Figure 1 shows the effect of tetracaine on the amplitude of contraction and associated calcium transients in field-stimulated rat ventricular myocytes. Application of 100 μM tetracaine transiently reduced the amplitude of contraction. Contraction then gradually recovered towards the control level over a period of 1–2 min, despite the maintained presence of the drug. The magnitude of peak contraction was also more variable in the presence of tetracaine. Removal of tetracaine was associated with a transient elevation of contraction amplitude above the control level, which subsequently recovered to the control level. Similar effects of tetracaine addition and removal were seen on the systolic Ca2+ transient record (Fig. 1A, lower trace). This suggests that changes in contraction amplitude are a consequence of changes in the Ca2+ transient magnitude, and do not reflect effects of the drug on the contractile apparatus. The amplitude of the Ca2+ transient does not recover to the control level, while the contraction appears to demonstrate recovery to the control level. We cannot exclude the possibility that the lack of complete recovery of the Ca2+ transient is due to errors in correction for the intrinsic fluorescence of tetracaine (see Methods). However, the changes in the amplitude of the Ca2+ transient which occur during the maintained presence of tetracaine, and the overshoot on removal of tetracaine must be due to changes of [Ca2+]i rather than artifactual changes of fluorescence.
It is likely that the effects of tetracaine are a result of reduced calcium-induced calcium release (CICR). However, tetracaine has well-documented effects on sodium and calcium currents across the sarcolemma (INa and ICa, respectively) (Hille, 1977; Carmeliet et al. 1986). The former will decrease excitability, and this effect can be eliminated by using voltage-clamped cells. The latter will directly decrease Ca2+ entry. The experiment illustrated in Fig. 2 shows a typical result in a voltage-clamped cell. The effects of tetracaine on contraction (Fig. 2A) are qualitatively similar to those seen in field-stimulated cells: tetracaine produces an initial decrease of contraction followed by recovery and then an overshoot on removal of tetracaine. The transient overshoot of contraction amplitude on removal of the drug, in this case, was accompanied by spontaneous oscillations, indicating some degree of calcium overload at this point in the experiment. Figure 2B shows specimen records of current and contraction. The immediate major depression of contraction (b) is accompanied by a modest decrease (to 83 %) of the peak magnitude of the calcium current. This is associated with an increase in the integral of the Ca2+ current (see later). However, while contraction recovers towards control levels, there is no recovery of the calcium current (c). Similarly, the overshoot in contraction amplitude on removal of tetracaine (d) is not accompanied by an increase in peak calcium current above the control level. Changes in the peak amplitude of the calcium current, therefore, cannot explain the recovery of contraction amplitude during continued superfusion with tetracaine, nor the transient overshoot of contraction observed on its removal. On average, in twelve cells, the minimum level of contraction reached in 100 μM tetracaine was 40.0 ± 4.5 % of the control level and this recovered to 96.2 ± 2.6 % in the steady state. This level of contraction during steady-state exposure to tetracaine was not significantly different from that in control (P > 0.1). On removal of tetracaine the mean peak level of contraction reached was 170.8 ± 16 % of the control level.
Figure 2. The effects of tetracaine on contraction and membrane current in a voltage-clamped cell
A, time course of changes of contraction. Tetracaine (100 μM) was applied for the period indicated by the bar. The membrane potential was held at −40 mV, and 200 ms duration depolarizing pulses to 0 mV were applied at a frequency of 0.5 Hz. B, specimen records of membrane current (top) and contraction (bottom) obtained at the times (a-d) indicated in A.
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In an attempt to gauge to what extent inhibition of the L-type calcium current may account for changes in contraction, in Fig. 3 we compared the effect of tetracaine with that produced by deliberately reducing the calcium current, by reducing the size of the depolarizing step (from 40 to 30 mV). Reducing the size of the depolarization reduced the peak calcium current considerably more than did exposure to 100 μM tetracaine (here, to 54 % of the control peak ICa, cf. 83 % in tetracaine). However, the contraction elicited by the smaller pulse was only marginally reduced in magnitude, in contrast to the dramatic reduction of contraction amplitude observed during early exposure to tetracaine. This confirms that only a small proportion of the effects of tetracaine on contraction amplitude can be due to reduction of the L-type calcium current.
Figure 3. Comparison of the effects of tetracaine with those of decreasing the size of depolarization
In all panels the traces show (from top to bottom): membrane potential, current, cell length. In a and b, the depolarizing pulse was to 0 mV. Panel a, control; b, after 4 s exposure to tetracaine (100 μM); c, in the absence of tetracaine (depolarization to −10 mV). Membrane potential was held at −40 mV and depolarizing pulses of 200 ms duration were applied to elicit contraction.
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Measurement of Ca2+ flux balance in tetracaine
The transient elevation of contraction amplitude observed on removal of tetracaine may be indicative of an increase in the SR calcium load of the cells following exposure to tetracaine. This is further suggested by the presence of spontaneous oscillations in some cells immediately following removal of the drug (Fig. 2). Results presented later in this paper provide quantitative measurements of this increase of SR Ca2+ content. The aim of the series of experiments described below was to investigate the origin of this increase. In order to do this we have measured the sarcolemmal fluxes of calcium. As in our previous work (Negretti et al. 1995; Trafford, Díaz, Negretti & Eisner, 1997b), Ca2+ entry was measured by integrating the L-type Ca2+ current and Ca2+ efflux from the Na+-Ca2+ exchange current tail activated on repolarization (Fedida, Noble, Shimoni & Spindler, 1987; Bridge, Smolley & Spitzer, 1990). To facilitate the measurements, short (100 ms duration) pulses were used in order to minimize Ca2+ efflux during depolarization, when any Na+-Ca2+ exchange flux will be obscured by the L-type Ca2+ current. The contraction record in Fig. 4 again shows a transient reduction of amplitude followed by a recovery on application of tetracaine and overshoot on removal of tetracaine. Figure 4B illustrates sample current records. The integrated currents for this cell show that in control conditions (Fig. 4Ba) depolarization produces a gain of about 4 μmol (l cell volume)−1 Ca2+ via the L-type Ca2+ current. On repolarization there is a loss of calcium from the cell which is of the same magnitude as the initial gain. In other words, influx equals efflux. On average, in twelve cells, the Ca2+ entry during the Ca2+ current was 4.19 ± 0.43 μmol l−1 in comparison with average efflux of 4.55 ± 0.37 μmol l−1 (all fluxes expressed with respect to total cell volume). These are not significantly different (P > 0.10). The records illustrated in Fig. 4Bb show the early effects of tetracaine. Despite the reduction in the peak magnitude of the Ca2+ current, the integrated Ca2+ entry is greater (presumably due to reduced Ca2+-induced inactivation of the current, because of the smaller Ca2+ transient: Sipido, Callewaert & Carmeliet, 1995; Adachi-Akahane, Cleemann & Morad, 1996). The main effect of tetracaine on membrane current is, however, a decrease of the Na+-Ca2+ exchange current tail on repolarization due, presumably, to the decreased magnitude of the systolic Ca2+ transient. The net effect is that the cell has gained about 3 μmol Ca2+ l−1 at the end of the record shown. During the period of exposure to tetracaine, as the sizes of the systolic Ca2+ transient and contraction increase, so does that of the current tail on repolarization. Therefore, in the steady state in tetracaine (Fig. 4BC) Ca2+ entry on depolarization exactly balances the loss on repolarization, such that there is no net change of cell Ca2+. The mean data confirm that influx (4.00 ± 0.42 μmol l−1) and efflux (4.64 ± 0.38 μmol l−1) are not significantly different and, therefore, balance in tetracaine (P > 0.05) once a steady-state level of contraction is achieved. When tetracaine is removed there is a small decrease of the integrated calcium current. This is accompanied by a much larger increase of Ca2+ loss on repolarization due to the larger Ca2+ transient. On this pulse there is a net loss of cell calcium of almost 4 μmol l−1. Although not shown, as the contraction and systolic Ca2+ transient decrease towards control levels, the Ca2+ loss on repolarization decreases to control levels and Ca2+ flux balance is once again achieved. This post-tetracaine depletion of calcium presumably accounts for the gradual reduction of contraction towards the control steady-state level.
Figure 4. Transient loss of Ca2+ flux balance during application and removal of tetracaine
A, time course of the effects on contraction of applying tetracaine for the period shown by the bar. B, specimen records of membrane current (top) and cumulative integral (bottom). Membrane potential was held at −40 mV and depolarizing pulses of 100 ms duration were applied at 0.5 Hz. The Ca2+ flux traces show the cumulative integral of the calcium current (initial upward deflection) followed by a downward deflection due to the Ca2+ efflux. The records were obtained at the times (a-d) shown in A. (See Methods for calculation of sacrolemmal Ca2+ movements.) The vertical positions of the current traces have been aligned to facilitate comparison. Tetracaine produced an outward shift of holding current of 7 pA and this has been removed to facilitate comparison between records.
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The net changes of calcium illustrated in Fig. 4 take place over each cycle of contraction and relaxation. It appears that exposure to tetracaine causes a net increase of calcium by the cell, and this is balanced by a loss of calcium from the cell on its removal. The total amount of calcium gained and lost in this way can therefore be calculated by summing the net calcium flux for each cycle. An example of this type of calculation is illustrated in Fig. 5. This calculation was performed using the same data as in Fig. 4. The bar illustrates the period of tetracaine superfusion. The upper panels represent the values of Ca2+ influx and efflux, respectively, calculated from the integrals of Ca2+ current and tail current, associated with each cycle of contraction. The bottom panel illustrates the cumulative difference between influx and efflux, i.e. the total amount of calcium gained or lost by the cell since the start of the record. The data presented earlier show that, on average, in either control or tetracaine, in the steady state Ca2+ influx and efflux are equal. However, any small differences will add up with this cumulative method. We have therefore calculated the mean steady-state values of calcium influx and efflux for control, tetracaine and recontrol. These steady-state values were subtracted from the integral of each pulse in the appropriate solution. As shown in Fig. 4, application of tetracaine is associated with a reduction in calcium efflux from the cell, and a transient elevation of calcium entry above the control level, probably as a consequence of reduced calcium-induced inactivation of ICa. Both these effects will contribute to increasing the calcium content of the cell. As the cell gains calcium, contraction recovers towards the control level, and with it efflux from the cell, activated by the increasing magnitude of associated Ca2+ transients. On removal of tetracaine there is a transient elevation of the efflux integral, producing a net loss of calcium from the cell. There is also a significant undershoot of calcium entry into the cell, which will further contribute to reducing cell calcium content. The net calcium loss on removal of tetracaine calculated in this way (35.5 ± 3.3 μmol l−1, n= 11) is similar to (P > 0.05) the calculated amount of calcium gained in the presence of tetracaine (33.7 ± 3.1 μmol l−1, n= 11).
Figure 5. Net accumulation of calcium during exposure to and removal of tetracaine
The traces show (from top to bottom): calculated Ca2+ entry via the L-type Ca2+ current, Ca2+ efflux on repolarization (calculated as shown in Fig. 4), cumulative change of cell Ca2+ content. Calcium content (presumably SR) is expressed per unit total cell volume.
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In the steady state, the flux of calcium into the cell in Fig. 4 is minimally affected by 100 μM tetracaine. This is reflected in the fact that contraction amplitude recovers towards the control level in the steady state (where influx and efflux are balanced). Inhibition of the calcium current may, however, contribute to any observed reduction in the steady-state amplitude of contraction below that observed under control conditions, during prolonged exposure to tetracaine. This effect is more obvious in Fig. 6, which illustrates the effect of 200 μM tetracaine on contraction and associated sarcolemmal currents and calcium fluxes. Changes in contraction amplitude during exposure to 200 μM tetracaine follow a similar, although somewhat exaggerated, pattern as observed with lower concentrations of the drug. Exposure to 200 μM tetracaine (indicated by the bar) eventually produces a new steady-state level of contraction, which is below the control level. In this case, recovery time is also considerably prolonged. The associated ICa (Fig. 6B, top panel) is inhibited to a greater extent by tetracaine, thereby reducing calcium entry into the cell more significantly. In this case, the calcium current integral was reduced to 62.1 % of the mean control value by exposure to 200 μM tetracaine. In the same cell a reduction in the magnitude of ICa to 82.6 % of the mean control value was observed during exposure to 100 μM tetracaine. The decrease in the Ca2+ current integral during exposure to 200 μM tetracaine was reflected by a reduction in the steady-state amplitude of contraction to 54.8 % of the control level, in comparison with 85.5 % during exposure to 100 μM tetracaine.
Figure 6. The effect of 200 μM tetracaine on contraction and current
A, time course of cell shortening in response to electrical stimulation elicited by 200 ms depolarizing steps to 0 mV from a holding potential of −40 mV. Tetracaine (200 μM) was applied as indicated by the bar. B, specimen records of membrane current (top) and cumulative integral (bottom). The records were obtained at the times (a-d) shown in A. The records in a and b are the means of 10 and 5 pulses, respectively. Single pulses are shown in c and d.
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