time constants of Ca-activated current decay. Control cells ( -l) in (a) and (b) (n = 18 cells). (a) Cells exposed to 1 mm caffeine (0. n = 6 cells); 10mM caffeine (0, n = 6 cells). (b) Cells exposed to 1 M ryanodine (0, n = 10 cells); 10pM ryanodine (S, n = 6 cells). In the presence of 1 mm caffeine time constants were not significantly different from control except at 12ms (P
,'-.
Macmillan Press Ltd, 1990
The effects of ryanodine and caffeine on Ca-activated current in guinea-pig ventricular myocytes 'Edward White & 2Derek A. Terrar University Department of Pharmacology, South Parks Road, Oxford, OX1 3QT 1 Action potentials from guinea-pig single ventricular myocytes were interrupted by application of a 300ms voltage clamp to -40mV in order to evoke the Ca-activated tail current which is thought to be carried by Na: Ca exchange. Stimulation frequency was 1 Hz and temperature 36°C. 2 The actions of ryanodine (1 ,UM and 10 UM) and caffeine (1 mm and 10 mM) on Ca-activated tail currents were investigated. 3 Exposure to 10 mm caffeine and ryanodine reduced tail currents associated with very abbreviated (12 ms duration) action potentials and greatly reduced the difference between first and steady-state tail currents at this action potential duration. These observations were interpreted in terms of suppression of Ca release from the sarcoplasmic reticulum (SR) stores. 4 Tail current decay during the voltage clamp is thought to reflect the fall in [Ca]i which accompanies muscle relaxation. Current decay is dependent on Ca extrusion via Na: Ca exchange and on Ca accumulation by the SR stores. Time constants of tail current decay were seen to decrease with increasing action potential duration. This relationship was not affected by 1 mm caffeine or 1 pM ryanodine. Ryanodine at 10puM and 10nmm caffeine abolished this relationship and increased the time constants of current decay. An increase in the time constant of tail current decay was thought to reflect a reduction in the rate of Ca accumulation by the sarcoplasmic reticulum. 5 The actions of caffeine and ryanodine on the Ca-activated tail currents are consistent with a dosedependent leakage of Ca from the SR Ca stores. The Ca-activated tail current appears to be a useful tool in the study of Ca homeostasis.
Introduction A Ca-activated 'tail' current has been demonstrated by several groups in single myocytes from amphibian hearts (Hume & Uehara, 1986; Cambell et al., 1988) and in mammalian hearts following either voltage clamp depolarizations (Fedida et al., 1987; Mitchell et al., 1987; Giles & Shimoni, 1989) or interruption of action potentials (Egan et al., 1989; Terrar & White, 1989). There is general agreement that the major component of the tail current is carried by electrogenic Na: Ca exchange operating in the Ca extrusion mode. However, the presence of a Ca-activated non-specific cation conductance (Ehara et al., 1988) in some situations cannot be discounted. Properties of Na: Ca exchange in single guinea-pig ventricular cells have been described by Kimura et al. (1987), BarcenasRuiz et al. (1987) and Beuckelmann & Wier (1989). In the heart Na:Ca exchange is thought to be a major mechanism in Ca homoeostasis (Mullins, 1979; Chapman, 1983; Noble, 1986). The time course of the decay of the Ca-activated current is thought to reflect the fall in cytosolic Ca which accompanies relaxation of cardiac muscle. The fall in cytosolic Ca is believed to result from the actions of Na:Ca exchange (which extrudes Ca from the cell) and the sarcoplasmic reticulum (SR) adenosine-5'-triphosphatase (ATP-ase, which loads Ca into the SR for re-release); see for e.g. Hilgemann & Noble (1987). It is predicted that the tail current will be sensitive to actions which influence these mechanisms. In support of this hypothesis, it has been shown that the non-specific Na:Ca exchange inhibitor dichlorobenzamil can slow the decay of tail current (Lipp & Pott, 1988). Agents active upon SR function also influence tail current decay, e.g. adrenaline speeds the decay of the tail current (Fedida et al., 1987). Caffeine slows the tail current decay (Giles & Shimoni, 1989; Terrar & White, 1989) 1 Present address: Lab. d'Electrophysiologie et de Pharmacologie Cellulaires, Universit6 de Tours, Parc de Grandmont, F-37200, Tours, France. 2 Author for correspondence.
but the effects of ryanodine are less clear (compare Fedida et al., 1987; Giles & Shimoni, 1989; White & Terrar, 1990). In contrast the cardiac glycoside strophanthidin (which is not thought to exert a direct influence on SR function) can increase the magnitude of the tail current without influencing the decay (White & Terrar, 1990). Clearly the Ca-activated tail current should be useful in studying processes which modulate the systolic intracellular Ca transient of cardiac tissue. Ryanodine and caffeine are important pharmacological tools in the study of Ca regulation in the heart (for ryanodine see Sutko & Willerson, 1980; Sutko et al., 1985; Meissner, 1986; Rousseau et al., 1987: for caffeine, Blinks et al., 1972; Chapman & Loety, 1976; Hess & Weir, 1984; Rousseau & Meissner, 1989). To date there is no quantitative comparative study of the effects of ryanodine and caffeine on the Caactivated tail current in single myocytes. The major aim of this work was to make such a study. A second aim was to explore the possibility that the study of Ca-activated current in the presence of these drugs might shed further light on the roles of Na: Ca exchange and SR function in Ca homeostasis.
Methods Experiments were performed on single cardiac ventricular myocytes isolated from guinea-pig. Guinea-pigs were stunned then killed by cervical dislocation. Hearts were excised and single cells isolated as previously described by Powell et al. (1980) and Mitchell et al. (1984a). After isolation cells were superfused with a oxygenated solution at 36°C containing (mM): NaCl 118.5, NaHCO3 14.5, KCl 4.2, K2H2PO4 1.2, MgSO4 1.2, glucose 11.1 and CaC12 2.0. Ca-activated tail currents were evoked by use of the technique of interrupted action potentials. This procedure was used to produce an envelope of tail currents. Currents were analysed with the VCAN software package provided by Dr. J. Dempster. Both these techniques have been described in detail previously (Terrar & White, 1989; White & Terrar, 1990).
400
E. WHITE & D.A. TERRAR
Briefly, action potentials were evoked at a frequency of 1 Hz and were interrupted at various times after the upstroke by application of a 300 ms voltage clamp to -40 mV. The pattern of stimulation was always eight full action potentials followed by eight interrupted action potentials. The onset of the voltage clamp evoked a Ca-activated tail current which decayed during the voltage clamp pulse. By fitting a single exponential to the decay of the current (using the VCAN package) the magnitude of the current at the point of interruption of the action potential together with the time constant of the decaying current could be estimated. This technique allowed the separation of Ca-activated tail current from the rapid current transient (thought to consist of capacity currents and current carried by rapidly de-activating Ca channels) which is also evoked by onset of the voltage clamp. In some control cells the first tail current associated with an interrupted action potential duration of 12ms was seen to rise for a few ms before falling. In these cases a single exponential described the falling section of the current. Current amplitude was extrapolated by eye (White & Terrar, 1990). The rise in tail current following repolarization is thought to reflect continued release of Ca from the SR and has also been described by Egan et al. (1989) and Giles & Shimoni (1989). Throughout this work a tail current 'transient' refers to the relationship between current amplitude and action potential duration (e.g. Figures 1 and 2). 'Decay' of a tail current refers to the fall in current during a single voltage clamp pulse at -40mV (e.g. Figures 5 and 6). Cells were exposed to caffeine (1 mM or 10 mM) or ryanodine (1 fiM or 10uM). These drug concentrations were chosen as they represent those most commonly used to achieve an effect on SR function (see references in the final paragraph of Introduction). Exposure was achieved by inclusion of the agent in the superfusing solution and was therefore continuous unless otherwise indicated. It was assumed that under these conditions the effects of both caffeine and ryanodine had reached a steady state before the records presented in the results were collected. It should be stressed in particular that the responses to caffeine were distinct from the 'caffeine contractures' evoked upon initial exposure to this agent (e.g. Chapman & Tunstall, 1983). It was a constant concern that the experiments should be performed at as close to physiological conditions as the experimental design would allow. For this reason the action potential was used to generate the calcium transient in favour of more controllable voltage clamp steps. As the processes under study are know to be temperature sensitive (see, for example, Egan et al., 1989; Giles & Shimoni, 1989), it was felt important that these studies be carried out at 360C. Statistical tests were made by either paired or unpaired t tests as appro-
priate. Results
Tail current transients Action potentials were stimulated at a frequency of 1 Hz. After eight normal action potentials, eight action potentials were interrupted by a 300ms voltage clamp pulse to -40mV. This procedure evoked the Ca-activated tail current which is thought to be carried by Na:Ca exchange. Varying the time of action potential interruption allowed the generation of an envelope of tail currents. Such an envelope is shown in Figure 1. It can be seen that in this cell, current amplitude (following the initial rapid current transient) decreased with increasing action potential duration. The tail current decayed to a steady state level within the 300 ms period for which the voltage
clamp was applied. Amplitude of the first evoked tail current for each action potential duration gives a tail current transient which is thought to reflect qualitatively the cytosolic Ca transient (Egan et al., 1989; Terrar & White, 1989). Figure 2 (c and d,
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9
lOO1
Figure 1 Membrane potential (upper traces) and membrane current (lower traces) in guinea-pig ventricular myocytes. Envelope of Caactivated tail current, constructed by superimposing records for the first tail currents evoked by interrupting action potentials after 12, 52, 102 and 152ms with a 300ms voltage clamp to -40mV. In all figures zero current is indicated by the level of current before and after the voltage clamp. Stimulation frequency 1 Hz, 360C.
dotted lines) describes mean tail current transients from 18 cells. Several characteristic features about the time course of this transient should be noted: (1) even very short (12 ms) action potentials can evoke a large tail current; (2) the transient peaks approximately 50ms into the action potential; (3) there is a rapid fall in the transient to a low level before the end of action potential repolarization. An explanation of our hypothesis of events which determine the shape of this 'control' transient is crucial to the interpretation of the results. It is predicted that the ability of short (12 ms) action potentials to trigger tail currents of almost equal amplitude to those found at the peak of the tail current transient is due to a substantial release of Ca from intracellular SR stores. The fall in the transient after the peak is due to a combination of Ca removal from the cytoplasm by extrusion from the cell by Na:Ca exchange and Ca re-uptake into SR stores. If these assumptions are correct then the tail current transients and the time course of tail currents during the voltage clamp should be affected by ryanodine and caffeine as both are thought to influence SR function. Figure 2c gives transients for cells exposed to 1 or 10mM caffeine (6 cells in each case). The transient in cells exposed to 1 mm caffeine was similar to control except that the fall in the transient appeared to be slowed (cf. points after 152 ms). In the presence of 10mM caffeine the transient was altered in a manner described by Terrar & White (1989) under slightly different conditions (2.5 mm Ca, 0.3 Hz). Currents associated with 12 ms action potentials were reduced (in both absolute amplitude and relative to peak current) and the fall in the transient was greatly delayed. It should also be noted that while exposure to 10 mm caffeine resulted in action potentials with a characteristic delayed phase of repolarization (see Terrar & White, 1989) this was not observed in cells exposed to 1 mm caffeine. If it is assumed that exposure to 10 mm caffeine results in an absence of normal SR function, then the ability of cells to generate Ca-activated tail currents of comparable magnitude to control cells may be associated with the large increase in Ca influx via Ca channels associated with exposure to 10mM caffeine (Terrar & White, 1989). Figure 2d shows the transients from cells exposed to 1 CM (10 cells) or 1pOaM ryanodine (6 cells). The transients reveal a major depression of current associated with the shortest action potentials and a reduction of peak current, although current appeared to remain close to peak levels for longer than control before falling. The early depression of current is consistent with the removal of a stores component of Ca release which normally allows rapid development of the tail current transient (presumably reflecting a rapid rise of cytosolic Ca). Tail currents in the presence of 1 ,UM ryanodine were
Ca-ACTIVATED CURRENT IN GUINEA-PIG VENTRICLE
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Action potential duration (ins) Action potential duration (ins) Figure 2 Representative action potentials in guinea-pig ventricular myocytes. (a) Control (0), in the presence of 1 mm caffeine (0) and 1O mm caffeine (0). (b) Control (El), in the presence of 1 FM ryanodine (0) and 10pUM ryanodine (0). Mean Ca-activated tail current transients for control cells are represented by (El --- El) in (c) and (d) (n = 18 cells) and for cells exposed to: (c) 1 mM caffeine (0. n = 6 cells); 10mM caffeine (0, n = 6 cells); (d) 1UM ryanodine (0, n = 1O cells); 10pIM ryanodine (0, n = 6 cells). Note depression of tail currents at 12ms in cells exposed to ryanodine and 1OmM caffeine. Mean tail currents at 202 ms were calculated from those cells which had not repolarized beyond -40 mV after this time (control, n = 7; 1 mm caffeine, n = 3; 10mM caffeine, n = 6; 1fM ryanodine, n = 2 only and hence not included; 10pgM ryanodine, n = 4). In the presence of 1 mm caffeine tail currents were significantly larger than control at 102ms (P < 0.05), 152ms (P < 0.01) and 202 ms (P < 0.05). Tail currents in 10mM caffeine were smaller than control at 12 ms (P < 0.001), larger at 102 ms (P < 0.05), 152 ms and 202 ms (P < 0.001). In the presence of 1jUM ryanodine tail currents were smaller than control at 12ms (P < 0.001) and 52ms (P < 0.01). In 10pM ryanodine, currents were smaller at 12ms and 52ms (P < 0.001) and larger at 152ms (P < 0.05) and 202ms (P < 0.01), P calculated by unpaired t tests. Exposure to 10mM caffeine and 10pM ryanodine prolonged the transient. In (c) and (d) vertical lines indicate s.e.
not significantly different from those in the presence of 1Opam ryanodine up to 152 ms.
First/steady state currents It was consistently observed that in control cells the level of tail current associated with the first action potential of 12ms duration could not be sustained by the following similarly abbreviated action potentials (P < 0.001) (Figure 3a). It may be that the first shortened action potential can trigger Ca release from stores (filled by the preceding full action potentials). As a consequence of reduced Ca entry through Ca channels and increased Ca-extrusion via Na:Ca exchange (which both result from the early repolarization), this high level of stores filling may not be maintained during the subsequent shortened action potentials and hence a reduction in current occurs. Figure 3b shows the differences between first and steady state current remains despite exposure to 1 mm caffeine (P < 0.001). This supports the hypothesis that in these experiments controlled release of Ca persists in cells treated with 1 mm caffeine. In contrast the response is abolished or reduced in cells exposed to either 10mm caffeine (Figure 3c), 1aum ryanodine (Figure 3d) or 10puM ryanodine (Figure 3e) (P > 0.05 in all 3 treatments). Figure 4 gives mean first and steady state currents. Responses in the control and 1 mm caffeine cells are clearly different from those during other treatments.
Although the magnitude of currents in the presence of ryanodine is much smaller than control there was a reduction of current at the steady state in some cells (5 out of 10 cells exposed to 1 atm ryanodine and 5 out of 6 of those exposed to 10pM). One explanation is that this observation reflects a small ryanodine insensitive component of stores release. In some cells no difference in first and steady state current was observed but in others a difference was seen despite prolonged (over 1 h) exposure to ryanodine. It was also observed that despite prolonged exposure a difference in first and steady state current was often seen when cells were first stimulated.
This difference disappeared or reduced with further stimulation and was thought to be related to the use-dependent nature of ryanodine action (Mitchell et al., 1984b; Malecot & Katzung, 1987). For this reason cells were stimulated for at least 1 min before collection of tail current records. From these results it seems that the modulation of a large first steady-state response in very abbreviated action potentials may be a useful tool in the study of Ca release by the SR.
Decay of tail currents After the onset of the voltage clamp pulse the Ca-activated current decayed at a steady state. It has been shown that the decay of the current speeds up as the length of the associated action potential (Terrar & White, 1989) or voltage clamp depolarization (Giles & Shimoni, 1989) increases. This effect is demonstrated by the records in Figure 1. Time constants of current decay decreased with increasing action potential duration as follows: 36, 27, 18, 6ms. This observation has been explained in terms of increasing activity of the Ca uptake procedure by Giles & Shimoni (1989). Figure 5 shows tail currents recorded after action potentials of 52 ms for control and cells exposed to caffeine and ryanodine. The greatest divergence from control is seen in the cell exposed to 10 mm caffeine (Figure Sc). Mean relationships between current decay and action potential duration in the presence of caffeine are shown in Figure 6a. The decrease in time constant with increasing action potential duration was maintained in cells exposed to 1 mm caffeine but not in the presence of the higher dose of the drug. Exposure to 10mm caffeine greatly increased time constants of currents associated with action potentials of over 12 ms duration. Figure 6b gives values for cells exposed to ryanodine. Time constants appeared little affected by 1 aUM ryanodine. In the presence of 10Mm ryanodine time constants were qualitatively similar to those in the presence of lOmm caffeine though faster. The very large standard error of current decay associated with 12 ms action potentials in the presence of 10,uM ryanodine is probably due to the difficulty in fitting
402
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Figure 3 First (arrows where distinguishable) and steady state tail currents associated with action potentials interrupted after 12ms for:
(a) control; (b) 1mM caffeine; (c) 10mM caffeine; (d) 1pM ryanodine; (e) 10pM ryanodine. Note responses in (a) and (b) are clearly different from others. Stimulation frequency 1 Hz, 36°C.
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Figure 4 Mean first (open columns) and steady state (solid columns) Ca-activated current associated with action potentials interrupted after 12 ms for control (n = 18 cells); 1 mM caffeine (Caff, n = 6 cells); 10mM caffeine (n = 6 cells); 1pM ryanodine (Ryan, n = 10 cells) and 10pM ryanodine (n = 6 cells). *** P < 0.001, NS = not significant, paired t tests. Bars show s.e.mean.
Figure 5 Ca-activated tail currents associated with action potentials interrupted after 52 ms. Time constants of current decay (r) are given. (a) Control; (b) 1 mm caffeine; (c) 10mM caffeine; (d) 1pM ryanodine and (e) 10pM ryanodine. Stimulation frequency 1 Hz, 36°C.
an exponential to such small currents, given the signal to noise ratio of these records. One further point should also be noted: it was often observed that in control cells the time constant of the first tail current associated with action potentials of 12 ms was greater than that of the steady state (P < 0.05); for example, time constants of currents in Figure 3a were 36 and 30 ms for first and steady state respectively. The first abbreviated action potential is thought to be associated with a large release of SR Ca but reduced Ca entry. It would be predicted that this situation leads to a net extrusion of Ca from the cell by Na: Ca exchange until (during subsequent abbreviated action potentials) a steady state is established (at which point Ca entry must equal Ca extrusion, Hilgemann & Noble, 1987). It is possible that the increased amount of Ca extruded during the first abbreviated action potential results in a slower time constant of current decay as compared with the steady state. In order to test the above observations more fully we also recorded tail currents in some cells which were subsequently exposed to either 1 pM ryanodine or 10 mm caffeine (3 cells for each treatment). In all cases the observed changes in action potential shape, tail current transients, first steady-state and time constant of current decay were consistent with the above observations. This was also true in the case of 3 cells which
Ca-ACTIVATED CURRENT IN GUINEA-PIG VENTRICLE a 100
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Figure 6 Mean time constants of Ca-activated current decay. Control cells ( -l) in (a) and (b) (n = 18 cells). (a) Cells exposed to 1 mm caffeine (0. n = 6 cells); 10mM caffeine (0, n = 6 cells). (b) Cells exposed to 1 M ryanodine (0, n = 10 cells); 10pM ryanodine (S, n = 6 cells). In the presence of 1 mm caffeine time constants were not significantly different from control except at 12ms (P
